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hemifacial spasm is a disorder of the seventh cranial nerve , and is characterized by irregular , involuntary and recurrent tonic and clonic contractions of the ipsilateral facial expression muscles . each spontaneous motor paroxysm starts with eye twitching and progresses to involve the other muscles innervated by the facial nerve . the episode characteristically begins with a series of twitches that increase in frequency and intensity , followed by a sustained spasm . the disorder is typical during adulthood and is commonly attributed to vascular compression of the facial nerve emergence at the brain stem . altogether , posterior fossa or cerebellopontine angle tumors are rare causes of hemifacial spasm , reported in less than 1% of a large series of adult patients . in children , the association of hemifacial spasm with tumors is also rare , with very few cases reported . a 6-year - old girl without any significant past medical history was admitted with intermittent involuntary twitching of the right orbicular region ; the twitching had occurred for 18 months . over time , the spasms became more tonic , and after 4 months , the spasms also involved the right orbicularis oris area . she was noted to be in good overall condition , showing a normal physical examination without fever or any other signs of infection . her height was 133 cm , and she weighed 39 kg , with a body mass index of 22.4 . the neurological examination was noteworthy for dysarthria and right hemifacial spasm [ figure 1 ] . sustained right hemifacial spasm laboratory evaluations , including a complete blood cell count , electrolytes , blood urea nitrogen and liver enzymes were within the normal range . cranial magnetic resonance imaging ( mri ) revealed a right - sided heterogeneous lesion from the midbrain to the pons , extending to the cerebellopontine angle , with a slight mass effect on the fourth ventricle [ figure 2 ] . after the neuroimaging procedures , a cerebral biopsy was performed , which revealed a pilocytic astrocytoma . the hospital ethic commission approved this case report , and the patient 's parents gave informed consent for publication . ( a ) t2-weighted coronal and ( b ) flair axial mri images showing a right - sided heterogeneous high signal lesion from the midbrain to the pons , extending to the cerebellopontine angle , with a slight mass effect on the fourth ventricle . ( c ) t1-weighted aspect of the lesion , with heterogeneous low signal and ( d ) t1-weighted post - gadolinium sagittal image showing its enhanced pattern these causes include venous sinus thrombosis , masses of the fourth ventricle , pilocytic astrocytoma , congenital or acquired cholesteatoma , tuberculous meningitis , thickening of the arachnoid membrane , neurosurgical facial nerve injury , accommodative esotropia and mandibular prognathism . masses may directly impinge on the facial nerve depending on the size and location of the tumor . some studies described cases in which the facial nerve was compressed against bony structures or tethered to arachnoid adhesions . this case shows the importance of a thorough neurological evaluation because our patient presented with subtle clinical signs of hemifacial spasm as the initial manifestation of a pilocytic astrocytoma . children who present with hemifacial spasm , even if very mild , should undergo further investigation . varicella zoster virus ( vzv ) is a neurotropic dna alpha herpesvirus that infects more than 95% of the world population . usually , varicella results from primary infection . the virus then becomes latent in ganglion neurons along the entire neuroaxis , and after reactivation , vzv moves transaxonally to the skin resulting in zoster . less often , vzv reactivation leads to neurologic complications more common in immunocompromised patients , such as vzv vasculopathy . vasculitis has , however , been reported in patients with dermatomal herpes zoster infection without trigeminal nerve involvement . the average time from rash to neurologic symptoms and signs is 4.1 months , but sporadically the patient can present with rash and stroke simultaneously . nonetheless , approximately one third of patients have no history of rash . in recent years , the number of recognized vzv vasculopathies has grown , but the diagnosis is not easy because , frequently , clinical presentation and imaging features are not specific of this entity . antiviral and antithrombotic treatment should probably be considered in stroke associated with vzv vasculopathy , but data on which to base therapeutic decisions are lacking . a 31-year - old right - handed female was admitted to the emergency department because of headache associated with visual disturbance . she had no previous history of headache and no known vascular risk factors but she used an oral contraceptive pill . on admission , she reported a 9-hour progressive pulsatile headache , with nausea and photophobia , followed by a sudden left visual field defect . neurological examination a head computed tomography ( ct ) scan was judged to be normal ( fig . the next day , the patient was observed at an outpatient consultation and subsequently admitted in the neurology ward because the headache had worsened and she felt weakness and numbness of the left limbs . she also recalled a history of thoracic rash 1 month before and she reported having had varicella at the age of 7 years . neurological examination at that time revealed slight drowsiness , mild left hemiparesis , left hemihypesthesia and homolateral homonymous hemianopia . a new ct scan showed an ischemic infarct on the territory of the right posterior cerebral artery ( fig . two days after admission , the neurological picture worsened with acute onset of left central facial palsy , visual and tactile inattention and worsening of the left motor deficit . a new ct scan showed enlargement of the ischemic lesion with involvement of the right thalamus ( fig . 1c ) and new areas of infarction involving different arterial territories , further depicted on mri ( fig . mr angiography showed an occlusion of the right posterior cerebral artery 5 mm after its origin ( fig . 2c ) . transthoracic and transesophageal echocardiography and holter ecg were within the norms , as were the of laboratory blood tests ( including hiv antibodies ) . clinical deterioration continued with further motor compromise , and although no cardiac or large vessel embolic source had been documented , facing neurological worsening , acetylsalicylic acid was substituted by low molecular weight heparin ( 60 mg subcutaneously twice a day ) 6 days after admission . the patient remained stable for a week , but then a new episode occurred , with headache and slight drowsiness associated with hemorrhagic transformation involving the thalamus but sparing the cortical territory of the posterior cerebral artery ( fig . because of the medical history of recent vzv reactivation , it was then hypothesized that the stroke could be secondary to vzv vasculopathy . however , at that time , evidence of increased intracranial pressure contraindicated the lumbar puncture . acyclovir and prednisolone were started empirically ( acyclovir 750 mg three times daily , and prednisolone 60 mg once a day for 21 days ) , with slow but steady improvement . digital subtraction angiography ( dsa ) showed narrowing at the origin of the right posterior cerebral artery and demonstrated an irregular lumen of the posterior segment of the pericallosal artery with beading - like pattern appearance ( fig . she underwent a lumbar puncture 11 days after initiation of therapy ( 27 days after admission ) . cerebrospinal fluid ( csf ) protein content was slightly increased ( 48 mg / dl ) , glucose 59 mg / dl , and a discrete mononuclear pleocytosis was detected ( 6 cells ) . vzv - dna was not detected by polymerase chain reaction , but there was an increased csf / serum ratio of vzv igg ( 3.3 ) , confirming intrathecal production of anti - vzv antibodies . motor function improved , but the patient was discharged with persistent left homonymous hemianopia and spatial inattention , unable to walk without assistance [ modified rankin scale ( mrs ) score 4 ] . after antiviral therapy , she restarted acetylsalicylic acid 150 mg once a day . on the 6-month follow - up , only a mild to moderate paresis of the left limbs was present . the patient was able to walk unassisted and look after her own affairs ( mrs score 2 ) . recognition of vzv vasculopathy was not straightforward because this is an uncommon entity and strokes of other etiologies may present with a similar neurologic picture and imaging abnormalities . on the other hand , finally , vzv - induced central nervous system ( cns ) disease seems to be rare in immunocompetent patients . despite no previous history of headache and sudden presentation of the visual deficit , migrainous infarction could be considered in our patient on account of presentation with pulsatile progressive headache in a young female and the known predilection of this complication for the posterior circulation . cryptogenic stroke , which may be responsible for as much as 50% of strokes in young adults [ 6 , 7 ] , was also considered in the absence of findings that supported cardiac embolism , atherosclerosis , arterial dissection , inflammatory or drug - induced etiologies . although there was no evidence to support the decision , anticoagulation was considered because of the stepwise deterioration of the neurological status under aspirin , with new ischemic areas in different territories , and a mr angiography not indicative of vasculitis . looking backwards , anticoagulation may not have been the most suitable therapeutic choice and should probably be avoided with in - patients with stroke and recent vzv infection , as hemorrhagic transformation occurred probably in the context of vessel wall damage but may also have been triggered by anticoagulation . the biological mechanisms are probably multifactorial : inflammation associated with systemic infection may lead to endothelial dysfunction ; in addition , the vzv virus spreads along nerve fibers and directly involves the vessels . vasculitis has been described in patients with dermatomal herpes zoster infection without trigeminal nerve involvement , as in our case . the vasculopathy typically involves the anterior circulation , but vzv vasculitis involvement of the vertebrobasilar system has also been reported . these studies suggest that vzv may enter the cns not only from trigeminal ganglia but also from other pathways . as suggested in previous reports , this case highlights the usefulness of dsa and csf / serum ratio of vzv igg for the diagnosis of vzv vasculopathy . vzv dna was not present in csf , but several studies with serial analysis revealed its presence only during the first 2 weeks of disease , while anti - vzv igg antibodies become detectable during the second week after infection . the diagnostic value of detecting anti - vzv igg antibody in csf is greater than that of detecting vzv dna . the multifocal nature of vzv vasculopathy makes biopsy a test with low sensitivity and high morbidity . when the diagnosis of vzv vasculopathy was considered , a lumbar puncture was contraindicated due to increased intracranial pressure ; nevertheless , we began treatment with intravenous acyclovir and steroids . although there are no controlled trials to access the optimal treatment schemes , we continued treatment during 21 days . duration of antiviral treatment as well as acyclovir dosage and benefit of steroids are issues that deserve further research . in conclusion , our case underlines that vasculopathy and stroke related to vzv infection should be borne in mind when dealing with stroke in young adults . in fact , available data suggest that this complication of a frequent infection may be more common than usually reported , not only after herpes zoster or varicella but also in cases of a stroke of unknown origin [ 1 , 2 ] . dsa seems to be important to the diagnosis of vzv vasculopathy , which is confirmed by intrathecal synthesis of anti - vzv igg antibody . the authors declare that there are no conflicts of interest and that no funding was received for this report .

hemifacial spasm is a disorder of the seventh cranial nerve , which is characterized by irregular , involuntary and recurrent tonic and clonic contractions of the ipsilateral facial expression muscles . this disorder affects mainly adults , and there are few cases reported in childhood . the main etiologies are vascular problems , although tumors are an important cause of hemifacial spasm via a direct or an indirect mass effect . we report a 6-year - old girl who presented with right hemifacial spasm . magnetic resonance imaging showed a lesion in the cerebellopontine angle , extending from the midbrain to the pons with a slight mass effect on the fourth ventricle . the histological examination revealed a pilocytic astrocytoma . ischemic and hemorrhagic stroke are recognized complications of varicella zoster virus ( vzv ) infections , although uncommon and poorly documented . the authors report the case of a 31-year - old woman admitted with acute ischemic stroke of the right posterior cerebral artery and a history of a thoracic rash 1 month before . aspirin and simvastatin were prescribed , but the patient suffered a stepwise deterioration the following days , with new areas of infarction on brain imaging . despite no evidence of cardiac or large vessel embolic sources , anticoagulation was started empirically 6 days after stroke onset . one week later , symptomatic hemorrhagic transformation occurred . the diagnosis of vzv vasculopathy was then considered , and treatment with acyclovir and prednisolone was started with no further vascular events . cerebrospinal fluid analysis and digital subtraction angiography findings corroborated the diagnosis . the patient was discharged to the rehabilitation center with a modified rankin scale ( mrs ) score of 4 . on the 6-month follow - up , she presented only a slight disability ( mrs score 2 ) . in conclusion , vzv vasculopathy needs to be considered in young adults with stroke . a high index of suspicion and early treatment seem to be important to minimize morbidity and mortality . anticoagulation should probably be avoided in stroke associated with vzv vasculopathy .

the definition of street children differs across countries and cultures.1 one way to identify this group is by categorizing the children s background according to their relationship to their family of origin , as follows:2,3 1 ) children on the street : this category comprises children working on the street , but who maintain more or less regular ties with their families ; 2 ) children of the street : children in this category maintain only weak relations with their families ; and 3 ) abandoned children : children in this category have no ties with their biological families and are completely on their own . with increasing awareness of governments and international agencies to the phenomenon of street children , this group of children has been identified as a highly vulnerable group that needs special attention and care . it has been reported that the number of street children range from 10 million to 100 million ; the majority of them are living in major urban areas in developing countries.1 in amman , jordan , it is estimated that 3% of children aged 1018 years were working , which is the highest percentage noted in the capital.4 according to the palestinian central bureau of statistics , 3.5% of palestinian children were working in 2001.5 more than one factor plays a role in driving children onto the streets . these include dropping out of school,2 family economic problems,3 child abuse and neglect,68 urbanization,7 broken families,9,10 peer pressure,11 as well as the role of media in promoting this phenomenon.7 the world health organization has identified that street children are prone to developing malnutrition disorders,12 sexually transmitted diseases,1 dental problems,13 psychiatric problems,14 cognitive and learning problems , unplanned pregnancies,1 as well as being prone to being trafficked.1 due to its longstanding situations of war and economic sanctions , this phenomenon is not unknown in iraq . however , there is no clear estimate of the size of the problem in iraq . since formal education is compulsory for the age group ( 612 years ) , the only clue to the magnitude of this problem is the school drop - out rate , which was found to be 1.9 million out of 4.3 million primary school - aged children.6,15 there is no evidence , neither in duhok nor in the kurdistan region , on the demographic characteristics or the psychopathology of street children in this country . this study aims to explore the demographic data and psychiatric morbidity among street children in duhok . a cross - sectional study was conducted from march 2004 to may 2005 at the zewa center for street children ( duhok , iraq)the only drop - in daycare center for street and working children in the governorate of duhok , which has about 1,300,000 inhabitants ; 60% are under 18 years of age . at the time of the study , the illiteracy rate among men was 11.6% , and among women it was 26.4%.16 almost all of the children found on the street in duhok were registered with the police directorate and social affairs directorate , and they were referred to this center for follow - up and support . this registration by these directorates only served an administrative purpose to enlist those children who worked on the street . the zewa center aimed at involving these children in educational programs , while providing social and psychological support . the focus of the center was to encourage children to attend the center on a voluntary basis . the activities in the center included drama , creative art , and music sessions that complemented the school curriculum . the center also provided medical care and follow - up services , as well as family visits . a total of 107 of children were eligible to participate ; all were included in the study except for seven children ( 6.5% ) who refused to complete the diagnostic interviews . the study was ethically evaluated by the ethical committee at duhok university ( duhok , iraq ) . a letter was sent by a social worker to the parents for consent before the child was included . the study instruments were administered by a trained social worker . a modified family map ( genogram)1719 was used to obtain the demographic and background information of the children , such as their age , gender , education level , work type , number of working hours , reasons for working ( they were asked whether family need / pressure or peer pressure were among the reasons that drove them to work on the street ) , number of years on the street , socioeconomic status , number of family members ( all members of family living in the same house were counted ) , and history of disabilities and medicosurgical problems . parental information regarding their own education ( never attended school or illiterate , or completed primary school , secondary / high school , or a university degree ) , work type , illness history , parent s death and reason of death , and child s age when the parent died was also collected . a composite score was used to determine the socioeconomic status ; this score was based on : 1 ) whether the father is employed or not ; 2 ) the house is owned , belongs to a relative , or is being rented ; 3 ) monthly income is below or above average , or no income ; 4 ) number of family members working in an income - generating activity ; and 5 ) other sources of income . every item mentioned above had a maximum score of 2 and a minimum score of 0 . a total score between 04 was considered low , 57 was average , and 810 was considered good . trained social workers were involved in the study , and they used the genogram , while also collecting information about each child s background characteristics . the children were given time to recall and answer questions ; if they were not able to answer , one of the parents or siblings was approached . children were not forced to answer questions , particularly about the death of their parents . the structured international neuropsychiatric interview for children and adolescent ( mini - kid ) tool was used to identify childhood psychiatric disorders.20 for every diagnosis assessed in the instrument , there is a key question that indicates the exclusion of the diagnosis if the child answers negatively . the mini - kid tool showed high interrater and test retest reliability , and it was good at screening for all psychiatric disorders except for dysthymia in children and adolescents aged 617 years.21 the harvard uppsala trauma questionnaire for children ( hutq - c)19 was administered to children to measure the exposure of traumatic events and their severity . children were asked to recall traumatic events , regardless of whether they were experienced , witnessed , or heard of . one score was given for every reported event , regardless of whether the child was exposed to , witnessed , or heard about the event . if the same type of event occurred more than three times during the same year , it was given a maximum score of 3 . the trauma level was determined by the sum of scores experienced , witnessed , and heard of . children were identified as being mildly traumatized ( scores 14 ) , moderately traumatized ( scores 59 ) , or severely traumatized ( scores > 9 ) . children who were found to have mental health problems were referred by the researcher to the mental health center in duhok for further management . the statistical package for the social sciences ( spss for windows , version 10.0 ; ibm corporation , armonk , ny , usa ) was used for the analyses . the study found that all of the children belonged to the children on the street category . the background characteristics of the studied street children revealed that most of them had parents who were illiterate ( 90% of fathers and 95% of mothers ) . fourteen percent of children had at least one parent that died , and 1% reported that both parents had died . ninety - eight percent reported that they worked because there was family need and/or pressure . the phenomenon was also common in children of low socioeconomic status ( 84% ) , and when there was a higher number of siblings . two - thirds of the children reported selling a variety of items ( table 1 ) . the mini - kid interviews indicated that 61% of the children had at least one psychiatric disorder ; 16% fulfilled the criteria for two or three diagnoses , and 8% for more than three diagnoses . fifty - seven percent of the children suffered from anxiety disorders , 29% fulfilled the criteria of posttraumatic stress disorder ( ptsd ) , and 10% for depression , 7% for conduct disorders , 5% for attention deficit hyperactivity disorder ( adhd ) , and 2% for tic disorders 1 ) . the study revealed no cases of psychotic disorders , drug abuse , or bipolar affective illnesses . eighteen percent of them were categorized as being mildly traumatized , 49% as moderately traumatized , and 29% as severely traumatized , as shown in figure 2 . the current study was the first to describe the phenomenon of street children in duhok . though the actual statistics pertaining to the number of street children in duhok was not known , the sample could be interpreted as being representative of the street children in the duhok governorate . at the time of the study , almost all street children who attended the zewa center were registered by the social affairs directorate and police directorate , which tracks working children . the study revealed an extremely high level of parental illiteracy when compared to what was reported in other studies conducted in developing countries . for instance , in bangladesh , only 70% of fathers and 76% of mothers of street children were illiterate.22 this high level of illiteracy , as well as the low socioeconomic status , observed among 48% of street children could explain why the children work on the street ; 98% of the children indicated that their reasons for being on the street included family need and pressure . similarly , in middle eastern countries such as sudan , syria , yemen , egypt , tunisia , and jordan , low socioeconomic status pushes children to work on the street.5 in addition , in duhok ( as is evident in other middle eastern countries ) , some cultures have dominating religions , such as islam . both these characteristics encourage children learn to take responsibilities at early ages for their family s survival and income - generating activities . the situation is different in north america , where two other background factors among street children prevail as the main causes that lead children to work on the street : low levels of parent support and increased feelings of rejection and hostility , as well as difficulties and failure at school.23 the high rate ( 61% ) of reported psychiatric problems has no relation to whether the reason for the child working on street was due to neglect , poverty , or family need and pressure.3,23,24 rather , the reasons why children work on the street are related to genetic predisposition or exposure to traumatic events , as experienced by these children . this is supported by the prevalence of ptsd among these children ; one - fourth of the children meet all of the criteria for ptsd.3,5,23,24 high levels of ptsd symptoms among the street children were consistent with the traumatized children that were noted in previous studies conducted in kurdistan.19,25,26 however , these findings have to be regarded as hypothesis - generating rather than conclusive . in addition , these children will be at risk of experiencing failures in nurturance , which are likely to be complicated by abuse and derogation on the street . furthermore , half of the street children suffered from anxiety disorders a rate that was much higher than that reported among the general population ( 10%15%),27 but this is consistent with what was reported in a previous study.24 rates of depression were also higher among the street children ( 10% ) when compared to the prevalence rate of 2%8% observed among the general population . literature evidence has indicated that there is a gender difference in depression rates , with a lower prevalence observed among males.29 since the study subjects in the current study were only comprised of boys , it is expected that the rate of depression would be closer to that of the general population , which is a rate that accounts for depression in both males and females . it was slightly higher than the prevalence of conduct disorders observed among the general population ( 5.6% ) . this may be due to high levels of stress experienced on the street , with bad friends , and due to low levels of social support.30 the study showed that 5% of children on the street had adhd , which is similar to the rate observed in the general population ( 3%5%).30 there was an absence of psychotic disorders , and this can be explained by difficulties of diagnosis or misdiagnosis with other pervasive disorder and social dysfunction of psychotic disorders in contrast to the selected sample . the nonreporting of bipolar affective illness among the studied sample could be partly due to its misdiagnosis as adhd.31 the study revealed that there was no substance abuse noted among the subjects , which was in contrast to our expectations . however , during the years of 20002003 , several cases of glue sniffing were identified among street children in duhok city , but the local authorities took active steps to prevent glue sniffing , which can partly explain the absence of glue sniffing cases among our sample.24 street children in duhok seem to be mostly comprised of children working on the street . the study concluded that family needs and financial pressures drive children to work on the street . lack of psycho - social support and a nurturing environment will lead to a child s exposure to many health problems , both mental and somatic . special attention to this risky group of children by their families , communities , schools , and the government is essential . provision of good environments at the family level , with the availability of community - based education to increase psycho - social awareness , can help decrease the amount of suffering experienced among street children . school support for street children , the provision of psychological support , as well as involving families and street children in family care programs through the adoption of hosting centers ( similar to the zewa center ) are urgently needed . however , further research is needed to test the hypotheses generated by this study , as the findings were not conclusive . it should be noted that this study was a descriptive study , and the findings were not representative of all street children and their families in duhok . sepsis is the systemic inflammatory response to infection and represents a major cause of morbidity and mortality in patients admitted to intensive care units ( icus ) . however , despite decades of research , the pathophysiology of sepsis remains incompletely understood . a critical limitation of tissue oxygen delivery due to macrocirculatory or microcirculatory failure may play a role , especially in the early phase of the disease process before resuscitation has been initiated . nonetheless , a growing body of evidence suggests that multiple organ failure ( mof ) may develop during sepsis mainly as a consequence of impaired cellular oxygen utilization . supportive data in patients include the following findings : total body oxygen consumption falls progressively with increasing severity of sepsis ; skeletal muscle tissue oxygen tension is abnormally high but normalizes during the recovery phase ; necrotic and apoptotic cell death is minimal , if it occurs at all , in most dysfunctioning organs ; and organs with limited regenerative capabilities , such as kidney , are usually able to recover to such an extent that long - term support is usually not needed . sepsis - induced mof may thus be related to a potentially reversible impairment in cellular function rather than any permanent structural damage . cellular energy production depends on three interconnected pathways : glycolysis within the cytoplasm , the krebs cycle and the electron transport chain within the mitochondria ( figure 1 ) . glycolysis is a sequence of reactions that degrade glucose to pyruvate . in the presence of oxygen , pyruvate and other fuel molecules such as fatty acids and amino acids enter the mitochondria , where they are completely oxidized within the krebs cycle . the reduced nicotinamide ( nadh ) and flavin ( fadh2 ) adenine dinucleotides transfer electrons to the respiratory enzyme complexes located in the inner mitochondrial membrane ( electron transport chain ) for the process of atp generation by oxidative phosphorylation . the electrons then flow via coenzyme q ( ubiquinone ) to complex iii , and are then transported via cytochrome c to reach complex iv ( cytochrome oxidase ) . at this final stage electron transfer through complexes i , iii and iv generates a proton gradient across the inner mitochondrial membrane that is used by atp synthase ( complex v ) to generate energy by phosphorylating adp . the complete oxidation of one molecule of glucose yields 3036 molecules of atp , two of which come from glycolysis and two from the krebs cycle . however , when oxygen is lacking , pyruvate can no longer be further oxidized within the mitochondria and is thus metabolized to lactate within the cytoplasm . glycolysis represents a much less efficient metabolic pathway compared with the krebs cycle and oxidative phosphorylation , because there is net synthesis of only two molecules of atp per molecule of glucose . because mitochondria utilize more than 90% of total body oxygen consumption to produce atp , the abnormalities in oxygen consumption described during sepsis are likely to be associated with evidence of mitochondrial dysfunction . studies conducted during the early phase of sepsis ( within the first few hours ) have produced conflicting results . nonetheless , mitochondrial structure and function were consistently shown to be impaired in a severity - dependent manner in animal models lasting at least 1216 hours . of note , atp levels were variably affected , depending on the balance between energy production and consumption , the model and possibly the tissue under investigation . in septic shock patients studied within 24 hours of icu admission , the degree of skeletal muscle mitochondrial dysfunction was associated with the severity of the disease . in this work , tissue atp levels were significantly lower in nonsurvivors than in an orthopaedic surgical control population , but they were maintained in those who survived sepsis . a reduction in energy consumption implies a reduction in cellular metabolism , which manifests clinically as organ dysfunction . rather than being viewed negatively as ' failure ' , an alternative paradigm may be advanced whereby this metabolic shutdown represents an adaptive cellular strategy . in the face of persisting mitochondrial dysfunction and reduced atp production , the cell may shift its focus to survival rather than aiming to continue normal functioning . nitric oxide ( no ) , reactive oxygen species and other inflammatory mediators are produced in excess and can directly inhibit mitochondrial respiration . no competes with oxygen in binding to cytochrome oxidase ( complex iv ) , thereby decreasing the activity of the enzyme . superoxide will react with no to generate peroxynitrite and other nitrogen species that are able to alter the structure and function of several other mitochondrial proteins , notably complex i . early cellular hypoxia may favour the competitive no - mediated inhibition of cytochrome oxidase , contributing to the earlier , if not greater , development of mitochondrial dysfunction . endocrine changes that occur during sepsis are also likely to play a role . among others , thyroid and sex hormones , insulin , glucocorticoids and leptin positively modulate mitochondrial energy production , protein synthesis and biogenesis [ 13 - 17 ] . increased incidences of the low tri - iodothyronine ( t3 ) syndrome , hypogonadism , insulin resistance , adrenal insufficiency and decreased circulating leptin levels in nonsurvivors compared with survivors have been reported during prolonged sepsis and critical illness . accordingly , depletion of respiratory complex proteins has been described in the diaphragm in a rat model of sepsis . a further mechanism could be represented by the down - regulated synthesis of new mitochondrial protein . in human volunteers , administration of bacterial endotoxin decreased blood leucocyte expression of mitochondrial respiratory chain complexes and atp synthase genes . assuming that the pathogenesis of mof during sepsis is contingent on development of mitochondrial dysfunction and cellular energetic failure , recovery is likely to occur when damaged organelles are repaired or replaced . preliminary results have shown an association between progressive improvement in mitochondrial respiration and organ function in patients who survive their episode of septic shock . strategies aimed at preventing or reversing mitochondrial dysfunction and cellular energetic failure may thus represent a new therapeutic option in the treatment of sepsis ( figure 2 ) . mitochondrial dysfunction in sepsis can occur even with aggressive fluid resuscitation and adequate tissue oxygenation . derangement in liver metabolism possibly due to mitochondrial damage was recently reported in a hyperdynamic , normotensive , mechanically ventilated , antibiotic - treated septic animal model , despite preserved microvascular perfusion . nonetheless , early cellular hypoxia can further limit aerobic production of atp and contribute to the development of energy failure . optimization of oxygen delivery can ameliorate cellular energetic failure provided that mitochondria retain their ability to produce energy . patients with severe sepsis or septic shock whose global oxygen delivery was optimized early after admission to an emergency room experienced better outcomes than did conventionally managed patients . conversely , no benefit or even harm was reported when a similar approach was adopted after admission to icu , when organ failure had already become established . the same intervention , performed at different time points , had very different clinical impacts . in the early phase , when the cellular energetic machinery is still likely to be functional and oxygen supply may represent a limiting factor , reversal of tissue hypoxia may ameliorate the impending cellular energetic failure and reduce the incidence / severity of organ dysfunction . in a later phase , when mitochondrial damage has occurred and the cell has become intrinsically unable to utilize oxygen to produce atp , a similar strategy may not provide any benefit . lack of improvement in oxygen consumption despite a re - established oxygen supply has been associated with unfavourable outcomes in patients with sepsis syndrome or septic shock . hyperglycaemia and insulin resistance are common among critically ill patients and represent an additional potential threat to mitochondrial integrity . acute hyperglycaemia can dramatically increase the production of reactive oxygen species in normal bovine aortic endothelial cells . maintenance of normo - glycaemia with intensive insulin therapy during critical illness has been demonstrated to preserve hepatocyte mitochondrial ultrastructure and function and improve outcome in both medical and surgical intensive care patients . reactive oxygen and nitrogen species are over - produced during sepsis , whereas mitochondrial antioxidants ( reduced glutathione and manganese superoxide ) are depleted . the membrane permeable glutathione ethyl ester can protect complex i from oxidative and nitrosative damage in an early phase . manganese - based superoxide dismutase mimetics may exert a similar protective effect , scavenging superoxide anions and preventing them from further reacting with no to generate peroxynitrite within the mitochondria . once permanent mitochondrial dysfunction has developed , cellular optimization of any residual ability to produce energy and/or reduce metabolic requirements may prevent the atp level from dropping below the threshold that stimulates initiation of cell death pathways . electron donors that are able to ' bypass ' defective components of the respiratory chain may help in attaining the former objective . within the inner mitochondrial membrane , complex ii works in parallel with complex i , albeit to a lesser extent , transferring electrons from fadh2 produced during the oxidation of succinate to coenzyme q. unlike complex i , the activity of complex ii is relatively preserved during sepsis . when complex i is inhibited , the administration of succinate may increase electron flow through the respiratory chain and thus increase generation of atp , provided that any inhibition of the electron transport chain distal to complex ii has not become rate - limiting . preliminary data from our laboratory confirm this action . in two different animal models of sepsis , the infusion of succinate dimethyl ester prevented the fall in liver atp content and prolonged survival time . another possible strategy that could be pursued in the face of a severe and extended impairment in mitochondrial energy production is to reduce cellular energetic expenditure . hibernating and aestivating animals reduce their metabolic rate in the face of climate change or drought . similarly , oxygen - conforming organisms such as turtles and frogs can tolerate prolonged periods of hypoxia by suppressing atp turnover . humans do not hibernate or aestivate and have only a limited tolerance to inadequate oxygenation . nonetheless , patients with chronic coronary artery disease frequently develop a myocardial contractile dysfunction termed myocardial hibernation that may represent an adaptive response to ischaemia , rather than depend on an ongoing energetic deficit , which will recover on restoration of adequate perfusion . carbon monoxide and no may mediate the active decrease in energy demand that occurs in cells that lack oxygen . the natural peptide ' hibernation induction trigger ' , its synthetic analogue [ d - ala , d - leu ] enkephalin ( dadle ) and other -opioids can also reduce cellular metabolism and protect organs against ischaemia . rapid induction of profound cerebral hypothermia in animals that do not normally hibernate may guarantee protection during prolonged cardio - circulatory arrest . mice exposed to hydrogen sulphide experience a dramatic decrease in their metabolic rate : within 6 hours , oxygen consumption and carbon dioxide production drop by around 90% , and body core temperature approaches that of the environment . such a suspended animation - like state fully reverses when the hydrogen sulphide is discontinued , without any permanent behavioural or functional damage . it is conceivable that , even during sepsis , induced hibernation may protect the organism from prolonged energetic failure and enable faster recovery on resolution of the inflammatory insult . for example , the hyperthermic response to infection activates the expression of cytoprotective heat shock proteins and may therefore be considered beneficial . premature stimulation of cellular metabolism before mitochondria have regained their ability to respond adequately in terms of energy production may lead to cellular compromise . examples of harmful therapeutic approaches that may be invoked are the use of high - dose dobutamine , thyroxine and growth hormone . repair and replacement of damaged mitochondria are probably controlled at a transcriptional level , but proximal steps in the signalling pathway still need to be elucidated . no was recently suggested to play a major role . long - term exposure to a low concentration of the gas triggered expression of transcriptional factors that regulate mito - chondrial proliferation and significantly increased mito - chondrial mass in different cells in culture . the large quantity synthesized by the inducible isoform of nitric oxide synthase ( nos ) during the acute inflammatory response to sepsis blocks mitochondrial respiration and can be cytotoxic . on the other hand , the smaller amounts of no produced by the specific constitutive endothelial nos may trigger mitochondrial biogenesis in a later phase . taken together , these results suggest that recovery from mito - chondrial dysfunction may depend on a no - dependent signalling pathway . specific inhibition of inducible nos during sepsis may represent a potential therapeutic strategy [ 52 - 55 ] , although dose selection will be critical . this is pertinent to the dose - related increase in mortality reported in a phase iii trial of a nonspecific nos inhibitor in septic shock patients . indeed , the overall negative outcome of this study camouflages the survival benefit seen with low doses . in contrast to the acute response , persistently low circulating levels of t3 during the prolonged phase of critical illness may be due to neuroendocrine dysfunction . replacement hormonal therapy given at the right time , when cells have regained the ability to both restore mitochondrial activity and increase metabolic rate , may beneficially arouse the cell and promote earlier organ recovery . however , as described above , thyroxine supplementation may prove dangerous , and so the right conditions must be in place . other hormones that could be considered in the treatment of sepsis are leptin and oestrogen . it regulates food intake and energy balance to maintain constancy of total body fat mass . in diabetic fatty rats , ectopic hyperleptinaemia triggered mitochondrial proliferation , transforming white adipocytes into mitochondria - rich , fat - oxidizing cells . administration of oestrogen or antiandrogen agents after trauma / haemorrhage also increased mitochondrial enzyme activities , protein synthesis and atp levels relative to those in sham - operated controls . this is a reversible , low - growth state well recognized in mycobacteria such as mycobacterium tuberculosis . micrococcus luteus can be aroused from its quiescent phase by an endogenous protein named ' resuscitation promoting factor ' . as mitochondria descend from a bacterial endosymbiont , the identification and application of a similar protein that can specifically stimulate mitochondrial activity may well yield beneficial results . mitochondrial dysfunction occurs during sepsis and may play a major role in the development of mof . prevention and correction of mitochondrial dysfunction and cellular energetic failure represent novel strategies that may improve clinical outcomes of septic patients . timing of any intervention appears to be critical and the possibly adaptive role of some changes currently viewed as pathological must be considered . the regulated induction of a hypometabolic state resembling hibernation may help the cell in facing a reduced capacity to generate energy . the stimulation of mitochondrial activity and biogenesis during the late phase of sepsis may accelerate the recovery process . this increasing insight into underlying mechanisms promises to be an exciting era of novel therapeutic developments . fadh2 = flavin adenine dinucleotide , reduced ; icu = intensive care unit ; mof = multiple organ failure ; nadh = nicotinamide adenine dinucleotide , reduced ; no = nitric oxide ; nos = nitric oxide synthase ; t3 = tri - iodothyronine . ms receives research funding from the medical research council and wellcome trust to undertake basic science research on mitochondria . electrons donated from nadh and fadh2 pass down the electron transport chain with oxygen being the terminal acceptor at complex iv . this movement of electrons results in a shift of protons across the inner mitochondrial membrane , generating the energy necessary for atp synthase to produce atp from adp . fadh2 , flavin adenine dinucleotide , reduced ; nadh , nicotinamide adenine dinucleotide , reduced .

backgrounddue , in part , to family constraints in dealing with the economical burden of raising a family , a wave of street children is sweeping the developing world . such children are prone to both somatic and mental illnesses . this is the first ever study that has been conducted to explore the psychopathology among street children in the duhok governorate.methodsthe study was conducted between march 2004 and may 2005 in duhok city among street children who attended the zewa center the only center for street children in the region at the time of the study . among a total of 107 eligible children , 100 agreed to participate ( 93% response rate ) . a modified family map ( genogram ) was used to obtain demographic data from the children and their caregivers through semi - structured interviews . in addition , the mini international neuropsychiatric interview for children and adolescents ( mini - kid ) structured interviews were conducted with the children.resultsthe study found that 98% of children worked on the street because of the economic need and pressure on their families . there was high rate of parental illiteracy ( 90% of fathers and 95% of mothers ) , and 61% of respondents were shown to have at least one psychiatric disorder . a high percentage ( 57% ) of these children suffered from anxiety disorders including posttraumatic stress disorders ( 29% ) . ten percent had depression , and 5% had attention deficit hyperactivity disorder.conclusionstreet children in duhok seem to be working children due to their families needs . the pathogenesis of sepsis - induced multiple organ failure may crucially depend on the development of mitochondrial dysfunction and consequent cellular energetic failure . according to this hypothesis , interventions aimed at preventing or reversing mitochondrial damage may have major clinical relevance , although the timing of such interventions will be critical to both ensuring benefit and avoiding harm . early correction of tissue hypoxia , strict control of glycaemia , and modulation of oxidative and nitrosative stress may afford protection during the initial , acute systemic inflammatory response . the regulated induction of a hypometabolic state resembling hibernation may protect the cells from dying once energy failure has developed , allowing the possibility of functional recovery . repair of damaged organelles through stimulation of mitochondrial biogenesis and reactivation of cellular metabolism may accelerate resolution of the multiple organ failure syndrome .

an efficient photocatalyst minimizes recombination of photoexcited electron hole pairs and maximizes electron and hole transfer to the adsorbate upon consecutive surface reactions . in general , the photocatalytic activity of a given particle system depends on multiple factors . for a reliable photoactivity assessment , relative contributions from the particles bulk and interfaces have to be sorted out , and their impact to the overall performance requires a careful evaluation . a major limitation of any photocatalytic process originates from charge carrier recombination . an important approach to counteract charge carrier recombination in photocatalytic particle systems is based on the coupling of different semiconductor components with desirable matching of their electronic band structures . in the case of composite nanoparticle systems , synergistic properties and those that are enhanced with respect to the individual components can only emerge if the particles are mixed intimately enough . for that reason , researchers have explored efficient and cost - effective methods for combining different metal oxide nanoparticles . chemical ways could offer great control in this respect . while a variety of experimental approaches leading to the coupling of two semiconductors have been successfully employed for particles that were grown in colloidal solutions , there are very few reports about the charge separation properties of dry particle systems that feature corresponding types of interfaces . with this study we present a simple as well as versatile approach that involves surface charging of multiple particle systems inside the same aqueous dispersion medium to achieve blended nanoparticle systems of superior mixing quality and , thus , a maximum concentration of functional interfaces between different particle types . ( a ) schematic showing the principle of hydration - dehydration induced particle aggregation and solid solid interface formation . ( b ) adjustment of opposite surface charges on multiple particle types is expected to favor preferential attraction between different particles and yields good mixing qualities . we have shown in previous work that water - mediated aggregation of tio2 and zro2 nanoparticles and subsequent dehydration procedures under high vacuum conditions are effective in the generation of solid solid interfaces . the mechanistic steps of the underlying material transformation process are as follows ( figure 1 ) : in aqueous dispersion , mutual attraction and agglomeration of metal oxide nanoparticles takes place . upon vacuum treatment dehydration / dehydroxylation processes ( evaporation of liquid phase ) in conjunction with particle condensation lead to the formation of chemical interparticle metal oxygen bonds . annealing - induced aggregation of particles during thermal treatment results in interconnected particles but , according to n2 sorption measurements , does not reduce the specific surface area in comparison to the unprocessed nanoparticle powders . schematic energy diagram showing the positions of conduction and valence band edges of tio2 and sno2 . expected vectorial charge transfer directions are indicated for electrons ( top arrow ) and holes ( bottom arrow ) . for the exploration of surface charge - induced heteroaggregation , we chose the tio2sno2 system . both materials are central to photocatalysis , solar water splitting , and dye sensitized solar cells . as a result of composite formation upon generation of heterointerfaces , light - induced charge separation and vectorial charge transfer ( figure 2 ) the conduction band position of sno2 is lower than that of tio2 and such that it is incapable of reducing oxygen molecules to form superoxide anions . the band offsets between tio2 and sno2 will promote charge separation across the interfaces . using electron paramagnetic resonance ( epr ) spectroscopy , we address the vectorial transfer of separated charges to generate paramagnetic o radicals as well as paramagnetic ti sites . another reason for selecting this system has been the exclusion of sn ti o compound formation . for sno2tio2 core shell nanostructures , it was found that the phase boundary between the two oxides remains abrupt up to t = 1200 k without substantial chemical transformations in this region . thus , heteroaggregated sno2tio2 nanoparticle networks represent an ideal model system to explore the potential of surface charge - induced heteroaggregation with regard to the generation of heterojunctions that enhance the separation of photogenerated charges . tio2 and sno2 nanoparticles were prepared by metal organic chemical vapor synthesis ( mo - cvs ) based on the decomposition of either titanium(iv ) isopropoxide ( aldrich , 99.999% trace metals basis ) or tetra - n - butyltin ( aldrich , technical grade , > 93% ) vapor at t = 1073 k in a flow reactor system . for purification , the obtained powder samples were subjected to thermal treatment under high vacuum conditions . first , the powder sample was heated to t = 870 k using a rate of r 5 k min . subsequent oxidation with o2 at this temperature followed by cooling in o2 atmosphere was successfully applied to remove organic remnants originating from the precursor material , on the one hand , and to guarantee the stoichiometric composition of the oxide on the other . the average particle size determined by transmission electron microscopy ( tem ) and nitrogen sorption was 13 nm for tio2 and 10 nm for sno2 nanoparticles . for the preparation of mixed ensembles of tio2 and sno2 nanoparticles , 250 mg of the powder samples were dispersed in 100 ml of c = 10 moll formic acid solution for 30 min under ultrasonication ( hielscher sonifier 200s ) and simultaneous agitation by a magnetic stirrer . cooling with an ice centrifugation and subsequent drying in vacuum at room temperature resulted in the formation of monolithic pieces , which were transferred to a quartz glass cell and annealed to t = 873 k at p < 10 mbar prior to spectroscopic investigations . prior to liquid exposure zeta potentials and agglomerate size distributions of suspensions of 2.5 g oxide nanoparticlesl formic acid solution were derived from dynamic light scattering measurements on a malvern zetasizer nano zs . nitrogen sorption isotherms were obtained at t = 77 k using nitrogen adsorption / desorption ( quantachrome nova 4000e ) . samples were degassed for 2 h in the degas unit of the adsorption apparatus at t = 473 k under vacuum prior to analysis . the bet surface area sbet was evaluated using adsorption data in a relative pressure range p / p0 from 0.05 to 0.2 . scanning electron microscopy ( sem ) measurements were performed on a zeiss gemini ultra 55 microscope operating at 20 kv equipped with an energy dispersive x - ray emission ( edx ) detector . to characterize the mixing quality inside the nanoparticle networks we used edx to track compositional homogeneity changes in the range of a few hundreds of nanometers . the local resolution of chemical information for edx analysis is limited to the penetration depth and scattering of the primary electrons and therefore to the volume where characteristic x - rays are emitted of the sample . with acceleration voltages of 20 kv , the sampled volume in edx studies is on the order of a few cubic micrometers for bulk tio2 or sno2 samples . consequently , the determination of absolute ti and sn concentrations via edx at the nanometer scale is not feasible . nevertheless , by scanning the aggregate with a linescan , the change in the ratio between ti and sn can be tracked with a very high spatial resolution of approximately 100 nm . for epr measurements , the powder sample was contained within a suprasil quartz glass tube connected to an appropriate high vacuum pumping system with a base pressure p = 10 mbar . the light beam was passed through a water filter to exclude ir contributions from the excitation spectrum . it was held constant at pirr = 0.9 mwcm for the energy range 3.2 ev < e < 6.2 ev throughout all experiments . during uv exposure , the samples were held at a temperature between t = 90 k and t = 140 k in order to keep uv - induced heating effects constant . x - band epr measurements were performed on a bruker emx micro spectrometer using a bruker er 4119 hs resonator . for measurements in the temperature range between t = 90 k and t = 140 k , an er 4131 vt variable - temperature accessory was used . the procedure of particle aggregation as outlined by figure 1 aims at the conversion of aerosol metal oxide nanoparticle powders , of high purity and well characterized particle surface properties , into binary metal oxide nanoparticle networks of high mixing quality and therefore with high concentrations of solid solid heterointerfaces ( figure 1b ) . prior to the process , aggregation between particles must be avoided in the starting mixture in order to achieve nanoparticle networks of different and perfectly intermixed metal oxides . dynamic light scattering experiments on dispersions of vapor phase - grown tio2 nanoparticles with an average particle size of 13 nm as determined by tem clearly demonstrate that metal oxide nanoparticles that were grown by mo - cvs can be efficiently deagglomerated by ultrasonic treatment in aqueous dispersion ( figure 3 ) . as a consequence of ultrasonification , the agglomerate size distribution maximum shifts from 100 nm ( figure 3a ) to 20 nm ( figure 3b ) . after discontinuation of ultrasonic treatment , particles reagglomerate , and the agglomerate size distribution maximum shifts to approximately 40 nm ( figure 3c ) . size distribution profiles determined by dynamic light scattering experiments on dispersions of vapor phase - grown tio2 nanoparticles with an average particle size of 13 nm in c = 10 moll formic acid . ultrasonic treatment breaks up particle agglomerates and leads to the shift of the agglomerate size distribution maximum from ( a ) 100 nm to ( b ) approximately 20 nm . after 15 min of reagglomeration , which occurs after discontinuation of ultrasonic treatment , the maximum increases to approximately 40 nm ( c ) . zeta potential measurements reveal that both oxides carry a negative surface charge in neutral aqueous dispersion ( figure 4 ) . as a result of particle aggregation , tio2 and sno2 form a nonuniform network which has a significant fraction of tio2tio2 and sno2sno2 interfaces . self - assembly of oppositely charged particles in colloidal dispersion and , in consequence , the generation of desired tio2sno2 heterointerfaces is achievable via control of the surface charge . in comparison to inorganic acids , which potentially contaminate the particle surface with ions such as cl , carboxylic acids are eligible candidates for surface charge adjustment since they can be efficiently eliminated from the particle network via oxidation at elevated temperatures . the zeta potential dependence of tio2 and sno2 nanoparticles on the formic acid concentration ( figure 4 ) prompted us to choose a formic acid concentration of c = 10 moll ( indicated by gray arrow ) where the zeta potential of tio2 and sno2 particles is + 24 mv and 9 mv , respectively . zeta potentials of tio2 and sno2 nanoparticle samples in aqueous dispersion as a function of formic acid concentration . to zeta potential measurements , all metal oxide samples were subjected to vacuum annealing and subsequent oxidation treatment at elevated temperatures to eliminate unintended effects that could originate from surface impurities . surface charge adjustment for dispersed particles in combination with control over the ph in solution shows a complex functional dependence on the formic acid concentration . this is due to the formate adsorption equilibria at the metal oxide particle surfaces which , in turn , are subject to particle concentration in the dispersion and on the concentration of the acid and , concomitantly , on the ph . ( for further details please see supporting information , figure s1 . ) dehydration and dehydroxylation as well as oxygen treatment of the obtained particle networks at elevated temperatures were applied in order to eliminate organic surface groups that previously lead to surface charging and heteroaggregation . x - ray diffraction , tem , and n2 sorption measurements were carried out to identify potential process - induced structural changes that may result from hydration- and annealing procedures . all methods unambiguously reveal that primary particle properties size , structure , and morphology have been retained ( supporting information , figure s2 ) . changes in the adsorption desorption hysteresis of nitrogen sorption experiments ( left panel of figure 5 ) clearly demonstrate that loose nanoparticles transform into a mesoporous network , which are made up from interconnected particles and show a distinct pore size distribution ( right panel of figure 5b ) . sorption isotherms ( left ) and bjh pore size distributions ( right ) of sno2tio2 nanoparticle ensembles before ( a ) and after formation of heterointerfaces the sorption isotherms and pore size distributions are shifted on the ordinate scale for 300 cmg and 3 cmgnm , respectively , for clarity . we employed sem to characterize the composition and mixing quality inside the nanoparticle networks and used edx to track compositional homogeneity changes in the range of few hundreds of nanometers . a typical edx line scan on nanoparticle networks obtained from surface charge - induced heteroaggregation in aqueous formic acid solution ( c = 10 moll ) is shown in figure 6a and reveals that the ratio of the two elements should remain constant across the scanned distance ( gray line ) . the mixing quality and thus the concentration of sno2tio2 heterojunctions is expected to be significantly higher than in the water processed networks . here , deviations of the desired ti to sn ratio point to a nonuniform distribution of the two elements ( gray line in figure 6b ) and therefore to the low mixing quality of the network . characteristic edx linescans of tio2sno2 networks obtained by heteroaggregation in ( a ) aqueous formic acid solution ( c = 10 moll ) and ( b ) water . scanned paths are shown in the sem insets . the black and red lines show the distribution of ti and sn within the scanned distance . the here presented procedure provides a very good measure of the local mixing quality within the nanoparticle networks . to obtain statistical meaningful information throughout different nanoparticle network samples , we evaluated the deviation of the desired ratio of ti / sn for approximately 600 sample spots ( figure 7 ) . deviations of 0% indicate that the chosen value of one tio2 particle per sno2 particle has been achieved . it can be shown that the nanoparticle network that originates from processing in pure water displays a very broad distribution of ti / sn deviation , while the network which was prepared in aqueous formic acid exhibits excellent mixing quality , i.e. , relatively small deviations from desired ti / sn ratio . deviations of the desired ti / sn ratios within line scans of several micrometers across the particle networks are plotted in the diagram . the samples were prepared in c = 10 moll aqueous formic acid solution ( hcooh / high mixing quality ) and water ( h2o / low mixing quality ) . a good mixing quality of sno2 and tio2 nanoparticle mixtures represents a necessary requirement for a high concentration of heterojunctions inside the resulting particle network . to verify that these solid solid interfaces are truly suitable for interfacial charge transfer , we spectroscopically probed the photoelectronic materials properties . for this purpose we utilized epr to track the photogeneration of surface trapped hole centers o , unpaired electrons in shallow trap states ti or adsorbed o2 ions ( supporting informations ) . as demonstrated by previous studies this technique provides site specific information about charge trapping sites . band gap and conduction band edge energies determine the pathway of electrons or holes from one oxide to the other ( figure 2 ) . the relative positions of the conduction band minima favor electron transfer from tio2 to sno2 . on the other hand , the valence band positions of the two oxides are such that photogenerated hole centers become accumulated on tio2 nanoparticles . epr spectra of metal oxide samples after 30 min uv / vis light exposure ( pirr = 0.9 mwcm for the energy range 3.2 ev < e < 6.2 ev ) . ( a ) simulated epr spectrum of o and ti centers on tio2 nanoparticles . ( b ) experimental epr spectrum of o and ti centers on tio2 nanoparticles . all spectra were acquired at t = 90 k and p < 10 mbar using a microwave power of pmw = 6.32 mw . in figure 8a , a simulated epr spectrum of o and ti centers is shown in comparison to an experimental spectrum ( b ) , which was acquired on dehydroxylated tio2 nanoparticles after 30 min of uv / vis exposure under high vacuum conditions . the separation of photoexcited states ( eq 1 ) is followed by persistent trapping of electrons and holes upon formation of ti and o centers , respectively ( eqs 2 and 3).123 the corresponding epr spectrum of sno2 nanoparticles shows no significant signals after irradiation and is therefore not presented here . after irradiation of a nanoparticle network of low mixing quality , the corresponding epr spectrum reveals the presence of o and ti stabilized on tio2 nanoparticles ( figure 8c ) . however , the network prepared from surface charge induced heteroaggregation in aqueous formic acid solution shows a strong signal for o stabilized on tio2 nanoparticles but no ti centers ( figure 8d ) . their absence points to an efficient interfacial electron transfer from tio2 particles to sno2 particles ( figure 2 ) upon formation of epr silent states . for corroboration of this assumption , we exposed the samples after uv exposure to molecular oxygen ( p = 10 mbar ) . in the case of tio2 nanoparticles , photogenerated electrons transfer to o2 and form paramagnetic superoxide anions ( o2 ) ( eqs 4 and 5 ) , which remain stabilized on the particle surface and give rise to characteristic epr signal signatures ( table 1 , figure 9a).45 epr spectra of preirradiated metal oxide samples after oxygen exposure ( p(o2 ) = 10 mbar ) . ( a ) epr spectrum of o and o2 species stabilized on mo - cvs tio2 nanoparticles . the spectra were acquired at t = 90 k and p < 10 mbar using a microwave power of pmw = 0.2 mw . for better comparison the redox potential of o2/o2 ( figure 2 gray dotted line , e = 4.22 ev ) is slightly above the conduction band minimum of sno2 ( ecb = 4.5 ev ) , which inhibits the transfer of sno2 related conduction band electrons to molecular oxygen and , thus , the formation of surface adsorbed o2 ions . figure 9 shows epr spectra of the preirradiated metal oxide samples after 15 min oxygen exposure at t = 90 k and subsequent pumping to p < 10 mbar . in the case of tio2 nanoparticles ( a ) , additional epr resonances are observed on the network of low mixing quality ( b ) and attributed to o2 radicals , which are stabilized on sno2 surfaces ( table 1 ) . for the network of high mixing quality , no o2 stabilized on tio2 are observed in the epr spectrum ( c ) . from the absence of o2 ions we infer that all photogenerated electrons are drained into sno2 ( figure 2 ) . this is different from tio2 , where photogenerated electrons , irrespective of whether they localize in shallow trap states ( ti ) or whether they remain in the conduction band , readily transfer to molecular oxygen . for particle systems that were only in contact with pure water and obviously , related networks contain too many regions where tio2 or sno2 particles are aggregated in such a way that interfaces between identical metal oxide particles types prevail . corresponding solid solid interfaces were found to actually enhance the recombination of photogenerated charge carriers . the quantitative analysis of persistently trapped photogenerated charges on the three different powder samples shows no concentration increase of stabilized hole centers ( o radicals ) for the water prepared network with low mixing quality in comparison to the pure mo - cvs tio2 nanoparticles ( figure 10 ) . however , a strong enhancement of the concentration of o radicals was observed for the sample of high mixing quality . the metal oxide samples were irradiated for 15 min ( pirr = 0.9 mwcm for the energy range 3.2 ev < e < 6.2 ev ) at t = 90 k and p < 10 mbar . the quantitative analysis of the yield of photogenerated charges clearly shows that the adjustment of surface charge during particle network formation allows for the achievement of high mixing qualities and enables the realization of a high concentration of heterojunctions that are vital for the separation of photogenerated electrons and holes . the here presented results clearly underline the necessity to characterize and annihilate charge recombination on photoactive materials . surface charge - directed aggregation of different types of particles and the subsequent introduction of functional interfaces is a cheap and versatile particle engineering approach to generate high concentrations of heterojunctions inside particle - based devices for photochemical application and energy production . we intentionally introduced functional particle interfaces between two types of metal oxide nanoparticles and quantitatively determined the yield of photogenerated and trapped charges . to yield blended nanoparticle systems hosting high concentrations of heterointerfaces , it is vital to prepare nanoparticles that subsequently can be easily deagglomerated in colloidal dispersion and to control the process of interface formation via surface charge induced heteroaggregation . as shown in this study , mo - cvs - grown particles can be simply dispersed by use of ultrasonic treatment to the single particle level . controlled interface formation was achieved via heteroaggregation , i.e. , via the self - assembly of oppositely charged particles in colloidal dispersion using formic acid for the adjustment of surface charge . in comparison to mixed particle systems prepared in pure water via surface charge - directed aggregation - prepared mixed particle systems show a substantially increased yield for photogenerated hole centers . the extremely high concentration of sno2tio2 heterojunctions actually gives rise to an enhanced cross section for the separation of photogenerated charges , which results from an effective interfacial charge transfer across the particle particle interfaces . the repercussions and clinical impact of this minimally invasive technological advance have been broadly highlighted in the international scientific literature and more recently expanded to pulmonology and thoracic oncology . endobronchial ultrasound ( known as ebus ) has faced greater technical bottlenecks , related to the smaller diameters of the bronchoscope , its working channel , the patients ' airways and , especially , the interface between the ultrasound and air.1 the first sectorial echobronchoscope was launched on the international market only in the middle of the first decade of 2000 . gastrointestinal endosonography ( known as eus ) , on the other hand , has been in use as a routine procedure for more than 15 years at large hospitals performing high - complexity procedures , including those in brazil.2 - 4 its diagnostic and therapeutic range has been well established for pancreatic and pelvic diseases ; mediastinal lesions can also be approached through the intrathoracic esophagus.5,6 fine - needle aspiration ( fna ) of masses and lymph nodes through the esophageal wall has been performed at specialized centers , with minimal risks of infection or bleeding and without great technical difficulty.5 - 8 the importance and usefulness of eus for the mediastinal staging of primary lung cancer has been well known since 1996.6 the main limitation of eus is its inability to access the anterior mediastinum because of the interference of air present in the trachea.1,7,8 this study aims to assess the performance of eus - guided fna in diagnosing mediastinal tumor lesions ( including lymph node enlargements ) and to describe some advantages and particularities of the technique . this observational , retrospective , and cross - sectional experience analysis reports the experience of a single endosonography service linked to a private hospital in sao paulo city between february 1997 and january 2011 . all clinical data ( including copies of radiological and endosonography images ) were obtained from the service 's computerized database . the demands for eus for mediastinal assessment purposes were spontaneous because the patients ' own physicians referred them due to pathological findings on chest - computerized tomography ( ct ) and , in some more recent cases , on positron emission tomography ( pet ) scans . for the sake of this study , patients were classified into two groups according to the purpose of the examination : group 1eus - fna performed for mediastinal staging of previously known malignant tumors ; and group 2eus - fna performed for diagnostic definition of lymph nodes or suspected mediastinal masses . no technical or logistic differences occurred when the procedure was accomplished in both groups , which always followed the same service routine . all examinations took place in an outpatient setting , under general anesthesia , starting with conventional upper digestive endoscopy . then echoendoscopy was used to identify the mediastinal lesions previously detected on radiology exams . under a direct and real - time ultrasound view , one single lesion ( the largest in cases of multiple identified lesions ) was punctured with a dedicated 22-gauge endoscopic needle . once guided into the target lesion , the needle was moved back and forth within the mass while applying suction with a 20-ml syringe . frozen - section examination was not performed during the procedure in any of the cases . the aspirated material was fixed in formaldehyde and analyzed through the cell - block technique . in case of inconclusive cytopathology results , the patient 's physician - in - charge was asked for further information on clinical monitoring , other diagnostic methods , and the respective final diagnosis in each case . approval for this study was obtained from the local institutional review board in compliance with the national health council resolution 196/96 . out of 1,639 gastrointestinal endosonographies performed during the study period , 51 ( 3.1% ) looked for mediastinal lesions . this series involved 37 ( 72.5% ) men and 14 women between 26 and 87 years old ( median 65 years ) . out of these 51 patients , 23 ( 45.1% ) manifested thoracic symptoms ( dysphagia , dyspnea , thoracic pain ) , 22 ( 43.1% ) reported nonspecific signs and symptoms ( fever and weight loss ) , and 6 ( 11.8% ) were asymptomatic . the forwarding physicians included 22 ( 43.1% ) oncologists , 18 ( 35.3% ) clinical pulmonologists and thoracic surgeons , and 11 ( 21.6% ) others ( general clinicians , digestive surgeons , and cardiologists ) . it should be highlighted that out of the 51 eus performed for mediastinal assessment purposes , 23 ( 45.1% ) happened in the final four years of the research period , and these cases were mostly referred by pulmonologists and thoracic surgeons . endoscopic alterations ( extrinsic compression ) were found in 24 ( 47.1% ) patients , three of whom already displayed esophageal stenosis . in group 1 ( with previously known malignant disease , forwarded for mediastinal staging ) , 17 patients were included , 9 with primary lung tumors , 4 with breast tumors , 2 with kidney tumors , 1 with a colon tumor , and 1 with a bladder tumor . out of these 17 patients , a previous pet scan had been done in only 4 , all of whom were considered the diameter of the punctured lesions ranged from 1.1 to 6.8 cm , with an average of 3.7 cm . their location ( mountain , 1997)9 corresponded to stations 2r ( 2 cases ) , 2l ( 1 ) , 4r ( 1 ) , 4l ( 2 ) , # 5 ( 1 ) , # 6 ( 1 ) , # 7 ( 3 ) , # 8 ( 2 ) , 10r ( 1 ) , and 10l ( 3 ) . eus - fna demonstrated metastatic involvement in 15 out of 17 ( 88.2% ) patients in group 1 . the respective lymph node stations sampled by eus were the paraesophageal ( # 8 ) and the left hilar ( 10l ) . both cases were later submitted to classical cervical mediastinoscopy , which identified metastases in lower paratracheal lymph nodes ( # 4 ) that were previously undetected through eus . group 2 ( undiagnosed lymph node enlargements or mediastinal masses ) comprised 34 patients . the diameter of the punctured lesions varied from 1.6 to 9.8 cm ( average 4.0 cm ) . their location ( mountain , 1997)9 corresponded to stations 2l ( one case ) , 4r ( 3 ) , 4l ( 1 ) , # 5 ( 11 ) , # 6 ( 5 ) , # 7 ( 6 ) , # 8 ( 6 ) , and 10l ( 1 ) . among the 34 patients in group 2 , 22 ( 64.7% ) new tumors were diagnosed through eus - fna , including epidermoid carcinoma ( 10 ) , adenocarcinoma ( 5 ) , oat - cell ( 3 ) , lymphoma ( 2 ) , sarcoma ( 1 ) , and neuroendocrine carcinoma ( 1 ) . other diagnoses established in this group included tuberculosis ( 5 ) and duplication cyst ( 2 ) . cytology was not malignant ( but without a specific diagnosis ) in three cases and inconclusive in two others these five patients were later submitted to mediastinoscopy , which identified two other cases of oat - cell carcinoma , one non - hodgkin b - cell lymphoma , and one ganglionic cryptococcosis , in addition to confirming one case of non - specific reactive lymphadenitis . figure 1 displays a flow chart that summarizes the procedures and diagnoses in this study . figure 2a illustrates a clinical case from group 1 ; figure 2b illustrates a clinical case from group 2 . despite its technical and commercial availability , eus is still rather underused in the treatment of thoracic illnesses . aside from its well - established importance for lung cancer staging,6,10 its indication extends to other clinical situations , such as mediastinal lymph node enlargement of unknown causes or primary tumor masses and cystic lesions ( for diagnostic or symptom relief purposes).11 considering each patient 's final diagnosis as the gold standard , the general sensitivity of eus - fna in our study was 88.0% , with 11.7% false negative cases . these rates still apply if the sample is limited to the 17 cases in group 1 . in a recent meta - analysis6 restricted to lung cancer cases , the general eus - fna sensitivity was 84% for metastasis detection ( n2 and/or n3 ) , with a global false - negative rate of 19% . the only other brazilian publication found that addressed this issue12 assessed 25 eus - fna performed for the sake of diagnostic clarification of mediastinal masses and lymph nodes . normal lymphatic tissue was obtained in three cases ( 12% ) and , in four others ( 16% ) , insufficient material was sampled . no data are available on other complementary methods used to define the diagnosis for inconclusive cases . the comparison between different methods ( eus , ebus , and surgical mediastinoscopy ) in mediastinal staging for primary lung cancer has been a recurrent and widely discussed theme . this technical choice depends , among other factors , on the patient 's clinical condition , the degree of suspected mediastinal involvement , the location of the primary tumor , the histological type , diameter and level of the biopsied lymph nodes , the number of samples obtained and , most importantly , the availability of different methods at each institution , as well as the respective results the local team has achieved.6,13 a larger number of recent eus have been performed at the request of chest physicians we believe this change resulted from these specialists ' recent contact with the large - scale dissemination of ebus in the international literature , particularly regarding clinical repercussions . both eus and ebus are recommended by the main thoracic oncology guidelines on the invasive mediastinal staging of primary lung cancer.6,14,15 yet other facts and peculiarities should be reminded : any invasive sampling method is more specific than ct scan and pet scan alone.16 the association between eus and ebus in the same patient reaches accuracy levels of more than 95%.17,18 these rates are quite encouraging , but combining both sets of equipment , logistics , training , and the availability of human and technical resources can hardly be justified in commercial terms . mediastinoscopy continues to be an obligatory complementary method whenever the above techniques reveal a negative result.6,14,15 some authors defend the position that if the main goal is the diagnostic confirmation of suspected metastatic disease detected through ct or pet scan , then endosonography methods ( eus and/or ebus ) , if available , are an excellent alternative , with high sensitivity and low morbidity levels . however , if the main goal of invasive staging is to confirm the absence of mediastinal involvement , in most cases , surgical mediastinoscopy seems to be the best option.13 based on mountain 's former lymph node map9 ( which was the gold standard used during the study period ) , eus can assess and obtain samples from the upper and lower paratracheal levels ( stations # 2 and # 4 ) , aortic window ( # 5 ) , subcarinal level ( # 7 ) , paraesophageal level ( # 8) , inferior pulmonary ligament ( # 9 ) , and pulmonary hilum ( # 10 ) . it should be noted that eus also permits staging ( and biopsying ) of primary pulmonary lesions when located near ( or eventually invading ) the mediastinum.19 it has also been capable of detecting ( and biopsying ) metastatic disease in subdiaphragmatic lesions , such as those affecting the left adrenal gland , celiac lymph nodes , and liver.6,8 the experience reported here includes a considerable number ( six cases ) of samples obtained from the para - aortic level ( station # 6 ) , which deserves more careful and detailed analysis . strictly speaking , station # 6 corresponds to the lymph nodes anterior and lateral to the ascending aorta , between a line tangent to the superior border and another to the inferior border of the aortic arch.9 hence , although station # 6 can indeed be visualized through eus , it is rather difficult to obtain samples through the esophageal route , as that would imply transection of the pulmonary artery or the aorta itself with the puncture needle . this location may therefore have not been very precise under the eus view , so that some lymph nodes attributed to the para - aortic position ( if not all of them ) may include lesions from stations 4l , # 5 and even # 8 . because esophageal endosonography does not offer easy anatomical reference points , the endoscopist 's experience and knowledge of regional topography are fundamental for a successful examination . identifying and sampling lesions located at the subcarinal level ( # 7 ) , for example , will hardly represent any difficulty because of its central position , which is always anterior to the middle esophagus . it is known that fna of station # 7 guided by endosonography techniques ( both eus and ebus ) does not obtain better results than a simple , blind transtracheal puncture.20 the pulmonary hilar levels ( # 10 ) , on the other hand , are frequently mixed up with the inferior paratracheal stations ( # 4 ) , especially on the right . such inadequate staging can radically change therapeutic decisions this means a possible n2 false - positive result . in lung cancer patients , if the lymph nodes of stations 10 r / l ( classified as n1 ) are unintentionally interpreted as belonging to stations 4 r / l ( classified as n2 ) , a malignant aspirate may exclude the option for a radical surgical resection and the potential for cure.8,13 there are further issues related to the routine adopted at our service , which remains limited to the puncturing of a single suspect lesion ( the largest in case of multiple identified lesions ) . hence , it is recommended that , in all cases , samples be obtained from at least two lymph node zones as recently mapped by the international association for the study of lung cancer ( ialsc),21 always including the subcarinal zone ( station # 7 ) , to improve prognostic definitions . equipment costs ( especially disposable needles ) and the learning curve for use of the technique are highlighted as the main difficulties that ebus will still have to face before achieving greater availability and widespread use.1 international and brazilian experiences with eus - fna , on the other hand , have already demonstrated the method 's ability to avoid surgical procedures ( mediastinoscopy , videothoracoscopy or even exploratory thoracotomy ) in a considerable number of patients86.3% of cases in this study . not only endoscopists but also oncologists , pulmonologists , and thoracic surgeons should consider its reliable potential and current availability .

we explored the impact of interfacial property changes on aggregation behavior and photoinduced charge separation in mixed metal oxide nanoparticle ensembles . tio2 and sno2 nanoparticles were synthesized by metal organic chemical vapor synthesis and subsequently transformed into aqueous colloidal dispersions using formic acid for adjustment of the particles surface charge . surface charge - induced heteroaggregation was found to yield blended nanoparticle systems of exceptionally high mixing quality and , after vacuum annealing , to extremely high concentrations of heterojunctions between tio2 and sno2 nanoparticles with dehydroxylated surfaces . for tracking charge transfer processes across heterojunctions , the photogeneration of trapped charge carriers was measured with electron paramagnetic resonance ( epr ) spectroscopy . on blended nanoparticles systems with high concentrations of sno2tio2 heterojunctions , we observed an enhanced cross section for interparticular charge separation . this results from an effective interfacial charge transfer across the interfaces and gives rise to substantially increased concentrations of electrons and hole centers . the here presented insights are key to the rational design of particle - based heterojunctions and mesoporous nanoparticle networks and help to engineer composite nanomaterials for photocatalysis and solar energy conversion . objectives : to disseminate transesophageal ultrasound - guided fine needle aspiration ( eus - fna ) as an alternative to investigate mediastinal tumoral lesions because it is an underused modality that has been available in brazil for more than 15 years.methods:descriptive analysis of a single endoscopy service 's experience since 1997 in the accomplishment of eus - fna for mediastinal staging of previously known malignancies ( group 1 ) or diagnostic definition of suspect lymph nodes and masses ( group 2).results : eus - fna was performed in 51 patients between 26 and 87 years of age . the diameter of the lesions ranged between 1.1 and 9.8 cm ( mean 3.9 cm ) . their location corresponded to the following stations : higher paratracheal ( 4 cases ) , lower paratracheal ( 7 ) , aortic window ( 12 ) , para - aortic ( 6 ) , subcarinal ( 9 ) , paraesophageal ( 8) , and hilar ( 5 ) . in group 1 , 17 patients had previously diagnosed primary lung ( 9 ) , breast ( 4 ) , kidney ( 2 ) , colon ( 1 ) , and bladder ( 1 ) cancer . fifteen of these punctures were positive for malignity . two others were later submitted to mediastinoscopy , which identified metastases not detected by eus - fna . group 2 comprised 34 patients . among these patients , eus - fna diagnosed 22 neoplasms , five cases of tuberculosis and two duplication cysts . cytology was inconclusive or without a specific diagnosis in five other cases . mediastinoscopy identified two undiagnosed cases of oat - cell carcinoma , one lymphoma and one cryptococcosis , and confirmed one reactive lymphadenitis . there were no complications related to the method.conclusions:eus-fna obviated the need for surgical procedures in 86.3% of cases . therefore , oncologists , pulmonologists , and thoracic surgeons should always remember the technique 's potential and availability .

prostate cancer is one of the most common cancers diagnosed in men in the united states : there were 238 590 new cases and 29 720 deaths recorded in 2013 . prostate specific antigen ( psa ) is currently used as a diagnostic biomarker of prostate cancer . psa is an androgen - regulated serine protease and a member of the tissue kallikrein family . the concentration of psa is elevated in the blood serum of prostate cancer patients . this is due to the loss of basal cells , basement membrane , and lumen architecture , thus permitting propsa to have direct access to the peripheral circulation . the simplicity of psa testing , which measures the concentration of psa in blood serum , has prompted its wide use to detect and screen prostate cancer . however , psa testing in the diagnostic gray zone does not clearly distinguish between benign prostate hypertrophy and prostate cancer . to develop new candidate biomarkers for prostate cancer , a deeper understanding of the biochemical properties of psa glycosylation is one of the most common protein post - translational modifications ( ptms ) , and approximately 8% of psa by weight is composed of n - glycans that occupy a single glycosylation site at asn69 . several papers have reported a variable degree of sialylation of psa in sera between healthy subjects and those with prostate cancer . the major isoform , consisting of over 90% of psa , has an isoelectric point ( pi ) of 6.9 , whereas the minor one has a pi of 7.2 . the glycosylation pattern at high pi was observed to have a high level of sialylation . these properties were similarly observed in a study of psa isoenzymes from the serum of patients diagnosed with prostate cancer and the serum of patients diagnosed with benign prostate hyperplasia . psa n - glycans were also reported to be mostly core fucosylated and to have a minor presence of galnac residues with the increasing pi of the psa fraction . these different glycosylation patterns at different pi s could be employed to develop new diagnostic biomarker for detecting different prostate diseases . to effectively characterize the n - glycosylation of psa , a widely used method is liquid chromatography interfaced with mass spectrometry ( lc ms or lc ms / ms ) . the applicability of these methods to investigate aberrations in glycosylation associated with several diseases has already been demonstrated . enrichment techniques , such as lectin affinity chromatography or hydrazide chemistry , and peptide labeling have also been used in conjunction with lc ms / ms to characterize and quantify the glycosylation sites of glycoproteins . here , we report the comprehensive qualitative and quantitative glycoproteomic studies of two psa isoforms by lc ms / ms . the overarching goal is to effectively identify the different glycoforms of psa prior to prostate cancer - related glycoproteomic study . our results were compared with findings of 2012 abrf glycoprotein research group ( gprg ) study , of which we were one of the participating laboratories . the purpose of the 2012 abrf study was to establish a global overview of the approaches and methodologies in glycoproteomics using a consensus among interlaboratory data . we found a comparable pattern / level of glycosylation between our results and those of the abrf study . psa and psa - high pi isoform ( psah ) samples were obtained from lee biosolutions ( st . louis , mo ) . sodium chloride and disodium phosphate were obtained from mallinckrodt chemicals ( phillipsburg , nj ) . d , l - dithiothreitol ( dtt ) , iodoacetamide ( iaa ) , and ms - grade formic acid were purchased from sigma - aldrich ( st . louis , mo ) . hplc - grade solvents , including methanol and isopropanol , were purchased from fisher scientific ( pittsburgh , pa ) . mass spectrometry grade trypsin gold was obtained from promega ( madison , wi ) . a 20 g aliquot of psa and 5 g aliquot of psah were prepared in 50 mm pbs buffer ( ph 7.5 ) ( phosphate buffered saline containing 50 mm disodium phosphate and 150 mm sodium chloride ) . in both cases , denaturation was performed at 65 c for 1 h. the samples were then reduced by adding by adding a 1.25 l aliquot of 200 mm dtt prior to incubation at 60 c for 45 min . those reduced samples were then alkylated with the addition of a 5 l aliquot of 200 mm iaa and incubated at 37.5 c for 45 min in the dark . excess iaa was consumed through the addition of a second 1.25 l aliquot of 200 mm dtt . the reaction was allowed to proceed at 37.5 c for 30 min in the dark . trypsin was added into the samples using an enzyme / substrate ratio of 1:25 w / w , and the samples were subjected to overnight incubation at 37.5 c for 18 h. to complete the enzymatic digestion , samples were subjected to microwave digestion at 45 c and 50 w for 30 min before adding a 0.5 l aliquot of neat formic acid to the samples . finally , the samples were dried and suspended in 0.1% formic acid prior to lc lc ms / ms was carried out on dionex 3000 ultimate nano - lc system ( dionex , sunnyvale , ca ) interfaced with an ltq orbitrap velos mass spectrometer ( thermo scientific , san jose , ca ) equipped with a nano - esi source . the psa and psah digests were initially online - purified using a pepmap 100 c18 cartridge ( 3 m , 100 , dionex ) . a 2 g aliquot of psa and 0.4 g aliquot of psah digests were injected into the trapping cartridges . the purified peptides were then separated using a pepmap 100 c18 capillary column ( 75 m i.d . the separation was achieved at a 350 nl / min flow rate using the following gradient conditions : 010 min , 5% solvent b ( 98% acn with 0.1% formic acid ) ; 1040 min , ramping of solvent b from 5 to 45% ; 4045 min , ramping of solvent b from 45 to 80% ; 4550 min , maintaining solvent b at 80% ; 5051 min , decreasing solvent b to 5% ; and 5160 min , sustaining solvent b at 5% . the first scan event was a full ms scan of 3802000 m / z at a mass resolution of 15 000 . the collision induced dissociation ( cid ) and higher - energy collision dissociation ( hcd ) ms / ms were performed on the eight most intense ions seen from the first ms scan event . the second scan event was the cid ms / ms of parent ions selected from the first scan event with an isolation width of 3.0 m / z , a normalized collision energy ( ce ) of 35% , and an activation q value of 0.250 . the third scan event was set to acquire hcd ms / ms of the parent ions selected from the first scan event . the isolation width of hcd experiment was set to 3.0 m / z , and the normalized ce was set to 45% with an activation time of 0.1 ms . the protein sequence and purity of prostate specific antigen were confirmed using mascot and scaffold . proteome discoverer , version 1.2 , software ( thermo scientific , san jose , ca ) was used to generate a mascot generic format file ( * .mgf ) which was subsequently employed for database searching using mascot , version 2.3.2 ( matrix science inc . , parent ions were selected from a mass range of 30010 000 da with a minimum peak count of 1 . the parameters from mascot daemon were set to search against uniprotkb ( uniprot release 2014_06 ) . oxidation of methionine was set as a variable modification , and carbamidomethylation of cysteine was set as a fixed modification . tandem ms ions were searched within a 0.8 da mass tolerance , and the peptide sequences were identified within 10 ppm . the results from mascot were imported into scaffold 3 ( proteome software , inc . , the protein identifications were based on an ion score cutoff higher than 30 and a minimum number of peptides of two . the identification of psa and psah glycopeptides was achieved by glypid 2.0 and glycoseq with additional manual confirmation . glypid 2.0 used a combination of hcd / cid scoring and precursor mass - based searching to detect and characterize glycopeptides . because trypsin is well - known to produce incomplete cleavage at c - terminal lysine residues , we searched glycopeptides for theoretical psa sequon - containing peptides . three sequences , nksvillgr , avcggvlvhpqwvltaahcirnk , and avcggvlvhpqwvltaahcirnksvillgr , have valid identifications of glycopeptides in tryptic digest psa samples . a default glycan library was modified to consider sulfated / phosphorylated versions of glycans . it contains 413 glycan compositions with combinations of glcnac , man , fuc , and neuac . glycoseq ( available as open source software at http://sourceforge.net/projects/glycoseq/ ; manuscript in preparation ) was used to determine glycan sequences ( i.e. , topology or cartoon - graph representation ) using a de novo sequencing algorithm . briefly , starting with the y1 ion , the glycan topology is grown using cid fragmentation peaks while adhering to n - linked glycan synthesis rules . all identified glycopeptides and associated glycan structures were manually confirmed . this strategy was also applied for other glycoprotein impurities that are present in the samples . the findings of these searches are discussed in detail in the results and discussion . although psa possesses a single glycosylation site at asn69 , competitive ionization of co - eluting peptides still presents a challenge that limits the ms / ms identification of glycopeptides because a lower number of glycopeptides would be subjected to tandem ms . therefore , we created an inclusion list consisting of all theoretical m / z values possibly associated with three peptide backbones , thus ensuring the acquisition of a large number of tandem ms data to facilitate identification of more glycopeptides . the theoretical m / z values were based on every theoretical peptide backbone sequence using the glycomod tool from the expasy web site . these m / z values were used to create extracted ion chromatograms ( eics ) using xcalibur qual browser 2.1 ( thermo scientific , san jose , ca ) . a mass accuracy of 5 ppm or better was then applied to confirm ions prior to manual evaluation of tandem ms data . however , only 9% of the detected ions depicted a mass accuracy of 7 ppm . the mass range was set to full ftms scan with 7-point smoothing enabled and a mass tolerance of 10 ppm allowed . prostate specific antigen in the samples analyzed here was identified with sequence coverages of 85.6 and 86.5% from psa and psah samples , respectively ( data not shown ) . peptides containing the glycosylation sequon ( n69ks ) were not detected in the proteomics analysis , thus suggesting that the n69 residue is glycosylated . protein identifications in proteomics analysis were based on an ion score cutoff higher than 30 and a minimum number of peptides of two . according to spectral count data generated by scaffold software , spectral counts of psa accounted for 86% of the total spectral count data ( 15 identified peptides ) , whereas the spectral counts of psah accounted for 78% of the total spectral count data ( 17 peptides ) . other proteins that were detected in the case of the psa sample were triosephosphate isomerase ( 6% of total spectral counts , three peptides ) , prostaglandin - h2 d - isomerase ( 3% of the total spectral count data , two peptides ) , and growth / differentiation factor 15 ( 5% of total spectral count data , two peptides ) . other proteins detected in the case of the psah sample were triosephosphate isomerase ( 12% of the total spectral count data , eight peptides ) , prosaposin ( 5% of the total spectral count data , two peptides ) , prostaglandin - h2 d - isomerase ( 2% of the total spectral count data , two peptides ) , and growth / differentiation factor 15 ( 3% of the total spectral count data , two peptides ) . furthermore , glypid analysis using these proteins did not identify any valid glycopeptides associated with above - mentioned proteins , yet they all have the glycosylation sequences . therefore , all glycopeptides reported here are unique to psa and are not originating from the other proteins observed in the lc ms / ms analysis . additionally , the y1 ions reported here are unique to psa and are not from any contaminant proteins observed in the lc since psa is known to have only one n - linked glycosylation site at asn69 , it is adequate to interpret its glycosylations with cid or hcd tandem ms . if the cid or hcd spectra include glycan fragmentation or glycopeptide diagnostic ions in addition to the expected y1 ion ( peptide + glcnac ) , then the glycopeptides associated with asn69 were confirmed . for example , a y1 ion associated with asn69 detected at an m / z value of 602 or 601 corresponded to doubly charged nksvillgr + glcnac . it is observed in low abundance ( < 8% ) when y1 ion intensities are incredibly high . however , hexose rearrangement is not the case in this study because y1 ion intensities associated with the psa glycosylation site are not that high , as can be seen in cid ms / ms scans ( supporting information figure 1 ) . in cases when the theoretical m / z values of glycopeptides were used , they were confirmed by matching the retention times of the glycopeptides that were confirmed by tandem ms . using a 5 ppm cutoff mass accuracy allowed glycopeptides that have similar molecular weights for example , a glycopeptide possessing 2 fuc residues is only 1 da different from that possessing an neuac residue , which can be differentiated using this 5 ppm cutoff . moreover , their retention time differences were also used to distinguish them , since a glycopeptide containing neuac is retained longer by lc than a counterpart glycopeptide containing 2 fuc . overall , the use of the aforementioned criteria results in an overall averaged mass accuracy of 3.05 ppm for all identified glycopeptides . in total , tryptic digestion of the samples resulted in the formation of three peptide backbones containing the glycosylation site , namely , nksvillgr ( 1723 min ) , avcggvlvhpqwvltaahcirnk ( 2621 min ) , and avcggvlvhpqwvltaahcirnksvillgr ( 3136 min ) , as shown in figure 1a . there are 56 glycopeptides observed for psa , as summarized in table 1 . because 55 of the structures were identified on the nksvillgr backbone , the discussion will be mainly focused on this peptide backbone . one missing glycoform associated with the nksvillgr backbone is hexnac3hex3dhex1neuac1 , which was detected on the avcggvlvhpqwvltaahcirnk backbone . figure 1b d illustrates averaged full ms with observed n - glycans detected on the nksvillgr there were 11 neutral n - glycans observed at 17.5 min ( figure 1b ) , 36 monosialylated and 2 sulfated / phosphorylated n - glycans were observed at 19.5 min ( figure 1c ) , and 6 disialylated the m / z values ( charge state = 3 ) of these glycopeptides were listed with confirmed glycan structures if they were supported by tandem ms data . otherwise , the most likely structures were described as previously reported . there were 32 n - glycans confirmed by tandem ms data , as shown in table 1 . total ion chromatogram of psa digests with different retention times of three peptide backbones possessing the asn69 glycosylation site ( a ) . different retention times correspond to the detection of neutral glycoforms ( b ) , sulfated / phosphorylated ( s / p ) neutral glycoforms and monosialylated glycoforms ( c ) , and disialylated glycoforms ( d ) . the identifications of glycan structures were supported by ms / ms , which are included in supporting information figure 1 . for psah , similar retention times were detected with different types of n - glycans , as mentioned above ( figure 2a ) . in total , 57 n - glycans all of the n - glycans were detected on the nksvillgr backbone except hexnac6hex4dhex2neuac1 , which was detected only on the avcggvlvhpqwvltaahcirnk backbone . figure 2b d depicts an averaged full ms with identified n - glycans associated with the nksvillgr backbone . there were 15 neutral n - glycans observed at 17.5 min ( figure 2b ) , 29 monosialylated and 2 sulfated / phosphorylated n - glycans observed at 19.5 min ( figure 2c ) , and 10 disialylated n - glycans detected at 22 min ( figure 2d ) . manual annotations of the cid ms / ms of 37 glycopeptides are depicted in supporting information figure 1 . total ion chromatogram of psah digests with different retention times of three peptide backbones possessing the asn69 glycosylation site ( a ) . different retention times correspond to the detection of neutral glycoforms ( b ) , sulfated / phosphorylated ( s / p ) neutral glycoforms and monosialylated glycoforms ( c ) , and disialylated glycoforms ( d ) . the identifications of glycan structures were supported by ms / ms , which are included in supporting information figure 1 . assigning glycan structure from cid ms / ms is achievable because the mass difference between the glycan residues of glycopeptides , such as hex , hexnac , fuc , or neuac , is rather large . also , sequencing n - glycan structures follows some rules because specific glycosyltransferases / exoglycosidases are involved in trimming or attachment of each glycan residue during the biosynthesis of n - glycans . the precursor m / z values of glycopeptides were confirmed using a 5 ppm cutoff mass accuracy because the detection of precursor ions was achieved by an orbitrap ftms analyzer . the intriguing features of n - glycans associated with psa are the presence of an galnac residue instead of gal followed by glcnac and sulfation / phosphorylation . in this study , many n - glycans with these characteristics were detected , some of which were confirmed by tandem ms , as depicted in figure 3 . the glycan fragments of a glycopeptide with m / z 1034.1406 were assigned with diagnostic ions originating from glycan residues ( figure 3a ) . the annotation of this tandem ms demonstrates that this glycopeptide is core fucosylated and monosialylated followed by either a gal or glcnac residue . the core fucosylation was confirmed on the basis of the existence of a y1 ion ( peptide backbone + glcnac ) with fucose , which has an m / z value of 675.3 . one of the fragment peaks with an m / z value of 1303.95 represents the loss of a galnac residue . also , we confirmed that there are two co - eluting isomeric glycopeptides . in the low m / z region , diagnostic ions with m / z values of 495.16 and 698.35 confirmed galnac + neuac and glcnac + galnac + neuac glycan structures , respectively . in addition , the presence of a 657.19 m / z value affirms the presence of a glcnac + gal + neuac glycan structure . therefore , the sialylation followed by gal and galnac residues co - eluted , as confirmed in this single tandem ms . also , on the basis of the assignment of fragment ions of glycans , we distinguished between the attachment of galnac followed by antenna glcnac and bisecting structures . when a series of hexnac fragmentation were detected as associated with antenna glcnac without assignment of a glc residue , regarding bisection structures , the presence of 2glcnac + man + glcnac , 2glcnac + 2man + glcnac , and 2glcnac + 3man + glcnac was detected , which correspond to m / z values of 886 , 967 , and 1048 , respectively . two examples of identified glycoforms whose structures were confirmed by cid ms / ms . the glycan fragments of hexnac5hex4dhex1neuac1 glycopeptide with m / z 1034.1406 ( a ) support the presence of a galnac residue followed by a glcnac residue on its antenna . the annotation of a sulfated / phosphorylated ( s / p ) glycopeptide , determined as hexnac5hex4dhex1s / p1 glycopeptide with m / z 963.7589 ( b ) , suggests the occurrence of sulfation / phosphorylation on the glcnac residue before the galnac residue . figure 3b illustrates annotated tandem ms of a sulfated / phosphorylated glycopeptide corresponding to m / z 963.7589 . the sulfation / phosphorylation on a glcnac residue was determined on the basis of the detection of fragment ions at m / z 1303.87 and 1344.45 , the difference of which corresponds to a doubly charged sulfate or phosphate ion . because the peak at m / z 1344.45 originated from the loss of a galnac residue , this suggests that the occurrence of sulfation / phosphorylation is on a glcnac residue . also , the presence of m / z values of 1221.05 and 1262.04 supports the occurrence of sulfation / phosphorylation on a glcnac residue . the difference between these two assignments of sulfation / phosphorylation was 0.5 da , which fits well within the mass accuracy of cid ms / ms . on the other hand , no diagnostic ions representing a sulfated / phosphorylated glycan residue were observed from hcd ms / ms , as shown in supporting information figure 2 . overall , more of core - fucosylated and/or disialylated glycoforms were identified in psah than in normal psa . moreover , highly branched glycan structures such as tri- or tetra - antennary structures were more abundant in psah than in normal psa there was a difference in the number of identified n - glycans from the three peptide backbones , as shown in tables 1 and 2 . this might be due to the fact that the ionization efficiency of peptides becomes substantially higher as the mobile phase s organic content increases . as a result , some of the glycopeptides with minor intensities would not be subjected to tandem ms experiments . quantitative results of all identified glycopeptides associated with the three peptide backbones are illustrated in supporting information figure 4a , b for psa and psah , respectively . glycopeptides with relatively low intensities associated with the nksvillgr backbone were not detected in the other two peptides backbones , which is believed to be mainly due to competitive ionization . the quantitation of all identified glycopeptides associated with the three detected peptide backbones is summarized in tables 1 and 2 . average peak areas and standard deviation values ( std ) were measured from technical triplicates . figure 4 shows the quantitation results of n - glycans on the nksvillgr backbone . there are 66 common n - glycans with 68 total identified glycoforms that were quantitatively compared between psa and psah samples . the abundances of two glycopeptides associated with the peptides avcggvlvhpqwvltaahcirnk and avcggvlvhpqwvltaahcirnksvillgr were excluded because their ionization efficiencies might be different from those of other glycopeptides associated with the peptide nksvillgr . figure 4b depicts a separate comparison of two glycoforms that demonstrated a higher abundance for psah relative to that in all other structures . the most abundant glycoform for psa was determined to be hexnac3hex4dhex1neuac1 ( 13.3% ) . four glycopeptides possessing different glycan structures , namely , hexnac3hex6neuac1 ( 9.3% ) , hexnac4hex4neuac1 ( 7.9% ) , hexnac4hex4dhex1neuac1 ( 6.8% ) , and hexnac5hex5neuac1 ( 5.7% ) were detected as the next most abundant ions . on the other hand , glycopeptides with hexnac4hex5dhex1neuac1 ( 28.9% ) and hexnac4hex5dhex1neuac2 ( 27% ) were observed at higher intensities in the psah sample . glycopeptides possessing hexnac5hex5neuac1 ( 6.8% ) , hexnac5hex4dhex1neuac2 ( 6.6% ) , hexnac5hex5neuac2 ( 5.3% ) , and hexnac5hex4dhex1neuac1 ( 5.2% ) glycans were observed as the next most abundant ions . the glycoform of hexnac5hex4dhex1s / p was identified in both psa and psah with different abundances ( 0.52 and 0.28% , respectively ) . an interesting observation related to this structure is that the summation of the relative abundances of hexnac5hex4dhex1 and hexnac5hex4dhex1s / p from psah was determined to be comparable with the intensity of hexnac5hex4dhex1s / p from psa . on the other hand , quantitative values ( relative abundance ) of n - glycans on the nksvillgr backbone . ( b ) relative abundances of two glycoforms , which shows low - abundance glycoforms for psah by separating the extremely high abundances of hexnac4hex5dhex1neuac1 and hexnac4hex5dhex1neuac2 in psah . an image representing a glycoform was added to the side for which higher quantities were observed . the abundances of the 46 common n - glycans were compared between the psa and psah samples . for example , the glycopeptides possessing hexnac3hex6neuac1 ( > 21.7-fold ) , hexnac4hex4neuac1 ( > 13.8-fold ) , hexnac4hex3dhex1neuac1 ( > 8-fold ) , hexnac3hex4dhex1neuac1 ( > 6.5-fold ) , and hexnac5hex4neuac1 ( > 5-fold ) were detected with higher intensities in the psa sample compared with that of the psah sample . on the other hand , the glycopeptides with hexnac5hex4dhex1neuac2 ( > 9.2-fold ) , hexnac4hex5dhex1neuac2 ( > 4.8-fold ) , and hexnac4hex5dhex1neuac1 ( > 3.2-fold ) were observed with higher abundances in psah than that in psa . as one of the 26 global laboratories that participated in the 2012 abrf glycoprotein research group ( gprg ) study ( the 2012 abrf study ) , the goal of the 2012 abrf study was to provide a global overview of glycoproteomics methods using a consensus among interlaboratory data . as a result , 57 twenty six data sets collected from the participating laboratories were classified into four clusters from a to d based on data similarity . the major cluster is c , where the data were summarized from 21 laboratories , including our laboratory . according to abundances , n - glycans were grouped into three categories , including major , intermediate , and minor abundance structures . they were also an abrf - participating laboratory that identified 42 glycoforms of intact psa / psah glycoprotein using hr esi / tof ms . as shown in supporting information figure 5a , the three studies reported 85 total glycoforms associated with psa and psah , with 29 common glycoforms . seventeen additional glycoforms were commonly identified between the 2012 abrf study and this study . three glycoforms were reported by behnken et al . and were observed in our study but were not described in the abrf study . our study and ten glycoforms were described in behnken et al.s study but were not observed in our study , of which four were described in the abrf study . this discrepancy may be due to the use of different methods : bottom - up gycoproteomics in this study and top - down glycoproteomics in the behnken et al . study . as shown in supporting information figure 5b , the relative abundances of 18 major / intermediate n - glycans appeared to be comparable with that in the 2012 abrf study . in particular , the n - glycans hexnac3hex4dhex1neuac1 , hexnac4hex5dhex1neuac2 , hexnac5hex3dhex1neuac1 , and hexnac5hex4dhex1neuac1 were observed with a very high overlap in intensities . from minor n - glycans , we noticed that 6 of the 13 missing structures were sulfated / phosphorylated n - glycans with very low intensities . one sulfated / phosphorylated glycoform , hexnac5hex4dhex1s / p , was reported in the 2012 abrf study as being less than 1% , but it is present in psa at approximately twice the amount as that in psah . this particular glycoform was observed in our analysis and with a similar intensity profile variation . behnken et al . reported a single analysis of quantitation ; thus , no variation was inserted ( supporting information figure 5c ) . among the 32 common glycoforms that were observed in our study , most of them showed a comparable trend of intensities between psa and psah . with regard to the 18 major / intermediate glycoforms defined by the 2012 abrf study , 16 glycoforms were observed by behnken et al . , whereas 2 major / intermediate glycoforms were not reported , including hexnac2hex5 and hexnac4hex4dhex1 . most of the major / intermediate glycoforms show comparable abundance profiles except for hexnac4hex5dhex1neuac2 , in which its abundance in psa appeared to be 4.60-fold lower when comparing this study and the 2012 abrf study . the comprehensive qualitative and quantitative glycoproteomic studies of two psa isoforms were successfully achieved by lc , there were 56 n - glycans detected for the psa sample , whereas 57 n - glycans were observed for psah . in total , three sulfated / phosphorylated neutral n - glycans were identified , which were supported by tandem ms . moreover , several galnac residues that were monitored were followed by glcnac residues on the antenna . from the quantitative results , the levels of the glycosylation patterns the glycopeptide possessing hexnac3hex4dhex1neuac1 ( 13.3% ) was observed with the highest intensity from psa , whereas the ones with hexnac4hex5dhex1neuac1 ( 28.9% ) and hexnac4hex5dhex1neuac2 ( 27% ) were predominantly present from psah . a comparable pattern / level of glycosylation was observed between the cumulative results from the 2012 abrf study and those from a specific participating laboratory . these results can be further investigated with the aim of distinguishing glycosylation patterns between healthy and prostate cancer patients . the national diabetes epidemic continues to expand , with about 1.6 million new cases each year and an overall prevalence of 23.6 million people . additional 57 million american adults are at high risk for type 2 diabetes mellitus ( t2 dm ) . the annual diagnosed diabetes incidence is projected to almost double from 8 cases per 1,000 in 2008 to about 15 in 1,000 by 2050 , with a potential total prevalence projected to increase one - fifth of the us population by 2050 . currently , 10% of americans have t2 dm , and 20% to 25% are considered prediabetic with impaired glucose tolerance and elevated fasting glucose measurements . the long - term complications of uncontrolled hyperglycemia in diabetics include neuropathy , nephropathy , retinopathy , and other chronic issues . the in - hospital morbidity of diabetics and new - onset hyperglycemics is well documented . . showed that for inpatients with new hyperglycemia or known diabetes there was a higher in - hospital mortality , increased length of stay , increased intensive care unit ( icu ) stay , increased transitional or nursing home care , a higher rate of infections , and more neurologic events compared to a normoglycemic control group . in addition , diabetes and inpatient hyperglycemia increase mortality for patient with an acute myocardial infarction [ 5 , 6 ] . the role of diabetes in outcomes of the critically ill , intensive care unit patient continues to be debated [ 710 ] . diabetics are increasingly undergoing elective and emergent surgery ; however , whether the presence of diabetes compromises surgical outcomes remains unclear . it is accepted that diabetics have an increased risk of cardiovascular disease compared to nondiabetics . have shown that diabetics undergoing general and vascular surgery with postoperative hyperglycemia exceeding 150 mg / dl had increased rates of infection . moreover , bower et al . , in a study of prospective patient registries , showed increased morbidity in known diabetic patients with and without malignancy and that diabetes was related to degree of postoperative complications in a quantitative fashion . in addition , the detriment of uncontrolled hyperglycemia in surgical patients has been investigated [ 14 , 15 ] . in specific surgical populations , particularly cardiac and vascular patients , the negative effect of diabetes on postoperative outcomes is clear . patients undergoing surgery are subject to altered carbohydrate metabolism , increased production of glucose , and increased resistance to insulin , leading to a stress - induced hyperglycemia . in cardiac surgery patients , maintenance of perioperative blood glucose levels between 125 and 200 mg dl resulted in fewer episodes of atrial fibrillation and recurrent ischemia , as well as a shorter length of stay ( los ) [ 16 , 17 ] . similarly , in the vascular population suboptimal hgb a1c levels in diabetics and non - diabetics predicted poor postoperative outcomes . diabetics undergoing lumbar spine surgery fared worse than non - diabetics for spine - specific outcomes postoperatively . however , the neurosurgical literature reveals only modest benefits linked to strict control of glycemic indexes during acute periods of brain ischemia and in specific surgical situations such as spine and tumor surgery . a diagnosis of diabetes has not been shown to be an independent risk factor for poor outcomes in general surgery . studies investigating overall surgical outcomes of patients with a known diagnosis of diabetes , irrespective of perioperative hyperglycemia , are sparse , and most of these are fraught with small sample sizes , are specific to only one surgical specialty , are or guided towards one specific outcome measure ( e.g. , surgical site infection ) . we asked the question of whether a diagnosis of insulin dependent or non - insulin dependent diabetics impacted overall postoperative morbidity and mortality in a large administrative general and vascular surgical population . we sought to answer this question through query of a comprehensive statewide surgical outcomes database . this study was categorized as exempt from review at both central michigan university ( cmu ) college of medicine and covenant healthcare . we used data from the michigan surgical quality collaborative ( msqc ) for this study . briefly , the msqc is a statewide quality improvement effort representing 52 member hospitals in the state of michigan . it is modeled after the american college of surgeons ' national surgical quality improvement program . over 200 variables are collected about surgical interventions using reliable and validated systematic sampling techniques to provide a representative sample of cases from each participating hospital . the msqc prospectively collects data on ( 1 ) thirty - day morbidity , ( 2 ) preoperative laboratory values , ( 3 ) readmissions and unplanned return to the operating room , ( 4 ) preoperative risk factors , ( 5 ) postoperative laboratory values and discharge variables , and ( 6 ) 30-day postoperative mortality . at larger hospitals , in addition , quarterly and twice - a year reports are generated for participating hospitals to aid in quality improvement . we included subjects from the msqc database from the years 2007 through 2011 who had general or vascular surgery and diabetes status recorded and were over 18 years of age . in addition , we created a secondary outcome variable , any major morbidity , from six variables in the msqc database detailing postoperative occurrences : wound , respiratory , urinary tract , central nervous system , cardiac , and other occurrences . subjects experiencing one or more of these post - operative occurrences were classified as having any major morbidity . we also used a range of variables from the msqc database to model our outcome variables . preoperative risk variables included history of diabetes , smoking , dependence on ventilator within 48 hours , copd , dialysis , steroid use , 10% weight loss within six months , and sepsis within 48 hours . subjects were classified as having a cardiac preoperative risk if they had any of the following : congestive heart failure ( chf ) within 30 days , history of myocardial infarction ( mi ) , previous percutaneous coronary intervention ( pci ) or percutaneous transluminal coronary angioplasty ( ptca ) , previous cardiac surgery , history of angina within 30 days , or on hypertensive medications . finally , we included or time , wound classification , asa class , whether cases were emergent , and type of surgery : general or vascular . chf within 30 days is defined by the msqc as chf , congestive heart failure , or pulmonary edema . a history of mi within past 6 months is defined as a history of a non - q wave or a q wave myocardial infarct in the six months prior to surgery . a history of previous pci / ptca includes balloon dilatation and stent placement ; however , it does not include valvuloplasty . previous cardiac surgery includes procedures classified as off - pump repair or utilizing cardiopulmonary bypass , valve replacement or repair , great thoracic vessel repair , cardiac transplant , repair of atrial or ventricular septal defects , left ventricular aneurysmectomy , insertion of left ventricular assist devices , and so forth ; however , it does not include insertions of pacemakers or automatic implantable cardioverter defibrillators . hypertension requiring medication is defined as a persistent elevation of systolic pressure over 140 mmhg or a diastolic pressure over 90 mmhg or requires an antihypertensive treatment , at the time the patient is being considered for surgery . emergency cases are defined as being performed as soon as possible and no later than 12 hours after hospital admission or onset of appropriate symptoms . steroid use is defined as the regular administration of oral or intravenous corticosteroids 30 days prior to surgery for a chronic condition and does not include topical , rectal , or inhalation corticosteroids or a course of short course steroids ( 10 days or less ) . we conducted binary logistic regression models for 30-day mortality ( table 2 ) for the entire sample ( model 1 ) , a subgroup of diabetics with general surgery ( model 2 ) , and a subgroup of diabetics with vascular surgery ( model 3 ) . similarly , we conducted binary logistic regression models for any morbidity ( table 3 ) for the entire sample ( model 4 ) , a sub - group of diabetics with general surgery ( model 5 ) , and a subgroup of diabetics with vascular surgery ( model 6 ) . all models were saturated models , that is , using all independent variables within each dependent variable ( mortality and morbidity ) to see which independent variables had the highest contribution to the desired outcome . all analyses were done using statistical product and service solutions ( spss ) version 20 ( ibm , armonk , ny ) . a complete demographic and variable summary of our sample is presented in table 1 . of 211,436 subjects , there were 177,430 ( 83.9% ) general surgeries and 34,006 ( 16.1% ) vascular surgeries . there were 175,639 ( 83.1% ) nondiabetics , 15,050 ( 7.1% ) insulin dependent diabetics , and 20,747 ( 9.8% ) diabetics who were dependent on oral medications . overall thirty - day mortality was two percent ( 4,083 , 1.9% ) and 13.0% ( 27,533 ) of cases were observed to have any type of major morbidity . in addition , 24% of subjects were smokers with the past 12 months , 6% had chronic obstructive pulmonary disease ( copd ) , and 50.8% had some level of cardiac risk . 84% of patients were either asa class 2 or 3 , and 52.7% of wounds were classified as clean . our goal was to determine if specific patient characteristics influenced mortality and morbidity and so we began by running regression analyses on these variables . interestingly , our preliminary analyses indicated that smoking is not associated with an increased risk of mortality . while this is unexpected based on most other studies that associate smoking with increased mortality , in our sample , there is a negative correlation between smoking and diabetes . in addition , smokers were more likely to have anesthesia types other than general anesthesia . in both cases ( diabetes and anesthesia other than general ) the risk of mortality is decreased and the association of smoking with these characteristics likely impacts the influence of smoking . due to the high multicollinearity of smoking with several other characteristics , we removed smoking as a variable from the regression analyses reported in section 3.2 . we regressed the 30-day mortality variable using three binary logistic regression models ( table 2 ) to determine the ability of different variables to predict mortality in diabetic subgroups of general and vascular surgery patients . model 1 uses the entire sample . in this case , those subjects who died within 30 days were less likely to be oral ( or = 0.78 , 95% ci 0.700.86 ) or insulin ( or = 0.83 , 95% ci 0.750.92 ) dependent diabetics than not diabetic . in our sample , diabetes does not increase the risk of 30-day mortality . vascular surgery ( compared to general surgery ) is a significant predictor of mortality ( or = 1.57 , 95% ci 1.421.72 ) for the entire group . due to our interest in the diabetic population , we conducted further subgroup analyses for 30-day mortality using only diabetic patients . model 2 ( table 2 ) is a subgroup analysis of diabetic , general surgery subjects . in this case model 3 ( table 2 ) is a subgroup analysis of diabetic , vascular surgery subjects . as with the general surgery subgroup , there is no difference between oral and insulin dependent vascular surgery , diabetics in survival . moving from the consideration of diabetes as a variable , we also included our other variables in the analyses . in general and as we would expect , ventilator dependence , copd , cardiac risk factors , current dialysis , steroid use , weight loss , sepsis , emergent cases , increased asa class , and any wound classification increase the risk of 30-day mortality . in summary , our models for 30-day mortality show that for our entire sample , the presence of diabetes does increase the risk of 30-day mortality . there do not appear be significant differences in variables that contribute to mortality , when comparing models for diabetic subgroups of general and vascular surgery and , as expected , ventilator dependence , copd , current dialysis , weight loss , sepsis , emergent cases , increased asa and any wound classification increase the risk of 30 day mortality in diabetic patients . we also regressed the any morbidity variable using three binary logistic regression models ( table 3 ) to determine the ability of different variables to predict any type of morbidity in different subgroups of surgery patients . subjects with any type of morbidity were more likely to be insulin dependent ( or = 1.11 , 95% ci 1.061.16 ) diabetics than oral dependent diabetics , or not diabetic . vascular surgery ( compared to general surgery ) is also a significant predictor of morbidity ( or = 1.69 , 95% ci 1.621.75 ) . model 5 ( table 3 ) is a subgroup analysis of diabetic , general surgery subjects . in this case , insulin dependent diabetics are more likely to have any morbidity ( or = 1.20 , 95% ci 1.121.29 ) when compared to diabetics who are dependent on oral medications . model 6 ( table 3 ) is a subgroup analysis of diabetic , vascular surgery subjects . again , we found that insulin dependent diabetics are more likely to have any morbidity ( or = 1.24 , 95% ci 1.121.38 ) when compared to oral medication dependent . again , considering other variables across all models for any morbidity , we found that in general , ventilator dependence , copd , cardiac risk factors , current dialysis , steroid use , weight loss , sepsis , emergent cases , increased asa class , and any wound classification increase the risk of any morbidity . in summary , models for any morbidity show that for our entire sample , insulin dependent diabetics have an increased risk for any morbidity ( or = 1.11 , 95% ci 1.061.16 ) when compared to non - diabetics . this increased risk is also present in subgroup analyses comparing insulin dependent diabetics to oral medication dependent diabetics for general ( or = 1.20 , 95% ci 1.121.29 ) and vascular ( or = 1.24 , 95% ci 1.121.38 ) surgeries . , there do not appear be significant differences in variables that contribute to morbidity , when comparing models for diabetic subgroups of general and vascular surgical patients . as expected , ventilator dependence , copd , current dialysis , weight loss , sepsis , emergent cases , increased asa and any wound classification increase the risk of any morbidity in diabetic patients . our objective was to understand the postoperative risk for morbidity and mortality in diabetics versus non - diabetic patients undergoing general or vascular surgery . we found that , with regard to mortality , the presence of diabetes is not predictive of mortality ; however , vascular surgery itself is predictive of mortality . there are no significant differences in variables that contribute to mortality , when comparing diabetic and non - diabetic subgroups for both general and vascular surgery . in our morbidity analysis , insulin dependent diabetics have an increased risk for any morbidity compared to non - diabetics or non - insulin ( oral ) dependent diabetics in the general and vascular surgery subgroups . our multivariate analyses confirm findings in the literature of risk factors in general surgery that contribute to morbidity and mortality , such as ventilator dependence , copd , cardiac risk factors , dialysis , steroid use , significant weight loss , sepsis , emergent cases , and increasing asa class . interestingly , notable differences were detected when analyzing separate groups of diabetics and non - diabetics undergoing general surgery . for instance , male non - diabetics undergoing general surgery have an increased risk for any morbidity as compared male diabetics . it is possible that higher risk male diabetics were more likely denied elective surgical intervention , as opposed to higher risk non - diabetic males , resulting in our finding . as in the mortality analysis , there do not appear be significant differences in variables that contribute to morbidity for diabetics and non - diabetics specifically undergoing vascular surgery . this is the first study in the literature of overall post - operative outcomes ( morbidity and mortality ) of diabetics undergoing general surgery with such a large population of patients . examined 55,000 diabetic patients in the veterans heath administration national surgical quality improvement program database and analyzed the effect of different levels of preoperative hyperglycemia on the rate of wound infection . however , in their cohort , there was no comparison of diabetics to a non - diabetic control group , as that was not the purpose of their study . within their study , they showed that higher preoperative serum glucose levels correlated with worse wound - related outcomes ; however , preoperative hba1c ( long - term glucose control in diabetics ) had no association with infection rates . in a study of preoperative hba1c levels , o'sullivan found that , after multivariate analysis , elevated hba1c levels were predictive of morbidity in non - diabetic patients , but not in diabetics , undergoing vascular surgery . the o'sullivan study does highlight the disturbing problem of undiagnosed diabetics undergoing surgery without proper control of their glycemia . the lack of correlation between diabetic diagnosis and mortality in general surgery in our study is not unique in the literature , where a clear consensus on the issue can not be found . . showed in a study of over 600 patients undergoing hepatic resection that multivariate analysis did not identify diabetes as an independent variable having an impact on mortality and that overall complications were equally frequent between diabetic and non - diabetic patients . diabetes was also shown not to be a statistically significant predictor of overall mortality or cardiac morbidity in patients undergoing open aortic aneurysm repair . in a matched cohort retrospective review , diabetes did not increase the mortality rates of cardiac surgery , but increased the risk for renal and neurological complications , blood transfusion , reoperation , and length of icu stay . however , a smaller study than ours from finland , with a long 7-year followup , showed quite convincingly that short - term and long - term mortality was higher in diabetics versus non - diabetics undergoing similar noncardiac surgeries . described a high mortality , due mainly to cardiovascular complications , in cohort of diabetics undergoing noncardiac surgery ; however , there was no non - diabetic control group in the study . our study revealed that the presence of cardiac risk predicted overall morbidity in diabetic and non - diabetics undergoing general and vascular surgery equally . however , cardiac risk predicted mortality only in non - diabetic general and vascular surgery patients . conversely , hollenberg et al . showed that diabetes was one of five risk factors predicted of cardiac events in patients undergoing non - cardiac surgery . one difference that may account for the results is that the latter study began from the premise of a population of patients that were already high risk for a cardiac event occurring and underwent non - cardiac surgery . in contrast , our study evaluated all patients from the perspective of being diabetic or not and assessing factors predictive of morbidity and mortality in diabetic versus non - diabetic , that is , a difference of study design to test a different hypothesis . most studies regarding surgical outcomes of diabetics involve studying the presence of perioperative hyperglycemia in diabetics or non - diabetics and its effect on morbidity and mortality . currently , the literature regarding optimal target glucose ranges for the perioperative patient is unclear . the ada recommends screening for diabetes preoperatively in select populations , especially in obese patients . the united states preventive services task force ( uspstf ) recommends screening only hypertensive adults for diabetes . some of the first studies to evaluate the benefits of tight glucose control were the leuven study in 2001 and 2006 , which sparked widespread interest in intensive insulin therapy in critically ill patients [ 33 , 34 ] . some researchers relate the benefits identified in this study to our current understanding of the bodies stress response . in the leuven study , the majority of the critically ill patients studied had spent more than five days in the intensive care unit . the effects of an anabolic steroid , such as insulin , may be more beneficial as the response to the acute injury resolves . however , during the acute onset of stressful stimulus such as surgery or possibly traumatic events the effect of treatment with these hormones has not been fully explained and may be harmful . despite the fact , improvements in surgical outcome have been seen in patients with tight intraoperative glucose control . as previously mentioned , a growing body of evidence suggests that even small changes in glycemic ranges are associated with impaired outcomes . in one study , in patients undergoing cardiac surgery , a 20 mg / dl increase in mean intraoperative glucose was associated with increased risk of more than 30% of adverse outcome . however , it is important to make the distinction that these findings can not be generalized to general surgical patients . the broad array of patient demographics and options for surgical care encompass an extensive variety of settings , and as many operations performed by the modern general surgeon are now performed in the ambulatory setting , the assessment and management of the dysglycemia patient continue to be a looming challenge . the msqc is an excellent source of data for studying general and specific 30-day morbidity for various surgical procedures on a larger scale . the administrative database provides risk and reliability adjusted reports for over 52 participating hospitals in the state of michigan . the other option to answer our question would have been to use the national surgical quality improvement program ( nsqip ) database or the national inpatient sample ( nis ) hospital discharge data . the nis system , although widely available and currently the largest all - payer database of hospital discharge records , poses a number of limitations . first , inconsistencies across states and providers in the data element reporting may compromise data quality . second , other data elements that would be needed to analyze risk factors for certain preoperative patient populations , such as race / ethnicity , detailed test results , whether a condition was present on admission , functional status , severity of illness , and behavioral risk factors , would be absent in many cases . first , collaboration in the msqc is purely voluntary , and these hospitals may have different characteristics from those who have chosen not to participate . therefore , our results may not be generalizable to the entire cohort of statewide hospitals . moreover , michigan hospital practices may not be representative of us hospitals , although this database includes academic and community as well as various sizes and geographic locations within the state . secondly , data in the msqc is collected retrospectively and analyzed by the end - user retrospectively and therefore will have the biases inherent to such a study design . third , the present analysis does not address procedure specific outcomes for diabetes . for example , although a cholecystectomy may not have the level of morbidity or mortality as a pancreatic operation for a diabetic , the volume of cholecystectomies in the database outweighs that of complex pancreatic operations . although cases are easily distinguished between vascular and nonvascular surgeries , we did not separate out or group the 3000 cpt codes to identify procedure specific risk . in summary , we found that a diagnosis of diabetes ( insulin or oral dependent ) did not affect outcomes in general surgical patients . however , the presence of insulin - dependent diabetes was associated with increased morbidity in the vascular and general surgery subgroups greater than non - diabetics or non - insulin dependent diabetics . however , the reassuring fact that the presence of diabetes did not have an overt effect on mortality , along with morbidity risk of insulin - dependent diabetes , may greatly impact the surgeon - patient preoperative dialogue and our ability to appropriately risk - stratify and risk - optimize these patients .

prostate specific antigen ( psa ) is currently used as a biomarker to diagnose prostate cancer . psa testing has been widely used to detect and screen prostate cancer . however , in the diagnostic gray zone , the psa test does not clearly distinguish between benign prostate hypertrophy and prostate cancer due to their overlap . to develop more specific and sensitive candidate biomarkers for prostate cancer , an in - depth understanding of the biochemical characteristics of psa ( such as glycosylation ) is needed . psa has a single glycosylation site at asn69 , with glycans constituting approximately 8% of the protein by weight . here , we report the comprehensive identification and quantitation of n - glycans from two psa isoforms using lc ms / ms . there were 56 n - glycans associated with psa , whereas 57 n - glycans were observed in the case of the psa - high isoelectric point ( pi ) isoform ( psah ) . three sulfated / phosphorylated glycopeptides were detected , the identification of which was supported by tandem ms data . one of these sulfated / phosphorylated n - glycans , hexnac5hex4dhex1s / p1 was identified in both psa and psah at relative intensities of 0.52 and 0.28% , respectively . quantitatively , the variations were monitored between these two isoforms . because we were one of the laboratories participating in the 2012 abrf glycoprotein research group ( gprg ) study , those results were compared to that presented in this study . our qualitative and quantitative results summarized here were comparable to those that were summarized in the interlaboratory study . aims . preoperative diabetic and glycemic screening may or may not be cost effective . although hyperglycemia is known to compromise surgical outcomes , the effect of a diabetic diagnosis on outcomes is poorly known . we examine the effect of diabetes on outcomes for general and vascular surgery patients . methods . data were collected from the michigan surgical quality collaborative for general or vascular surgery patients who had diabetes . primary and secondary outcomes were 30-day mortality and 30-day overall morbidity , respectively . binary logistic regression analysis was used to identify risk factors . results . we identified 177,430 ( 89.9% ) general surgery and 34,006 ( 16.1% ) vascular surgery patients . insulin and noninsulin diabetics accounted for 7.1% and 9.8% , respectively . insulin and noninsulin dependent diabetics were not at increased risk for mortality . diabetics are at a slight increased odds than non - diabetics for overall morbidity , and insulin dependent diabetics more so than non - insulin dependent . ventilator dependence , 10% weight loss , emergent case , and asa class were most predictive . conclusions . diabetics were not at increased risk for postoperative mortality . insulin - dependent diabetics undergoing general or vascular surgery were at increased risk of overall 30-day morbidity . these data provide insight towards mitigating poor surgical outcomes in diabetic patients and the cost effectiveness of preoperative diabetic screening .

recombinant human tgf-2 was obtained from r&d systems ( minneapolis , mn , usa ) . ( st . louis , mo , usa ) and calbiochem - emd millipore ( temecula , ca , usa ) , respectively . as to primary antibodies , fascin was from millipore ( temecula , ca , usa ) , e - cadherin from bd transduction laboratories ( lexington , ky , usa ) , active -catenin ( clone 8e7 ) from upstate ( lake placid , ny , usa ) , mmp9 and gapdh from abcam ( cambridge , ma , usa ) , -sma fluorescein isothiocyanate ( fitc ) conjugated and unconjugated from sigma - aldrich corp . , pan actin from abcam . all secondary antibodies for immunofluorescence staining were purchased from molecular probes ( invitrogen , eugene , or , usa ) ; secondary antibodies for western blots were obtained from li - cor biosciences ( lincoln , ne , usa ) . rats for this research were obtained and cared for in accordance with the institutional animal care and use committee and canadian council of animal care rules . all animal procedures were carried out in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the ethics committee on animal research of mcmaster university ( hamilton , ontario , canada ) . lens epithelial explants were obtained from 17- to 19-day - old wistar rats ( charles river laboratories , montreal , canada ) as previously described . briefly , rat lenses were isolated and placed in 35-mm culture dishes containing prewarmed , serum - free medium m199 , supplemented with antibiotics ( all from invitrogen ) . the posterior suture of the lens was located and a small incision was made to remove the posterior capsule . this revealed the fiber mass , which was then gently peeled off the anterior epithelium . once separated , the epithelium was then pinned to the culture dish with a blunt tool exposing lecs to culture medium . twenty - four hours after explanting , confluent epithelial explants were left untreated in serum - free m199 or treated with tgf-2 at 6 ng / ml for 48 hours . -catenin inhibitors icg-001 and pnu-74654 were dissolved in dimethyl sulphoxide ( dmso ) at a final concentration of 17.6 and 10 mm , respectively . the working concentration for icg-001 and pnu-74654 was 10 m ( 1.14 l/2 ml ) and 20 m ( 4 l/2 ml ) , respectively . the working concentration of icg-001 and pnu-74654 was determined based on dose response experiments performed on lens epithelial explants . for -catenin inhibitor studies , lens explants were pretreated with icg-001 ( 5 m ) or pnu-74654 ( 20 m ) for 1 hour before coincubating with tgf-2 ( 6 ng / ml ) for 48 hours . lens explants were fixed with 10% neutral buffered formalin for 15 minutes and washed with phosphate - buffered saline ( pbs ; invitrogen , carlsbad , ca , usa ) . explants were then detached from the culture dish and permeabilized at room temperature for 1 hour in 0.1% triton x-100 , 0.5% sodium dodecyl sulphate ( sds ) , and 5% donkey serum , followed by incubating overnight at 4c with primary antibody at a dilution of 1:200 in pbs . following further pbs washes , explants were incubated with corresponding secondary antibody , alexa fluor 488 or alexa fluor 568 ( invitrogen ) , at a dilution of 1:200 at room temperature for 1 hour , and washed three times with pbs ( 10 minutes for each wash ) . the explants were then mounted in prolong gold antifade reagent with 4,6-diamidino-2-phenylindole ( dapi , invitrogen ) to visualize nuclei . immunostained explants were analyzed using a zeiss 510 meta confocal microscope ( carl zeiss canada , toronto , canada ) or zeiss apotome inverted fluorescence microscope ( carl zeiss canada ) or leica dmra2 fluorescence microscope ( leica microsystems canada , richmond hill , canada ) equipped with a q - imaging retiga 1300i fast digital camera ( q - imaging , surrey , bc , canada ) . minor adjustments were made to the captured images using photoshop cs3 ( adobe systems , mountain view , ca , usa ) . lens explants ( 57 per sample ) were lysed in triton lysis buffer ( 50 mm tris , ph 8.0 , 150 mm nacl , 1% triton x-100 ) with complete mini , edta - free protease / phosphatase inhibitor cocktail ( roche , laval , qc , canada ) . equal amounts of total protein ( 5 g ) were loaded on a gel , as determined by dc protein assay ( bio - rad , mississauga , on , canada ) , and sds - page was performed . resolved bands were transferred onto a nitrocellulose membrane ( pall life sciences , pensacola , fl , usa ) . membranes were blocked with odyssey blocking buffer ( li - cor biosciences ) for 1 hour and incubated with primary antibody overnight at 4c . primary antibodies were diluted in tris - buffered saline with 0.1% tween-20 ( tbst ) at a dilution of 1:500 for fascin ( abcam ) , 1:1000 for e - cadherin ( millipore ) and mmp9 ( abcam ) , and 1:10,000 for -sma ( sigma - aldrich corp . ) and actin and gapdh ( abcam ) . blots were washed in tbst and probed with corresponding li - cor near - infrared respective secondary antibodies ( either 680- or 800-nm wavelengths ) at a dilution of 1:10,000 for 1 hour . the blots were then washed with tbst and scanned using odyssey near - infrared scanning system ( li - cor biosciences ) . densitometry on visualized bands was performed using fiji image processing software ( imagej , http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . the peaks from the densitometric scans ( imagej ) were used to calculate the average density and the ratio between protein of interest and loading control . the average band density was further used to make graphs and calculate standard deviation using graphpad prism software ( la jolla , ca , usa ) . unpaired tukey 's multiple comparison test was performed on the averages to calculate the p values using graphpad prism software . recombinant human tgf-2 was obtained from r&d systems ( minneapolis , mn , usa ) . ( st . louis , mo , usa ) and calbiochem - emd millipore ( temecula , ca , usa ) , respectively . as to primary antibodies , fascin was from millipore ( temecula , ca , usa ) , e - cadherin from bd transduction laboratories ( lexington , ky , usa ) , active -catenin ( clone 8e7 ) from upstate ( lake placid , ny , usa ) , mmp9 and gapdh from abcam ( cambridge , ma , usa ) , -sma fluorescein isothiocyanate ( fitc ) conjugated and unconjugated from sigma - aldrich corp . , pan actin from abcam . all secondary antibodies for immunofluorescence staining were purchased from molecular probes ( invitrogen , eugene , or , usa ) ; secondary antibodies for western blots were obtained from li - cor biosciences ( lincoln , ne , usa ) . rats for this research were obtained and cared for in accordance with the institutional animal care and use committee and canadian council of animal care rules . all animal procedures were carried out in accordance with the arvo statement for the use of animals in ophthalmic and vision research and were approved by the ethics committee on animal research of mcmaster university ( hamilton , ontario , canada ) . lens epithelial explants were obtained from 17- to 19-day - old wistar rats ( charles river laboratories , montreal , canada ) as previously described . briefly , rat lenses were isolated and placed in 35-mm culture dishes containing prewarmed , serum - free medium m199 , supplemented with antibiotics ( all from invitrogen ) . the posterior suture of the lens was located and a small incision was made to remove the posterior capsule . this revealed the fiber mass , which was then gently peeled off the anterior epithelium . once separated , the epithelium was then pinned to the culture dish with a blunt tool exposing lecs to culture medium . twenty - four hours after explanting , confluent epithelial explants were left untreated in serum - free m199 or treated with tgf-2 at 6 ng / ml for 48 hours . -catenin inhibitors icg-001 and pnu-74654 were dissolved in dimethyl sulphoxide ( dmso ) at a final concentration of 17.6 and 10 mm , respectively . the working concentration for icg-001 and pnu-74654 was 10 m ( 1.14 l/2 ml ) and 20 m ( 4 l/2 ml ) , respectively . the working concentration of icg-001 and pnu-74654 was determined based on dose response experiments performed on lens epithelial explants . for -catenin inhibitor studies , lens explants were pretreated with icg-001 ( 5 m ) or pnu-74654 ( 20 m ) for 1 hour before coincubating with tgf-2 ( 6 ng / ml ) for 48 hours . lens explants were fixed with 10% neutral buffered formalin for 15 minutes and washed with phosphate - buffered saline ( pbs ; invitrogen , carlsbad , ca , usa ) . explants were then detached from the culture dish and permeabilized at room temperature for 1 hour in 0.1% triton x-100 , 0.5% sodium dodecyl sulphate ( sds ) , and 5% donkey serum , followed by incubating overnight at 4c with primary antibody at a dilution of 1:200 in pbs . following further pbs washes , explants were incubated with corresponding secondary antibody , alexa fluor 488 or alexa fluor 568 ( invitrogen ) , at a dilution of 1:200 at room temperature for 1 hour , and washed three times with pbs ( 10 minutes for each wash ) . the explants were then mounted in prolong gold antifade reagent with 4,6-diamidino-2-phenylindole ( dapi , invitrogen ) to visualize nuclei . immunostained explants were analyzed using a zeiss 510 meta confocal microscope ( carl zeiss canada , toronto , canada ) or zeiss apotome inverted fluorescence microscope ( carl zeiss canada ) or leica dmra2 fluorescence microscope ( leica microsystems canada , richmond hill , canada ) equipped with a q - imaging retiga 1300i fast digital camera ( q - imaging , surrey , bc , canada ) . minor adjustments were made to the captured images using photoshop cs3 ( adobe systems , mountain view , ca , usa ) . lens explants ( 57 per sample ) were lysed in triton lysis buffer ( 50 mm tris , ph 8.0 , 150 mm nacl , 1% triton x-100 ) with complete mini , edta - free protease / phosphatase inhibitor cocktail ( roche , laval , qc , canada ) . equal amounts of total protein ( 5 g ) were loaded on a gel , as determined by dc protein assay ( bio - rad , mississauga , on , canada ) , and sds - page was performed . resolved bands were transferred onto a nitrocellulose membrane ( pall life sciences , pensacola , fl , usa ) . membranes were blocked with odyssey blocking buffer ( li - cor biosciences ) for 1 hour and incubated with primary antibody overnight at 4c . primary antibodies were diluted in tris - buffered saline with 0.1% tween-20 ( tbst ) at a dilution of 1:500 for fascin ( abcam ) , 1:1000 for e - cadherin ( millipore ) and mmp9 ( abcam ) , and 1:10,000 for -sma ( sigma - aldrich corp . ) and actin and gapdh ( abcam ) . blots were washed in tbst and probed with corresponding li - cor near - infrared respective secondary antibodies ( either 680- or 800-nm wavelengths ) at a dilution of 1:10,000 for 1 hour . the blots were then washed with tbst and scanned using odyssey near - infrared scanning system ( li - cor biosciences ) . densitometry on visualized bands was performed using fiji image processing software ( imagej , http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . the peaks from the densitometric scans ( imagej ) were used to calculate the average density and the ratio between protein of interest and loading control . the average band density was further used to make graphs and calculate standard deviation using graphpad prism software ( la jolla , ca , usa ) . unpaired tukey 's multiple comparison test was performed on the averages to calculate the p values using graphpad prism software . additionally , increased expression of fascin and its actin binding activity have also been demonstrated to play a key role in cell adhesion and migration during emt in cancer cells . to investigate the expression pattern of fascin during tgf-induced emt in our lens system , primary rat lens epithelial explants were treated with tgf-2 , a known emt inducer , followed by immunostaining for fascin . immunostaining revealed a low - level , basal expression of fascin in untreated lens explants ( fig . 1a ) , along with an absence of f - actin staining , the latter of which was determined by rhodamine phalloidin . in comparison , a significant increase in expression of fascin was observed in lens explants following treatment with tgf-2 for 48 hours , and this was correlated with the appearance of stress fibers ( f - actin filaments ) ( fig . importantly , total actin protein levels remained similar between untreated and tgf-2treated lens explants ( supplementary fig . interestingly , we observed a significant increase ( 13% , * p < 0.007 , n 1b ) in tgf-2treated explants , and this was specific to the filaments of fascin ( arrowheads , fig . finally , to quantify the increased expression of fascin upon tgf-2 stimulation , we performed western blot analyses on lysates from untreated and tgf-2treated explants . a significant increase in the expression of fascin protein was observed in tgf-2treated explants when compared to the untreated controls ( fig . stained slides were imaged using 63 water lens of zeiss lsm510 confocal microscope and analyzed using zeiss lsm software . ( b ) a graph for colocalization of fascin with actin was made by scoring at least six different areas of the untreated and tgf-2treated lens explants ( ( c ) western blot analysis for fascin and gapdh was carried out using protein lysates extracted from untreated lens explants and tgf-2treated lens explants . densitometric quantification ( lower ) shows fold increase in fascin normalized to untreated lens explants ( n = 4 , * wnt/catenin signaling has been shown to play a critical role during tgf-induced emt . in lens explants , -catenin translocates to the nucleus from the cell margin following treatment with tgf-. to corroborate our results with previous findings , we initially examined the status of -catenin following tgf-2 stimulation in the lens explants . immunofluorescence staining showed membranous localization of -catenin in untreated explants , whereas delocalization of -catenin from the cell membrane was observed in the lens explants at 20 hours post tgf-2 treatment ( inset , panel 2 , fig . additionally , an increase in nuclear accumulation of -catenin was observed in the explants incubated with tgf-2 for 20 hours ( inset , panel 2 , fig . a complete delocalization of -catenin from the cell membrane and concomitant increase in nuclear localization of -catenin were observed upon incubation of explants with tgf-2 at the later time point of 48 hours ( inset , panel 3 , fig . untreated lens explant shows membranous localization of -catenin ( inset , merge image , upper ) that is disrupted upon stimulation with tgf-2 starting at 20 hours post treatment . tgf-2 induces nuclear localization of -catenin ( inset , merge image , panels 2 , 3 ) . during tgf-induced emt , it has been proposed that -sma expression is regulated by the interaction between -catenin and smad3 , which is further dependent on the transcriptional coactivator cbp . inhibition of the -catenin / cbp complex by the inhibitor icg-001 prevents -catenin and smad3 interaction and has been shown to inhibit tgf-induced emt in other systems . we therefore utilized icg-001 to determine whether disruption in -catenin / cbp dependent signaling in tgf-2treated explants can prevent fascin expression and emt . lens explants were treated with tgf-2 in the presence or absence of icg-001 ( 10 m ) for 48 hours . upon stimulation with tgf-2 , lecs showed actin cytoskeleton remodeling , as demonstrated by a significant increase in both fascin and -sma expression ( inset , fig . interestingly , tgf-2induced expression of fascin was prevented by cotreatment with icg-001 , and this suppression in fascin expression was further confirmed by western blot analysis ( fig . 3b ) . a significant decrease in tgf-2induced expression of -sma was also observed upon disruption of -catenin / cbp dependent signaling by cotreatment with icg-001 when compared to tgf--treated lens explants ( inset , fig . 3a ; figs . untreated and icg-001treated explants did not exhibit any induction in either fascin or -sma expression . a previous report had shown that inhibition of wnt/catenin signaling by icg-001 induces cytotoxicity in multiple myeloma cells . however , we did not observe any significant increase in cell death in the lens explants treated with icg-001 alone or in combination with tgf-2 . effect of inhibition of -catenin / cbp dependent signaling by icg-001 on tgf-induced emt of lens explants . ( a ) untreated , icg - only , tgf-2only , and tgf-2 and icg-001-treated lens explants were immunostained for fascin and -sma . slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( b ) western blot analysis for fascin , -sma , and gapdh was carried out using protein lysates extracted from untreated , icg - treated , tgf-2treated , and tgf-2 and icg-001-treated lens explants . ( c ) densitometric quantification of -sma indicating fold reduction in -sma expression normalized to untreated lens explants ( n = 3 , * * p < 0.005 , * * * p < 0.0005 ) . since we had shown that fascin colocalizes with f - actin in tgf-2treated explants ( fig . 1 ) , we further examined f - actin expression following treatment with tgf-2 in the presence and absence of icg-001 . an increase in actin polymerization was observed in tgf-2treated explants , whereas coincubation with icg-001 completely inhibited tgf-2induced f - actin polymerization ( fig . 4a ) . inhibition of -catenin / cbp dependent signaling by icg-001 prevents stress fiber formation and tgf-induced e - cadherin delocalization . lecs incubated with icg only , tgf-2 only , and tgf-2 and icg-001 immunostained for actin and e - cadherin . stained slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( a ) actin staining reveals a decrease in tgf-2induced stress fiber formation ( panel 3 ) with inhibition of -catenin / cbp interaction by icg-001 ( panel 4 ) . ( b ) the tgf-2induced loss of membranous e - cadherin ( panel 3 ) was restored by icg-001 ( panel 4 ) . our lab and others have shown that during tgf-induced emt of lecs , e - cadherin is degraded , and its localization at the cell membrane is lost . loss of e - cadherin leads to a dissociation of the e - cadherin/catenin complex that results in translocation of -catenin to the nucleus and activation of its downstream targets . thus , we examined the effects of tgf-2 on e - cadherin expression in the presence of icg-001 . untreated lens explants showed hexagonal , cobblestone arrangement with e - cadherin exhibiting membranous localization ( inset , fig . 4b ) , which was also observed in cells treated with icg-001 alone . in comparison , explants treated with tgf-2 exhibited a substantial decrease in the localization of e - cadherin at the cell membrane . in comparison , coincubation with tgf-2 and icg-001 was able to partially rescue the tgf-induced e - cadherin delocalization ( fig . these results suggest the involvement of -catenin / cbp dependent signaling in e - cadherin localization downstream of tgf- signaling in lens explants . previous studies have speculated that -catenin / tcf dependent signaling may regulate tgf-induced cataract formation and emt of lens epithelial explants . to investigate this further , we inhibited the -catenin / tcf dependent signaling pathway utilizing pnu-74654 ( pnu ) , an inhibitor that selectively inhibits the interaction between nuclear -catenin and tcf4 . we did not observe any change in the cellular morphology in the lens explants cotreated with tgf-2 and pnu when compared to the cells of the explant incubated with tgf-2 . we first examined the change in expression of -sma following cotreatment of lens explants with tgf and pnu as compared to cells treated with tgf alone . we did not observe any change in expression of -sma positive stress fibers ( fig . these findings revealed that while untreated explants showed membranous localization of e - cadherin ( arrowhead , fig . 6a ) , explants treated with tgf-2 exhibited a decrease in the localization of e - cadherin at the cell membrane ( arrowhead , fig . as expected , we did not observe any change in the localization of e - cadherin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone . these results were confirmed by western blot analyses showing that cotreatment with pnu did not prevent the tgf-induced decrease in e - cadherin expression ( figs . c ) . inhibition of -catenin / tcf dependent signaling fails to protect tgf-induced -sma expression and f - actin polymerization . ( a ) lecs incubated with tgf-2 and tgf-2 and pnu for 48 hours immunostained for -sma , and with rhodamine phalloidin for f - actin , and imaged using 40 lens of leica dmra2 fluorescent microscope . scale bar : 100 m ( b ) representative western blot of -sma protein from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - treated lecs ( n = 3 ) . ( a ) e - cadherin stained untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs imaged using zeiss lsm 510 confocal microscope . ( b ) total protein from untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs was subjected to western blot analysis for e - cadherin and gapdh ( n = 3 ) . ( c ) graph showing average relative density sd of e - cadherin normalized to untreated lecs ( n = 3 , * p < 0.05 , * * p < 0.005 ) . previous studies have shown that fascin gene expression is regulated by the wnt/catenin signaling pathway , which promotes cancer cell migration and invasion . more specifically , the fascin promoter has been shown to contain binding sites for tcf / lef , transcription factors that are activated specifically by -catenin . to investigate the role of -catenin / tcf interaction on fascin expression , rat lens epithelial explants immunofluorescence results revealed no change in the expression of fascin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone ( fig . this finding was further confirmed using western blot analysis , where the protein extracts from lens explants cotreated with tgf-2 and pnu revealed no change in the levels of fascin protein when compared to explants treated with tgf-2 alone ( figs . ( a ) untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs were stained for fascin and imaged using 40 lens of leica dmra2 fluorescent microscope ( n = 3 ) . ( b ) western blot analysis for fascin and gapdh was carried out using protein lysates from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - cotreated lecs ( n = 3 ) . ( c ) graph representing the relative density sd of fascin normalized to untreated lecs ( n = 3 , * p < 0.05 ) . during emt , cells undergoing transformation express mesenchymal markers , such as the matrix metalloproteinases ( mmps ) , as they lean toward a mesenchymal phenotype . previous reports from our lab show an increase in mmp9 protein levels in the supernatants from tgf-2treated whole rat lenses . first , to show the mesenchymal phenotype of rat lens explants induced by tgf-2 and to investigate whether -catenin / cbp dependent signaling has a role in the regulation of mmp9 expression , as observed in other systems , we examined mmp9 expression in tgf-2treated lens explants in the presence and absence of icg-001 . western blot analyses revealed a significant 2.7 0.8-fold reduction ( p < 0.02 ) in tgf-2induced mmp9 protein levels upon inhibition of -catenin / cbp dependent signaling by icg-001 compared to explants treated with tgf-2 alone ( figs . next , we investigated the ability of the pnu inhibitor to block tgf-2induced mmp9 expression and found that it was ineffective ( data not shown ) . these results suggest a role for -catenin / cbp dependent signaling in regulating mmp9 expression during tgf-induced emt in lens epithelial explants . ( a ) untreated , icg-001treated , tgf-2treated , and tgf-2 and icg-001cotreated lens explants were subjected to western blot analysis for mmp9 and gapdh ( n = 3 ) . ( b ) graph representing the relative density sd of mmp9 normalized to untreated lecs ( n = 3 , * p < 0.02 across all samples ) . additionally , increased expression of fascin and its actin binding activity have also been demonstrated to play a key role in cell adhesion and migration during emt in cancer cells . to investigate the expression pattern of fascin during tgf-induced emt in our lens system , primary rat lens epithelial explants were treated with tgf-2 , a known emt inducer , followed by immunostaining for fascin . immunostaining revealed a low - level , basal expression of fascin in untreated lens explants ( fig . 1a ) , along with an absence of f - actin staining , the latter of which was determined by rhodamine phalloidin . in comparison , a significant increase in expression of fascin was observed in lens explants following treatment with tgf-2 for 48 hours , and this was correlated with the appearance of stress fibers ( f - actin filaments ) ( fig . importantly , total actin protein levels remained similar between untreated and tgf-2treated lens explants ( supplementary fig . interestingly , we observed a significant increase ( 13% , * p < 0.007 , n 1b ) in tgf-2treated explants , and this was specific to the filaments of fascin ( arrowheads , fig . finally , to quantify the increased expression of fascin upon tgf-2 stimulation , we performed western blot analyses on lysates from untreated and tgf-2treated explants . a significant increase in the expression of fascin protein was observed in tgf-2treated explants when compared to the untreated controls ( fig . stained slides were imaged using 63 water lens of zeiss lsm510 confocal microscope and analyzed using zeiss lsm software . ( b ) a graph for colocalization of fascin with actin was made by scoring at least six different areas of the untreated and tgf-2treated lens explants ( ( c ) western blot analysis for fascin and gapdh was carried out using protein lysates extracted from untreated lens explants and tgf-2treated lens explants . densitometric quantification ( lower ) shows fold increase in fascin normalized to untreated lens explants ( n = 4 , * wnt/catenin signaling has been shown to play a critical role during tgf-induced emt . in lens explants , -catenin translocates to the nucleus from the cell margin following treatment with tgf-. to corroborate our results with previous findings , we initially examined the status of -catenin following tgf-2 stimulation in the lens explants . immunofluorescence staining showed membranous localization of -catenin in untreated explants , whereas delocalization of -catenin from the cell membrane was observed in the lens explants at 20 hours post tgf-2 treatment ( inset , panel 2 , fig . additionally , an increase in nuclear accumulation of -catenin was observed in the explants incubated with tgf-2 for 20 hours ( inset , panel 2 , fig . 2 ) . a complete delocalization of -catenin from the cell membrane and concomitant increase in nuclear localization of -catenin were observed upon incubation of explants with tgf-2 at the later time point of 48 hours ( inset , panel 3 , fig . untreated lens explant shows membranous localization of -catenin ( inset , merge image , upper ) that is disrupted upon stimulation with tgf-2 starting at 20 hours post treatment . tgf-2 induces nuclear localization of -catenin ( inset , merge image , panels 2 , 3 ) . during tgf-induced emt , it has been proposed that -sma expression is regulated by the interaction between -catenin and smad3 , which is further dependent on the transcriptional coactivator cbp . inhibition of the -catenin / cbp complex by the inhibitor icg-001 prevents -catenin and smad3 interaction and has been shown to inhibit tgf-induced emt in other systems . we therefore utilized icg-001 to determine whether disruption in -catenin / cbp dependent signaling in tgf-2treated explants can prevent fascin expression and emt . lens explants were treated with tgf-2 in the presence or absence of icg-001 ( 10 m ) for 48 hours . upon stimulation with tgf-2 , lecs showed actin cytoskeleton remodeling , as demonstrated by a significant increase in both fascin and -sma expression ( inset , fig . interestingly , tgf-2induced expression of fascin was prevented by cotreatment with icg-001 , and this suppression in fascin expression was further confirmed by western blot analysis ( fig . 3b ) . a significant decrease in tgf-2induced expression of -sma was also observed upon disruption of -catenin / cbp dependent signaling by cotreatment with icg-001 when compared to tgf--treated lens explants ( inset , fig . untreated and icg-001treated explants did not exhibit any induction in either fascin or -sma expression . a previous report had shown that inhibition of wnt/catenin signaling by icg-001 induces cytotoxicity in multiple myeloma cells . however , we did not observe any significant increase in cell death in the lens explants treated with icg-001 alone or in combination with tgf-2 . effect of inhibition of -catenin / cbp dependent signaling by icg-001 on tgf-induced emt of lens explants . ( a ) untreated , icg - only , tgf-2only , and tgf-2 and icg-001-treated lens explants were immunostained for fascin and -sma . slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( b ) western blot analysis for fascin , -sma , and gapdh was carried out using protein lysates extracted from untreated , icg - treated , tgf-2treated , and tgf-2 and icg-001-treated lens explants . ( c ) densitometric quantification of -sma indicating fold reduction in -sma expression normalized to untreated lens explants ( n = 3 , * * p < 0.005 , * * * p < 0.0005 ) . since we had shown that fascin colocalizes with f - actin in tgf-2treated explants ( fig . 1 ) , we further examined f - actin expression following treatment with tgf-2 in the presence and absence of icg-001 . an increase in actin polymerization was observed in tgf-2treated explants , whereas coincubation with icg-001 completely inhibited tgf-2induced f - actin polymerization ( fig . 4a ) . inhibition of -catenin / cbp dependent signaling by icg-001 prevents stress fiber formation and tgf-induced e - cadherin delocalization . lecs incubated with icg only , tgf-2 only , and tgf-2 and icg-001 immunostained for actin and e - cadherin . stained slides were imaged using 40 lens of zeiss apotome microscope and analyzed using zeiss zen software . ( a ) actin staining reveals a decrease in tgf-2induced stress fiber formation ( panel 3 ) with inhibition of -catenin / cbp interaction by icg-001 ( panel 4 ) . ( b ) the tgf-2induced loss of membranous e - cadherin ( panel 3 ) was restored by icg-001 ( panel 4 ) . our lab and others have shown that during tgf-induced emt of lecs , e - cadherin is degraded , and its localization at the cell membrane is lost . loss of e - cadherin leads to a dissociation of the e - cadherin/catenin complex that results in translocation of -catenin to the nucleus and activation of its downstream targets . thus , we examined the effects of tgf-2 on e - cadherin expression in the presence of icg-001 . untreated lens explants showed hexagonal , cobblestone arrangement with e - cadherin exhibiting membranous localization ( inset , fig . 4b ) , which was also observed in cells treated with icg-001 alone . in comparison , explants treated with tgf-2 exhibited a substantial decrease in the localization of e - cadherin at the cell membrane . in comparison , coincubation with tgf-2 and icg-001 was able to partially rescue the tgf-induced e - cadherin delocalization ( fig . these results suggest the involvement of -catenin / cbp dependent signaling in e - cadherin localization downstream of tgf- signaling in lens explants . previous studies have speculated that -catenin / tcf dependent signaling may regulate tgf-induced cataract formation and emt of lens epithelial explants . to investigate this further , we inhibited the -catenin / tcf dependent signaling pathway utilizing pnu-74654 ( pnu ) , an inhibitor that selectively inhibits the interaction between nuclear -catenin and tcf4 . we did not observe any change in the cellular morphology in the lens explants cotreated with tgf-2 and pnu when compared to the cells of the explant incubated with tgf-2 . we first examined the change in expression of -sma following cotreatment of lens explants with tgf and pnu as compared to cells treated with tgf alone . we did not observe any change in expression of -sma positive stress fibers ( fig . these findings revealed that while untreated explants showed membranous localization of e - cadherin ( arrowhead , fig . 6a ) , explants treated with tgf-2 exhibited a decrease in the localization of e - cadherin at the cell membrane ( arrowhead , fig . as expected , we did not observe any change in the localization of e - cadherin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone . these results were confirmed by western blot analyses showing that cotreatment with pnu did not prevent the tgf-induced decrease in e - cadherin expression ( figs . c ) . inhibition of -catenin / tcf dependent signaling fails to protect tgf-induced -sma expression and f - actin polymerization . ( a ) lecs incubated with tgf-2 and tgf-2 and pnu for 48 hours immunostained for -sma , and with rhodamine phalloidin for f - actin , and imaged using 40 lens of leica dmra2 fluorescent microscope . scale bar : 100 m ( b ) representative western blot of -sma protein from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - treated lecs ( n = 3 ) . effect on e - cadherin by inhibition of -catenin / tcf interaction . ( a ) e - cadherin stained untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs imaged using zeiss lsm 510 confocal microscope . ( b ) total protein from untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs was subjected to western blot analysis for e - cadherin and gapdh ( n = 3 ) . ( c ) graph showing average relative density sd of e - cadherin normalized to untreated lecs ( n = 3 , * p < 0.05 , * * p < 0.005 ) . previous studies have shown that fascin gene expression is regulated by the wnt/catenin signaling pathway , which promotes cancer cell migration and invasion . more specifically , the fascin promoter has been shown to contain binding sites for tcf / lef , transcription factors that are activated specifically by -catenin . to investigate the role of -catenin / tcf interaction on fascin expression , rat lens epithelial explants immunofluorescence results revealed no change in the expression of fascin in lens explants cotreated with tgf-2 and pnu when compared to explants treated with tgf-2 alone ( fig . this finding was further confirmed using western blot analysis , where the protein extracts from lens explants cotreated with tgf-2 and pnu revealed no change in the levels of fascin protein when compared to explants treated with tgf-2 alone ( figs . ( a ) untreated , tgf-2treated , and tgf-2 and pnu - cotreated lecs were stained for fascin and imaged using 40 lens of leica dmra2 fluorescent microscope ( n = 3 ) . ( b ) western blot analysis for fascin and gapdh was carried out using protein lysates from untreated , tgf-2treated , pnu - treated , and tgf-2 and pnu - cotreated lecs ( n = 3 ) . ( c ) graph representing the relative density sd of fascin normalized to untreated lecs ( n = 3 , * p < 0.05 ) . during emt , cells undergoing transformation express mesenchymal markers , such as the matrix metalloproteinases ( mmps ) , as they lean toward a mesenchymal phenotype . previous reports from our lab show an increase in mmp9 protein levels in the supernatants from tgf-2treated whole rat lenses . first , to show the mesenchymal phenotype of rat lens explants induced by tgf-2 and to investigate whether -catenin / cbp dependent signaling has a role in the regulation of mmp9 expression , as observed in other systems , we examined mmp9 expression in tgf-2treated lens explants in the presence and absence of icg-001 . western blot analyses revealed a significant 2.7 0.8-fold reduction ( p < 0.02 ) in tgf-2induced mmp9 protein levels upon inhibition of -catenin / cbp dependent signaling by icg-001 compared to explants treated with tgf-2 alone ( figs . next , we investigated the ability of the pnu inhibitor to block tgf-2induced mmp9 expression and found that it was ineffective ( data not shown ) . these results suggest a role for -catenin / cbp dependent signaling in regulating mmp9 expression during tgf-induced emt in lens epithelial explants . ( a ) untreated , icg-001treated , tgf-2treated , and tgf-2 and icg-001cotreated lens explants were subjected to western blot analysis for mmp9 and gapdh ( n = 3 ) . ( b ) graph representing the relative density sd of mmp9 normalized to untreated lecs ( n = 3 , * p < 0.02 across all samples ) . understanding the mechanisms through which tgf- induces emt in the lens is important for developing novel therapeutic strategies for the prevention of cataract . in this study , we show that tgf-induced -catenin / cbp dependent signaling is critical during tgf-induced emt in the lens . in particular , we show that tgf-induced -catenin / cbp dependent signaling is responsible for the upregulation of fascin , an actin - bundling protein known to be involved in emt and migration and invasion in a number of different cancer cells . icg-001 , a specific inhibitor of -catenin / cbp interactions , prevented tgf-induced fascin , -sma , and mmp9 induction , demonstrating the requirement of cbp - dependent -catenin signaling in the regulation of proteins that facilitate tgf-induced emt in the lens . these data add to the growing list of mechanisms regulated during tgf-induced ocular fibrosis , and identify novel signaling events and molecular markers that contribute to tgf-induced emt , which could offer potential new therapeutic approaches to control fibrosis of the lens . mesenchymal transition plays a major role in the pathogenesis of numerous fibrotic diseases including fibrotic cataract . transforming growth factor- is the growth factor involved in emt of lecs following cataract surgery that results in the development of secondary cataract or pco . in addition , emt and the formation of anterior subcapsular plaques containing -sma expressing , spindle - shaped cells can be induced by tgf-. transforming growth factor-induced emt in lec explants involves a loss of epithelial characteristics with downregulation / degradation of epithelial markers , such as e - cadherin , and a transition to a mesenchymal / fibroblast phenotype characterized by an upregulation of myofibroblast markers , namely , -sma incorporated into f - actin stress fibers . formation of stress fibers requires the coordinated interaction between monomeric actin and its accessory proteins , such as fascin . fascin has been shown to facilitate formation of peripheral actin - rich protrusions through its actin - bundling activity that results in increased cell migration . recent reports have demonstrated more specific roles for fascin in the formation and turnover of adhesive structures of the cell . for example , fascin promotes cell migration by restricting the number and thickness of newly polymerized actin filaments into tight noncontractile bundles at the focal adhesions ; depletion of fascin results in thicker and more contractile actin filaments that generate higher tensile forces and slow turnover of focal adhesions , ultimately causing decreased cell migration . indeed , fascin along with other actin - modulating proteins helps cancer cells undergoing emt to change their cellular morphology , aiding in tumor cell migration and invasion . overexpression of fascin has also been shown to modulate emt during progression of breast cancer and pancreatic tumors ; however , its fate during tgf-induced emt in the lens had yet to be examined . in the current study , we demonstrate that stimulation of rat lens epithelial explants with active tgf- led to a significant increase in the expression of fascin ( figs . 1a , 1c ) . in accordance with the role of fascin discussed above , this increase in expression of fascin in our lens explant model system following tgf- treatment may contribute to the migratory phenotype of lecs often observed during pco . since our lens explant model often consists of a confluent culture of cells , migration is reserved only to the edges of the explant . thus , future studies examining the role of fascin in the cellular protrusions of migratory lens explant cells will be important . it is well known that tgf- signaling in the lens is mediated through a canonical , smad - dependent pathway ; however , smad - independent pathways are also involved in regulating emt . our group has demonstrated that overexpression of tgf- in the lens leads to the formation of asc in the absence of smad3 . this finding indicates that smad3 signaling alone is not required for tgf- to induce a lens fibrotic response . transforming growth factor-induced smad signaling has also been shown to cross - talk with other pathways , such as wnt/-catenin . in other epithelial cell systems , -catenin has been shown to form a complex with smad3 that then translocates to the nucleus , and cooperatively promotes the transcription of emt target genes . the translocation of -catenin from cell margins to the nucleus in tgf-treated lens epithelial explants as shown in this study ( fig . 2 ) , and as reported previously , suggests the role of -catenin dependent signaling during tgf-induced emt in our lens explant model system . it is possible that tgf- may be initiating -catenin signaling through upregulation of wnts and their receptors , frizzled ( fz ) , in our lens explant model system , as has been observed previously by others using a tgf- transgenic mouse model . the formation of -catenin / smad3 complex is further dependent on the -catenin transcriptional coactivator , cbp , which differentially regulates a subset of target genes . , we demonstrate that in the presence of icg-001 , tgf-induced -sma expression ( fig . 4a ) were completely inhibited in lens epithelial explants , indicating the prevention of a contractile , myofibroblast phenotype . further , the drastic decrease in tgf-induced expression of fascin in the presence of icg-001 ( figs . 3a c ) indicates a novel role of -catenin / cbp dependent signaling in regulation of cofactors that are essential for cell migration and invasion . these observations show a complete inhibition of the tgf-induced expression of emt - related proteins such as -sma by icg-001 in lecs . the morphology of lens explant cells cotreated with tgf-2 and icg-001 showed a cobblestone arrangement of cells resembling that of untreated control explants , indicative of a prevention of tgf-induced transformation of lecs ( supplementary fig . as well , the known membrane delocalization of e - cadherin induced by tgf- signaling was partially restored in the presence of icg-001 ( fig . however , since the inhibition was not complete , it is possible that tgf-induced loss of e - cadherin localization is also regulated by -catenin independent repressive activity of transcription factors such as slug on e - cadherin . nonetheless , our findings suggest the importance of -catenin / cbp dependent signaling during tgf-induced emt in the lens . dependent signaling has also been implicated in emt in other model systems . in the lens , it is speculated that -catenin / tcf signaling plays a role in regulating emt / fibrosis . to further understand the role of -catenin / tcf signaling during tgf-induced emt in lecs , a specific inhibitor of -catenin / tcf interaction , pnu-74654 , was used . the morphology of lens explant cells cotreated with tgf-2 and pnu resembles that of tgf-2treated lecs , indicating that pnu was unable to prevent tgf-induced transformation of lecs ( supplementary fig . the observed appearance of contractile , -sma expressing fibers and presence of polymerized f - actin / stress fibers in our lens explant cells in the presence of tgf- and pnu indicates that -catenin / tcf dependent signaling is not required in the regulation of tgf-induced -sma expression and stress fiber formation ( fig . these observations are in line with those reported in other emt model systems , in which -catenin / cbp dependent signaling as compared to -catenin / tcf dependent signaling is more critical during tgf-induced emt . the icg-001 inhibitor has , however , in some cases been shown to interfere with -catenin / tcf dependent signaling . hence , we can not rule out the possibility that -catenin / tcf dependent signaling may have partially contributed to tgf-induced emt in lecs . however , -catenin / cbp dependent signaling , as opposed to -catenin / tcf dependent signaling , has been shown to be required for -sma expression and , importantly , the cis elements of the -sma promoter do not possess -catenin / tcf binding sites , but instead are regulated by transcription factors that are controlled by -catenin / cbp / smad3 signaling . interestingly , inhibition of -catenin / cbp dependent signaling by icg-001 , and not inhibition -catenin / tcf dependent signaling by pnu , partially inhibited tgf-induced nuclear translocation of -catenin ( supplementary fig . neither icg-001 nor pnu treatment alone resulted in the decrease in -catenin expression ( supplementary fig . / tcf complex , has been shown to coincide with -sma expression during tgf-induced emt in other epithelial cells , such as tubular cells . indeed , further investigation is needed to elucidate the role of the interaction between -catenin / cbp and smad3 in the regulation of -sma expression during tgf-induced emt in lecs . the presence of fascin in the lens explants cells incubated with both tgf- and pnu ( fig . ahr signaling , as opposed to -catenin / tcf dependent signaling , is responsible for the regulation of fascin . furthermore , incubation of lens explants with pnu did not prevent tgf-induced downregulation of e - cadherin ( figs . c ) , a key component of adherens junction , which is in agreement with the previous report showing tgf-induced smad - dependent signaling , but not -catenin / tcf dependent signaling , as the key mechanism involved in downregulation of e - cadherin during tgf-induced emt . overall , the presence of -sma , fascin , and e - cadherin shows that inhibition of -catenin / tcf dependent signaling by pnu was unable to prevent the tgf-induced contractile , myofibroblast phenotype and emt in lecs . during tgf-induced emt , epithelial cells begin to express mesenchymal markers such as mmps as they lean toward a more mesenchymal phenotype . previously , we have shown that in the absence of mmp9 expression , tgf-induced emt - like changes , such as induction of -sma in lecs , are prevented . in tubular epithelial cells , it has been demonstrated that the inhibition of -catenin / cbp interactions by icg-001 decreases active levels of mmp9 , as assessed by gelatin zymography of supernatants , thereby completely preventing the mesenchymal transition . here , we found that inhibition of -catenin / tcf interactions by pnu was not able to prevent tgf-induced mmp9 expression , suggesting that -catenin / tcf dependent signaling may not be required in the regulation of mmp9 during tgf-induced emt in lecs ( data not shown ) . interestingly , inhibition of -catenin / cbp dependent signaling by icg-001 showed a significant decrease in tgf-induced mmp9 expression along with suppression of the mesenchymal transition of lecs ( fig . a decrease in tgf-induced mmp9 expression upon inhibition of -catenin / cbp interactions also shows the novel role of -catenin / cbp dependent signaling in regulation of mmp9 during tgf-induced emt . taken together , our findings have revealed a -catenin mediated mechanism underlying the regulation of fascin , -sma , and mmp9 expression during tgf-induced emt in the lens . we propose that tgf-mediated downregulation of e - cadherin results in disruption of e - cadherin/catenin complex that leads to increased cytosolic -catenin , which interacts and forms a complex with cbp ( fig . we demonstrate for the first time that -catenin / cbp dependent signaling is indispensable in tgf-mediated emt in the lens . -catenin / cbp complex may further interact with other regulators of emt and regulate tgf-induced emt of lecs . the interaction between -catenin and smad3 is known to promote transcription activity of myocardin - related transcription factor ( mrtf ) , the master regulator of cytoskeletal genes , during tgf-induced emt in tubular epithelial cells . interestingly , our previous finding suggests translocation of mrtf to the nucleus during tgf-induced emt in lecs . hence , it is possible that the interplay between -catenin , smad3 , and mrtf may be playing a crucial role in tgf-mediated emt in lecs . indeed , a detailed molecular investigation is needed to dissect out the role of -catenin , smad3 , and mrtf interactions in tgf-induced emt in the lens . in conclusion , the inhibition of tgf-induced emt by icg-001 may represent a new therapeutic approach to target ocular fibrosis of the lens . tgf-induced downregulation of e - cadherin leads to disruption of e - cadherin/-catenin complex that results in interaction of -catenin with cbp . free -catenin may interact with either smad in a cbp - dependent manner or tcf , and regulate tgf-induced -sma , fascin , and mmp9 expression . inhibition of interaction between -catenin and tcf fails to inhibit tgf-induced emt - like changes in lecs . however , inhibition of interaction between -catenin and cbp prevents tgf-induced emt in lens explants . direct coronary stent implantation is an elegant technique for coronary artery revascularization.1 however , calcified coronary lesions , often seen in older patients suffering from diabetes mellitus , renal failure and hypertension , are challenging to deal with , as they require optimal lesion preparation prior stenting for avoiding stent underexpansion which is related to in - stent restenosis , target lesion revascularization and subsequent stent thrombosis.2 several strategies and technologies have been developed to address the problem of heavily calcified coronary lesions . these include simple dilatation using standard non - compliant balloon , cutting balloon and plaque modification using rotational atherectomy . we report on the management of an underexpanded bare - metal stent in a patient with heavily calcified lesion not amenable to high - pressure balloon - dilatation . a 72-year old man suffering from progressive angina over the past 8 weeks presented to our chest pain unit . he had previously documented insulin - dependent diabetes , alimentary obesity , hyperlipidemia and arterial hypertension . an ambulatory performed myocardial perfusion scintigraphy revealed a reduced tracer - uptake in the apex , left posterior and antero - lateral wall during physical examination ( 100 watt - cycling ) . coronary angiography , which was performed via right radial access using a 5f sheath , revealed a 50% stenosis of the left anterior descending artery ( lad ) and ramus posterolateralis sinistra ( rpls ) while the right coronary artery ( rca ) had a critical 90% stenosis ( fig . due to the patients symptoms and the angiographic findings we decided to perform a percutaneous coronary intervention ( pci ) . the patient received 600 mg clopidogrel , 500 mg aspirin and 5000u heparin followed by primary pci and direct stenting of a bare - metal stent ( bms ) ( coroflex blue 3.5 mm/8 mm , b. braun , melsungen , germany ) with 18 atm for 30 sec ( fig . post - pci angiography revealed a 75% stenosis in the mid - portion of the stent ( fig . 1c ) . a subsequent dilatation with a semi - compliant balloon ( pantera 3,5/10 mm with [ biotronik , berlin , germany ] 18 atm over 30 sec ) , a non - compliant balloon ( quantum 3,5/8 mm [ boston scientific , natick , usa ] with 20 atm over 30 sec ) and a cutting - balloon ( 3,0/10 mm [ boston scientific , natick , usa ] with 18 atm over 30 sec ) could not expand the stent further ; pointing out the heavily calcified nature of this lesion . due to the fact that an underexpanded stent is a predictor for worse clinical outcome we decided on rotablation . additionally we introduced a 5f sheath in right femoral vein and inserted a transient pacemaker lead . after passing across the stenosis with the 0.009 rotawire we ablade the heavily calcified stenosis as well as the stent struts ( stentablation ) ( fig . all ablations were performed with a 1.75 mm burr with at least 150,000 rpm and ablation times < 30 sec without a decrease in rotational speed of > 5,000 rpm . the procedure was free of complications and we continued with dilatation with a non - compliant balloon ( quantum 3,5/8 mm with 20 atm over 30 sec ) and a cutting - balloon ( 3,0/10 mm with 16 atm over 30 sec ) . with complete expansion of the balloons the procedure was continued with implantation of a drug - eluting stent ( taxus libert 4.0/12 mm [ boston scientific , natick , usa ] with 16 atm over 30 sec ) ( rotastenting ) ( fig . finally , there was timi 3 without evidence of dissection or residual stenosis ( fig . 2c ) . following uneventful hospital stay without evidence of myocardial necrosis the patient was discharged after 3 days on 100 mg aspirin , 75 mg clopidogrel , 5 mg bisoprolol , 5 mg of ramipril and 40 mg simvastatin with a recommendation for dual antiplatelet therapy of 1 year without any change in his extra - cardiovascular medications . a routine coronary angiography performed 6 months after index - pci revealed a good result with a mild ( 25% ) restenosis ( fig . 2d ) . direct stenting is the implanation of stents in coronary lesions without predilatation.1 from animal restenosis models , direct stenting without the need for predilatation appears to reduce vessel trauma , in particular as a result of less endothelial denudation , resulting in less neointimal hyperplasia subsequently.1 pci of calcified and complex lesions has been associated with lower success rates , an increased frequency of acute complications , and higher restenosis rates than pci of simple lesions.2 as seen in our case , delivering the stent may be difficult and stent expansion may be inadequate in heavily calcified lesions , resulting in smaller acute gain compared to non - calcified lesions.2 it is widely accepted that achieving postprocedural residual stenosis is a major determinant of restenosis during follow - up and optimal stent expansion is a crucial factor in minimizing the risk of stent thrombosis pointed out by the fact that only 22% of patients that experienced subacute stent thrombosis have an acceptable pci result as assessed by ivus.2,3 a variety of strategies and technologies have been developed to address the problem of an underexpanded stent . the postit trial revealed that in case of using only the stent delivery balloon over 70% of patients did not achieve optimal stent deployment.4 use of non - compliant balloon to achieve full distension in resitant lesions is a reasonable first - step . however , focal points of resistance within a lesion result in non - uniform balloon expansion and characteristic dog - boning with overexpansion in the more compliant segments . in this non - uniform expansion may cause vessel dissection and rupture acutely as well as restenosis due to deep - wall injury in the follow - up . cutting - balloon , designed to score the vessel longitudinally rather than causing uncontrolled plaque disruption , have been used successfully in the treatment of undilatable lesions.5 in our case , none of these techniques were successful in reducing the underexpansion , demonstrating the nature of the heavily calcification , which was not assumed on initial fluoroscopy . thus , despite the existence of limited data,6 we decided to rotablade the remaining calcification and the underexpanded stent struts to avoid aforementioned complications . high - speed rotational atherectomy preferentially cuts hard plaque , increases plaque compliance and thereby renders the lesion more amenable to balloon dilatation.7 the rotablator is able to ablate inelastic tissue selectively while maintaining the integrity of elastic tissue due to the principle of differential cutting . these particles are small enough to pass through the coronary microcirculation and ultimately undergo phagocytosis in the liver , spleen , and lung.7 the procedure performed in our case was uneventful with no dissection , slow - flow , heamodynamic compromise or myocardial necrosis . we had applied a transient pacemaker via the right femoral vein to overcome possible conduction disturbances when handling in the right coronary artery . several observational studies have confirmed that rotational atherectomy prior to stent deployment in severely calcified lesions does facilitate stent delivery and expansion , but incidence of restenosis remains unsatisfactory ( 23% ) when bms are used.8 there is limited information about rotational atherectomy followed by des implantation , but initial results seem promising.9 a comparison of bms ( n = 84 ) and des ( n = 213 ) after rotablation with cardiac death and recurrent myocardial infarction being defined as primary endpoint and binary restenosis as secondary endpoint revealed lower rates for primary endpoint in des group ( 2.3% versus 7.1% ; p = 0.04 ) during a follow - up of 1300 days.10 despite our procedural success and good midterm result , there are no data on long - term follow - up after stentablation and rotastening . thus , it should be emphasized that a better lesion preparation is needed to avoid stent underexpansion in undilatable lesions .

purposetransforming growth factor-induced epithelial mesenchymal transition ( emt ) is one of the main causes of posterior capsular opacification ( pco ) or secondary cataract ; however , the signaling events involved in tgf-induced pco have not been fully characterized . here , we focus on examining the role of -catenin / cyclic amp response element binding protein ( creb)-binding protein ( cbp ) and -catenin / t - cell factor ( tcf)-dependent signaling in regulating cytoskeletal dynamics during tgf-induced emt in lens epithelial explants.methodsrat lens epithelial explants were cultured in medium m199 in the absence of serum . explants were treated with tgf-2 in the presence or absence of the -catenin / cbp interaction inhibitor , icg-001 , or the -catenin / tcf interaction inhibitor , pnu-74654 . western blot and immunofluorescence experiments were carried out and analyzed.resultsan increase in the expression of fascin , an actin - bundling protein , was observed in the lens explants upon stimulation with tgf- , and colocalized with f - actin filaments . inhibition of -catenin / cbp interactions , but not -catenin / tcf interactions , led to a decrease in tgf-induced fascin and stress fiber formation , as well as a decrease in the expression of known markers of emt , -smooth muscle actin ( -sma ) and matrix metalloproteinase 9 ( mmp9 ) . in addition , inhibition of -catenin / cbp dependent signaling also prevented tgf-induced downregulation of epithelial cadherin ( e - cadherin ) in lens explants.conclusionswe show that -catenin / cbp dependent signaling regulates fascin , mmp9 , and -sma expression during tgf-induced emt . we demonstrate that -catenin / cbp dependent signaling is crucial for tgf-induced emt in the lens . calcified coronary lesions are challenging to deal with , as they require optimal lesion preparation . direct stenting in this scenario is associated with risk of stent - underexpansion , which is related to in - stent restenosis , target lesion revascularization and stent - thrombosis . we report on the interventional management of an underexpanded bare - metal stent not amenable to high - pressure balloon dilation and cutting - balloon . by using rotablation we could abrade the underexpanded stent struts and the calcification with subsequent implantation of a drug - eluting stent . follow - up of 6 months revealed good results without evidence of significant restenosis . our clinical experience and case reports in the literature suggest that this strategy might be an option for underexpanded stents not amenable to conventional techniques .

the emergency care system in denmark has been reorganized during recent years , and a danish quality database on emergency care has been established to assess , monitor , and improve quality . the danish quality database for prehospital emergency medical services ( qems ) is one of the three national sub - databases . the aim of the qems is to assess , monitor , and improve the quality of prehospital emergency medical service ( ems ) care in the entire patient pathway and acts as basis for clinical and health - service research . provision of prehospital emergency care is subject to variations in organization among ems providers , with domestic and regional as well as international differences . there are few uniform quality indicators and standards based on high - level evidence , and most standards are based on expert panel opinions.14 some specific diagnostic groups benefit from quality indicators and standards , such as cardiac arrest,5,6 myocardial infarction ( mi),7,8 stroke,9,10 and severe injury . the major challenge and difficulty in emergency care , especially in the prehospital setting , is that the vast majority of patients present with symptoms and not diagnoses . thus , ems care has to be initiated and delivered on the basis of the symptoms with limited knowledge about the patient s objective condition and medical history . in 2013 , a working group was established by the danish regions with medical and administrative representatives from all five health regions , with the task of formulating recommendations on indicators and standards of ems quality . the working group consisted of medical directors and administrative professionals from prehospital organizations from each of the five regions . the selection of quality indicators was a consensus process , based on literature and the actual availability of prehospital data in our system and national registries on specific emergency conditions , cardiac arrest , mi , and stroke . the danish clinical registries a national improvement program was established in 2014 with medical representatives from the five public regional ems organizations , a professor in prehospital and emergency medicine and representatives from the danish society of cardiology , the danish society of neurology , and a clinical epidemiologist and a statistician from the danish clinical registries . an initial pilot test of indicators is currently underway in 2015 . due to lack of high evidence , we could not define all standards a priori . a process of defining the remaining standards will follow evaluation of the pilot test . ultimately , aggregated data at a regional level will be available monthly , and national and open - source annually , and provide stimulus for ems improvement . the aim of this review is to describe the design and the implementation of qems , a new danish clinical quality database . in denmark , access to health care is a tax - financed service , and access to ems and emergency hospital care is free of charge . during the last 7 years five regional health care regions were established and were responsible for the pre hospital ems in their own region . these regional ems organizations are responsible for the entire prehospital patient care pathway , from the 112 call made to the recently established emergency medical coordination centers ( emcc ) , through the care given on scene and during transport , and until the patient arrives at hospital , or is taken care of without being brought to hospital . at the emcc , the national emergency number 112 covers both ems , fire and police , and is answered by the police , except in the capital region . here all decisions regarding medical prehospital responses are made at the emcc , based upon a focused , structured interview of the person calling 112 . assessment is performed according to a criteria - based dispatch protocol , the danish index for emergency care ( danish index).11 the danish ems is a two- or three - tier system , with ambulances as the basic - level response , an intermediate level with paramedic- or nurse - manned cars , and an advanced level with dedicated prehospital doctors ( consultant anesthesiologists ) in cars ( mobile emergency care unit ) or helicopter ( helicopter emergency medical service [ hems ] ) in a rendezvous model . the danish hems has only recently been added to the system , established as national hems in 2014 . ambulances are run by municipal or private companies and staffed with emergency medical technicians or paramedics , educated and trained to national minimum standards according to national regulations.12 the regional ems organizations contract with ambulance providers after european union - regulated tenders . the regional ems is responsible for the quality of care delivered in the entire ems pathway , including the care delivered by private ambulance contractors . all ems providers nationwide are to use the same electronic prehospital patient record system , under implementation in 2015 . the study population consists of all 112 patient contacts defined as patients in need of emergency help , where access to the health care system is a 112 call forwarded to the emcc . . the corresponding number of 112 patient contacts will be lower , estimated to be 300,000350,000 as there might be more 112 calls on the same patient . the database includes data acquired from each of the five regional emcc and when fully implemented data from the prehospital electronic patient record . data from the danish database for acute hospital contacts will also be included in the future . emcc call - takers assess the level of emergency and respond in accordance with the danish index for emergency care , which consists of 37 main symptoms . the index defines five levels of urgencies : a : life- threatening or potentially life - threatening condition , immediate response required ; b : urgent , but not life - threatening condition ; c : nonurgent condition that needs an ambulance ; d : nonurgent supine patient transport ; and e : other service or advice / instruction including taxi transportation ( no ambulances dispatched ) . the emcc call - taker assigns the letter appropriate for the level of urgency coupling it with a specific number to the case according to the danish index criteria code that corresponds to the main symptom and specific subgroup symptom . the indicators aim at describing quality at the regional ems organizational level and the quality of the medical care . as the prehospital electronic patient record has not yet been fully implemented , only some of the indicators are monitored in the initial pilot test phase . indicator numbers 13 describe quality at an ems organizational level ; these include only responses of level of urgency a ( table 1 ) . time from arrival until the patient is handed over to the hospital personnel ( indicator 3 ) awaits the electronic patient record . concerning the three medical emergencies , cardiac arrest , st - elevation mi , and stroke , all these await full implementation in the electronic patient record . in the meantime , data will be obtained from the national research database , the danish out - of - hospital cardiac arrest registry , and regional and national databases.5 indicator number 4 , the proportion of 112 calls coded according to danish index , was included to assure registration of the initial symptom as this is the only patient information available at time of the decision on level of urgency . indicator number 5 was defined in order to monitor the proportion of patients calling 112 classified as having no need for an ambulance and/or no need for hospital care , who died or called 112 again within 24 hours . in denmark , each citizen is assigned a unique ten - digit civil registration ( cpr ) number , universally used in all patient records and danish registries , and enables unambiguous linkage between these registries . thus , the registration of this number , as indicator 9 , is essential as a key to patient care and for follow - up . table 3 describes the variables included in the primary data set . apart from being an identifier for an event and person , the variables comprise mainly date and time stamps identifying when the 112 call was received at the emcc , when the dispatcher sent a prehospital resource , and when the first resource arrived . the danish index , including urgency level , is also included in the data set . it is , however , a fact that the cpr number is missing in a substantial proportion of patients because it is not always possible to extract this information from the caller . patients with a valid danish cpr number are followed from the date of 112 call and until death for up to 2 days after the task has been completed using the danish civil registration system.13 the database is under establishment , and no studies have been performed yet , but there are previous studies using some of similar data from three regions comparing the danish index with diagnosis and outcome after hospitalization.11,14,15 data will be available to researchers after application to the steering committee , provided that permissions from the danish data protection agency and/or ethical committee were obtained . the qems is an approved clinical quality database ( the danish data protection agency record no 2013 - 58 - 0026 , the central denmark region record no 1 - 16 - 02 - 508 - 14 , and the state serum institute record no 14/18767 ) funded by the danish regions and receives administrative , epidemiological , and biostatistical support from the danish clinical registries ( danish regions ) . aggregated data at regional level will be reported annually in a published report , and data will be provided monthly to the danish regions for use in each regions information system . aggregated data at a regional level will be available monthly , and national and open - source annually , providing incentive for improvement of patient care pathways in the prehospital setting . recent advances in array - based and sequence - based instrumentation have opened up new ways to monitor the inner molecular world of the cells and tissues that might be relevant to human diseases . yet it is far from evident how these large datasets should be analyzed and how they can be integrated with other sources of data in order to become informative . conversely , the medical community expects nothing less than a list of predictive biomarkers reflecting the risk of disease or its progression and an understanding of the cellular mechanisms involved in disease . however , comparing microarray samples from healthy and diseased individuals using a differential gene expression protocol generates a list of thousands of genes , and it is not clear which genes are important for what . a key idea , originating from engineering science in general and computer science in particular , is the notion of ' divide and conquer ' , which refers to first breaking down a problem into smaller sub - problems that are simple enough to allow an analysis and then combining the solutions to the subproblems , which gives the solution to the original problem . modular analysis of genomic data implements this strategy by dividing the original genomic data into smaller number of modules and then conquering the reduced complexity by using these modules for prioritization to give a shorter list of disease - associated genes . such genes could either be causal drivers of disease or secondary reactions to disease that could potentially be useful biomarkers . benson and colleagues , in a recent paper in bmc systems biology , have used a modular approach to study allergic asthma . they managed to divide the complexity and arrive at the gene encoding the interleukin-7 receptor ( il7r ) as a putative key regulator in allergic asthma . here , i put their analysis in the context of other modular approaches and discuss the possible use of this methodology for finding and prioritizing useful candidates for therapeutics . not surprisingly , there are several different ideas on how to divide and conquer high - throughput functional genomics data . i will restrict my discussion here to gene expression data , although similar remarks could be made for sequence data . one is : given a module of disease - associated genes , how can we compute and/or experimentally predict which genes are good candidates for therapeutics ? before discussing this problem i will first give an overview of different approaches to the other problem : identifying a module of genes . a module is a group of genes that are related in some way to each other and therefore a module is effectively a measure of similarity . for example , if similarity is defined as the distance in a network , then a graph theoretical calculation will be used . however , if gene functional associations are used , then gene similarity will be measured in terms of gene ontology ( go ) or correlation in gene expression values . therefore , different algorithms are used for dividing the genes into modules , a fact that could be confusing for the clinical researcher . the need to reduce the complexity of the original high - throughput gene expression data applying established engineering concepts , such as principal component analysis ( pca ) and singular value decomposition ( svd ) , reduced the dimensionality of the data . instead of analyzing scattered points ( the samples ) in a high - dimensional space equaling the number of genes , the data could thereby be projected into a two- to four - dimensional space . however , it turned out to be difficult to make a biological interpretation of the resulting linear combinations of large numbers of genes . this problem forced the development of different strategies in which the available knowledge on a limited number of genes could be used to predict the functions of as - yet uncharacterized genes . the use of hierarchical clustering in the classic compendium study on yeast data by rosetta inpharmatics grouped genes ( shown as rows ) by their similarity of expression across several experimental conditions ( columns ) . novel gene function was then predicted by inspecting genes in the same cluster as genes with known functions . subsequent work by eran segal and colleagues developed more statistically sound procedures for identifying robust modules using a bayesian formalism applied to microarray data generated from cancer samples . it became clear , however , that a similarity measure based only on correlations was insufficient , because the clusters ( modules ) or bayesian modules did not have an internal network structure that could be used for a more refined analysis . as a consequence , a large number of studies addressing this problem appeared in the literature at the beginning of 2004 . the idea was that if we could identify the wiring within cellular networks , various different algorithms could be applied to find ' connected groups ' in such networks . now , how can such networks be found using only a small number of experimental samples with a large number of genes ? this is an impossible problem from the point of view of engineering system identification , because the number of possible networks consistent with the data is prohibitively large . the key simplifying insight came from ideker and lauffenburger and was later developed by nicolas luscombe and colleagues in a pioneering paper . here , the edges ( or connections ) in the network were simply defined by transcription factor binding experiments , and gene expression data were used to select the subsets of edges that were active under different conditions . this idea of defining edges in a network using a static scaffold has since been reused using various data types ( protein - protein interaction data , pathways from a database , text mining and dna variants ) . the network of interest is then defined by combining the gene expression data with the scaffold , leaving only the active edges . by searching through such an active network using graph algorithms it is then possible to define ' more ' connected parts in a well defined manner , thereby providing modules with an intrinsic network structure . all the above approaches basically begin with a large , complex dataset , which is then simplified by dividing the data into smaller modules . they instead began with four well characterized breast cancer genes and , by using these ideas , constructed a module in which the genes were ' close ' as defined by expression and proteomic data in several species . benson and colleagues have now contributed to a disease - oriented modular analysis by combining several of the above ideas in a novel manner , as summarized in the flow chart in figure 1 . first , because allergic disease involves multiple cells in different tissues and because no prior characterization of key genes was available , they turned to several different sets of gene expression microarray data in order to find a reference disease - associated gene around which they could construct a module . using the idea that disease - associated genes tend to interact , they could search for other disease - associated genes that were ' close ' . for this purpose , the authors used a graph algorithm that identified a connected clique of 103 disease - associated genes from the microarray data . integration of several public gene expression datasets revealed a group of shared ( blue ) and closely connected clique ( red and black ) disease - associated genes . a subset of these genes were found to share the t - cell receptor signalling pathway , an observation that was then validated by independent experimentation . to identify a transcription factor ( gata3 ) regulating one of this subset , the itk gene , the final module of 37 disease - associated genes consisted of genes listed in public databases as having relevant expression patterns and interacting with gata3 . the t - cell receptor signaling pathway turned out to be a pathway shared by these 103 genes , as detected by the ingenuity pathway analysis tool , which identifies physical , transcriptional and enzymatic interactions from the literature . experimental analysis of this pathway in patient - derived cells revealed strong activation of the itk gene , which is also known to be located in the genomic susceptibility region for allergy . combining a promoter analysis of the itk gene with expression data revealed that the transcription factor gata3 regulated itk . the expression data were used to filter out 10 inactive genes , thus leaving a final module of 37 disease - associated genes around the gata3 transcription factor . the construction of this module was accompanied by several experimental tests at various stages , providing confidence to the analysis . the problem of selecting therapeutic targets within a module has not received much attention in studies that have used a modular approach for reducing complexity . there are various ideas from graph theory on how to compute mathematically defined properties , such as clustering and connectivity in large networks , which then could suggest which nodes are essential . however , essentiality is not necessarily equivalent to disease association . experimental investigators have instead performed target selection using the full dataset in combination with extensive experimental testing . the analysis by benson and colleagues is important because it highlights the difficulty of selecting a disease - associated target from a module of 37 genes despite the elegant prior reduction of complexity . they resorted to using a connectivity criterion , selecting the il7r gene because it had the largest number of connections , and they were also able to demonstrate that perturbing the il7r gene affected other genes and the t - cell phenotype . there are probably several other disease - associated genes in their module that warrant further experimental investigation . benson and colleagues have introduced a useful procedure for defining a module of disease - associated genes . as with most complex diseases , the study of allergy is complicated by the fact that the disease affects several cell types and tissues . the process of identifying such modules therefore requires the kind of stringent experimental validation as was performed by the benson team . despite their careful analysis , because there are other transcription factors for the itk gene that are active in the expression datasets there is a significant risk that several disease - associated genes remain that were not captured in their module . the second step of selecting a gene for therapeutics from a module is even more problematic because we are currently lacking systematic tools for this selection problem . furthermore , it is not unlikely that an efficient therapy could require targeting of several disease - associated genes simultaneously . however , the number of combinations of three genes that can be chosen from a small ten - gene module , for example , quickly exceeds what is experimentally feasible to study . in conclusion , benson and colleagues have devised an interesting method for finding disease - associated genes , but it needs to be evaluated on other complex diseases . their study also makes clear that the problem of prioritizing disease - associated genes within a module for therapeutic studies in the clinic is still unsolved .

aim of databasethe aim of the danish quality database for prehospital emergency medical services ( qems ) is to assess , monitor , and improve the quality of prehospital emergency medical service care in the entire prehospital patient pathway . the aim of this review is to describe the design and the implementation of qems.study populationthe study population consists of all 112 patient contacts defined as emergency patients , where the entrance to health care is a 112 call forwarded to one of the five regional emergency medical coordination centers in denmark since january 1 , 2014 . estimated annual number of included 112 patients is 300,000350,000.main variableswe defined nine quality indicators and the following variables : time stamps for emergency calls received at one of the five regional emergency medical coordination centers , dispatch of prehospital unit(s ) , arrival of first prehospital unit , arrival of first supplemental prehospital unit , and mission completion . finally , professional level and type of the prehospital resource dispatched to an incident and end - of - mission status ( mission completed by phone , on scene , or admission to hospital ) are registered.descriptive datadescriptive data included age , region , and danish index for emergency care including urgency level.conclusionqems is a new database under establishment and is expected to provide the basis for quality improvement in the prehospital setting and in the entire patient care pathway , for example , by providing prehospital data for research and other quality databases . complex diseases such as allergy change gene expression in several cell types and tissues . benson and colleagues have now shown , in a paper in bmc systems biology , that this complexity can be studied effectively using an integrated experimental and computational modular analysis . their strategy revealed a core of allergy - associated genes of potential therapeutic value .

the efficiency and function of the left ventricle ( lv ) as a pump are represented as systole and diastole . the systolic function is energy dependent ; the diastolic function is an important part of cardiac output ( as preload ) and also includes active processes such as the suction effect , which is also energy dependent and can be weakened by ischemia . thus , diastolic dysfunction ( ddf ) is an early marker of ischemia , because it is sensitive to impaired perfusion . most physicians pay attention to the lv ejection fraction ( ef ) for systolic function in the preoperative echocardiographic evaluation of patients about to undergo surgery ; however , lv ddf is often overlooked in such cardiac risk evaluation . lv ddf , and even diastolic heart failure ( dhf ) , are usually not significant problems in the daily life of patients who do not suffer from exercise intolerance , dyspnea , or pulmonary edema . however , when they reach the ward after an operation , they often end up in the intensive care unit ( icu ) with unstable vital signs in combination with pulmonary edema , which are not responsive to the normal use of intravenous ( iv ) epinephrine or norepinephrine . this can lead to poor outcomes , caused by lv ddf regardless of lv systolic dysfunction ( sdf ) , with hemodynamic instability developing due to surgery - induced adrenergic stimulation and anesthesia - induced sympathetic attenuation , resulting in a lengthy stay in the icu or even death . in american heart association - american college of cardiology ( aha - acc ) guidelines , lv ddf is still underestimated in preoperative risk assessment and few clinical data supporting its importance are available . in particular , such patients need more appropriate anesthetic management to prevent postoperative complications and to aid in recovery from anesthesia . because overlooking patients with lv ddf or lv dhf during the perioperative period , regardless of lv systolic function , can be very dangerous and cause poor outcomes , it is important that anesthesiologists are familiar with the pathophysiology , management , and surgical outcomes and are able to notice their phenotypic characteristics , and diagnostic parameters . regardless of lv systolic function and the patient 's symptoms , ddf indicates impaired filling of the ventricles at low pressure and abnormalities in diastolic distensibility , filling , or relaxation , which are usually associated with hypertension , diabetes , or ischemia . the diagnosis of ddf can come from cardiac catheterization and doppler patterns of lv diastolic filling . ddf shows increased lv diastolic pressure ( > 16 mmhg ) with preserved systolic function and lv volume . ddf itself is a physiological or preclinical state that can be compensated for with an increase in left atrial pressure , so there is essentially an asymptomatic period . in addition to echocardiographic or angiographic evidence , when accompanied by symptoms and signs that can be confused with chronic obstructive pulmonary disease such as exercise intolerance , exertional dyspnea , and pulmonary edema with normal ef , it could be considered dhf . dhf is also characterized by a stiff and thickened ventricle , with a small cavity and low cardiac output . dhf can also show a normal ef ; this is called heart failure with a normal ejection fraction ( hfnef ) or preserved ef ( hfpreef ) in preclinical ddf , hypertension , hyperlipidemia , coronary artery disease , and renal dysfunction are prevalent . in one study , the 2-year cumulative probability of developing any symptom was as high as 31% and the probability of cardiac hospitalization was 21% . asymptomatic ddf is generally common in patients without heart failure , increases with age , and is particularly seen in elderly women with hypertension and hypertrophy of the ventricle . ddf alone can predict worse outcomes in patients and their prognosis worsens according to its increasing degree . moreover , more than half of all patients with heart failure have a normal ef . compared to sdf , the incidence and prevalence of ddf are increasing , and in geriatric patients in particular , it can cause hospitalization , increasing hospital costs , and even mortality . a study of two cohorts of patients with decompensated heart failure showed that 35% had preserved lv ef and that they were elderly and mainly women . phillip et al . reported that ~60% of elderly patients with normal ef presenting for geriatric cardiac and non - cardiac surgery had lv - filling abnormalities . moreover , ~15% of these patients had moderate to severe ddf in the absence of symptoms . in patients undergoing high - risk vascular surgery , dhf increased proportionally with age , up to 70% in those aged > 70 years . there are also age - related changes in the cardiovascular system including coronary artery disease , systemic hypertension , and hypertrophic or infiltrative cardiomyopathy . the effects of these conditions can also increase filling pressure due to abnormalities in relaxation and decreased compliance of the lv . diastole can be divided into four stages : isovolumetric ventricular relaxation , rapid passive ventricular filling , diastasis , and atrial contraction . these can also be subdivided into early and late phases . in the isovolumetric relaxation period , after mitral valve closure , calcium is sequestered back into the sarcoplasmic reticulum ; this is an active process for the suction effect , which needs adenosine triphosphate ( atp ) . in ddf , the breakdown of this process causes the ventricle to fail to relax and become stiff / less compliant . this dysfunction can be caused by impaired lv compliance , a passive process , or by changes in lv active relaxation . anatomical diastole occurs from aortic valve closing to mitral valve closing , but at the molecular level , relaxation of the lv starts during the early systolic period , because it starts with the dissociation of actin - myosin cross bridges . because lv segmental and atrio - ventricular synchronization are indispensable in guaranteeing effective relaxation , loss of the atrial kick in atrial fibrillation not only changes lv filling but also slows myocardial relaxation . before doppler - derived variables , the gold - standard measurement of lv diastolic function was the invasive measurement of lv pressures using cardiac catheterization . this can show pressure / volume loops , lv pressure decline , and the time constant of isovolumetric relaxation ( ) . , lv ddf shows a diastolic loop ( compliance curve ) shifted to the left and upwards . in such cases , lv end - diastolic volume is normal but end - diastolic pressure is increased , resulting in pulmonary congestion . however , sdf causes the end - systolic slope to shift to the right and downwards ( fig . 1 ) . in active diastole with isovolumetric relaxation , which is energy dependent , abnormalities in relaxation can be caused by myocardial ischemia , hypertension , aortic stenosis , and hypertrophic cardiomyopathy . in contrast , passive diastole can be impaired by increased stiffness of the lv chamber , caused by an infiltrative disorder ( amyloidosis ) , myocardial fibrosis , and progression from impaired relaxation . age - related changes in the cardiovascular system can be condensed into a reduction of cardiovascular reserve , which includes increased arterial and myocardial stiffness , impaired -adrenergic responsiveness and lv relaxation , and reduced sinus nodal function and baroreceptor responsiveness . aging is related to ventricular and arterial stiffness , which increase and exacerbate myocardial oxygen consumption for cardiac output . apoptotic myocytes are not replaced , but decrease in number with an increased amount of connective tissue matrix . the afterload causes increased arterial stiffness that affects lv relaxation , resulting in ventricular hypertrophy to preserve systolic function . age - related arterial stiffening has two important consequences : decreased aortic distensibility and increased pulse wave velocity , which result in higher pulse pressure ( pp ) and prolonged systolic contraction , respectively . increased pp is an established cardiovascular risk factor and the early reflected wave , because of prolonged systolic contraction , impinges on early diastole , which can cause greater myocardial stroke work , wall strain , and oxygen consumption such as a mistimed intra - aortic balloon pump . beyond this , impaired calcium homeostasis and reduced -receptor responsiveness also cause lv ddf . at the cellular level , reduced sarcoplasmic / endoplasmic reticulum calcium atpase 2 ( serca2 ) activity and increased phospholamban activity contribute to impaired lv diastolic function . the signal dampening of -receptors may be due to receptor down - regulation or diminished coupling of -receptors with intracellular adenylate cyclase activity . consequently , despite increased circulating catecholamines , there is a limited increase in heart rate and contractility in response to endogenous and exogenous catecholamines , so there is impaired baroreflex control of blood pressure ( bp ) . apoptosis , fibrosis , fatty infiltration , and calcification of pacemaker and his - bundle cells can result in conduction block , sick sinus syndrome , and arterial fibrillation , with loss of the atrial kick for preload in the end - diastole stage . arrhythmias ( e.g. , atrial fibrillation ) and myocardial ischemia affecting diastolic timing can decompensate pre - existing ddf further . treatment with blockers or calcium channel blockers has been recommended to control tachycardia or arrhythmias to improve lv filling . patients with ddf in the operating room have some common phenotypic characteristics such as being elderly women and having peripheral edema , hypertension , and obesity . they also tend to have limitations in functional capacity caused by reduced chronotropic , vasodilator , and cardiac reserve . in addition to a functional capacity evaluation , risk factors and phenotypes for heart failure should be checked in the preoperative assessment for ddf because a heart failure history alone is associated with increased morbidity and mortality after non - cardiac surgery ( table 2 ) . biomarkers of ddf are important as prognostic parameters in patients with myocardial ischemia , atrial fibrillation , aortic stenosis , and general heart failure . cardiac catheterization can show increased ventricular diastolic pressure ( > 16 mmhg ) with preserved systolic function and normal ventricular volumes in ddf . beyond the analysis of pressure / volume curves from cardiac catheterization , echocardiographic assessment of diastolic function becomes very important in perioperative risk stratification , surgical care , and anesthetic management of patients undergoing operations . however , before considering echocardiographic findings , the different application of variables in transthoracic echocardiography ( tte ) and transesophageal echocardiography ( tee ) in the outpatient clinic from intraoperative tee should be acknowledged because of differences in the postures of patients and their ventilation status , as well as the effects of anesthetic drugs . the classification and assessment of ddf comes from the initial , transmitral flow ( tmf ) examination with a pulsed - wave doppler ( pwd)-based evaluation of la and lv filling and its abnormalities , according to current guidelines . however , because of the known limitations of the rigid application of conventional doppler indices , the most recent guidelines no longer recommend determining e and a waves on tmf with pwd as the initial step in the decision for grading ddf with normal lv ef . new guidelines based on tte have been simplified and focus on the increase in left atrial pressure ( lap ) rather than grading diastolic dysfunction . in patients with normal lv ef , more than half of the following should be positive to determine the presence of ddf : average e / e > 14 , lateral e < 10 cm / s , tricuspid regurgitation ( tr ) velocity > 2.8 m / s , and la volume index > 34 ml / m . in patients with depressed lv ef and with normal lv ef but myocardial disease , the mitral inflow ratio and the criteria above are used for evaluating lap and grading ddf ( table 3 ) . some doppler variables are valuable for the assessment of myocardial diastolic function including tmf with pwd ( e / a wave , deceleration time [ dt ] , a duration ) , pulmonary venous inflow with pwd ( s / d , atrial reversal [ ar ] ) , tissue doppler image ( tdi ) with pwd ( e/a wave ) , and transmitral color m - mode ( tmf propagation velocity [ vp ] ) , usually on midesophageal ( me ) four - chamber and me long - axis views . conventionally , the lv filling pattern of tmf ( e and a wave , e / a ratio ) and dt are most commonly used in echocardiographic examinations regarding ddf , but these , along with pulmonary venous flow patterns , are dependent on preload variation and heart rate and rhythm disturbances . in this regard , the patient 's preload can be reduced using nitroglycerin ( ntg ) or with the use of the valsalva maneuver in an awake state ( or reverse trendelenburg position or a prolonged lung recruit maneuver in a patient under general anesthesia ) potentially unmasking an e , a pattern and impaired relaxation to discriminate normal from a pseudo - normal state . in contrast , tissue doppler imaging of the mitral annulus is considered a more sensitive tool in the assessment of ddf , in which measurements of e indicate the myocardial velocity , and not blood flow velocity . e may be influenced by preload variation in the normal heart , but becomes preload - independent in a heart with ddf . the abnormal range of e values is also influenced by age : an e < 10 cm / s in those < 50 years and e < 8 in those > 50 . the value of e is higher at the lateral annulus than the septal annulus , because the motion of the lateral annulus is freer than that of the septal annulus due to the surrounding structures . it is convenient to use the lateral e in intraoperative tee and the septal e in tte . measurements of e represent the early diastolic active relaxation phase , but do not represent lv compliance for which the e / e ratio ( the ratio of the transmitral e wave velocity to the mitral annular velocity ) normalizes early transmitral lv filling to mitral annular motion . this is used to estimate mean left atrial pressure , with values > 15 representing elevated filling pressures and < 8 reflecting normal filling pressures . with a ratio between 8 and 15 , this measurement has been considered accurate and relatively independent of lv systolic function , rhythm abnormalities , lv hypertrophy , and functional mitral regurgitation . the prediction of lv filling pressure using doppler modalities is important in patients with ddf . elevated lv filling pressures may be the most important factor in poor outcomes , rather than merely the presence of delayed relaxation . after cardiac surgery , it can also predict a prolonged icu / hospital stay and mortality independently of systolic function . when evaluating diastolic function with echocardiographic tools , measurements should be performed multiple times , considering multiple doppler variables ( table 4 ) . according to these echocardiographic variables , based on tee , it might be suggested that predictors for increased perioperative cardiovascular risk would include e / a of 12 , dt of 150200 ms , e < 8 cm / s , e / e > 15 , vp < 40 cm / s , an enlarged la , and lv hypertrophy in tte . in fact , tdi is of limited use in analyzing regional wall motion abnormalities ; however , there are some recent advances in color tdi , such as strain , strain rate , speckle tracking , and velocity vector imaging that may allow a better appreciation of lv filling dynamics and a more accurate classification of diastolic function . brain natriuretic peptide ( bnp ) and n - terminal probnp ( nt probnp ) are recognized as specific markers of heart failure ( hf ) with acute dyspnea and are elevated in both hfpreef and hf with reduced hf ( hfref ) . these markers tend to be higher in hfref than hfpreef , but do not distinguish hfpreef from hfref effectively . suggested levels for a diagnosis of acute hf are bnp > 100 pg / ml or nt probnp > 300 pg / ml , which are independent predictors of adverse cardiovascular events in hfpreef . other reasons for elevated bnp factors include renal failure and pulmonary hypertension , caused by lung disease , and obesity , which decrease bnp levels . as mentioned above , it is important not to overlook patients with asymptomatic lv ddf in the preoperative evaluations , and risk factors for heart failure should also be considered . although not specific to geriatric patients , the latest acc / aha guidelines should be used for the perioperative care of cardiac patients undergoing general surgery , and if needed , preoperative cardiovascular risk factors should be evaluated and treated by cardiologists . in ischemic heart disease - related procedures , bare - metal stenting can be performed with dual anti - platelet therapy for 46 weeks . statins should not be discontinued before surgery . because the renin - angiotensin - aldosterone system plays an important role in the development of dhf and particularly in myocardial remodeling and fluid retention , angiotensin - converting enzyme inhibitors , angiotensin receptor antagonists , and aldosterone antagonists as aforementioned mentioned , drugs for the treatment of pre - existing heart failure , arrhythmias , hypertension , and ischemic heart diseases ( e.g. , diuretics , -blockers , calcium channel blockers , antiplatelet agents ) should be maintained during the perioperative period . the principles of anesthetic management for patients with ddf must be addressed on a case - by - case basis . in addition to standard monitoring tools , monitoring the volume status in these patients is important , because they are very sensitive to volume changes due to surgical bleeding and sympathetic tone changes due to anesthesia in the perioperative period . thus , instituting an arterial cannula is necessary for invasive arterial monitoring , indirect assessments of preload by stroke volume variance , and frequent blood sampling ; the timing of each should be based on experience and local practice . in major or vascular surgery , central venous catheterization , pulmonary artery catheterization , and/or tee may be needed , which can contribute to evaluating intravascular volume during anesthesia . during anesthesia , patients with ddf or dhf are commonly elderly and may have hemodynamic instability because of general and regional anesthesia disturbing the baseline sympathetic tone , thereby leaving the patient with a higher resting sympathetic tone and altered -receptor sensitivity . in addition , the frequent use of diuretics due to pre - existing diseases , a reduction in the thirst response in the elderly , and age - related renal function changes can aggravate the reduction of preload , increasing the sensitivity to volume status . moreover , direct effects of anesthetics on cardiac inotropy and lusitropy as well as arterial and venous vasodilation can not be ignored with regard to unstable hemodynamics either . the choice of regional versus general anesthesia is still debated and there are no definitive recommendations either way . however , epidural anesthesia is preferable to spinal anesthesia because of slower hemodynamic changes from the slow onset time and the smooth removal of sympathetic tone . in addition , for general anesthesia , iv induction and maintenance with balanced anesthesia of volatile agents and opioids are recommended . sevoflurane during spontaneous ventilation preserves diastolic relaxation better than propofol , but there are no differences between them regarding diastolic function during positive pressure ventilation through balanced anesthesia . the common volatile agents ( sevoflurane , desflurane , isoflurane ) , iv anesthetics ( propofol , midazolam ) , and opioids ( morphine , remifentanil ) have no significant effects on diastolic performance in healthy people , unlike some animal models . however , in high - risk dhf patients , anesthesiologists should avoid acute changes in hemodynamic load conditions , heart rate , and myocardial oxygen balance . to achieve this , a 3050% reduced dose of induction agents should be used , followed by waiting for their delayed effects due to the reduced cardiac reserve and slower circulation time , as well as titrating the anesthetics . intraoperative bp control is important , in that systolic bp must be within 1020% of the baseline value and pp should be kept below the diastolic bp . an increased pp increases ventricular wall stress , alters ventricular contraction time , and impairs early diastolic filling , increasing the cardiac workload . the rule of 70s is useful for controlling bp : age > 70 , diastolic bp > 70 , hr = 70 , and pp < 70 . given the effects on cardiac work by controlling pp , the combined use of low - dose ntg ( 0.54 g / kg / min ) and phenylephrine titration ( from 0.25 g / kg / min ) is applicable and helpful , but either agent alone can worsen hemodynamics . this protocol preserves vascular distensibility , and prevents reductions in preload and coronary perfusion pressure , thereby maintaining stroke volume with minimal cardiac work . in the postoperative period in patients with known ddf , hypoxemia and/or atrial fibrillation are the most common complications , because restored vascular sympathetic tone due to emergence and resolution from general or regional anesthesia causes a volume shift to central blood volume . the subsequent volume overload can result in pulmonary edema and/or atrial fibrillation due to decompensation . because these events can happen acutely in a patient with an initially stable appearance , continued and careful monitoring are essential . maintenance of ntg at a low dose ( 25 g / min ) is also useful for the pulmonary vasculature , and it is important to avoid the risk factors that predispose patients to hf ( table 5 ) . among these factors , a hypertensive crisis can be managed with sound anesthetic practices , planning for appropriate postoperative analgesia , preventing shivering by control of body temperature during the operation , and using iv calcium channel blockers ( nicardipine , diltiazem ) and high doses of iv ntg . specific drugs with positive lusitropic effects are often invaluable in circulatory failure related to ddf and include phosphodiesterase iii inhibitors ( milrinone ) and calcium channel sensitizers ( levosimendan ) , both of which are inodilators . milrinone has inotropic , vasodilatory , and lusitropic ( particularly in a heart failure state ) effects with minimal chronotropy , by increasing calcium ion uptake in the sarcoplasmic reticulum . it can be used at a bolus dose of 50 g / kg over 10 min and an infusion of 0.3750.75 g / kg / min . levosimendan sensitizes the contractile elements to calcium and has positive inotropic effects , modulating the interaction between troponin and calcium . it also has vasodilatory effects by opening atp - sensitive potassium channels and improving both systolic and diastolic functions . ddf of the lv and dhf with preserved ef are not rare pathologies , but are frequently underestimated . in the preoperative evaluation of asymptomatic patients undergoing high - risk surgery , echocardiographic assessment of lv diastolic function should be included in cardiovascular assessments , as should consideration of the risk factors for dhf . early detection of and not overlooking preclinical lv diastolic dysfunction can be useful for the choice of appropriate surgery , appropriate bp control , timely monitoring tools , and proper volume management to maintain normovolemia on a case - by - case basis . although anesthesiologists may not appreciate the stages of ddf , those who are involved in the perioperative care of these patients with cardiovascular disorders need to be able to manage them appropriately . because there is not yet an evidence - based treatment of choice regarding acute decompensated dhf , its management should focus on resolving known precipitating factors such as myocardial ischemia , hypertensive crisis , arrhythmias , and pulmonary congestion . the results of imaging studies are negatively affected by patients movements ; hence , patient s cooperation is required during imaging . children s movements and lack of cooperation are common indications for pediatric procedural sedation during imaging studies such as computed tomography ( ct ) scans ( 1 ) . different rates of success have been achieved by various sedation regimens such as diphenhydramine , propofol , ketamine , midazolam ( 2 ) , chloral hydrate ( 3 ) , pentobarbital ( 4 ) , and dexmedetomidine ( 5 ) ; therefore , the efforts to find an ideal regimen are continued . the selected sedative should have a rapid onset of action , few adverse effects , short and sufficient duration of action , self - maintenance of a patent airway , minimal effects on respiration or hemodynamics , and rapid recovery ( 6 ) . over the last few years , researchers have shown a special interest in finding effective , nonparenteral , sedative agents that do not have injection problems ( 3 ) . ketamine is a noncompetitive antagonist of the n - methyl - d - aspartate receptor ( nmdar ) , which is used for premedication , sedation , and induction as well as maintenance of general anesthesia . quick onset , short duration of action , and maintenance of laryngeal reflexes have made it a popular sedative choice for pediatric patients in the emergency department ( 7 ) . although ketamine is known as a parenteral agent , some researchers have successfully used it as an oral sedative drug ( 8 , 9 ) . midazolam is a potent benzodiazepine with rapid onset and offset effects as well as anxiolytic and amnestic properties , which can be administered through different routes ( i.e. oral , intravenous , intramuscular , rectal , sublingual , and intranasal ) . oral midazolam ( om ) is a safe and effective choice for sedation in children ( 10 ) . in previous studies , the authors have used a combination of oral midazolam and ketamine ( omk ) as premedication and sedative regimens ; besides , some studies reported that combination therapy has higher efficacy without any additional adverse effects in comparison to the methods using each drug alone ( 11 , 12 ) . to the best of our knowledge , limited number of studies have reported the use of om during imaging , which had reported lower effectiveness rate in comparison with chloral hydrate ( 13 , 14 ) ; however , omk has not been used for this purpose , yet . therefore , this study was conducted to compare the effects of omk with om as procedural sedatives in pediatric patients undergoing ct imaging . study design and setting this randomized , double - blinded , clinical trial was conducted from november 2012 to november 2013 in two teaching hospitals ( ayatollah kashani hospital and alzahara hospital ) affiliated to isfahan university of medical sciences , isfahan , iran . the study protocol was approved by ethics committee of the isfahan university of medical sciences . all parents or guardians were informed of the study 's protocol , risks , and benefits and were asked to sign an informed written consent . we included six - month to six - year old children with medium - risk minor head trauma who were scheduled to undergo brain ct scan ( 15 ) . medium - risk minor head trauma was defined as initial glasgow coma scale score of 15 with any history of brief loss of consciousness , posttraumatic amnesia , vomiting , headache , or intoxication . all of these patients were classified as status i or ii according to american society of anesthesiologists ( asa ) physical status classification . children with neurological disorders , anomalies of the cardiovascular system , known allergy to midazolam , gastritis , any serious systemic diseases , those on long - term treatment with hepatic enzyme - inducing drugs or those receiving erythromycin concurrently ( due to drug interaction ) , and patients who had received medication within the preceding 48 hours were excluded . patients were randomly allocated to two groups ; om group received 0.5 mg / kg midazolam ( produced by ; tehran shimi , tehran , iran ) orally and omk group received 0.2 mg / kg midazolam and 5 mg / kg ketamine ( produced by ; rotexmedica , trittau , germany ) orally . demographic and basic characteristics such as age , height , gender , weight , body mass index ( bmi ) , and history of any medical condition were recorded before administration of study drugs . then a nurse , who was blinded to the study , mixed both of the medications with 5 ml of sugar syrup to make it palatable . pulse rate and oxygen saturation ( sao2 ) were continuously monitored by a portable pulse oximeter . blood pressure , heart rate , respiratory rate , and sao2 were recorded at baseline ( just before drug administration ) and every 30 minutes until the patient was discharged from the radiology department . the level of sedation of patients after drug administration was assessed using ramsay sedation scale ( rss ) ( 16 ) . patients who did not show satisfactory response to the sedative drugs within 40 minutes and those who were awakened or moved during the imaging were excluded from further analysis . the interval between administration of sedative drugs and achieving rss of four was considered as the time to achieve adequate sedation . once the patients achieved adequate sedation , they were transferred to a scanner room and the imaging was performed according to the protocol . after the scan , the patients were transferred to another room in the radiology department to monitor and observe their conditions . the time to discharge from radiology department was defined as the interval between the start of sedative administration and return to the baseline alertness and spontaneous breathing . statistical analyses all statistical analyses were performed using the spss 19.0 ( spss inc . , two - way anova was used to compare changes of vital signs during 180 minutes after drug administration between two groups . consort flow diagram of study inter - group differences of vital signs at baseline and at various time points after administration of midazolam and combination of midazolam - ketamine ( df : 18 , p > 0.05 for all comparisons ) . demographic characteristics of the participants a , data are presented as mean standard deviation . the outcomes of patients in midazolam and midazolam - ketamine groups om : oral midazolam ; omk : combination of oral midazolam - ketamine ; rd : radiology department . the mean age of participants was 2.8 1.6 years ( range : 6 months to 6 years ) and 54.5% of the participants were male . table 1 shows the basic characteristics of patients . there was no significant difference between the two groups in age , height , weight , and male to female ratio ( table 1 ) . adequate sedation ( rss of 4 ) was achieved in five patients ( 15.2% ) in the om group and 15 patients ( 45.5% ) in the omk group , which showed a statistically significant difference ( p = 0.015 ) . no significant difference was found between the groups in time of achieving adequate sedation ( p = 0.854 ) and the time of discharging from radiology department ( p = 0.223 ) . adverse effects observed in two patients ( one with nausea and another with vomiting ) in the om group and three patients ( two with nausea and one patient with vomiting ) in the omk group . complications were minor and transient and did not differ between study groups ( p > 0.05 ) . systolic blood pressure , sao2 level , pulse rate , and respiratory rate showed no significant inter - group differences at baseline and at various time points after administration of the drugs ( for all analysis : df = 18 , p > 0.05 , figure 2 ) . our investigation showed that in comparison to om , omk successfully induced sedation in 45% of the patients . om failed to sedate the patient in 85% of the cases and led to administration of additional medication and longer time to obtain the scan . ketamine is used for premedication , sedation , and induction as well as maintenance of general anesthesia with minimal effect on respiration and tendency to preserve autonomic reflexes ( 7 ) . it has been reported that 10 mg / kg of ketamine provides effective sedation and analgesia in young children undergoing wound repair processes ( 9 ) . moreover , 6 mg / kg of ok was reported to be effective in sedation for outpatient pediatric dental surgeries ( 10 ) . om is the most commonly used premedication in the united states ( 18 ) , which has been used as a safe and effective sedative in pediatrics , mainly in pediatric dentistry ( 10 , 17 ) ; however , intravenous midazolam has been found to induce more sedation ( 18 ) . intravenous or nasal midazolam has gained widespread popularity as a sedative for children undergoing radiographic studies such as ct scan ( - ) . the omk and om alone are safe , effective , and practical approach to manage children for minor dental procedures ( 22 , 23 ) . the efficacy of om and ketamine , alone or in combination has been studied in pediatric dentistry sedation and premedication ( - ) . reported better sedative effects during suturing lacerations with ok ( 10 mg / kg ) in comparison to om ( 0.7 mg / kg ) ( 27 ) . although our study was consistent with the previous studies regarding the higher efficacy of omk in comparison to om , most of the previous studies reported success rates of 60% to 90% for om ( 11 , 17 , 28 ) and 46% to 95% for omk ( 11 , 22 ) . showed that the combination of oral midazolam ( 0.5 mg / kg ) and ketamine ( 5 mg / kg ) led to deeper sedation in comparison with om ( 0.5 mg / kg ) alone in children requiring laceration repair . in addition , 27% of patients in the om group and 6% in the omk group needed further intravenous sedation ( 11 ) . ( 12 ) compared the efficacy of om ( 1 mg / kg ) and the omk ( midazolam , 0.5 mg / kg ; and ketamine , 3 mg / kg ) for guiding the behavior of children undergoing dental treatment and reported higher efficacy of omk . on the other hand , there are several studies reporting lower success rate , which are similar to our findings . ( 6 ) compared the sedative effects of intravenous midazolam with pentobarbital during brain ct imaging in children aged six months to six years . they administered pentobarbital to 29 patients ( 53% ) and midazolam ( mean dose , 0.2 0.03 mg / kg ) to 26 patients ( 47% ) . in the midazolam group , only five patients ( 19% ) were successfully scanned with midazolam alone and the remaining 21 patients ( 81% ) required additional medication and took a longer time to scan . another investigation reported a failure rate of 60% in administrating intranasal midazolam ( 0.2 mg / kg ) for sedation of children undergoing ct scan ( 3 ) . ( 29 ) used 0.4 mg / kg of om 20 minutes before the induction of general anesthesia and reported a mild or no sedative effect in 76% to 84% of patients . in this study , the onset time of adequate sedation was 33 minutes in om group and 32 minutes in omk group . ( 27 ) reported an onset time of 20 minutes for ok and 43 minutes for om . other studies reported that the time to reach optimal sedation level would be 15 minutes for om ( 10 , 17 ) . studies , which used om ( 0.5 - 1 mg / kg ) as premedication reported that the best time for optimal preoperative sedation would be 30 to 45 minutes before scan ( 30 ) . barkan observed that the needed time to achieve adequate sedation was 17 minutes for om and 14 minutes for omk . the patients ' blood pressure , respiratory rate , pulse rate , and sao2 levels did not show any significant changes during the sedation . although ketamine and midazolam affect the respiratory and hemodynamics responses ( 11 ) , our results were in line with the findings of previous studies that reported minimal effects of low - dose om and ok on these parameters ( 17 , 31 ) . although further studies are needed to confirm or refute our results and attention to the pharmacological aspects is necessary , lower efficacy of om in this study could be explained as follows : first , we used these drugs for ct imaging that needs a deeper level of sedation in comparison with some other studies that used those drugs in other settings as premedication . second , in the previous studies , intravenous forms of these drugs were used , which might bring about unexpected results ; the same drugs in different studies should be compared with caution . third , the ethnic differences in response to these sedative agents might affect the results . a major cause of variations in drug responses is hepatic cytochrome p450 oxidase ( cyp450)-mediated drug metabolism ( 29 ) . midazolam is almost exclusively metabolized by cyp450 3a ( cyp3a ) isoenzymes ( 32 ) . although previous studies have shown that these parenteral forms can be used orally , the oral form of drugs might bring about results that are more precise . second , the number of participants who were included in the final analysis ( as sedated patients ) was small . this study demonstrated that in comparison with om , omk was more effective in producing a satisfactory level of sedation in children undergoing ct examinations without additional complications ; however , none of these two regimens fulfilled clinical needs for procedural sedation . all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors .

anesthesiologists frequently see asymptomatic patients with diastolic dysfunction or heart failure for various surgeries . these patients typically show normal systolic function but abnormal diastolic parameters in their preoperative echocardiographic evaluations . the symptoms that are sometimes seen are similar to those of chronic obstructive pulmonary disease . patients with diastolic dysfunction , and even with diastolic heart failure , have the potential to develop a hypertensive crisis or pulmonary congestion . thus , in addition to conventional perioperative risk quantification , it may be important to consider the results of diastolic assessment for predicting the postoperative outcome and making better decisions . if anesthesiologists see female patients older than 70 years of age who have hypertension , diabetes , chronic renal disease , recent weight gain , or exercise intolerance , they should focus on the patient 's diastologic echocardiography indicators such as left atrial enlargement or left ventricular hypertrophy . in addition , there is a need for perioperative strategies to mitigate diastolic dysfunction - related morbidity . specifically , hypertension should be controlled , keeping pulse pressure below diastolic blood pressure , maintaining a sinus rhythm and normovolemia , and avoiding tachycardia and myocardial ischemia . there is no need to classify these diastolic dysfunction , but it is important to manage this condition to avoid worsening outcomes . introduction : motion artifacts are a common problem in pediatric radiographic studies and are a common indication for pediatric procedural sedation . this study aimed to compare the combination of oral midazolam and ketamine ( omk ) with oral midazolam alone ( om ) as procedural sedatives among children undergoing computed tomography ( ct ) imaging . methods : the study population was comprised of six - month to six - year old patients with medium - risk minor head trauma , who were scheduled to undergo brain ct imaging . patients were randomly allocated to two groups : one group received 0.5 mg / kg midazolam ( om group ; n = 33 ) orally and the other one received 0.2 mg / kg midazolam and 5 mg / kg ketamine orally ( omk group ; n=33 ) . the vital signs were monitored and recorded at regular intervals . the primary outcome measure was the success rate of each drug in achieving adequate sedation . secondary outcome measures were the time to achieve adequate sedation , time to discharge from radiology department , and the incidence of adverse events . results : adequate sedation was achieved in five patients ( 15.2% ) in om group and 15 patients ( 45.5% ) in omk group , which showed a statistically significant difference between the groups ( p = 0.015 ) . no significant difference was noted between om and omk groups with respect to the time of achieving adequate sedation ( 33.80 7.56 and 32.87 10.18 minutes , respectively ; p = 0.854 ) and the time of discharging from radiology department ( 89.60 30.22 and 105.27 21.98 minutes , respectively ; p=0.223 ) . the complications were minor and similar among patients of both groups . conclusion : this study demonstrated that in comparison with om , omk was more effective in producing a satisfactory level of sedation in children undergoing ct examinations without additional complications ; however , none of these two regimens fulfilled clinical needs for procedural sedation .

the common occurrence of these tumors is prevalent among males in their second or third decades of life with site predilection toward trunk and extremities , especially in the forearm . however , the incidence of al is extremely rare in the intraoral region , with only 21 reported cases in english literature . a 90-year - old woman with no significant medical history was referred to our department for evaluation of painless mass arising from oral cavity . the tumor was noticed by the patient 20 years ago as a small nodule on the right buccal mucosa and since then it gradually increased in its size . the growth reached a size , which could not accommodate in mouth , and due to constant irritation from the tumor , the patient coughed the growth for 5 years . since then on extraoral examination , a solitary , smooth , nontender and nonpulsatile pedunculated growth of size 14 cm 10 cm with variable consistency was evident with intraoral origin [ figure 1 ] . clinical image shows preoperative tumor on intraoral examination , single , smooth , firm and nontender stalk - like pedicle of diameter 2 cm was seen originating from the right buccal mucosa . based on the history and clinical examination , we provisionally diagnosed it as a benign growth . surgical excision of the tumor was done under general anesthesia [ figures 2 and 3 ] . clinical image shows excised tumor mass clinical image shows postoperative frontal view histopathology confirmed it as noninfiltrating al [ figures 46 ] . one - year follow - up of the patient revealed no evidence of recurrence . ethical approval from the institutional review board was obtained for the publication , and patient release form was signed by the patient . clinical image shows histopathology section ( 4 magnifications ) clinical image shows histopathology section ( 10 magnifications ) clinical image shows histopathology section ( 40 magnifications ) lipomas are the most frequently found soft - tissue benign tumors , but their occurrence in head and neck region is rare . al , in the oral cavity , was first documented by davis et al . as a growth on the hard palate . history of trauma , lipomatous differentiation by hormones during puberty , vascular proliferation of a congenital lipoma and fatty degeneration of a central hemangioma have been implicated as possible etiological factors . the mean diameter at the largest portions of all the 21 documented als occurring in oral region was 3 cm , and the mean onset age of the patients was about 29 years old . although diagnostic modalities such as magnetic resonance imaging , computed tomography , ultrasonography and aspiration biopsy have been used to differentiate between hemangioma , lipomas and al , the diagnosis of these tumors is confirmed only by histopathology . the standard treatment of choice is surgical excision for the noninfiltrating als and complete surgical excision with a clear surgical margin for poorly encapsulated infiltrating als to avoid recurrence . the case we have presented showed the typical clinical and histological findings of a noninfiltrating al . its most striking and unique features were its occurrence in a 90-year - old female with a 20-year long history and pedunculated mass of 14 cm 10 cm . with regard to the size and its typical pedunculated appearance , our present case can be considered to be the first of its kind in literature . attention deficit hyperactivity disorder ( adhd ) is a behaviorally defined disorder characterized by a persistent pattern of inattention and/or hyperactivity impulsivity that is more frequent and severe than typically observed in individuals at a comparable level of development ( american psychiatric association , 2013 ) . substantial heterogeneity at both the genetic and phenotypic level has significantly impeded our understanding of the underlying biological mechanisms of adhd . as the past two decades of research on causal genetic and neurobiological risk factors for this illness have been met with limited success ( for reviews , see elia and devoto , 2007 ; franke et al . , 2009 ) , one promising strategy for investigating the pathogenesis of adhd involves the investigation of a disorder with known genetic etiology that shares similar phenotypic characteristics an approach that is supported by findings that large , rare chromosomal deletions and duplications ( copy number variants ; cnvs ) are implicated in adhd etiology ( williams et al . , 22q11.2 deletion syndrome ( 22q11ds ; velocardiofacial syndrome ) is a particularly compelling model , as it is a relatively common cnv that is highly penetrant for adhd ; approximately 40% of 22q11ds patients meet clinical criteria for the disorder ( green et al . , 2009 ) . however , it is not yet known whether the neural substrates of attentional dysfunction in 22q11ds are shared with idiopathic adhd . impaired response inhibition ( ri ) has been consistently implicated as a hallmark of idiopathic adhd ( dickstein et al . , 2006 ; durston , 2003 ; epstein et al . , 2007 ; schulz et al . , 2004 ; tamm et al . , 2004 ; van rooij et al . , 2015 ) . most commonly measured with either go / no - go or stop - signal task paradigms , successful inhibition of a prepotent response has been shown to rely upon engagement of a right - lateralized fronto - striatal network in healthy individuals ( aron and poldrack , 2006 ; aron et al . studies on the neural underpinnings of ri in adults with adhd have yielded inconsistent results , with some studies reporting less ri - related activation in adhd patients than controls ( e.g. , mulligan et al . , 2011 ; sebastian et al . , 2012 ; van rooij et al . , 2015 ) and , 2012 ; congdon et al . , 2010 ) or greater activity for adhd patients as compared to controls ( dillo et al . , 2010 ; karch et al . , however , the bulk of the evidence suggests a disturbed pattern of ri - related neural activation in adults with adhd . discrepancies in previous findings may be attributable to several factors , including differences in task parameters , medication status , symptom severity , and small sample size . consistent with findings of impaired performance on ri - related behavioral tasks in 22q11ds ( shapiro et al . , 2013 ) , using functional magnetic resonance imaging ( fmri ) we previously found that young adults with 22q11ds show significantly less activation in ri - related regions during successful ri relative to healthy controls ( montojo et al . , 2015 ) . further , we found that ri - related activity within the left middle frontal gyrus and basal ganglia was associated with the severity of self - reported cognitive impulsivity , suggesting that reduced engagement of ri - related regions in 22q11ds may be relevant to the behavioral manifestations of the disorder . despite symptomatic overlap between 22q11ds and idiopathic adhd , it is not yet known whether disturbances in the neural circuitry underlying inhibitory control difficulties are shared between these clinical populations . using fmri , we examined neural activity in patients with 22q11ds , idiopathic adhd , and demographically comparable healthy controls during performance of a stop - signal task . we hypothesized that , relative to controls , individuals with 22q11ds and those with idiopathic adhd would show reduced recruitment of fronto - striatal regions involved in inhibitory control . in addition , we explored the relationship of neural activity during task performance to dimensional measures of cognitive and behavioral impulsivity , to determine whether brain behavior associations were similar across groups . based on prior literature in 22q11ds and adhd ( montojo et al . , 2015 ; van rooij et al . , 2015 ) we hypothesized that , while 22q11ds and idiopathic adhd patients would share similar dysfunction within ri - related neural systems , this shared dysfunction would show different relationships to behavior . specifically , we hypothesized that 22q11ds patients would show an inverse relationship between neural activity during ri and cognitive impulsivity ( montojo et al . , 2015 ) . given the phenotypic and genetic heterogeneity known to exist within the adhd population , we hypothesized that we would not observe a significant relationship between neural activity and cognitive or behavioral impulsivity within this group . seventy - two participants ( 19 patients with 22q11ds , 23 with an adhd diagnosis , and 30 healthy adults ) were included in the study . 22q11ds participants include individuals with a molecularly confirmed diagnosis of 22q11.2 deletion syndrome recruited from an ongoing longitudinal study at the university of california los angeles ( ucla ) . individuals with adhd were recruited from the los angeles area as part of the consortium for neuropsychiatric phenomics ( cnp ) at ucla . the current study includes a partially overlapping subset of patients with 22q11ds and adhd that were included in previous publications ( congdon et al . , 2014 ; montojo et al . , four 22q11ds participants whose data were collected for the stop - signal task were excluded from analysis for the following reasons : low signal - to - noise ratio ( n = 1 ) , too much translational movement ( n = 1 ) , and poor registration between functional and anatomical scans ( n = 2 ) . thus , our final sample consisted of 15 patients with 22q11ds , 23 with adhd , and 30 healthy adults . all diagnoses were determined using the structured clinical interview for the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) ( scid ; first et al . , 2004 ) by trained raters who participated in an ongoing quality assurance program ( ventura et al . , adhd diagnoses in the cnp sample were confirmed with the adult adhd interview ( a structured interview derived from the kiddie schedule for affective disorders and schizophrenia , present and lifetime version ( ksads - pl ) ( kaufman et al . , 1997 ) ) , in order to provide a more detailed characterization of lifetime history of adhd in adults . prior to conducting diagnostic interviews , clinicians had to achieve good to excellent reliability with a set of gold standard ratings ( kappa coefficients of .90 or greater for categorical diagnoses and intra - class correlation coefficients ( iccs ) ranging from 0.85 to 1.00 for quantitative symptom ratings ) . diagnostic formulations for all 22q11ds cases were additionally reviewed in consensus diagnosis meetings led by the study director , a board - certified psychologist ( ceb ) . training , reliability and ongoing quality assurance procedures for psychiatric diagnostic assessments are detailed in a prior publication ( jalbrzikowski et al . , 2013 ) . exclusion criteria for all study participants included the following : neurological or medical condition that might affect performance , insufficient fluency in english , substance or alcohol abuse and/or dependence with the past 6 months , any contraindications to scanning , and left - handedness ( further details are provided in the supplementary material ) . the scid was used to ensure that healthy controls did not meet criteria for any current major mental disorder ( see the supplementary material and thakkar et al . , 2014 for additional details of inclusion / exclusion criteria ) . stable medications were permitted in 22q11ds and adhd participants , as it was not ethically permissible to ask participants to stop taking physician - prescribed medication in order to participate in the study . the ucla institutional review board ( irb ) approved all study procedures and informed consent documents . in both studies , after screening and neuropsychological assessment , participants took part in a behavioral testing session immediately prior to a 1-hour scan . in the behavioral testing session , participants received training on the stop - signal task in the form of one initial demonstration and trial run before completing one experimental run while inside the scanner . stimulus presentation and timing of all stimuli and response events were achieved using matlab ( mathworks ) and the psychtoolbox ( http://www.psychtoolbox.org , brainard , 1997 ) on an apple powerbook . for the experiment block administered in the scanner , each participant viewed the task through mri - compatible goggles and responded with his or her right hand on an mr - compatible button box in the scanner . participants completed a tracking version of the stop - signal task ( logan , 1994 ) enabled isolation of neural activation associated with the inhibition of an already - initiated motor response , and calculation of an individualized measure of inhibitory control ( stop - signal reaction time , ssrt ) . participants were instructed to respond quickly when a go stimulus was presented on the computer screen ( which consisted of left- or right - ward pointing arrows ) , except on the subset of trials where the go stop signal ( a 500 hz tone presented through headphones ) , in which case participants were instructed to withhold their response . the onset of the stop - signal , or stop - signal delay ( ssd ) , was adjusted according to the participant 's performance such that the ssd increased following a successful inhibition ( making the next trial more difficult ) and decreased following a failed inhibition ( making the next trial easier ) which ensured that subjects successfully inhibited on approximately 50% of stop trials . a complete description of the task and fmri acquisition parameters is presented in the supplementary material . the barratt impulsiveness scale ( bis ) version 11 ( bis-11 ; patton et al . , 1995 ) is a 30-item questionnaire assessing aspects of impulsivity which has been applied to various clinical groups , including those with adhd ( crunelle et al . in addition to calculating a total bis score , we used the revised , two - factor scoring method of the bis , which has been proposed as an alternative multidimensional structural representation of impulsivity ( reise et al . , 2013 ) . the revised method of scoring the bis results into two correlated factors : ( 1 ) cognitive impulsivity , reflecting difficulties in attentional control , concentration , careful and deliberate thinking , and planning ( e.g. , not a steady thinker , no self - control / concentration and not planful ) , and ( 2 ) behavioral impulsivity ( with some cognitive elements ) , which reflects acting impulsively , changing jobs , moving residences relatively often , and a scattered quick - paced cognitive tempo ( e.g. , extraneous racing thoughts , acts impulsively , and changes , moves around ) . we note that while the bis-11 measures an individual 's subjective view of his / her own behavior , there is a strong correlation between scores on this measure and clinical diagnoses of adhd ( malloy - diniz et al . , 2007 ) . for both scales , higher scores reflect higher levels of trait impulsivity . supervised clinical psychology doctoral students or phd staff administered a comprehensive neurocognitive battery . iq data were acquired for all participants using the wechsler abbreviated scale of intelligence ( wasi ; wechsler , 1999 ) or the wechsler adult intelligence scale ( wais - iv ; wechsler , 2008 ) . stop - signal task data were analyzed as has been previously described ( congdon et al . , 2010 , 2012 ) and as detailed in the supplementary material . briefly , ssrt was estimated using the quantile method ( band et al . , 2003 ) , with longer ssrt values reflecting poorer inhibitory control . additional performance measures included mean and standard deviation of reaction time ( rt ) on go trials , percent inhibition on stop trials ( i.e. , successful inhibition ) , and percent correct on go trials . to examine demographic differences between the three groups ( 22q11ds vs. adhd vs. controls ) we conducted univariate anovas for continuous variables or chi - square tests for categorical variables . pearson correlations were conducted to assess the relationship between demographics and stop - signal task performance . to examine group differences with regard to stop - signal task performance and bis-11 scores as iq and years of education showed significant differences between groups , ancovas were also conducted with years of education included as a covariate , in order to verify anova results ( iq was omitted given the high correlation between years of education and iq : r(68 ) = 0.577 , p < 0.01 ) . age was also included as a covariate given that this variable showed associations with task performance ( see results section ) . analyses were performed using tools from the fmrib software library ( http://www.fmrib.ox.ac.uk/fsl ) , version 5.0 ( s.m . smith et al . , 2004 ) , and preprocessing steps are outlined in the supplementary material . for each subject , stop successful null , and stop unsuccessful stop successful contrasts were computed , and the output from the subject - specific analyses was then analyzed using a mixed - effects model with flame for between - group comparisons . to examine between - group differences , group - level statistics images were thresholded with a cluster - forming threshold of z > 2.3 and a cluster probability of p < 0.05 , corrected for whole - brain multiple comparisons using the gaussian random field theory . as our primary goal was to examine between - group differences in ri - related activation , and as patterns associated with ri have been previously reported in each group alone ( congdon et al . 2015 ) , we present below only the results from stop successful go between - group comparisons . the whole - brain , between - group contrasts included the following : controls > 22q11ds , controls > adhd , 22q11ds > adhd , and adhd > 22q11ds . results for additional task contrasts are presented in the supplementary material . in order to identify differences in neural activity during successful ri that were common to both clinical groups relative to controls , we used activity maps for the contrasts of controls > 22q11ds and controls > adhd and performed a conjunction analysis to define a set of significantly overlapping regions between these two contrasts . we then extracted percent signal change values from this overlapping set of regions in order to visualize the patterns of group differences in neural activation for these regions of overlap ( for details , see methods for visualizing group differences below ) . in order to investigate correlates of ri - associated neural activity in these clinical groups , we characterized relationships between neural activity and bis-11 scores using whole - brain regressions , within 22q11ds and adhd patients only , given the limited range of bis-11 scores within control participants . first , to test for differences in the relationship between ri - related activation and bis-11 scores between the 22q11ds and adhd groups ( i.e. , an interaction effect ) , whole - brain regressions were defined for the stop successful go contrast with either bis cognitive or behavioral impulsivity subscale scores and group membership as covariates of interest , including age and sex as covariates of no interest . second , to further explore the pattern of association between ri - related activation and bis-11 scores within the 22q11ds and adhd patient groups separately without regard to group differences in these relationships we defined whole - brain regressions for the stop successful go contrast with either bis cognitive or behavioral impulsivity subscale scores as covariates of interest , including age and sex as covariates of no interest . each whole - brain regression was thresholded with a cluster - forming threshold of z > 2.3 and a cluster probability of p < 0.05 , corrected for whole - brain multiple comparisons using the gaussian random field theory . this approach allowed us to : 1 ) test whether there were differences in the association between neural activity during task performance and bis scores between groups ( 22q11ds > adhd ; adhd > 22q11ds ) ; and 2 ) subsequently investigate the associations between neural activity and bis scores within each group alone , in order to understand the directionality of any significant interaction effects . in order to visualize : 1 ) the common regions of reduced ri - related neural activity across both patient groups relative to controls ; and 2 ) the relationship between ri - related neural activity and bis-11 scores within each group , we extracted percent signal change values corresponding to a 1-s stimulus convolved with a double - gamma hemodynamic response function from each contrast of interest ( following mumford and poldrack , 2007 ) following inspection of group contrasts . for the conjunction analysis , after inspecting clusters of activation that significantly differed between both patient groups as compared with controls , we intersected the group conjunction map with an anatomical atlas ( the fsl harvard oxford probabilistic atlas thresholded at 25% ) to extract percent signal change from anatomically - constrained regions that contained the clusters of activation identified in the conjunction map . the anatomically constrained regions that contained these clusters of activation included the anterior cingulate cortex ( acc ) , cerebellum , inferior parietal lobe , middle frontal gyrus , middle temporal gyrus ( posterior ) , paracingulate gyrus , superior frontal gyrus , and left and right thalamus . by intersecting our group conjunction map with an anatomical atlas , we were able to extract percent signal change from multiple clusters within anatomically - defined regions . in order to visualize the relationship between stopping - related activation and self - reported impulsivity within each group , percent signal change was extracted from significant clusters identified in each whole brain regression of stop successful go activity on impulsivity scores . follow - up analyses were conducted to investigate : 1 ) the effects of psychostimulant use and iq on fmri results , and 2 ) the effects of group differences in performance on go trials on ri - related neural activity ( see the supplementary material ) . the total sample consisted of 68 participants ( 15 22q11ds , 23 adhd , and 30 healthy adults , age 1838 years old ) . as shown in table 1 , 22q11ds , adhd , and control groups were matched on all demographic factors except for years of education and iq . adhd patients and controls had significantly higher iq and more years of education than 22q11ds patients . there was no significant difference in years of education or iq between adhd patients and controls . 33% ( 5 out of 15 ) had a clinical diagnosis of adhd , and 20% ( 3 out of 15 ) were taking psychostimulants . in adhd patients , 40% ( 8 out of 23 ) reported currently taking psychostimulants . as shown in table 2 , there were no significant differences in ssrt between the three groups and given the tracking nature of the task design percent inhibition on stop trials was close to 50% in each group . however , there were significant differences in percent correct on go trials and median reaction time ( rt ) on go trials , with adhd patients and controls showing higher accuracy and faster response times on go trials as compared to 22q11ds patients . there were no significant differences between patients with adhd and controls in accuracy or median rt on go trials . these results remained unchanged when controlling for age and years of education . across the three groups , there was a significant relationship between percent correct on go trials with age ( r = 0.365 , p < 0.01 ) and years of education ( r = 0.575 , p < 0.01 ) . participants who were older or had more years of education performed with higher accuracy on go trials relative to younger or less educated participants . there was no association between any of the other demographic or performance variables ( see the supplementary material ) . , there were significant group differences in cognitive impulsivity . specifically , 22q11ds and adhd patients showed significantly elevated cognitive impulsivity scores relative to controls , while there were no significant differences between adhd and 22q11ds patients . there were also significant group differences in behavioral impulsivity . specifically , adhd patients showed significantly elevated behavioral impulsivity scores relative to 22q11ds patients and controls , while there was no significant difference between 22q11ds patients and controls . these results remained unchanged when controlling for years of education ( see the supplementary material ) . as shown in fig . 1 and table 3 , a direct comparison between controls and 22q11ds patients during successful ri ( stop successful go contrast ) revealed significantly increased activation in controls relative to 22q11ds patients in the bilateral medial frontal cortex / acc / paracingulate gyrus , bilateral middle / inferior / superior frontal gyrus , bilateral occipital and parietal regions , right middle temporal gyrus , posterior cingulate cortex ( pcc ) , bilateral basal ganglia ( thalamus ) and cerebellum . a direct comparison between controls and adhd patients revealed a similar pattern of increased activation for controls relative to adhd patients in the medial frontal cortex / acc paracingulate , right middle frontal gyrus , bilateral inferior parietal cortex , bilateral middle temporal gyrus , bilateral occipital cortex , bilateral basal ganglia ( thalamus ) , and cerebellum , while there were no regions showing greater activation for adhd patients as compared to controls . as controls showed greater ri - related activation than both 22q11ds and adhd patients across multiple brain regions , we conducted a conjunction analysis in order to determine the regions common to both clinical groups ( price and friston , 1997 ) . as illustrated in fig . 1 , conjunction regions that showed overlap for 22q11ds patients and adhd patients compared to controls included the medial frontal cortex / anterior cingulate / paracingulate gyrus , right middle frontal gyrus , left inferior parietal cortex , cerebellum ( bilaterally ) , thalamus ( bilaterally ) , and right middle temporal gyrus . corresponding percent signal change plots are illustrated in fig . 2 , showing that controls show greater activity than both adhd and 22q11ds patients within ri - related regions , including the acc , bilateral cerebellum , left inferior parietal cortex , right middle frontal gyrus , right middle temporal gyrus , paracingulate gyrus , superior frontal gyrus , and bilateral thalamus . as shown in fig . 3 and table 3 , a direct comparison between adhd and 22q11ds patients revealed significantly increased activation that was specific to the bilateral middle frontal gyrus for patients with adhd relative to those with 22q11ds . there were no regions showing greater activation for 22q11ds patients as compared with adhd patients . since we observed a significant difference in performance for go trials , which may affect the neural activity findings for the stop successful go contrast ( see the supplementary material for details ) . to further explore differences in ri - related activation between clinical groups , we examined the relationship between ri - related activation and trait impulsivity as a function of group status ( 22q11ds vs. adhd ) . 4a and detailed in table 4 , a significant interaction was observed between group and cognitive impulsivity , such that 22q11ds patients showed an inverse correlation between activity in the medial frontal cortex and precuneus with cognitive impulsivity , while adhd patients showed a positive correlation in these same regions . follow - up regressions within 22q11ds patients alone confirmed a negative association between cognitive impulsivity and ri - related activation in the medial frontal gyrus , precuneus / posterior cingulate cortex , and inferior parietal cortex ( table 4 ) . no regions were positively associated with cognitive impulsivity in the 22q11ds patients , and no significant correlation with cognitive impulsivity was evident in the adhd group alone after correction for multiple comparisons . as illustrated in fig . 4b and detailed in table 4 , a significant interaction in the opposite direction was observed between patient group and behavioral impulsivity , 22q11ds patients showed a positive correlation between activity in the left motor cortex and behavioral impulsivity , while adhd patients showed an inverse correlation . follow - up regressions within 22q11ds patients alone confirmed a positive association between successful stopping - related activation in the left motor cortex and behavioral impulsivity , such that increased neural activity in this region during successful stopping was associated with more severe self - reported behavioral impulsivity . no brain regions were inversely correlated with behavioral impulsivity in the 22q11ds patients , and no significant correlation with behavioral impulsivity was evident in the adhd group alone after correction for multiple comparisons . in a follow - up analysis , we excluded one outlier that was evident within the 22q11ds sample for the behavioral impulsivity score . while the interaction was no longer significant , the 22q11ds within - group correlation between ri - related activity and behavioral impulsivity remained significant to our knowledge , this is the first study to directly compare the neural substrates of response inhibition ( ri ) across distinct clinical populations associated with attentional dysfunction ( 22q11ds and idiopathic adhd ) and healthy controls . we hypothesized that , while 22q11ds and idiopathic adhd patients would share similar dysfunction within ri - related neural systems , this shared dysfunction would show different relationships to behavior . specifically , we anticipated that 22q11ds patients would show an inverse relationship with cognitive impulsivity ( montojo et al . , 2015 ) , whereas we would not observe a significant relationship between neural activity and cognitive or behavioral impulsivity within the adhd cohort . supporting our hypotheses , our fmri conjunction findings indicate that , during performance of the stop - signal task , 22q11ds and adhd patients show reduced ri - related neural activity compared to healthy controls within multiple overlapping brain regions , specifically the acc , middle and superior frontal gyri , middle temporal gyrus , inferior parietal cortex , paracingulate gyrus , bilateral thalamus and cerebellum . in addition to this shared pattern of hypoactivation across both clinical groups , 22q11ds patients showed reduced ri - related neural activity relative to adhd patients within the bilateral middle frontal gyrus . also in agreement with our predictions , in 22q11ds patients ( but not adhd patients ) , reduced neural activity within the medial frontal gyrus , precuneus / posterior cingulate , and inferior parietal cortex was associated with greater self - reported cognitive impulsivity . this measure of cognitive impulsivity was elevated in 22q11ds patients , in agreement with prior findings regarding the nature of adhd symptomatology in this population ( antshel et al . , 2008 ; niklasson et al . , these findings suggest that 22q11ds and idiopathic adhd patient groups share a pattern of reduced neural engagement during ri , relative to healthy controls , but that 22q11ds and idiopathic adhd patient groups differ with regard to the relationship of ri - related neural activity to trait impulsivity . specifically , these findings suggest that neural dysfunction during ri in 22q11ds is associated with variability in cognitive impulsivity , and not behavioral impulsivity . this relationship was not observed in patients with adhd . in the current study , both 22q11ds and idiopathic adhd patients showed significantly less engagement of several ri - related regions that have been implicated in prior research ( aron et al . , 2007 ; aron and poldrack , 2006 ; whelan et al . , 2012 ) . the use of a tracking stop - signal task ensured that participants successfully inhibited responses 50% of the time , which results in an equal number of successful stop trials for comparison and controls for difficulty level across participants . as such , findings of reduced activation in these cortical regions and the thalamus during successful inhibition in both patient groups are consistent with models of ri suggesting that the right inferior frontal cortex , via connections with a prefrontal - basal ganglia network , implements inhibition of a prepotent motor response ( aron et al . , 2014 ) . furthermore , findings of reduced ri - related neural activation in these patient groups , both of which are characterized by deficits in inhibitory control , are consistent with prior research ( mulligan et al . , 2011 ; sebastian et al . , 2012 ; van rooij et al . , 2015 ) . with regard to behavioral performance , it is somewhat surprising that there were no differences in ssrt between groups , given that deficits in response inhibition are widely reported in adhd . along these lines , we observed non - significantly increased accuracy on go trials in our adhd sample as compared with controls . because this study included adult participants with adhd who were able to complete up to 2 days of testing and from whom we have complete , usable data , it is possible that these individuals are not characterized by pronounced deficits in behavioral indices of response inhibition , or that they have acquired compensatory mechanisms to overcome response inhibition deficits . from that standpoint , the findings of anomalous patterns of neural activity during ri in the adhd group we previously found that healthy controls showed significantly greater activation than 22q11ds patients within the frontal cortical and basal ganglia regions during successful ri ( montojo et al . , 2015 ) . one prior study on adolescent 22q11ds patients showed greater activation in the left parietal regions compared to healthy controls during performance of a go / no - go task ( gothelf et al . however , in addition to a difference in tasks used , the different findings may be accounted for by age differences , as differences in neural activity during this age range may reflect changes in neural trajectories during this developmentally sensitive period . in terms of ri - related activation in idiopathic adhd patients , although reports are mixed , the bulk of the evidence supports a pattern of reduced neural activation during ri as compared with controls ( cortese et al . , 2012 ; mulligan et al . , 2011 ; sebastian et al . , 2012 ; strong support comes from a recent large - scale study reporting a pattern of relative hypoactivity in 185 adolescents with adhd , as well as in their unaffected siblings , as compared with healthy controls . employing the stopsignal task , the authors found reduced activation during successful ri seen in the left inferior and superior frontal gyri in adhd patients as compared with unaffected siblings and controls , and reduced ri - related activation seen in the left supramarginal gyrus , right postcentral gyrus , and right temporal parietal junction in both adhd patients and their unaffected siblings as compared with controls ( van rooij et al . , 2015 ) . these findings suggest that ri - related deficits in activity within these regions may be useful endophenotypes that extend beyond the affected individuals of the family . while there are also reports of greater fronto - striatal activation ( dillo et al . , 2010 ; karch et al . , , 2012 ; congdon et al . , 2014 ) , during ri in adhd patients as compared with controls , a review of this heterogeneous literature suggests that differences in the cognitive paradigm employed ( e.g. , stop - signal task vs. go / no - go task ) , medication status , symptom severity , and small sample size may account for these discrepant findings . our findings of common deficits in neural activity during ri between idiopathic adhd and patients with a specific genetic mutation that is highly penetrant for the illness suggest that this pattern of hypoactivation during ri may be a valuable neural endophenotype underlying attentional dysfunction . the direct comparison in ri - related neural activity between 22q11ds and adhd patients was of particular interest to determine whether the clinical groups differed in ri - related neural circuitry . this analysis revealed that adhd patients showed greater activity in the bilateral middle frontal gyrus regions compared to 22q11ds patients . although the right inferior frontal cortex has been shown to be central to ri , it does so through a network of prefrontal basal ganglia regions ( aron et al . , 2014 ) and the middle frontal gyrus is often engaged during suppression of a prepotent response ( e.g. , dambacher et al . , neuroimaging studies have additionally implicated the middle frontal gyrus in the processing of higher order information , including functions of working memory ( leung et al . , 2002 ; mccarthy et al . , 1994 ) and episodic retrieval ( rajah et al . , 2011 ) . our findings indicate substantial overlap across clinical groups in terms of hypoactivation in ri - related neural circuitry relative to controls , but with additional dysfunction in the middle frontal gyrus in 22q11ds patients . impulsivity is a key symptom of adhd ( robbins et al . , 2012 ) and has been defined as a trait that leads to actions which are poorly conceived , prematurely expressed , unduly risky or inappropriate to the situation and that often result in undesirable consequences the complexity of defining impulsivity has led researchers to suggest that impulsivity may not be a unitary construct ( dalley et al . , 2011 ) . in line with this , a recent study proposed that a two - factor model including cognitive and behavioral impulsivity may best describe the multidimensional structural representation of self - reported trait impulsivity as measured by the bis-11 ( reise et al . , 2013 ) . in terms of trait impulsivity , both 22q11ds and adhd patients showed greater cognitive impulsivity compared to healthy controls , as measured by the bis-11 . the elevated cognitive impulsivity scores that we observed within the 22q11ds group are of interest given that 1 ) the cognitive impulsivity subscale specifically captures difficulties in attentional control ( reise et al . , 2013 ) , and 2 ) adhd diagnoses in 22q11ds are primarily of the inattentive subtype ( antshel et al . , 2008 ; niklasson et al , we observed that while adhd patients showed elevated behavioral impulsivity relative to both 22q11ds patients and controls , 22q11ds patients did not show increased behavioral impulsivity relative to controls . thus , these findings are in general agreement with previous literature and suggest that 22q11ds and adhd patients share elevated cognitive impulsivity , while adult adhd patients exhibit elevations in both cognitive and behavioral impulsivity . our findings further indicate that deficits in ri - related neural activation are associated with increased cognitive impulsivity in 22q11ds patients , but not behavioral impulsivity . specifically , as cognitive impulsivity increases , ri - related neural activity decreases within the medial frontal cortex , precuneus / posterior cingulate , and inferior parietal cortex . we have previously reported an inverse correlation between ri - related activation and cognitive impulsivity in 22q11ds patients ( montojo et al . , 2015 ) within a set of a priori ri - related regions of interest ( primarily frontal and basal ganglia regions ) . here , we extend those findings in a whole - brain regression analysis , to demonstrate an association between ri - related neural activity and cognitive impulsivity in a broader set of regions . together , these complementary analysis approaches provide convergent evidence that self - reported cognitive impulsivity is inversely associated with ri - related neural activation in multiple brain regions . in contrast , the finding of a positive association between behavioral impulsivity and ri - related neural activity in the left motor cortex in 22q11ds patients is novel . although this association may reflect a compensatory mechanism in patients required to achieve sufficient ri , the presence of an outlier driving the interaction effect leads us to exercise caution in interpreting these findings and suggests that replication is necessary . while pathophysiologic models of adhd have primarily focused on dysfunction within fronto - striatal neural circuitry ( arnsten , 2009 ; cortese et al . , 2012 ; precuneus interactions may constitute a new locus of dysfunction in adhd ( castellanos and proal , 2012 ; castellanos et al . , 2008 ) . specifically , a prior study reported reduced functional connectivity between the anterior cingulate cortex and precuneus / posterior cingulate cortex regions during resting state within adhd patients ( castellanos et al . , 2008 ) . we extend those findings here to patients with attentional dysfunction associated with homogeneous genetic etiology ( 22q11.2 deletions ) , by reporting an inverse correlation between ri - related neural activity and cognitive impulsivity symptoms within the precuneus / posterior cingulate . in addition , 22q11ds patients show decreased cortical volume and surface area within the precuneus ( jalbrzikowski et al . , 2013 ) . these findings are in agreement with the idea of this region as another locus of dysfunction , with variability in neural activity being related to the severity of trait impulsivity . with regard to the medial prefrontal cortex , both primate and human studies implicate this region in cognitive control functions ( ridderinkhof et al . , 2004 ) , including : the detection of unfavorable outcomes ( ito et al . , 2003 ; shidara and richmond , 2002 ; stuphorn et al . , 2000 ) , response errors ( gemba et al . , 1986 ; ito et al . , 2003 ) , response conflict ( botvinick et al . , 2001 ) , and decision uncertainty ( botvinick et al . , the inferior parietal cortex is activated during go / no - go tasks ( braveret al . , 2001 ; garavan et al . , 1999 ; kiehl et al . , 2000 ) and has been related to response conflict ( braver et al . , 2001 ; carter et al . , 2000 ) and , more generally , multiple aspects of sensory processing and sensorimotor integration ( caspers et al . , 2006 ; clower et al . , 2001 ) . we suggest that reduced engagement of neural circuitry within these regions has potential significance for the clinical presentation of 22q11ds . despite clear elevations in trait impulsivity in idiopathic adhd , the relationship between impulsivity and ri - related neural activation is still an active area of investigation . in a large - scale study of adhd patients , van rooij et al . ( 2015 ) reported an inverse correlation between adhd symptom count ( based on hyperactive / impulsive and inattentive symptoms according to dsm - iv criteria ) and neural activation in the inferior frontal gyrus during successful ri . here , our within - group analyses did not reveal an association between ri - related neural activity and cognitive or behavioral impulsivity for adhd patients that survived correction . this may be related to the relatively small sample of adhd patients included in our study . given the genetic and phenotypic heterogeneity within adhd patients , it may be necessary to include a larger sample to detect neural function behavior relationships . these findings also suggest that there may be more power to detect associations between dysfunctional ri - related neural activity and trait impulsivity within 22q11ds patients , given the more homogeneous genetic etiology . notably , the 22q11.2 locus includes the catechol - o - methyltransferase ( comt ) gene , which codes for an enzyme involved in prefrontal dopamine metabolism ( egan et al . , 2001 ; fallgatter and lesch , 2007 ; gothelf et al . , 2007 ; lachman et al . , 1996 ) which may be relevant to behavioral manifestations of inattention and impulsivity in 22q11ds ( cole et al . , 2013 ; cools et al . , 2007 ; gothelf et al . , 2007 ; shashi et al . , 2006 ; soeiro - de - souza et al . , 2013 ) . investigation of the relationships between impulsive behavior , response inhibition - related neural circuitry , and allelic variation and expression of comt and other genes involved in dopaminergic function is warranted in future , larger - scale studies of this syndrome . the findings reported here help to elucidate the neurobiological basis of attentional dysfunction in the following ways . first , we directly compared patients with adhd , a group that traditionally exhibits substantial heterogeneity at both genotypic and phenotypic levels , with 22q11ds patients , a group representing a relatively homogeneous genetic etiology with common downstream phenotypic characteristics . 22q11ds offers a compelling model for the investigation of the neurobiological substrates of attentional dysfunction in adhd . direct comparison of our adhd and 22q11ds patients allowed us to demonstrate a shared deficit in ri - related neural activation between groups , as compared with controls , but circumscribed differences in adhd patients as compared with 22q11ds patients . second , we investigated the relationship between ri - related neural activity and different aspects of trait impulsivity cognitive and behavioral . we used a dimensional approach to assess these aspects of impulsivity symptoms , which is in agreement with the rdoc initiative ( cuthbert and kozak , 2013 ; insel et al . , 2010 ) that aims to characterize psychopathology on dimensions of observable behaviors or neurobiological measures rather than traditional diagnostic categories . as such , our results provide initial evidence for how dimensional assessments of impulsivity may characterize distinct etiologies of downstream adhd diagnosis . finally , our ability to detect significant within - group correlations between impulsivity measures and ri - related neural activation in 22q11ds patients , but not adhd patients , has implications for future efforts , as there may be more power to detect associations between dysfunctional ri - related neural activity and trait impulsivity within 22q11ds patients , given the more homogeneous genetic etiology . taken together , these findings suggest a pattern of reduced neural engagement during ri in both idiopathic adhd and 22q11ds , seen in the acc , cerebellum , inferior parietal cortex , middle frontal gyrus , middle temporal gyrus , paracingulate gyrus , superior frontal gyrus , and bilateral thalamus . notably , 22q11ds patients differed relative to idiopathic adhd patients in that they showed hypoactivation of the bilateral frontal regions . our results further indicate that deficits in ri - related activation are associated with increased cognitive impulsivity in 22q11ds patients , but not increased behavioral impulsivity , consistent with the behavioral presentation of the disorder . these results offer initial evidence that similar underlying ri - related deficits in adhd and 22q11ds may manifest as different forms of real - world trait impulsivity between patient groups .

we present an extremely rare case of noninfiltrating angiolipoma ( al ) of the buccal mucosa in a 90-year - old patient reaching a size of 14 cm 10 cm . al is rare in the soft tissues of oral cavity , and till date , only 22 cases have been reported , including our case . if size be taken into consideration , our case can be considered to be the first in literature to reach this massive size . surgical excision of the tumor mass was performed . the histopathological findings confirmed the diagnosis of noninfiltrating al . follow - up for 1 year revealed no signs of recurrence . highlights22q11ds offers a compelling model to understand the neural substrates of attentional dysfunction.first study directly comparing neural function in 22q11ds vs. adhd patients22q11ds and adhd patients show a shared deficit in ri - related activation.adhd patients showed greater activity in the middle frontal gyrus than 22q11ds during ri.neural activity is inversely correlated with self - reported cognitive impulsivity in 22q11ds .

gingival recession is defined as the partial denudation of the root surface due to the apical migration of the gingival margin . etiological factors include trauma from tooth brushing , malposition of teeth , ectopic insertion of frenum , and muscle attachments . the major therapeutic goals in mucogingival surgery are a correction of esthetic problems and management of hypersensitivity . numerous surgical procedures have been described to achieve soft tissue coverage of exposed root surfaces including coronally repositioned flaps , pedicle grafts , free gingival grafts , subepithelial connective tissue grafts ( ctg ) , and guided tissue generation ( gtr ) were commonly used procedures . the treatment of isolated or multiple buccal recessions with different surgical procedures depends on many factors such as defect size , presence or absence of keratinized tissue adjacent to the defect , and thickness of the gingiva . since the patients are concerned about their esthetic appearance , every effort should be made to achieve complete root coverage up to the cementoenamel junction ( cej ) . originally sullivan and atkins described a technique for coverage of exposed root surfaces using the free gingival autogenous graft . the graft survival over large expanses of avascular root surfaces was unpredictable , and complete root coverage was rarely achieved . demonstrates that the underlying connective tissue has a direct bearing on the type of epithelium that is superimposed upon it . edel showed that a significant increase in the volume of gingiva can be achieved by grafting gingival connective tissue alone . langer and langer described the ctg technique in root coverage on both single and multiple adjacent teeth . the advantage is the dual blood supply from the overlying flap and palatal connective tissue , which maximizes graft survival . when multiple adjacent teeth with gingival recessions are present in esthetic regions of the mouth , the preferred surgical technique should be such the one , which provides the possibility of achieving maximum root coverage . one of the problems with multiple root coverage grafting is the unavailability of the large blood supply of donor tissue . if connective tissue supply is limited , more than one surgical procedure may be needed . the purpose of the present study was to evaluate the effectiveness and the predictability of expanded mesh ctg ( e - mctg ) procedure for the treatment of multiple adjacent gingival recession defects . the study population 16 patients , ( age range 2055 years mean age 37 years ) with either dentin hypersensitivity or esthetic problems caused due to the recession defects were included in the study . prior to initiation of the study , ethical approval was obtained from institution ethical committee . all the patients agreed to the study protocol , and signed informed consent was obtained prior to inclusion in the study . the inclusion criteria are ( 1 ) the presence of at least three adjacent miller 's class i or class ii gingival recession on the buccal / facial aspect with recession depth ( rd ) of 2 mm , ( 2 ) probing depth ( pd ) of 3 mm , ( 3 ) a minimum width of keratinized gingival ( kg ) of at least 1 mm . nine subjects contributes three sites , and seven subjects contributed four sites [ figure 1 ] . preoperative facial view of gingival recession the exclusion criteria are ( 1 ) the presence of severe cervical abrasion / root caries , ( 2 ) the presence of abnormal frenal attachment , ( 3 ) current smokers , ( 4 ) medically compromised patients , ( 5 ) miller 's class iii and iv gingival recession . the patients initially completed a plaque control program , so as to achieve a full mouth plaque score ( fmps ) < 25% . the following clinical measurements were taken by a single examiner at baseline and 3 months , 12 months postoperatively . ( 1 ) rd measured from the cemento - enamel junction ( cej ) to the gingival margin , ( 2 ) recession width ( rw ) measured across the buccal surface at the cej level , ( 3 ) pd measured from the gingival margin to the bottom of the gingival sulcus , ( 4 ) width of keratinized tissue ( kt ) measured from the gingival margin into the mucogingival junction , ( 5 ) clinical attachment level ( cal ) measured from cej to the bottom of the gingival sulcus . all measurements were performed at the mid buccal level using a william 's periodontal probe ( hu - friedy ) and rounded to the nearest 0.5 mm . all surgical procedures were done by the same operator . following the induction of local anesthesia ( lignocaine hydrochloride with 1:100,000 adrenaline ) , an intra - crevicular incision was made through the bottom of the crevice and horizontal incision was placed at the level of cej extending 3 mm on either side of the involved tooth including their papilla . two vertical incisions were placed from the end point of the horizontal incision to the alveolar mucosa to establish a trapezoidal flap [ figure 2 ] . line diagram depicting the incisions a full thickness flap was elevated to 34 mm apical to the bone dehiscence followed by a split thickness flap and all muscle interferences were eliminated in order to facilitate its coronal advancement . the root surface was mechanically instrumented using gracey curettes followed by conditioning with 1 ml tetracycline hydrochloride solution for 3 min with subsequent rinsing with saline [ figure 3 ] . split thickness trapezoidal flap was elevated ctg was harvested from the molar- premolar area of the palate on one side [ figures 4 and 5 ] . the donor site was then sutured with 4 - 0 black silk to ensure primary intention healing . alternating incisions were then made on each edge of the harvested graft to expand it [ figure 6 ] so that it would cover the recipient bed completely , which was 1.5 times larger than the graft [ figure 7 ] . subsequently , the graft was positioned at the cej with interrupted 5 - 0 vicryl bioabsorbable sutures [ figure 8 ] . the mucogingival flap was coronally repositioned without tension to cover the e - mctg with 4 - 0 silk sutures [ figure 9 ] . the area was re - examined to ascertain that the graft was completely covered by the flap . a periodontal dressing ( coe - pak ) was placed over the recipient site and removed after a week . original size of the connective tissue graft was harvested from palatal mucosa original size of the connective tissue graft was positioned in the recipient area the expanded mesh connective tissue graft the expanded mesh connective tissue graft was positioned in the recipient area initial stabilization of the graft with 5 - 0 absorbable suture coronal repositioning of the flap with complete closure all patients were instructed to discontinue tooth brushing in the surgical site for 1-week so as to avoid trauma or pressure at the surgical site . a 0.12% chlorhexidine digluconate mouth rinse analgesics ( ibu profen and paracetamol tds for 5 days ) and antibiotics ( amoxycillin 500 mg tds for 5 days ) were prescribed . mechanical tooth cleaning of the treated areas using a soft toothbrush and a careful roll technique was resumed following the removal of periodontal dressing . the patients were recalled for oral prophylaxis after 2 , 4 weeks , and every 3 months for 1-year . the postoperative clinical measurements were taken at the end of 3 and 12 month [ figures 10 and 11 ] . postoperative facial view at the end of 3 month [ compare with figure 1 ] postoperative facial view at the end of 12 month [ compare with figure 1 ] the data were collected , and statistical analysis was performed using statistical package for social science version 16 ( spss , ibm corp . , chicago , usa ) . the following clinical measurements were taken by a single examiner at baseline and 3 months , 12 months postoperatively . ( 1 ) rd measured from the cemento - enamel junction ( cej ) to the gingival margin , ( 2 ) recession width ( rw ) measured across the buccal surface at the cej level , ( 3 ) pd measured from the gingival margin to the bottom of the gingival sulcus , ( 4 ) width of keratinized tissue ( kt ) measured from the gingival margin into the mucogingival junction , ( 5 ) clinical attachment level ( cal ) measured from cej to the bottom of the gingival sulcus . all measurements were performed at the mid buccal level using a william 's periodontal probe ( hu - friedy ) and rounded to the nearest 0.5 mm . all surgical procedures were done by the same operator . following the induction of local anesthesia ( lignocaine hydrochloride with 1:100,000 adrenaline ) , an intra - crevicular incision was made through the bottom of the crevice and horizontal incision was placed at the level of cej extending 3 mm on either side of the involved tooth including their papilla . two vertical incisions were placed from the end point of the horizontal incision to the alveolar mucosa to establish a trapezoidal flap [ figure 2 ] . line diagram depicting the incisions a full thickness flap was elevated to 34 mm apical to the bone dehiscence followed by a split thickness flap and all muscle interferences were eliminated in order to facilitate its coronal advancement . the root surface was mechanically instrumented using gracey curettes followed by conditioning with 1 ml tetracycline hydrochloride solution for 3 min with subsequent rinsing with saline [ figure 3 ] . split thickness trapezoidal flap was elevated ctg was harvested from the molar- premolar area of the palate on one side [ figures 4 and 5 ] . the donor site was then sutured with 4 - 0 black silk to ensure primary intention healing . alternating incisions were then made on each edge of the harvested graft to expand it [ figure 6 ] so that it would cover the recipient bed completely , which was 1.5 times larger than the graft [ figure 7 ] . subsequently , the graft was positioned at the cej with interrupted 5 - 0 vicryl bioabsorbable sutures [ figure 8 ] . the mucogingival flap was coronally repositioned without tension to cover the e - mctg with 4 - 0 silk sutures [ figure 9 ] . the area was re - examined to ascertain that the graft was completely covered by the flap . a periodontal dressing ( coe - pak ) was placed over the recipient site and removed after a week . original size of the connective tissue graft was harvested from palatal mucosa original size of the connective tissue graft was positioned in the recipient area the expanded mesh connective tissue graft the expanded mesh connective tissue graft was positioned in the recipient area initial stabilization of the graft with 5 - 0 absorbable suture coronal repositioning of the flap with complete closure all patients were instructed to discontinue tooth brushing in the surgical site for 1-week so as to avoid trauma or pressure at the surgical site . a 0.12% chlorhexidine digluconate mouth rinse analgesics ( ibu profen and paracetamol tds for 5 days ) and antibiotics ( amoxycillin 500 mg tds for 5 days ) were prescribed . mechanical tooth cleaning of the treated areas using a soft toothbrush and a careful roll technique was resumed following the removal of periodontal dressing . the patients were recalled for oral prophylaxis after 2 , 4 weeks , and every 3 months for 1-year . the postoperative clinical measurements were taken at the end of 3 and 12 month [ figures 10 and 11 ] . postoperative facial view at the end of 3 month [ compare with figure 1 ] postoperative facial view at the end of 12 month [ compare with figure 1 ] the data were collected , and statistical analysis was performed using statistical package for social science version 16 ( spss , ibm corp . , chicago , usa ) . at baseline , mean rd was 2.56 mm 0.62 mm , mean rw was 3.44 mm 0.44 mm , mean kt was 2.13 mm 0.73 mm , mean pd was 1.15 mm 0.23 mm , and a mean cal was 3.71 mm 0.61 mm . three months following surgical intervention , the mean rd reduced from 2.56 mm 0.62 mm to 0.61 mm 0.63 mm ( mean 1.96 mm 0.66 mm ) , the mean root coverage was 86% , the mean rw reduced from 3.44 mm 0.44 mm to 1.52 mm 1.40 mm ( mean 1.92 1.34 ) , the mean kt increased from 2.13 mm 0.73 mm to 3.55 mm 0.69 mm ( mean 1.42 0.62 ) , the pd from 1.15 mm 0.23 mm to 1.22 mm 0.25 mm ( 0.07 0.33 ) , and cal increased from 3.71 mm 0.61 mm to 1.83 mm 0.70 mm ( 1.88 0.69 ) . on statistical analysis , there was a significant reduction in rd and rw , and kt and clinical attachment gain at 3 months ( p < 0.001 ) compared to the baseline [ table 1 ] . mean and sd of baseline and end of 3 months postoperative parameters ( n=55 sites ) at the end of 12 months , the mean rd reduced from 2.56 mm 0.62 mm to 0.35 mm 0.54 mm ( mean 2.22 0.68 ) , the mean root coverage was 93.5% , the mean rw reduced from 3.44 mm 0.44 mm to 0.77 mm 1.16 mm ( 2.71 1.23 ) , the mean kt increased from 2.13 mm 0.73 mm to 3.87 mm 0.80 mm ( 1.75 0.78 ) , the pd from 1.15 mm 0.23 mm to 1.20 mm 0.25 mm ( 0.06 0.34 ) , and cal increased from 3.71 mm 0.61 mm to 1.51 mm 0.61 mm ( 2.20 0.68 ) . all parameters were statistically significant ( p < 0.001 ) compared to base line except pd ( p > 0.05 ) but there was no statistical significance improvement between 3 months to 12 months interval ( p > 0.05 ) [ table 2 ] . mean and sd of baseline and end of 12 months postoperative parameters ( n=55 sites ) at the end of 12 months postoperatively , favorable results were obtained using the e - mctg procedure . totally , 44 out of 55 sites , 44 ( 80% ) sites showed 100% root coverage 7 out of 55 sites , 7 ( 12.7% ) sites showed 90% root coverage 4 out of 55 sites , 4 ( 7.3% ) sites showed 65.5% root coverage at the end of 12 months [ figure 12 ] . gingival recession involves groups of adjacent teeth and is seldom localized to a single tooth . when multiple recession defects affecting adjacent teeth in esthetic areas of the mouth are present , they should all be treated at the same time to help ensure the best esthetic results . autogenous ctg have been extensively used for root coverage procedures in teeth and implants . whereas subepithelial ctg was extensively used for one or two adjacent gingival recession defects excellent result with color matching . harris 's study proposed that the use of acellular dermal matrix graft would improve the gingival color , reduce patient morbidity , provide a uniform thickness of material and eliminate the need for multiple surgeries because of unlimited availability . in the present study , a new approach of the ctg technique the most common problem for root coverage with ctg procedure is the amount that can be harvested . the rugae area is not suitable for graft material , and an extensive palatal wound will be uncomfortable for the patient . because of the high rate of complications and a limited amount of palatal mucosa available for grafting , it is advisable to refrain from covering large or multiple defects . formerly e - mctg was used for free gingival grafts and was generally applied to increase the width of keratinized tissue without root coverage . e - mctg provides more graft material since it can be expanded as much as 50% to cover a larger area . totally , 44 ( 80% ) sites showed 100% root coverage , 7 out of 55 sites , 7 ( 12.7% ) sites showed 90% root coverage , 4 out of 55 sites , and 4 ( 7.3% ) sites showed 65.5% root coverage at the end of 12 months regardless of the number of patients treated . , 94.68% ; romangno - genon 84.84% ; rosetti et al . , 95.6% ; and harris 97.7% . however , it has some limitations and complications such as color match , painful postoperative wound healing , and scar tissue formation in the donor area . it has been reported that using ctg to increase kt has a more rapid , maturation , and less traumatic healing of the graft in the recipient site . similar clinical observations were noticed in our study with the use of the e - mctg technique . as mentioned in previous studies , an increase in kt may be great when there is a narrow band of attached gingival ( 1 mm ) apical to the defects . in this study , a significant increase of kt was obtained after surgery and maintained over time for 12 months , and this was comparable to other studies . on the other hand , we did not achieve complete coverage of the graft in four cases where there were more than three adjacent gingival recession sites and the average amount of kt was 1.5 mm at baseline . it is suggested that attempting to cover completely the graft should be avoided when the initial height of keratinized tissue is poor . the mean gain in cal was 2.20 mm 0.68 mm at 12 months follow - up . histological studies have demonstrated that the blood supply from the periosteum and overlying flap results in a more rapid re - establishment of circulation . in the present study , we took care to place the graft with the periosteal side facing the root surface . borghetti and gardella are documented that creeping attachment may continue for 1-year postoperatively , when thick grafts were used . in this study , there was no statistically significant creeping attachment between the 3 and 12 months interval . however , five patients did show a 1 mm improvement in attachment from the 3 to 12 month . recently , an acellular dermal matrix has been shown to be effective in root coverage procedures as a substitute for ctg . tat et al . and wei et al . have shown that acellular dermal matrix was not as successful as the autogenous free graft and connective tissue free graft in increasing the kt , and a histologic report suggested that placing an acellular dermal matrix does not increase kt . ctg is the gold standard for treatment of gingival recession , but the disadvantages are the inadequate graft availability . the results of the present study demonstrated that the e - mctg procedure was an effective and predictable treatment modality for the management of multiple adjacent gingival recessions in terms of root coverage in the treatment of multiple adjacent gingival recessions . nowadays a highlighted issue for an increasing number of consumers is the problem of meat tenderness , as the result of its physicochemical and biochemical mechanisms acting mainly on myofibrillar structures postmortem . because calpastatin inhibits both calpain1 and calpain2 , it remains unclear whether one or both calpains are active in postmortem muscle . most of the studies that have used appropriate methodology have found the same effect of postmortem storage on the activity of the components of the calpain system [ 24 ] . this conclusion seems to be based on the finding that calpain2 is not autolyzed during postmortem storage [ 5 , 6 ] . but some studies also have now suggested that both unautolyzed calpain1 and calpain2 are proteolytically active [ 7 , 8 ] . the lack of expression data of the calpain2 gene in chicken makes it difficult to verify the role of calpain2 in control of meat quality and carcass traits . in this study , we aimed to ( 1 ) develop a convenient approach to quantify the abundance of the calpain2 transcripts in chicken tissues and ( 2 ) determine tissue distribution and ontogenic expression of this gene , particularly in muscle tissues . thirty - six chickens at different ages from two breeds / populations , mountainous black - bone chicken ( mb ) and a commercial chicken ( s01 ) from sichuan province , were used in this study . tissue samples ( including heart , liver , breast muscle , leg muscle , brain , and abdominal fat ) from mb chickens were collected at 0 , 2 , 4 , 6 , 8 , and 10 w , respectively ; we slaughtered four chickens at each time point . we also collected tissue samples from six s01 chickens at 10 w and six mb chickens at 12 w. tissue samples were quick - frozen in liquid nitrogen and then stored at 80c for total rna extraction . total rna was isolated from the heart , liver , brain , breast muscle , and leg muscle tissues by using the trizol reagent ( invitrogen ) . the quality of rna was determined by the a260/280 absorbance ratio ( 1.61.8 ) and the integrity of the 18s and 28s rrna bands on 1% formaldehyde agarose gel . isolated rnas were treated with 8 l dnase ( fermentas ) for 20 minutes at 37c and stored at 80c . the cdna was synthesized using the improm - ii reverse transcription system ( takara ) according to the manufacturer 's instructions . the reaction was performed in a volume of 10 l containing 5primerscript buffer , 10 mm of each dntps , 40 u/l rnase inhibitor , and 2.5 m oligo - dt primer . the reverse transcription was maintained at 30c for 10 minutes , 45c for 25 minutes , 99c for 5 minutes , and ended with a 4c for 5 minutes , then stored at 20c . according to the chicken capn2 mrna sequence in genbank ( accession number nm_205080.1 ) , a pair of primers were designed by using oligo 6.0 ( table 1 ) . the expression levels of chicken capn2 gene were detected by using the sybr green i assay on an iq5 real - time pcr thermal cycle instrument ( bio - rad , german ) and were normalized to the expression of the -actin gene ( actb ; genbank accession no . relative transcript quantification was performed using standard curves generated for the actb and capn2 genes based on a 7-fold serial dilution of the pooled cdna product prepared from a subset of the gastrocnemius samples . the cycling conditions consisted of an initial single cycle of 95c for 3 minutes , followed by 35 cycles of 15 s at 94c and 1 minute at 60c . reactions were performed in a volume of 25 l and included 2.0 l cdna template , 1.0 l of each specific primer ( table 1 ) , 12.5 l sybr green pcr master mix ( takara , japan ) , 1.0 l calibration liquid ( bio - rad ) , 7.5 l pcr - grade water , and 1.0 l cdna . all real - time quantitative pcr amplifications were performed in triplicate for each sample and were analyzed using the 2 method previously described . expression data were described as ( least square mean standard error ) and were analyzed using the sas 8.0 for windows software ( sas institute inc . , the expression levels of the capn2 gene between the tissue and age - related samples of the same breed and those samples from the two breeds with the same age were analyzed by the one - way anova and student 's t - test , respectively . relative mrna quantification was performed using standard curves generated for the actb and capn2 genes based on a serial dilution of cdna . in the current assay , the amplification efficiency of the actb and capn2 genes were 91.9% and 92.60% , respectively , which were approximately within the expected theoretical values . quantitative pcr analysis showed that the capn2 gene was expressed in all six mb chicken tissues . the capn2 gene transcript had the highest expression level in breast muscle and leg muscle whereas it had the lowest expression level in liver tissue . the mrna levels of the capn2 gene in breast muscle and leg muscle were higher than those in other tissues from the same chicken ( p < .05 ) except at 10 w ( table 2 ) . we analyzed the developmental changes of the capn2 mrna expression for each tissue in mb chickens with different ages . as shown in figure 1 , the capn2 mrna in breast muscle had the highest expression at 6 w and the lowest expression at birth . in leg muscle tissue , the highest expression of the capn2 gene was at 12 w and the lowest expression at birth . overall , the capn2 mrna levels exhibited a rise developmental change in all tissue . to characterize whether the expression of the capn2 gene had a breed - specific feature figure 2 presents the expression pattern of the capn2 mrna in the mb and s01 chickens at 10 w. the meat - type s01 chicken had a higher expression of the capn2 gene than that of mb chicken in all tissues . although previous studies on calpain protease activity indicate that the 2 calpains , calpain1 and calpain2 , have an important role in the postmortem proteolysis that increases meat tenderness , it remains unclear how the calpains function in postmortem muscle . in birds , and in particular the chicken breast muscle , the role of postmortem proteolysis is poorly documented , and the few studies performed did not take into account the particularities of the calcium - dependent proteases in these species [ 912 ] . in this study , we quantified the chicken capn2 gene tissue distribution and ontogenic expression . the capn2 mrna was expressed in all six different tissues studied in the current study , with a dominant expression in breast muscle and leg muscle tissues . the overall pattern of the capn2 expression in breast muscle was different from that of leg muscle . it is well known that the breast muscle of chicken is made of white fast oxidative glycolytic fibers that whereas the leg muscle has slow oxidative red aerobic fibers . we also found the relative expression level of the capn2 mrna in the s01 chicken was higher than that in the mb chicken . investigation of the role of the calpain 2 and its muscle protein substrates in these two chicken breeds may further explain the observed variation in meat tenderness . these results are necessary for knowing the effects of capn2 on the regulation of muscle protein metabolism and for defining the biological significance of degradation of the myofibrillar proteins in chicken , as well as potential applications in marker - assisted selection in chicken breeding . in conclusion , we have developed a highly sensitive real - time pcr method to detect tissue distribution and ontogenic expression of the capn2 mrna in chicken . future studies will be essential to determine the biochemical character of the muscles from the two chicken breeds and to discern the factors that contribute to differences in their meat quality , as well as to define the role of the calpain 2 .

background : multiple approaches have been used to replace lost , damaged or diseased gingival tissues . the connective tissue graft ( ctg ) procedure is the golden standard method for root coverage . although multiple sites often need grafting , the palatal mucosa supplies only a limited area of grafting material . to overcome this limitation , expanded mesh graft provides a method whereby a graft can be stretched to cover a large area . the aim of this study was to evaluate the effectiveness and the predictability of expanded mesh ctg ( e - mctg ) in the treatment of adjacent multiple gingival recessions.materials and methods : sixteen patients aged 2050 years contributed to 55 sites , each site falling into at least three adjacent miller 's class 1 or class 2 gingival recession . the ctg obtained from the palatal mucosa was expanded to cover the recipient bed , which was 1.5 times larger than the graft . clinical measurements were recorded at baseline and 3 months , 12 months postoperatively.results:a mean coverage of 1.96 mm 0.66 mm and 2.22 mm 0.68 mm was obtained at the end of 3rd and 12th month , respectively . twelve months after surgery a statistically significant increase in cal ( 2.2 mm 0.68 mm , p < 0.001 ) and increasing wkt ( 1.75 0.78 , p < 0.001 ) were obtained . in 80% of the treated sites , 100% root coverage was achieved ( mean 93.5%).conclusions : the results of this study demonstrated that multiple adjacent recessions were treated by using e - mctg technique can be applied and highly predictable root coverage can be achieved . we quantified chicken calpain 2 ( capn2 ) expression in two chinese chicken breeds ( mountainous black - bone chicken breed [ mb ] and a commercial meat type chicken breed [ s01 ] ) to discern the tissue and ontogenic expression pattern and its effect on muscle metabolism . real - time quantitative pcr assay was developed for accurate measurement of the capn2 mrna expression in various tissues from chickens of different ages ( 0 , 2 , 4 , 6 , 8 , 10 , and 12 weeks ) . results showed that the breast muscle and leg muscle tissues had the highest expression of capn2 compared to the other tissues from the same individual ( p < .05 ) . overall , the capn2 mrna level exhibited a rise developmental change in all tissues . the s01 chicken had a higher expression of the capn2 mrna in all tissues than the mb chicken . our results suggest that chicken capn2 expression may be related to chicken breeds and tissues .

acupuncture is a promising treatment approach in patients with low back pain ( clbp ) with nearly the same or even better results compared to conventional therapy . since in fact various acupuncture strategies are used in clinical practice , research in order to define the best acupuncture technique is of clinical interest for many medical practitioners as well as for clinical researchers designing acupuncture trials . allready for chinese acupuncture , representing the most widely used style , the specific manner in which acupuncture is applied ( treatment regimens , point selection , needle techniques ) appears to vary among , regions and countries . the same holds true for randomized clinical trials ( rct ) on clbp , with point selection being fixed for all patients , or points being selected on an individual basis according to trigger points , chinese meridians , syndromes , various microsystems to name a few . in rcts needle stimulation may range from non - existent to strong , needle insertion may be deep or shallow , the number of acupuncture treatments may vary from 1 to 15 , and the stimulation of ahshi points may be mandatory or forbidden . despite the multiplicity of existing approaches to acupuncture , in a previous study we ascertained that there is a high degree of concurrence on certain aspects ( minimal criteria ) of chinese acupuncture for the treatment of clbp . a systematic review by yuan et al . arrived at a similar conclusion , while detecting differences between chinese expert opinions , textbooks and acupuncture treatments in rcts . on the basis of a broad , international delphi survey the aim of this study was to find out how leading acupuncture experts from non - chinese countries would rate the acupuncture methods used in existing rcts of acupuncture for clbp . furthermore we calculated the correlation between the results of this expert survey and with a low back pain acupuncture score ( lbpas ) , derived from our recently published acupuncture questionnaire for low back pain . acupuncture experts in a number of countries were selected on the basis of personal contact and scientific publications in the field of acupuncture . additional criteria were clinical experience , a history of teaching , and textbook authorship . all experts were invited to recommend other experts to take part in the survey . those who had already taken part in our previous acupuncture survey establishing minimal criteria for chinese acupuncture in clbp were excluded from selection . twenty - five papers on rcts of acupuncture for clbp published between 1976 and 2007 , were identified via a computerbased search of the cochrane complementary field trials register , the cochrane controlled trials register , medline , embase and reference lists of articles . the acupuncture method for one of the 24 trials was published in two different papers ( papers 2 and 10 ) . for the purpose of this study we did not aim at completeness but sought to ensure that all major trials included in the recent meta - analyses of rcts on clbp were included . from each trial , the paragraph describing the acupuncture method was extracted , and supplemented by a table summarizing all relevant acupuncture treatment data for that trial ; a pdf document was created presenting this information for each trial in an anonymous way , thus minimizing the possibility that the surveyed experts would be able to identify the authors of the reported studies , which could be considered as a relevant source of bias . the pdf document was sent to the selected experts , asking them to answer the following three questions about the acupuncture treatment method of each trial .... please specify how you rate the acupuncture treatment for chronic low back pain in this trial .... 1 . ... according to your personal knowledge of chinese acupuncture as you have studied it from textbooks on chinese acupuncture . 2 . ... according to your personal concept of individualized point selection ( e.g. dry needling , trigger point stimulation , individual usage of chinese point selection rules such as ahshi points , channels , syndromes , pathogenic factors ... ) 3 . ... according to the acupuncture commonly performed in your country ( local style , not necessarily chinese style ) . answers were to be given on a likert scale : 1 = very poor ; 2 = poor ; 3 = fair ; 4 = good ; 5 = very good if the experts felt that the excerpt for a particular trial did not contain adequate information about the acupuncture treatment , they were asked to mark : " the trial does not show a sufficient amount of information for any further judgment . can not say " . recently we published the ' acupuncture questionnaire for low back pain ' based on an international expert survey . this survey revealed that a broad consensus exists with regard to the fundamental aspects of chinese acupuncture for clbp across different categories of practitioners and different countries . a typical treatment regime would consist of 11 sessions of 25 minutes each , given twice weekly . the practitioner would insert 12 needles , select points according to channels and syndromes , identify syndromes such as kidney deficiency ( yin and yang ) , cold dampness , and qi and blood stagnation . preferred points besides ahshi points are bl 23 , bl 40 , gb 34 , bl 54 , bl 60 , gb 30 and huatuo , although there is a high degree of variation concerning additional local and distal points . needling depth is dependent on the point location or other individual patient factors , varying from 0.5 mm in very sensitive patients to 10 cm for points such as gb 30 or bl 54 . acupuncture should be combined with other techniques of chinese medicine , the favourites being electrical stimulation and moxibustion . based on the acupuncture questionnaire for low back pain , a score was developed in such a way that the conformity of the acupuncture treatment applied in a given trial with the criteria derived from the acupuncture questionnaire for low back pain could be expressed in a single number from 0 to 8 , from 0 = very poor to 8 = very good ( table 1 ) . previously published data from 18 experts from 10 different countries showed that a typical acupuncture treatment regimen or treatment protocol should at least meet the above criteria . these results were the basis for the development of the low back pain acupuncture score this number is referred to as the low back pain acupuncture score ( lbpas ) . for the present study , we ( 2author ) calculated the lbpas for all 25 publications and compared it to the ies score . for each trial , the frequencies of the ratings given by the 15 experts were counted . for each question , the mode was defined as the true rating , since it had been selected by the majority of the experts . for all 25 papers we then counted how often experts missed the mode for each question ( false rating ) . the number of false ratings per expert is presented with its measures of average and variability . we calculated the spearman 's rank correlation coefficient of the expert 's true rating for the three questions of the ies . the same statistical procedure was used to calculate the correlation between the ies and the lbpas . acupuncture experts in a number of countries were selected on the basis of personal contact and scientific publications in the field of acupuncture . additional criteria were clinical experience , a history of teaching , and textbook authorship . all experts were invited to recommend other experts to take part in the survey . those who had already taken part in our previous acupuncture survey establishing minimal criteria for chinese acupuncture in clbp were excluded from selection . twenty - five papers on rcts of acupuncture for clbp published between 1976 and 2007 , were identified via a computerbased search of the cochrane complementary field trials register , the cochrane controlled trials register , medline , embase and reference lists of articles . the acupuncture method for one of the 24 trials was published in two different papers ( papers 2 and 10 ) . for the purpose of this study we did not aim at completeness but sought to ensure that all major trials included in the recent meta - analyses of rcts on clbp were included . from each trial , the paragraph describing the acupuncture method was extracted , and supplemented by a table summarizing all relevant acupuncture treatment data for that trial ; a pdf document was created presenting this information for each trial in an anonymous way , thus minimizing the possibility that the surveyed experts would be able to identify the authors of the reported studies , which could be considered as a relevant source of bias . the pdf document was sent to the selected experts , asking them to answer the following three questions about the acupuncture treatment method of each trial .... please specify how you rate the acupuncture treatment for chronic low back pain in this trial .... 1 . ... according to your personal knowledge of chinese acupuncture as you have studied it from textbooks on chinese acupuncture . 2 . ... according to your personal concept of individualized point selection ( e.g. dry needling , trigger point stimulation , individual usage of chinese point selection rules such as ahshi points , channels , syndromes , pathogenic factors ... ) 3 . ... according to the acupuncture commonly performed in your country ( local style , not necessarily chinese style ) . answers were to be given on a likert scale : 1 = very poor ; 2 = poor ; 3 = fair ; 4 = good ; 5 = very good if the experts felt that the excerpt for a particular trial did not contain adequate information about the acupuncture treatment , they were asked to mark : " the trial does not show a sufficient amount of information for any further judgment . can not say " . recently we published the ' acupuncture questionnaire for low back pain ' based on an international expert survey . this survey revealed that a broad consensus exists with regard to the fundamental aspects of chinese acupuncture for clbp across different categories of practitioners and different countries . a typical treatment regime would consist of 11 sessions of 25 minutes each , given twice weekly . the practitioner would insert 12 needles , select points according to channels and syndromes , identify syndromes such as kidney deficiency ( yin and yang ) , cold dampness , and qi and blood stagnation . preferred points besides ahshi points are bl 23 , bl 40 , gb 34 , bl 54 , bl 60 , gb 30 and huatuo , although there is a high degree of variation concerning additional local and distal points . needling depth is dependent on the point location or other individual patient factors , varying from 0.5 mm in very sensitive patients to 10 cm for points such as gb 30 or bl 54 . acupuncture should be combined with other techniques of chinese medicine , the favourites being electrical stimulation and moxibustion . based on the acupuncture questionnaire for low back pain , a score was developed in such a way that the conformity of the acupuncture treatment applied in a given trial with the criteria derived from the acupuncture questionnaire for low back pain could be expressed in a single number from 0 to 8 , from 0 = very poor to 8 = very good ( table 1 ) . previously published data from 18 experts from 10 different countries showed that a typical acupuncture treatment regimen or treatment protocol should at least meet the above criteria . these results were the basis for the development of the low back pain acupuncture score this number is referred to as the low back pain acupuncture score ( lbpas ) . for the present study , we ( 2author ) calculated the lbpas for all 25 publications and compared it to the ies score . for each trial , the frequencies of the ratings given by the 15 experts were counted . for each question , the mode was defined as the true rating , since it had been selected by the majority of the experts . for all 25 papers we then counted how often experts missed the mode for each question ( false rating ) . the number of false ratings per expert is presented with its measures of average and variability . we calculated the spearman 's rank correlation coefficient of the expert 's true rating for the three questions of the ies . the same statistical procedure was used to calculate the correlation between the ies and the lbpas . twenty - five publications on 24 rcts were selected and evaluated by 15 out of 18 experts from the following countries : australia ( 2 ) , austria ( 1 ) , england ( 3 ) , france ( 1 ) , germany ( 2 ) , korea ( 1 ) , norway ( 1 ) , spain ( 2 ) , and us ( 2 ) . three declined to take part , two because lack of time ( france , china ) , the other ( china ) reasoning that acupuncture treatment is a holistic approach and therefore information would have to be presented about every single patient 's tcm diagnoses for all complaints , not merely clbp . since ultimately we could not include a chinese expert in our survey , we restrict our results to non - chinese experts and countries . for each paper conformity of the acupuncture method with chinese textbook standards ( question 1 ) was rated good ( rating 4 or 5 ) in 6 trials , fair ( rating 3 ) in 8 trials , and poor or very poor ( rating 1 or 2 ) for 10 trials . five of the 6 trials rated good [ on question 1 ] received at least a good rating also in questions 2 , asking the experts to grade the degree to which the method in the trial accorded with their personally preferred style , or - question 3 - the country - specific acupuncture style . here too 6 of 10 trials that were rated poor or very poor received the same rating for all three questions . the results for each trial , differentiated by question , are presented in table 2 . questions : " please specify how you rate the quality of acupuncture treatment in this trial for chronic low back pain according to question 1 ... chinese acupuncture as you have studied it from textbooks on chinese acupuncture ? answers were to be given on a five point likert scale : 1 = very poor ; 2 = poor ; .3 = fair ; 4 = good ; 5 = very good . lbpas = low back pain acupuncture score , from minimum of 0 to a maximum of 8 points . lbpas rating : 1 to 4 = poor ; 5 and 6 = fair ; 7 and 8 = good and very good . we also counted how often each expert rated differently from the mode , which was defined as a false rating . , the experts made 8 false ratings in question 1 and 9 false ratings in questions 2 and 3 . the minimum number of false ratings was 4 for question 2 , and 5 for questions 1 and 3 . the maximum number of false ratings was 12 for question 2 , and 13 for questions 1 and 3 . the variability , as shown by the coefficient of variation , was low for all three questions and almost identical at nearly 24% . regarding papers 2 and 10 , the mode was 4 for each of the three questions in both papers . there was a high correlation between the rating of question 1 ( conformity with chinese textbook acupuncture ) , question 2 ( conformity with individual point selection ) and question 3 ( conformity with acupuncture as performed in the expert 's country ) . for 8 trials ( nos . 2 , 3 , 8 , 9 , 10 , 14 , 15 and 22 ) , the same rating was chosen for questions one , two and three . for all other trials the difference between the ratings was one mark only ( table 2 ) . the spearmann 's ranking correlation coefficient is 0.81 for question 1 to question 2 , and 0.80 for question 1 to question 3 . for question 2 to question 3 it is 0.72 ( two - sided p value < 0.0001 for all correlations ) . with the lbpas , the conformity of a given acupuncture method with the criteria derived from the low back pain survey published earlier is expressed as a single number from 0 ( very poor ) to 8 ( very good ) . two trials had an lbpas of 8 ( 15 , 25 ) , and two had an lbpas of 7 ( 2/10 , 11 ) . the lowest lbpas was 1 , for two trials ( 8 , 12 ) ( table 2 ) . a high correlation was found between the ies rating and the lbpas for questions 1 and 2 ( 0.85 , p < 0.0001 ) , and a slightly lower correlation was found for question 3 ( 0.66 , p < 0.0001 ) . twenty - five publications on 24 rcts were selected and evaluated by 15 out of 18 experts from the following countries : australia ( 2 ) , austria ( 1 ) , england ( 3 ) , france ( 1 ) , germany ( 2 ) , korea ( 1 ) , norway ( 1 ) , spain ( 2 ) , and us ( 2 ) . three declined to take part , two because lack of time ( france , china ) , the other ( china ) reasoning that acupuncture treatment is a holistic approach and therefore information would have to be presented about every single patient 's tcm diagnoses for all complaints , not merely clbp . since ultimately we could not include a chinese expert in our survey , we restrict our results to non - chinese experts and countries . for each paper conformity of the acupuncture method with chinese textbook standards ( question 1 ) was rated good ( rating 4 or 5 ) in 6 trials , fair ( rating 3 ) in 8 trials , and poor or very poor ( rating 1 or 2 ) for 10 trials . five of the 6 trials rated good [ on question 1 ] received at least a good rating also in questions 2 , asking the experts to grade the degree to which the method in the trial accorded with their personally preferred style , or - question 3 - the country - specific acupuncture style . here too 6 of 10 trials that were rated poor or very poor received the same rating for all three questions . the results for each trial , differentiated by question , are presented in table 2 . questions : " please specify how you rate the quality of acupuncture treatment in this trial for chronic low back pain according to question 1 ... chinese acupuncture as you have studied it from textbooks on chinese acupuncture ? answers were to be given on a five point likert scale : 1 = very poor ; 2 = poor ; .3 = fair ; 4 = good ; 5 = very good . lbpas = low back pain acupuncture score , from minimum of 0 to a maximum of 8 points . lbpas rating : 1 to 4 = poor ; 5 and 6 = fair ; 7 and 8 = good and very good . we also counted how often each expert rated differently from the mode , which was defined as a false rating . , the experts made 8 false ratings in question 1 and 9 false ratings in questions 2 and 3 . the minimum number of false ratings was 4 for question 2 , and 5 for questions 1 and 3 . the maximum number of false ratings was 12 for question 2 , and 13 for questions 1 and 3 . the variability , as shown by the coefficient of variation , was low for all three questions and almost identical at nearly 24% . regarding papers 2 and 10 , the mode was 4 for each of the three questions in both papers . there was a high correlation between the rating of question 1 ( conformity with chinese textbook acupuncture ) , question 2 ( conformity with individual point selection ) and question 3 ( conformity with acupuncture as performed in the expert 's country ) . for 8 trials ( nos . 2 , 3 , 8 , 9 , 10 , 14 , 15 and 22 ) , the same rating was chosen for questions one , two and three . for all other trials the difference between the ratings was one mark only ( table 2 ) . the spearmann 's ranking correlation coefficient is 0.81 for question 1 to question 2 , and 0.80 for question 1 to question 3 . for question 2 to question 3 it is 0.72 ( two - sided p value < 0.0001 for all correlations ) . with the lbpas , the conformity of a given acupuncture method with the criteria derived from the low back pain survey published earlier is expressed as a single number from 0 ( very poor ) to 8 ( very good ) . two trials had an lbpas of 8 ( 15 , 25 ) , and two had an lbpas of 7 ( 2/10 , 11 ) . the lowest lbpas was 1 , for two trials ( 8 , 12 ) ( table 2 ) . a high correlation was found between the ies rating and the lbpas for questions 1 and 2 ( 0.85 , p < 0.0001 ) , and a slightly lower correlation was found for question 3 ( 0.66 , p < 0.0001 ) . using excerpts of 24 rcts of chronic low back pain , we could show that there is a high correlation between how experts outside of china rate the acupuncture treatment compared to chinese textbook acupuncture standards , personally preferred acupuncture style , and local acupuncture style of their respective country . only a minority of 6 out of 24 trials ( 25% ) were rated good according to chinese acupuncture standards ; 10 were rated poor or very poor and 8 were rated fair . on average furthermore , the international expert evaluation correlates highly significantly with the lbpas , based on our recently published acupuncture questionnaire for low back pain , thereby providing an independent international validation of the lbpas score . nevertheless , the greatest difference in ratings was found between question 1 ( chinese acupuncture ) and question 3 ( acupuncture as performed in the respective country of the expert ) . this suggests that local styles do indeed differ from chinese textbook acupuncture in some aspects but that the ( non - significant ) differences are smaller then expected . the expert ratings of the methods used in the various rcts differed most strongly for rcts where the treatment was not described in sufficient detail according to the stricta criteria , thus adding further evidence to the argument that a complete description of acupuncture treatment in rcts is essential . we also found that rcts that made use of electro - acupuncture were generally rated higher than those that used only manual needle stimulation . recently published a systematic review of acupuncture treatment regimens used for low back pain , including nine chinese expert opinions . for chronic unspecific low back pain , the main findings , which were in line with ours , were the frequent use of bl23 , bl25 and bl40 as common acupuncture points , use of ashi and trigger points , de qi sensation , needling of about 10 points per treatment , with a needle retention time of about 20 minutes and a treatment number of 10 . in general chinese experts tended to use fewer points ( median 5 ) , treated a little longer ( median 25.5 minutes ) and our data are also in line with other major systematic reviews and meta - analyses on acupuncture for clbp , although in these reviews the rating either concerned only methodological characteristics of the study design , omitting an evaluation of the acupuncture treatment per se ( van tulder et al . ) , or judged the acupuncture treatment by three experienced acupuncturists , being coauthors , only ( furlan et al . ) . 1999 found , that only 2 of 11 studies met the level of " high quality " and furlan et al . it must be said that a high rating of the acupuncture treatment is one but not the only prerequisite of a high quality rct . while this might be self - evident for the majority of readers , large , high - powered randomized controlled trials of high methodological quality are often based on the particular acupuncture treatment standards of the authors , which are not necessarily compatible with chinese acupuncture itself . results of these trials therefore do not apply to chinese textbook acupuncture techniques , although this is not always clarified . future investigations should determine whether ratings attributed by major meta - analyses and systematic reviews have introduced a bias in favour of meeting methodology against commonly accepted acupuncture standards in rcts about clbp . it was collected using a one - step standard delphi survey of 15 acupuncture experts in nine different countries outside of china . it might be argued that 15 experts is not a sufficient number to be representative , that the respondents were not selected randomly , and that they were not equally distributed across the countries involved . while this is true , and we concede that other experts , e.g. from china , might come up with different ratings , we note that other systematic reviews or meta - analyses of acupuncture for clpb have selected between 0 and 3 acupuncture experts only , drawn from the pool of authors or the local region . these limitations aside , we know of no other study to work with as many non - chinese experts , drawn from as wide a range of different countries . furthermore , the correlations among experts , professions and countries are so high for the ratings of most rcts that it is unlikely that any other selection of experts would produce essentially different results . our data is specific to chinese acupuncture for clbp . it was collected using a one - step standard delphi survey of 15 acupuncture experts in nine different countries outside of china . it might be argued that 15 experts is not a sufficient number to be representative , that the respondents were not selected randomly , and that they were not equally distributed across the countries involved . while this is true , and we concede that other experts , e.g. from china , might come up with different ratings , we note that other systematic reviews or meta - analyses of acupuncture for clpb have selected between 0 and 3 acupuncture experts only , drawn from the pool of authors or the local region . these limitations aside , we know of no other study to work with as many non - chinese experts , drawn from as wide a range of different countries . furthermore , the correlations among experts , professions and countries are so high for the ratings of most rcts that it is unlikely that any other selection of experts would produce essentially different results . this international expert survey ( ies ) showed that only in a minority of rcts of acupuncture for low back pain the acupuncture quality was rated as good . among international experts there is a high consistency in the rating of acupuncture in respect chinese acupuncture textbook standards ; rating differences with regard to personally preferred methods or local styles of acupuncture do exist but are not significant . the ratings showed a high correlation with the low back pain acupuncture score ( lbpas ) , constituting an independent international validation of the score . we are thankful for the contributions of weihong li , david s. white ( australia ) , eva maria wolkenstein ( austria ) , hyangsook lee ( korea ) , jean - marc stephan ( france ) , michael koch , friedrich molsberger ( germany ) , panos barlas , george lewith , adrian white ( great britain ) , morten srlie ( norway ) , caridad ortega garcia , jose manuel aranda regules ( spain ) , remy coeytaux and n.n . the practice of psychiatry and the shift to noninstitutional care of severe psychiatric disorders are the result of antipsychotic medications , beginning with chlorpromazine in the early 1950s . approximately 10 other drugs , known as first - generation or typical antipsychotics , followed over the subsequent 30 years . these drugs were effective in treating positive symptoms of psychosis such as hallucination and delusion but did not alleviate the negative symptoms of withdrawal , apathy , cognitive impairment , or loss of affect . furthermore , they were associated with frequent extrapyramidal symptoms , including acute dystonia , akinesia , akathisia , tardive dyskinesia , and parkinsonism . a series of newer drugs began emerging in 1989 , referred to as second generation or atypical antipsychotics , thought to be more effective than the older agents in alleviating the negative , cognitive , and affective symptoms , with fewer extrapyramidal adverse effects . the antipsychotic drugs differ in their side effect profiles , as they affect different neuroreceptors ( histamine , -adrenergic , muscarinic , dopamine , or serotonin ) . the principal concern for endocrinologists with the newer drugs has been the metabolic effects of weight gain , glucose intolerance , hyperlipidemia , and hypertension . this is particularly important with the increasing use of these agents in pediatrics to treat bipolar disorder , schizophrenia , autism , oppositional and other behavior disturbances , tourette disorder , and pervasive developmental disorder . in 2003 - 2004 the importance and implications of the metabolic side effects of atypical antipsychotics for pediatric patients have been recently reviewed [ 1 , 4 ] . this paper will examine the side effect of hyperprolactinemia in children and adolescents treated with antipsychotic drugs . prolactin , a 198-amino acid polypeptide , is secreted by the anterior pituitary lactotroph cells in a pulsatile manner with 13 - 14 peaks per day , the peak amplitude ~60% above nadir . there is also a marked circadian variation with maximum secretion ~4 hours from sleep onset and minimum ~6 hours after waking . thus , there can be as much as a fourfold variation in level depending on the time of day or night sampling is done ; there are also transient mild increases related to meals , stress , and sexual activity . prolactin levels are higher during menstrual midcycle and the 2nd half of the cycle . during pregnancy , levels rise 1020-fold , reaching 200 g / l at term and 300 g / l during nursing . prolactin stimulates breast enlargement during pregnancy and milk production during lactation , while reducing libido and fertility , which may have evolutionary / survival significance . secretion of prolactin is inhibited predominantly by dopamine produced in the tuberoinfundibular neurons of the hypothalamus , released from nerve endings in the median eminence and carried through the portal hypophyseal circulation to the pituitary , there binding to dopamine d2 receptors on lactotrophs , inhibiting prolactin gene transcription . estrogens , binding to specific intracellular receptors in lactotrophs , enhance prolactin gene transcription and synthesis . they also inhibit dopamine synthesis in the tuberoinfundibular neurons and reduce d2 receptor levels on lactotrophs in animal models [ 10 , 11 ] . gonadotropin releasing hormone ( gnrh ) , released in a pulsatile manner from the hypothalamus , stimulates release of luteinizing hormone ( lh ) and follicle stimulating hormone ( fsh ) from the anterior pituitary . consequently , estrogen levels in women and testosterone levels in men are suppressed , with marked individual variability in the prolactin level causing gonadal hypofunction . in children and adolescents , hyperprolactinemia resulting from prolactinomas , which are rare , can result in galactorrhea , amenorrhea , gynecomastia , and maturational delay with growth failure . of concern is the potential effect of the induced hypogonadotropism state on the critical peak bone formation of adolescence and the maintenance of bone density through adulthood . psychiatric disorders may be associated with modest elevations in serum prolactin concentrations as a stress phenomenon . further prolactin elevation can be measured within minutes to hours after the start of treatment with first - generation antipsychotic drugs , with levels up to 10-fold after several weeks at therapeutic dosages . levels typically fall to normal within 2 to 4 days of stopping the drugs but may take up to 3 weeks to return to normal . in adult psychiatric patients clozapine and quetiapine did not raise plasma prolactin levels at any dosage . olanzapine only did so at higher dosages , but risperidone and amisulpride caused marked , sustained increase in serum prolactin levels in a substantial number of patients . aripiprazole , a relatively new atypical antipsychotic , also does not appear to increase prolactin levels . a review published in 2004 of 14 reports of the effects of both first- and second - generation antipsychotic agents in children and adolescents included 276 patients of whom 49 had prolactin elevations . a report of 35 patients aged 919 years found no prolactin elevation with clozapine , but in 9 of 10 with haloperidol and 7 of 10 with olanzapine . in another study of 11 outpatients aged 417 years treated with risperidone , 9 developed hyperprolactinemia of whom one had amenorrhea , and one had gynecomastia . a further study reported that prolactin levels were increased in all 34 patients aged 514 years treated with risperidone . hyperprolactinemia was also noted in pediatric patients using ziprasidone and olanzapine . as with adults , ziprasidone- associated prolactin elevation was mild and transient , but associated with mild gynecomastia and in one case with galactorrhea that resolved after drug discontinuation . two studies of the effects of quetiapine led to slightly differing results : one showed no increase in prolactin levels in 10 1215-year - old children while the other study of 15 1317-year - olds found a slight increase of no clinical significance , from a mean of 11.3 to 14.4 ng / ml . clozapine was not associated with increased prolactin levels , and the single patient who developed galactorrhea with risperidone had resolution of the problem when switched to clozapine . a retrospective study analyzed prolactin levels and hyperprolactinemia attributable side effects from 5 clinical trials involving 592 children and adolescents of subaverage intelligence with conduct or other disruptive behavior disorders aged 5 to 15 years treated with risperidone . there was a weak effect of risperidone on prolactin concentrations during short - term treatment and lesser effect with long - term treatment , with side effects of gynecomastia , amenorrhea , or galactorrhea in only 2.2% . these relatively benign findings have been attributed to low drug dosage for behavioral rather than psychiatric disorders and decreasing compliance over time . quite different findings emerged from a small double - blind placebo - controlled study of the effect of relatively low dose risperidone on prolactinemia in 10 children and adolescents with mental retardation and pervasive developmental disorders . prolactin levels approximately tripled , and this increase was sustained for a mean 33 weeks of treatment . three adolescents were reported with risperidone - induced hyperprolactinemia resulting in gynecomastia in one boy that cleared and did not recur with olanzapine , gynecomastia with galactorrhea in another boy with comparable prolactinemia that resolved when he was switched to clozapine , and amenorrhea and galactorrhea in the third patient that resolved when she was changed to quetiapine . their prolactin levels were 2100 , 1670 , and 1990 miu / l ( 58 , 46 , and 55 g / l ) when they were hyperprolactinemic and reduced to 63 , 90 , and 191 miu / l ( 2 , 2.5 , and 5.3 g / l ) after resolution . among 10 psychotic adolescents treated with risperidone , holzer and eap had 3 males developing gynecomastia and 2 females developing galactorrhea , along with 3 others having hyperprolactinemia without symptoms . sixteen adolescents aged 1317 years with subaverage intelligence and disruptive behavior disorders treated with olanzapine for 8 weeks had significant elevations in serum prolactin levels , from baseline 9.7 6.1 to 24.8 19.8 ( sd ) g / l without any symptoms or signs of hyperprolactinemia . a study from italy compared short- and long - term effects on prolactin of risperidone and olanzapine in 42 children and adolescents treated for a year . they found that after adjusting for dose and the greater potency of risperidone , the increase in prolactin levels during risperidone treatment was 10.7 times higher than that during olanzapine treatment . only one subject had a symptom of hyperprolactinemia , transient mild galactorrhea with risperidone which resolved without a change in therapy . similarly , a randomized comparison of quetiapine and risperidone in 22 1518-year - old adolescents with new - onset psychosis found prolactin elevation in 91% of those treated with risperidone versus 9% of those treated with quetiapine . dutch investigators have recently conducted an extensive literature review of studies of antipsychotic medication effects on prolactin level and associated side effects in children and adolescents . twenty of these were concerning risperidone , 7 olanzapine , 5 quetiapine , 4 haloperidol , 3 pimozide , 2 clozapine , and 1 ziprasidone . they found that all antipsychotics with the exception of clozapine , ziprasidone , and quetiapine increased the mean prolactin level from 8 to 2528 ng / ml . the incidence of hyperprolactinemia was 90% with haloperidol , 80% with pimozide , 62% with risperidone , 31% with olanzapine , and 12% with quetiapine . risperidone , olanzapine , and pimozide were seen to induce a persistent elevation in prolactin levels . associated gynecomastia , galactorrhea , or irregular menses were reported in 4.8% of the children and adolescents . interpretation of the numerous studies is confounded by variation in study design , diagnoses , dosages , and age distribution , varying use of concomitant medication , short duration of some studies , compliance uncertainty , lack of prolactin baseline values ( one fourth of the studies analyzed by roke et al . ) , and other missing data as noted in the table . there may also be publication bias , as all data on prolactin values from manufacturers ' files have not been published . the effects of hyperprolactinemia may be underestimated because they depend on self - report , and may be mistaken for common adolescent problems of gynecomastia and irregular menstrual cycles . the degree of hyperprolactinemia induced by short - term risperidone treatment in children and youth is dose dependent [ 17 , 33 ] . this dose dependency is linked to plasma concentrations of both risperidone and its active metabolite 9-hydroxyrisperidone . cyp2d6 is primarily responsible for the conversion of risperidone to 9-hydroxyrisperidone . in an examination of the possible role of activity of this enzyme in risperidone - induced prolactin release in children , troost et al . found a positive correlation of the fourfold elevation in serum prolactin level at 8 and 24 weeks with dose per kilogram body weight ( r = 0.65 , p < .001 ) , number of functional cyp2d6 genes , serum 9-hydroxyrisperidone concentration ( r = 0.66 , p < .001 ) and negative correlation with the risperidone/9-hydroxyrisperidone ratio ( r = 0.57 , p = .004 ) but not with risperidone concentration ( r = 0.24 , p = .26 ) . thus , more rapid cyp2d6 metabolism may be a risk factor for hyperprolactinemia with risperidone . polymorphic variation in the dopamine d2 receptor may be another pharmacogenetic factor determining risk for risperidone - induced hyperprolactinemia in children and adolescents . two variants were identified that were associated with higher prolactin concentration in a study of 107 patients treated for up to 3 years . because second - generation antipsychotics are being increasingly prescribed for children and adolescents with conditions that are not psychoses and that are also treated with stimulants , the potential mitigating effect of the stimulants on the side effects of the antipsychotics has been examined . subjects were 153 419-year - olds treated with antipsychotics , 71 of whom were coprescribed stimulants . the antipsychotic drugs included risperidone ( 33% ) , aripiprazole ( 30% ) , quetiapine ( 18% ) , olanzapine ( 12% ) , and ziprasidone ( 6% ) . in addition to no effect of cotreatment with stimulants on the side effect of hyperprolactinemia , there was no effect on body composition , metabolic parameters , sedation , or overall efficacy of the antipsychotic agent . antipsychotic - induced hyperprolactinemia in adults with schizophrenia has been associated with reduced bmd and increased fracture risk [ 36 , 37 ] . the initial report of the effect of psychotropic drugs on bmd in children was a cross - sectional study that involved 83 boys aged 7 to 17 years treated with risperidone for an average of 3 years and selective serotonin reuptake inhibitors ( ssris ) . with adjustment for the stage of sexual maturation , height , and body mass index , a negative association was found between serum prolactin level and trabecular volumetric bmd at the distal radius . furthermore , treatment with ssris was associated with lower trabecular bmd at the radius and bmd z - score at the lumbar spine . lumbar spine bmd z - score did not correlate with prolactinemia . in females with prolactin secreting tumors , hyperprolactinemia effects on bmd are mediated by hypogonadism . however , in this study , serum testosterone concentrations , adjusted for maturational stage , were not affected by hyperprolactinemia , suggesting a direct effect of the hyperprolactinemia on bone turnover . prolactin receptors have been found in osteoblasts , and animal studies indicate that hyperprolactinemia activates the phosphoinositide 3-kinase pathways via the prolactin receptors to suppress alkaline phosphatase activity . the finding that ssri treatment was associated with reduced bmd could reflect an effect on prolactinemia . however , no independent effect of ssris on prolactin concentration was found in an earlier study by these authors , and the negative association between ssris and bmd was found after adjustment for numerous covariates including prolactinemia . the study of calarge et al . is limited by the dependence on a single measurement of serum prolactin which can vary by time of day and stress level , and absence of measures of bone turnover . the authors recognize that it is premature to make any definitive conclusions about the effect of psychotropic medications on bone mineralization . noting higher - than - expected postmarketing reports of pituitary tumors associated with risperidone , szarfman et al . analyzed patterns of these tumors in the united states food and drug administration adverse event ( ae ) reporting system database . they sought disproportionate reporting patterns of pituitary tumor reports for antipsychotics with different affinities for blocking d2 receptors ( aripiprazole , clozapine , olanzapine , quetiapine , risperidone , ziprasidone , and haloperidol ) . the rank order of the strength of the association between the drug and the development of pituitary tumors corresponded to the affinity of these 7 drugs for the d2 receptor . in children 718 years of age , there was 1 ae of pituitary tumor associated with olanzapine and 3 with risperidone . the authors note the importance of these findings for children because the symptoms of a pituitary expanding mass may not be as readily evaluated in those with mental illness or serious behavioral problems and that delayed detection can result in hemorrhage or optic nerve compression . second - generation antipsychotics are being increasingly prescribed for children and adolescents with a wide range of behavioral disturbances in addition to psychoses , resulting in metabolic and hormonal changes of importance to the consulting pediatric endocrinologist . in addition to weight gain and associated comorbidities of insulin resistance , hyperprolactinemia is a common side effect resulting from the inhibition of dopamine action . first - generation antipsychotics , particularly haloperidol , and the second - generation antipsychotic drugs , most prominently risperidone , appear to be associated with the greatest risk for hyperprolactinemia ; some treated individuals developing hyperprolactinemia will have galactorrhea , amenorrhea , or gynecomastia . hyperprolactinemia may have a deleterious effect on peak bone mass attainment and increase long - term osteopenia risk , even in the absence of overt symptoms or signs of hyperprolactinemia . the suggestion of a greater risk for pituitary tumors related to drug affinity for d2 receptors also requires continued study . thus , in addition to surveillance for signs and symptoms of hyperprolactinemia in children and adolescents taking antipsychotic medications , monitoring serum prolactin concentrations is warranted . in the presence of hyperprolactinemia , cessation of antipsychotic therapy or changing to a formulation less likely to raise prolactin levels the author is a consultant to a law firm pursuing litigation with manufacturers of second - generation antipsychotic drugs .

backgroundacupuncture is a promising treatment approach in patients with chronic low back pain ( clbp ) but little is known about the quality of acupuncture in randomized controlled trials ( rct ) of acupuncture clbp.objectiveto determine how international experts ( ies ) rate the quality of acupuncture in rcts of clbp ; independent international validation of the low back pain acupuncture score ( lbpas).methodologyfifteen experts from 9 different countries outside china were surveyed ( ies ) . they were asked to read anonymized excerpts of 24 rcts of clbp and answer a three - item questionnaire on how the method of acupuncture conformed to 1 ) chinese textbook standards , 2 ) the expert 's personally preferred style , and 3 ) how acupuncture is performed in the expert 's country . likert scale rating , calculation of the mode for each answer , and spearman 's rank correlation coefficient between all three answers and the lbpas were calculated.resultson comparison with chinese textbook standards ( question 1 ) , 6 rcts received a good rating , 8 trials a fair and 10 trials a poor or very poor rating . 5 of the 6 trials rated good , received at least a good rating also in question 2 or 3 . we found a high correlation of 0.85 ( p < 0.0001 ) between the ies and lbpas ratings for question 1 and question 2 , and a correlation of 0.66 ( p < 0.0001 ) for question 3.conclusionthe international expert survey ( ies ) revealed that only 6 out of 24 ( 25% ) rcts of acupuncture for clbp were rated " good " in respect to chinese textbook acupuncture standards . there were only small differences in how the acupuncture quality was rated in comparison to chinese textbook acupuncture , personally preferred and local styles of acupuncture . the rating showed a high correlation with the low back pain acupuncture score lbpas . there is increasing use of antipsychotic drugs in pediatric and psychiatry practice for a wide range of behavioral and affective disorders . these drugs have prominent side effects of interest to pediatric endocrinologists , including weight gain and associated metabolic risk factors and hyperprolactinemia . the drugs block dopamine action , thus disinhibiting prolactin secretion . hyperprolactinemia is especially prominent with first - generation antipsychotics such as haloperidol and the second - generation drugs , most commonly risperidone , with some patients developing gynecomastia or galactorrhea or , as a result of prolactin inhibition of gonadotropin releasing hormone from the hypothalamus , amenorrhea . with concern about the long - term effects of antipsychotics on bone mass and pituitary tumor formation , it is prudent to monitor serum prolactin levels in antipsychotic drug - treated pediatric patients and consider treatment with an agent less likely to induce hyperprolactinemia .

experimental approaches such as x - ray and electron crystallography and microscopy have traditionally excelled at determining the structure of single folded proteins and large protein complexes . however , intrinsically disordered proteins ( idps ) are not amenable to these static structural determination methods . idps represent a new frontier in structural biology in that the idp structure must be characterized as a diverse ensemble of interconverting conformational substates , as opposed to a single dominant 3d structure . this necessitates an adjustment in the core methodology of protein structure determination for this class of protein . the experimental identification of proteins with global intrinsic disorder can be performed using various spectroscopic techniques including circular dichroism ( cd ) , nmr , infrared spectroscopy ( ir ) , uv spectroscopy , and fluorescence spectroscopy . cd and ir report on the amount of secondary structure , while lack of chemical shift dispersion in nmr spectra is a good indication of high flexibility . hydrodynamic techniques such as saxs , gel filtration , and dynamic light scattering can also aid in idp identification as they report on the radius of the protein , which is often larger for an idp or denatured protein than a folded protein of the same mass . lack of a cooperative folding transition , solubility at high temperatures , and proteolytic sensitivity are also attributes of idps that are useful in forming a complete picture of a certain protein s level of disorder . a subset of these techniques is generally employed to determine that a protein is an idp . recently increased importance has been placed on characterizing the conformational substates within idp ensembles since they each may have distinct functional roles or could lead to hypotheses about disease origin . in order to achieve both better ensemble classification and a detailed description of conformational substates , we must critically assess how we build these complex structural ensembles from experimental data and theoretical models . nmr is the experimental tool of choice for characterizing the solution structure and dynamics of biological molecules since it reports on the native distribution of conformations in an aqueous environment , and more importantly is a dynamical experiment that probes the nanosecond to millisecond time scales of conformational motion . observables from these experiments include chemical shifts , which are characteristic of functional groups and their surrounding environment , and spin spin couplings ( j - couplings ) , which independently report on backbone dihedral angles . dipole interactions give rise to the nuclear overhauser effect ( noe ) that reports on tertiary structure contacts , and more recently , residual dipolar couplings ( rdcs ) have been used to describe the relative orientation of spatially separated regions of a protein . paramagnetic relaxation enhancements ( pres ) , which can produce longer distance restraints than noes have also been used in the context of idps ; however , this measurement requires chemical modification of the protein with a nitroxide spin label or an amino - terminal copper binding motif , which sometimes requires sequence modification to attach the probe , and which may perturb the monomeric idp conformations . nanoseconds milliseconds time scale of the nmr experiment , leading to an averaging of the nmr observables across structural subpopulations . this uniform average hinders the structural characterization of all the conformational substates , and can even obscure the overall ensemble classification , as we will see for the amyloid peptides in this study . building the connection between the averaged nmr observables and the complete idp structural ensemble therefore depends critically on computational models . the goal of the computational model is to provide a properly weighted set of the diverse subpopulations of the idp most consistent with the nmr observables and perhaps other experimental measures such as circular dichrosim , small - angle x - ray scattering , or pres . thus , multiple types of nmr or other experimental observables are necessary for validation of the computational model . currently there are two primary but very different computational approaches to building an idp structural ensemble , which can be loosely contrasted as first principle or de novo molecular dynamics ( md ) methods versus knowledge - based approaches . the de novo approach implements md simulations based on the theoretical foundations of statistical mechanical sampling and model - derived potential energy force fields . de novo md generates a structural ensemble that is representative of given thermodynamic conditions according to the force field employed , i.e. , a boltzmann weighted ensemble of conformational subpopulations and their time scales , independent of experimental input . the md trajectories also allow calculation of the time correlation functions that underlie the nmr experiment . the complementary use of md and nmr data to determine structure and dynamics of folded and unfolded proteins has been a highly active area over the last two decades , particularly for relaxation measurements that require a dynamical interpretation of the nmr data at the picosecond and nanosecond time scales . for the de novo md method , multiple nmr or other experimental data are necessary to validate the md ensemble through direct back - calculation of observables , many of which depend on the time scales of motion , in order to directly compare to the experiment . once validated , md simulations provide a prediction of the complete idp structural ensemble , allowing overall classification as well as the study of individual conformational substates , which can be analyzed with some confidence . in contrast , we define knowledge - based approaches as those that use experimental nmr information directly to derive the structural ensemble . such methods are the foundation of nmr structure determination of folded proteins using experimentally derived conformational constraints based on chemical shifts , j - couplings , and noe data embodied in software packages such as candid , cyana , and x - plor - nih . while md is often used to generate atomistic predictions independent of nmr experimental input , as in our de novo method , a number of researchers have advanced the combination of applying knowledge from nmr to restrain the md ensemble . for example , md simulations have been combined with rdc restraint data for folded proteins that then allows for the analysis of other features of the ensemble , such as conformational fluctuations . nmr restrained md has also been applied to idps such as -synuclein , a disease protein indicted in parkinson s disease . this study incorporated distance restraints derived from pre experiments in order to guide the md so that the protein s radius of gyration distribution is in good agreement with the experimental value . other knowledge - based approaches for idps forego md simulations altogether and instead use an extensive set of statistical coil conformations ; this starting pool , which can be generated using a variety of heuristics , can be thought of as a basis set of structures . subsequently , the starting pool of structures is then culled for the subset of conformations that are in best agreement with experimental data to create the idp ensemble . in the energy - minima mapping and weighting ( emw ) method , stultz and co - workers used end - to - end distance restraints to develop a pool of conformations with varying radii of gyration ; they then selected , via monte carlo , a weighted ensemble of 15 structures to optimize the agreement with experimental c and n chemical shifts and j - couplings . blackledge and co - workers have developed the program flexible - meccano to create a pool of structures based on random coil backbone dihedral angles , on which they employ a genetic search algorithm in their asteroids software program to select structures that together best match experimental chemical shifts , pres , or rdcs . the ensemble method , developed by forman - kay and co - workers , typically defines the starting pool of idp conformational states as an ensemble of extended or random coil states generated using trades , with an option for biasing the secondary structure of the ensemble at certain places in the sequence that are known to be partially structured . structures are selected from this pool using a monte carlo selection algorithm with an energy - weighting scheme for each type of experimental input . the ensemble program includes modules for several different experimental data types including chemical shifts , rdcs , pres , j - couplings , and contact distances derived from noes , and is a user - friendly and publically available software package . although there are some specific differences , ensemble is largely representative of the knowledge - based approaches and is qualitatively equivalent to the combination of flexible - meccano and asteroids software . it is important to note that such techniques largely ignore the inherent dynamical information of certain types of the nmr data that can be important for discriminating between different idp structural ensembles . the primary objective of this work is to compare the de novo and knowledge - based approaches for deriving idp structural ensembles in context of the intrinsically disordered alzheimer s disease peptides amyloid- 140 ( a40 ) and amyloid- 142 ( a42 ) . we implement the ensemble knowledge - based method by building an ensemble from a pool of statistical coil structures , and compare this knowledge - based ensemble to md generated ensembles , which are qualitatively different in that they are comprised of mostly cooperative secondary structure and tertiary contacts . this comparison also exposes the relative utility of different types of nmr data for refining or validating the idp computational ensemble . we find that chemical shifts and j - coupling constants are not particularly useful for distinguishing between qualitatively different idp ensembles of the amyloid- peptides . finally we show that the combination of de novo md methods that provide boltzmann weighted samples with the ability to measure time correlation functions , and knowledge - based methods for conformation selection , provides the best agreement with the nmr data . in order to evaluate the alternative ensembles produced by knowledge - based and de novo approaches , we need a method of calculating the chemical shifts , j - coupling constants , rdcs , and h h noes as averages over the entire computationally generated structural ensemble for comparison with experimental values . general purpose chemical shift calculators such as shiftx and shifts describe the isotropic shielding of the applied magnetic field for the given atom , a quantity that depends sensitively on the local electronic structure environment . even for folded proteins with a dominant native conformer , each atom type can exist in many different local environments , and for disordered peptides and proteins the ensemble average reflects an even more diverse set of chemical environments . whether one uses shiftx ( used in ensemble ) or shifts to calculate chemical shifts , the results generated by the two programs are consistent when applied to amyloid- and averaged over the structural ensembles . we report results using shiftx in this work . to calculate the scalar coupling constants , jhnh , we used the karplus equation1where indicates the protein backbone dihedral angle , with coefficients a = 6.51 , b = 1.76 , and c = 1.60 corresponding to the parameter set by vuister and bax . however , sgourakis and co - workers and our own previous work on a42 found that the md results exhibited a systematic shift of calculated j - couplings with the experimental scalar couplings of wang and co - workers . we delved into this issue more deeply and derived an analytical correction , described in recent work , that must be applied to the original experimental j - coupling data from wang and co - workers . in particular , it has been corrected for a missing relaxation that makes scalar couplings determined from the hnh 3d experiment consistently lower than those from cosy splittings by a small amount ( from 15% ) . the j - coupling values are also averaged over all structures in the ensemble as in , and then the calculations can be compared to the corrected experimental jhnh values for both amyloid peptides . the standard method in the field for calculation of rdcs is the pales program , which we have used previously for the a40 and a42 md ensembles . the program computes the rdc by using steric properties of the molecule to generate a global alignment orientation . then , the angle between the backbone amide bond vectors and the external magnetic field is used to calculate the rdc for each conformation , and the rdcs are averaged over all conformations of a given ensemble . the ensemble program by contrast , evaluates rdcs using a local alignment program developed in the forman - kay lab , where 15 residue segments along the protein are aligned separately over the ensemble of structures . the local rdcs ( l - rdcs ) are also averaged over all conformations of a given ensemble . this local alignment has lower computational cost and has been shown to give similar results to pales , hence l - rdcs , rather than rdcs generated from a global alignment algorithm , are optimized in the standard implementation of the ensemble approach . similarly , the asteroids program by default employs a local alignment tensor to optimize ensemble agreement with experimental rdc data . we also note that the pales alignment and rdc calculation were developed for folded proteins , and their application to idps assumes individual idp conformations behave similarly to folded proteins during the rdc experiment , which may not be the case for idps such as amyloid-. we also evaluate the h h noesy ( or roesy ) spectra as we have described in previous studies by calculating the intensity of the noe cross - peaks2where x and are the eigenvectors and eigenvalues of the full relaxation matrix , composed of diagonal elements3aand off - diagonal elements3bthat are comprised of appropriate combinations of the spectral density functions4evaluated at the relevant larmor frequencies , , and where k is given by5h is the gyromagnetic ratio of h , 0 is the permeability of free space , and is planck s constant . reff is the distance between the hydrogen atoms raised to 6 power , averaged over all structures in the ensemble and then raised to the 1/6 power to convert back to units of distance . these calculations account for all hydrogen atoms explicitly ( including all methyl or methylene groups ) and hence reff and correlation functions for every pair of hydrogen atoms are evaluated . the spectral density function for each atom pair is calculated as the fourier transform of the correlation function for the pair vector and water proton coordinates are ignored , as is the standard assumption in the nmr experiment . n noes as we did in refs ( 14 and 48 ) by evaluating the steady state noe enhancement factor of the n spin by the h noe according to6where h and n are the gyromagnetic ratios of h and n , respectively . n cross - relaxation rate constant is given by7and the n self - relaxation by8 in this case , jhn( ) is the spectral density function for the h n covalently bonded pair . note that the homonuclear and heteronuclear noe calculations require correlation time information about the vector between each pair of atoms given by in eq 4 . this dynamic information is naturally supplied by the de novo md method , which allows direct measurement of the autocorrelation of the interatomic vector over the time of the simulation . however , dynamics are not considered in the generation of ensembles that are used in the knowledge - based approach . this is an inherent limitation of ensembles generated from a static perspective only , which we discuss further below . to determine an idp s level of disorder , we first generate several alternative ensembles , compare these ensembles to the available nmr data , and select the best validated ensemble . we consider the creation of three qualitatively different conformational ensembles that are typically used in the knowledge - based approaches for the a40 and a42 peptides . the common null hypothesis is that the disordered peptides can be well represented by a random coil ( rc ) ensemble . the second type of statistical coil ( pred - ss ) ensemble is also random , but incorporates bioinformatics - based knowledge about what secondary structure category is more likely for a given residue in the amino acid sequence . in this case , the random ensemble is biased to contain a statistical probability of predicted secondary structure on a per residue basis , but no cooperative secondary structure such as -helices , -hairpins , or -sheets are generated from the random secondary structure assignments . computational methods such as trades and flexible - meccano are used to generate these type of random or statistical coil ensembles . finally , a fully knowledge - based approach is considered , which culls the rc or , in this case , pred - ss ensembles to derive a subset of conformations that best agrees with the nmr data ( pred - ss - ens ) . the ensemble software package provides a working example of the knowledge - based approach that performs this biased selection and which has been successfully applied to a range of idps . each of the above three ensembles can then be compared against the ensembles generated by de novo md for both of the idps a40 and a42 . details of the de novo md approach applied to amyloid- can be found in other publications . we also consider an additional fifth ensemble ( md - ens ) that combines the knowledge - based and de novo md approaches , by using ensemble to select structures from the de novo md starting pool , and which is described in our recent study . table 1 shows the average radius of gyration ( rg ) values for each type of ensemble . we see that the order from most extended to most compact proceeds as pred - ss > rc > pred - ss - ens > md md - ens , and thus the alternative ensembles span a range of idp classifications by the rg measure . figure 1 provides the propensities for the pred - ss , pred - ss - ens , de novo md , and md - ens ensembles to form turns , antiparallel -strands , or helical structure by residue for a42 . we do not show the secondary structure profiles for the rc ensemble since it is similar to the pred - ss ensemble ( see the supporting information ) . percentage of a42 simulated ensemble in different types of secondary structure by residue for ( a ) the pred - ss , ( b ) pred - ss - ens , ( c ) de novo md , and ( d ) md - ens ensembles . the red line represents helix , the blue line for antiparallel sheet , and the black line for -turns . we note that the blue line represents only antiparallel sheet structure ( the most common ) and not all sheets . for the radius of gyration ( rg ) values , we report both the ensemble average and rmsd . for chemical shifts , we report that measures agreement between the computational ensembles and the experimentally measured chemical shifts : < 1 indicates no disagreement with experiment within shiftx calculator error . we also report the jhnh rmsd ( ) . some data reproduced from ref ( 14 ) . this plot emphasizes that the md - based ensembles are qualitatively different from the rc or pred - ss ensembles , in that the a40 and a42 peptides samples some type of structured conformations in 99% of the md ensemble , including complex strand formation . from this , we conclude that the radius of gyration trends stem from the much larger propensity for the md ensembles to form cooperative secondary structure and collapsed tertiary contacts , as opposed to the random or knowledge - based ensembles that do not generate contiguous blocks of secondary structure , and hence are more extended on average . although the secondary structure content of the md - ens ensemble resembles that of the md ensemble , figure 1 shows that there is some variation in the percentages with which certain residues adopt different types of secondary structure . for analysis of a40 and a42 idps considered here , we have utilized a wide range of previously published nmr data including chemical shifts from the zagorski group as well as j - coupling constants , rdcs , and heteronuclear h our group has collected h chemical shifts and noesy h h homonuclear spectra for the full length a40 and a42 peptides as reported elsewhere . the data for the longer peptides were processed as described in ref ( 48 ) in a similar approach to that used for the a2130 fragment . first we consider the chemical shift data , for which we note that the calculated chemical shifts have an uncertainty that is independent of the quality or type of structural ensemble , and results from approximations of the shiftx or shifts calculators themselves . other research groups have reported the uncertainty , ( ppm ) , for these calculators , with the value depending on the atom type and its bonding chemistry . therefore the best way validate the various idp ensembles with chemical shift data is to calculate the difference between the experimental chemical shift and the shift calculated from each of the structural ensembles , normalizing it by the calculator uncertainty , to generate values9reported uncertainties ( root mean squared difference , rmsd , from experiment ) for the shiftx calculator are = 0.23 ppm for h , = 0.49 ppm for hn , = 0.98 ppm for c , and = 1.10 ppm for c. any dominant error due to the underlying structural ensemble would then correspond to values of > 1 . table 1 displays the agreement between experimentally measured proton and carbon chemical shifts with those generated from each candidate ensemble for both a40 and a42 . experimental chemical shift data reported for the monomeric a40 and a42 peptides do not differ greatly from random coil values , and therefore the rc ensemble falls within uncertainty . since the pred - ss ensemble shows almost no dssp defined secondary structure ( figure 1a ) , it remains largely equivalent to the rc ensemble as deduced by chemical shifts . the de novo md structural ensemble is also in good agreement with the chemical shift data ; however , 99% of the md generated a conformations contain one or more elements of cooperative secondary structure somewhere along the peptide sequence ( figure 1b ) . the reason that the md ensemble is also in good agreement with the experimental chemical shifts is that averaging over a large ensemble of cooperatively formed secondary structure and tertiary contacts yields average chemical shifts that are consistent with random coil values . for example , averaging the chemical shifts of all folded proteins in the pdb results in averages very similar to random coil values . we have found that the ensemble optimization of the pred - ss and md starting pools improves the values , but all are within the calculator uncertainty . not surprisingly , if the knowledge - based ensemble approach were biased by chemical shift data alone , they would show little deviation from their starting soup , and the structural interpretation would be highly dependent on the starting ensemble . for this reason we conclude that nmr chemical shifts alone do not provide any qualitative discrimination between the alternative ensembles , at least not for the a40 and a42 disease idps . it may still be useful to apply chemical shift constraints in combination with other experimental observables to optimize an idp ensemble , as we have done when generating the md - ens ensemble . sanity check against ensembles that fit other observables , such as noes , but lead to unphysical chemical shift values . similarly , j - couplings alone also do not discriminate between random coil idps and those that are more structured with cooperative secondary structure and tertiary structure contacts . figure 2 illustrates this by plotting the agreement between experimentally measured j(hn , h ) , and those calculated from the rc , pred - ss , pred - ss - ens , de novo md , and md - ens ensembles for a40 and a4 . table 1 shows that all ensembles yield an rmsd across all residues of 0.601.09 hz , and also reports 10with j = 0.73 hz . j - couplings report on the backbone dihedral angle , and therefore could in principle distinguish between an unstructured peptide and a peptide with a defined secondary structure ; however , in the case of the disease idp a , the presence of diverse secondary structure in the md ensemble is not apparent from the calculated j - couplings . we believe that this stems from the fact that good agreement with scalar coupling data for idps can largely be predicted by sampling over the allowed regions of residue - specific ramachandran plots without needing to assume any structure adopted by the full length sequence . thus j - couplings also do not provide an experimental measure for discriminating among qualitatively different structural ensembles for the amyloid peptides . j - coupling constants for backbone amides for a40 and a42 . ( a ) a40 experimental j - coupling constants ( red squares ) compared to rc ( green triangles ) and de novo md ( solid blue circles ) . ( b ) a40 experimental j - coupling constants ( red squares ) compared to pred - ss - ens ( black diamonds ) and md - ens ( blue circles ) . ( c ) a42 experimental , rc , and de novo md j - coupling constants . ( d ) a42 experimental , pred - ss - ens , and md - ens j - coupling constants . has been corrected to account for t1sel relaxation and bring j - couplings determined from a hnh 3d experiment to be consistent with those from cosy splittings . table 2 provides the assessment of the five alternative ensembles for a40 and a42 using rdc values evaluated residue by residue using the pales program and l - rdcs based on local alignments . while the rc and pred - ss ensembles yield lower rmsd values , 1.31.5 hz , they are marginally better than the de novo md rmsd of 2.2 hz . this is in part due to the fact that experimental rdc uncertainties for idps are larger ( 0.9 hz for a40 and 0.5 hz for a42 ) than the uncertainty observed for folded proteins of 0.1 hz . in addition , there are large uncertainties in the accuracy of rdc calculators using programs such as pales . in fact , the reported rmsd of the pales calculator for folded proteins is 2.0 hz , on the same order as the rmsd for the de novo md ensemble . while the ensemble method does significantly lower the rmsd for l - rdcs for the pred - ss - ens and md - ens ensembles , the corresponding rmsd based on the global alignment using pales is marginally better than the pred - ss and de novo md starting pools . hence for this particular application on disordered amyloid peptides , we have found that rdcs are not a particularly good experimental metric for differentiating among the different ensembles , and substantial disagreement between rdcs based on local and global alignments are observed . we report rmsds for the rdc calculator pales and l - rdcs evaluated with ensemble using local alignments . the noes are back - calculated from the structural ensembles as described in section 4 . we evaluate the rmsd normalized by the largest noe intensity , rmsdn and ( correlation coefficient , r ) with the h2o and d2o experiments . some data reproduced from ref ( 14 ) . finally , we consider the performance of the different ensemble methods for reproducing h we have presented the noe data collection for the a42 peptide in which 700 cross - peaks are observed in the noe spectra , but only 200 can be uniquely assigned from experimental information alone . the remaining cross - peaks do not have a clear independent assignment ( and in fact require a computational model to interpret them ) . therefore we have only compared the different methods against the noe cross - peaks that can be assigned by experiment alone . we note that quantitatively reproducing noe intensities is a very high bar since peak volumes are extremely sensitive to r distance averaging , that also involve an appropriate time scale that is heterogeneous across proton pairs . geometric imperfections in the conformational ensemble where contact differences differ by a factor of 2 ( difference between 1 and 1.12 ) will double the corresponding intensity value , thereby driving up the rmsd error for all ensembles . large absolute noe intensities especially tend to dominate the rmsd error , and therefore we have mitigated this effect by normalizing the rmsd ( rmsdn ) by the experimental intensity for each noe as in ref ( 14 ) . table 2 shows that the predicted set of h h noes from de novo md is better than any other ensemble , with rmsdns that are lower than the rc and pred - ss ensembles values by 23 fold and with much higher correlation coefficients . the pred - ss - ens ensemble performs better for a40 and a42 than the randomly generated ensembles because the noe restraints are used in the knowledge - based ensemble selection . however , the pred - ss - ens ensemble still does not reproduce the data as well as the de novo md ensemble . the noe validation clearly indicates that the de novo md ensemble with its cooperative secondary structure is a better representation of a40 and a42 than are the rc , pred - ss , or pred - ss - ens ensembles , which have no cooperative secondary structure . since time information is not available for the static ensembles , we can only evaluate the noes for the statistical ensembles under the assumption of one uniform correlation time applied to all pairs of protons , for which we use a 1 ns correlation time , which is on the same order as those observed in the md simulations . of course the de novo md method can account for the time scales explicitly and more importantly for the fact that different pairs of hydrogen atoms do decay on different time scales . thus the statistically generated and knowledge - based ensembles agree relatively poorly with the noe observables since the heterogeneity in correlation times are unknown , and hence even the md - ens ensemble is in somewhat worse agreement with the experimental noes than the de novo md ensemble ( table 2 ) . the noe validation emphasizes that an idp s diverse set of conformations gives rise to a heterogeneous set of correlation times that must be described in order to validate against experimental noes . we further emphasize that the calculation of heteronuclear noes , being a purely dynamical measurement , is only possible with the de novo md method . n noe intensities , measured by yan and wang , and those derived from our md simulation for a42 and a40 , showing overall excellent agreement . unlike the h h noes , these assignments are unambiguous from experiment . we find that , as in the experiment , there is an increase in h n noe intensities calculated from simulation for residues 3540 for a42 compared to a40 , indicating that the longer peptide experiences slower dynamics at the c - terminus . n noes for a42 and a40 has previously been interpreted as evidence that a42 has greater structural rigidity in the c - terminus compared to a40 and we provide more analysis on this point in recently published work . agreement with experiment of simulated ( a ) a40 and ( b ) a42 h we have shown that the md and md - ens structural ensembles for the idps a40 and a42 previously characterized yield substantially better agreement with a range of nmr data than the random coil or statistical ensembles that are typically used with knowledge based approaches . the md ensembles are qualitatively different from random coil or statistical ensembles in that the subpopulations are richly structured , contain a diverse set of secondary structures including -helix , -turns , and -strands , and span the full range of compact to fully extended conformations . furthermore , while md generated ensembles are boltzmann weighted , the knowledge - based approaches give equal statistical weight to all conformations and thus are likely inconsistent with statistical mechanical weightings that are inherent to the nmr experiment . we have also shown that some types of nmr data may not be helpful for discriminating among qualitatively different structural ensemble of idps . in particular , averages over a diverse set of cooperative secondary structure conformations yield experimental values of chemical shifts that are superficially consistent with values expected from a random coil ensemble . furthermore , if the chemical shifts are not highly dispersed along the sequence of a particular idp , such as is found for the amyloid- peptides , then the chemical shifts have limited value as experimental refinement input or as a validation measure . j - couplings also do not provide discrimination between randomly generated conformations and a diverse population of cooperative secondary structure . in fact , we found that scalar couplings calculated as averages over the allowed regions of each residue - specific ramachandran plot gave as good agreement with the experimental j - couplings for a40 and a42 as did averages over the structural populations . unlike others who have used rdc data to help interpret idp or unfolded protein structural ensembles this may be due to limitations of rdc calculators such as pales , which were originally developed and successfully applied to folded proteins , but which are reported to have large uncertainties in their predicted rdc values . furthermore , calculated rdcs based on global alignment algorithms such as pales diverge significantly from rdcs evaluated from localized alignments for a40 and a42 , indicating that in cases like this the ensemble package should be employed using the pales calculator to fit rdcs , which is possible though not standard . more research may be necessary to apply programs like pales to disordered proteins , which likely do not align in an anisotropic medium in the same way as folded proteins , in part due to the time scale of interconversion of the conformational substates . for example , conventional methods for calculating rdcs can not be applied to the motion of multidomain biomolecules , and the local conformational sampling and long - range structure need to be simultaneously accounted for because they both affect the experimental rdc data . however , progress is being made in using rdcs to provide meaningful structural information for other idps . we speculate that success is greatest when all subpopulations of the idp ensemble are homogeneously classifiable ( as extended disordered conformations for example ) , so that the idp global alignment properties are uniform and resulting averages provide meaningful and consistent structural information . h noe intensities and heteronuclear n h noes are by themselves discriminating with regard to the tertiary contacts and backbone dynamics , respectively , that define the important validation of the md - based ensembles over the statistical coil ensembles . furthermore , a correct picture of the idp ensemble based on the experimental noe data would not be possible without a computational model providing both details of individual structures and the time scales for their interconversion . in turn , although the homonuclear noes are averaged over all subpopulations , they are still vital for deducing whether a given ensemble contains subpopulations of structure with the right tertiary contacts to give rise to the observed cross - peaks in the spectra . because these cross - peak intensities rely directly on the decay time scales of correlated proton distances , the noes for idps are reporting on a heterogeneous population of time scales . one of the primary limitations of the statically generated ensembles is that they are not associated with any information about motional time scales that can be used to calculate noe observables . relaxation times can be used with the ensemble method , although they are incorporated as structural rather than dynamic constraints . this dynamic information is a genuine strength of the de novo md methods , especially for n h noes , which can not be calculated from the static ensembles . we believe that the primary limitation of knowledge - based methods applied to the difficult amyloid case is 3-fold . we note that while there will be quantitative differences between ensemble and other knowledge - based approaches such as asteroids , qualitatively the problems will be similar . basis set of conformations to select the final ensemble from ; i.e. , these methods can not use the nmr data effectively to select for compact structures with elements of cooperative secondary structure if the initial pool of structures is largely composed of extended random coil structures . both ensemble and asteroids have relied on statistical coil ensembles as the starting pool of structures , and while some on - the - fly addition of new structures is possible with these methods , they do not yet support formation of complicated -sheet motifs . metrics of ensemble heterogeneity , such as those developed by the onuchic and stultz research groups , will continue to be useful as we explore the range of idps that can not be easily classified based on their level of disorder . second , for certain classes of idps such as amyloid optimization of structures to reproduce chemical shifts and scalar couplings does not discriminate among qualitatively different structural ensembles . third , the optimization phase of the knowledge - based approaches relies on approximations to nmr observables , which may diverge from a global property , as for l - rdcs , or from the dynamical origins of noe intensities . at the same time , the de novo md method is not quantitatively perfect , and therefore the md ensemble provides an excellent start state for subsequent refinement by knowledge - based approaches . an unambiguous future direction for the structural biology of idps is the combined use of knowledge - based approaches and md that supplies boltzmann weighted conformational substates as well as heterogeneous time scales of motion . all together , the productive interplay between nmr experiments , de novo md simulations , and knowledge - based approaches , along with supporting models , algorithms , and computer hardware , gives us an ability to accurately identify structures present in idp ensembles and use that knowledge to gain previously inaccessible functional insights . to further improve techniques for studying disordered proteins , we as a community could establish a high throughput computational infrastructure to predict idp structural ensembles using a combination of md and nmr . this would be similar to the establishment of x - ray crystallography beamlines for the rapid solution of folded protein structures that was launched during the structural genomics era . the ultimate goal in both cases is to use structural information to drive the formation of hypotheses about protein function . based on the success of using structural information for functional characterization of folded proteins and complexes , we hope and expect that structural knowledge of idp ensembles can provide similar insight into idp function and enable development of molecular hypotheses for disease idps . the protocol has been approved by the institutional review board at the national institute of cholera and enteric disease . briefly , fecal specimens were collected systematically from patients entering the hospital from november 2007 through february 2010 . of note , the previous study analyzed data through october 2009 ; however , the systematic sampling is still ongoing . the specimens were collected from every fifth patient with diarrhea on 2 randomly selected days each week . only patients with diarrhea ( defined by world health organization guidelines as passage of > 3 loose or liquid stools per day or more frequently than is normal for the person ) were eligible for inclusion in the study . each patient contributed 1 sample , and each sample was tested for all 26 common diarrheagenic pathogens . samples were collected in mccartney bottles ( using sterile catheters or rectal swabs ) containing cary - blair medium and examined ( within 2 hours of collection ) for bacterial , viral , and parasitic pathogens by a combination of conventional , immunologic , and molecular methods . the bacterial pathogens ( v. cholerae , v. parahaemolyticus , v. fluvialis , campylobacter jejuni , campylobacter coli , salmonella spp . , species and subtypes were confirmed by serotyping ( for v. parahaemolyticus , shigella spp . , and salmonella spp . ) with commercially available antiserum ( denka seiken , tokyo , japan ; biorad , marnes - la - coquette , france ) and by pcr ( for v. cholerae , v. fluvialis , enterotoxigenic e. coli [ etec , including heat - labile and heat - stable enterotoxin producers ] , enteropathogenic e. coli [ epec , typical and atypical ] , enteroaggregative e. coli [ eaec ] , enteroinvasive e. coli , and shiga toxin producing e. coli ) . rotavirus was detected by polyacrylamide gel electrophoresis and silver staining ( 8) . noroviruses ( groups i [ nvg1 ] and ii [ nvg2 ] ) , sapovirus , and astrovirus were detected by reverse transcription pcr with random primers for reverse transcription and specific primers for pcr ( 9 ) . adenoviruses were detected by the commercially available rotaadeno vikia kit ( biomrieux , marcy letoile , france ) . all samples were screened by using a highly sensitive antigen capture elisa ( techlab , inc . , blacksburg , va , usa ) of g. lamblia , cryptosporidium parvum , e. histolytica , and blastocystis hominis . to test for possible associations , we used the fisher exact test to compare pairs of pathogens ( 1 , both , or neither ) with an independent assortment based on the overall frequency with which pathogens were detected . to establish criteria for statistical significance , we calculated p values , odds ratios ( ors ) , and 95% confidence intervals ( cis ) . additional covariates were collected and examined for confounding and interaction . these included patient age , gender , residence , and religion and season of infection . seasons were defined as summer ( march june ) , monsoon ( july october ) , and winter ( november february ) . all analyses were conducted by using sas version 9.2 ( sas institute , cary , nc , usa ) . patient demographic characteristics are listed in table 1 . a large proportion ( 44% ) of patients were 1545 years of age , 13% were < 1 year of age , 80% resided in urban areas , 74% were hindu , and 25% were muslim . the following pathogens were detected in at least 1 sample : adenovirus , aeromonas spp . , astrovirus , b. hominis , c. jejuni , c. parvum , eaec , epec , etec , e. histolytica , g. lamblia , nvg1 , nvg2 , rotavirus , salmonella spp . , sapovirus , shigella spp . , v. cholerae o1 , v. cholerae o139 , v. cholerae non - o1 , v. cholerae non - o139 , v. parahaemolyticus , and v. fluvialis . no pathogens were detected in 766 ( 28% ) of the 2,748 samples ( table 1 ) , but test results were positive for the other 72% . one pathogen was found for 1,169 ( 43% ) samples and multiple pathogens for 813 ( 29% ) ( table 1 ) . the 2 most commonly detected pathogens were v. cholerae o1 and rotavirus , which were found in 24% and 22% of samples , respectively . * mean sd patient age 26 22 y. v. cholerae o1 was detected in 661 samples . v. cholerae was the sole pathogen in 379 samples ; however , it was isolated along with another diarrheagenic pathogen from 282 samples . the co - infection of v. cholerae and rotavirus was highly significant ( p = 1.12 10 ) . co - infection with v. cholerae and rotavirus was 5-fold less likely ( or 0.18 , 95% ci 0.130.25 ; figure , panel a ) to occur among those with than among those without v. cholerae infection . a negative association might be expected if a case of severe diarrhea caused by any given pathogen excluded other pathogens . consistent with this expectation , the presence of c. parvum , adenovirus , shigella spp . , etec , and v. parahaemolyticus was decreased significantly ( p = 7.87 10 to 1.32 10 ) and was 12.5-fold ( with v. parahaemolyticus , or 0.1 , 95% ci 0.020.33 ) to 2.44-fold ( with c. parvum ) less likely to occur among those with than among those without v. cholerae infection . however , antithetically , the rate of g. lamblia co - infection was significantly higher among v. cholerae o1positive than among v. cholerae o1negative fecal samples ( or 1.71 , 95% ci 1.322.21 ) . a significant difference in infection rates among those with and without v. cholerae o1 infection was not found for eaec , c. jejuni , v. fluvialis , e. histolytica , astrovirus , nvgii , and epec . tests for association were not performed for salmonella spp . , nvgi , aeromonas spp . , b. hominis , c. coli , sapovirus , v. cholerae non - o1 , v. cholerae non - o139 , and v. cholerae o139 because the low number of patients infected with those pathogens resulted in insufficient power . odds ratios ( ors ) showing odds of a ) vibrio cholerae or b ) rotavirus co - occurring with various other pathogens relative to the odds of v. cholerae or rotavirus co - occurring independently with various other pathogens at the frequency with which each is present in the entire sample . this standard forest plot indicates the best estimate and the 95% confidence intervals ( cis ) for each co - occurring organism . eaec , enteroaggregative escherichia coli ; epec , enteropathogenic e. coli ; etec , enterotoxigenic e. coli . rotavirus was detected in 594 of the fecal samples and was the sole pathogen found in 253 of them . rotavirus and at least 1 other gastrointestinal pathogen were found in 341 samples ; 119 samples were co - infected with rotavirus and > 2 other pathogens . when the effect of rotavirus co - infection with other pathogens was tested ( figure , panel b ) , shigella spp . were significantly less likely to be found in samples with rotavirus than in samples without rotavirus ( or 0.30 , 95% ci 0.170.52 ) . adenovirus were significantly increased in samples with rotavirus ( p = 6.15 10 to 1.61 10 ; ors 2.145.80 . a significant effect was not observed for g. lamblia , c. jejuni , epec , etec , v. parahaemolyticus , v. fluvialis , e. haemolyticus , astrovirus , and nvgii . tests for association were not performed for salmonella spp . , nvg1 , aeromonas spp . , b. hominis , c. coli , sapovirus , v. cholerae non - o1 , v. cholerae non - o139 , and v. cholerae o139 because the low number of patients infected with those pathogens resulted in insufficient power . analysis of samples from patients infected simultaneously with g. lamblia , v. cholerae , and rotavirus ( n = 41 ) revealed that the frequency of co - infection with g. lamblia was not significantly affected by co - infection with v. cholerae o1 and rotavirus ( p = 0.08 ) . analysis of covariates indicated that gender , religion , and residence largely had no effect on the associations between pathogens ; however , in some instances , age and season were identified as confounders or effect modifiers ( table 2 ) . to examine the effect of these covariates , we stratified the data by age and season and found that many associations remained significant ( table a1 ) . associations between rotavirus and adenovirus remained significant for all age and season strata except among children < 1 year of age . after adjusting for age and season by using logistic regression models , we found that co - infection with rotavirus and cryptosporidium spp . and co - infection with rotavirus and shigella spp . remained significant ( table 2 ) . the negative association between v. cholerae and adenovirus remained significant after adjustment by logistic regression for age and season ( or 0.36 ; 95% ci 0.210.64 ) ; associations between v. cholerae and many other pathogens remained significant within specific strata of age and season . assessing the effect of covariates for this reason , we did not include stratified results for co - infection with v. cholerae and v. parahaemolyticus in the table a1 . considered statistically significant at p<0.05 . or , odds ratio ; ci , confidence interval ; etec , enterotoxigenic escherichia coli ; eaec , enteroaggregative e. coli . our analyses revealed that co - occurrence of gastrointestinal pathogens in feces of patients with polymicrobial infections and severe diarrhea necessitating hospitalization was not in proportion to the pathogens presence in all patients with diarrhea . tests for association were performed with v. cholerae o1 and rotavirus because they were the most commonly detected pathogens and , hence , had the greatest power to detect an association with the other pathogens . some combinations of pathogens occurred less frequently than expected ( e.g. , v. cholerae and rotavirus [ or 0.18 , 95% ci 0.130.25 ] ) ; some combinations appeared more frequently than expected ( e.g. , rotavirus and adenovirus [ or = 5.8 , 95% ci 4.207.99 ] ) , and some combinations occurred at the same frequency whether with or without v. cholerae or rotavirus . after adjustment for age and season , these variables often acted as confounders or effect modifiers , but in general the associations remained significant . however , many of the stratified analyses had small numbers for comparison . v. cholerae o1 exhibited a positive association with only g. lamblia , suggesting that something may be unique about the co - occurrence of those 2 gastrointestinal pathogens . in support of that idea are 1 ) a report that co - infection with g. lamblia and v. cholerae results in g. lamblia being present in trophozoite form rather than in the cyst form found in feces of control patients ( 10 ) , and 2 ) a previous finding that g. lamblia trophozoites can bind cholera toxin ( 11 ) . alternatively , each is a pathogen with substantial environmental reservoirs , and the positive association may simply represent acquisition of both pathogens from the same environmental source . relatedness alone does not explain the apparent competitive inhibition or negative association that we found between v. cholerae and other pathogens . for example , although the closely related v. cholerae o1 and v. parahaemolyticus exhibited a 10-fold negative association , v. fluvialis , which is phylogenetically only slightly farther from v. cholerae o1 than is v. parahaemolyticus , did not show any inhibition in the presence of v. cholerae . also , although 2 members of the family enterobacteriaceae ( shigella spp . and etec ) were found less frequently than expected in combination with v. cholerae o1 , 2 other members of that family ( epec and eaec ) occurred in the expected proportion in samples from patients with mixed v. cholerae o1 infections . rotavirus had multiple strong positive associations ( ors > 2 ) with the other gastrointestinal pathogens detected in the fecal samples , even after considering age and season . the positive association of rotavirus , an rna virus that affects cells covered in microvilli , and adenovirus , a dna virus that affects cells that are dividing to generate new cells with microvilli , may represent an interaction between pathogens to cause more severe diarrhea ( in our sample all patients were hospitalized ) in patients 1545 years of age ( or 10.81 , 95% ci 5.6320.78 ) or a way to escape or circumvent immunity from previous exposures . in a previous study , koh et al . ( 12 ) found that among children in korea with virus - caused diarrhea , adenoviruses occurred preferentially in the presence of rotavirus and that rotavirus and norovirus , although most common , occurred in polymicrobial infections in proportion to their numbers in the samples . furthermore , bilenko et al . ( 13 ) observed that among bedouins , g. lamblia was frequently found in polymicrobial infections and , when present with rotavirus , produced less severe diarrhea than rotavirus alone . ( 14 ) found that among young children in so paulo , those with rotaviral and bacterial co - infections were more likely to have severe diarrhea than were children infected with either pathogen alone . however , the study had insufficient power to examine differences among bacterial pathogens , as did the rest of the studies of rotavirus infections mentioned in a recent review ( 15 ) . the presence of multiple pathogens in one third of patients with diarrhea has potential implications for treatment and raises several questions . do cases of diarrhea caused by v. cholerae or rotavirus and a second pathogen differ from those caused by v. cholerae or rotavirus alone ? do the pathogens behave synergistically to escape immunologic detection ? because the cross - sectional nature of our study did not enable us to investigate the temporal sequence of pathogen infection , future research is needed to provide more evidence concerning the causal pathway(s ) . also , the clinical significance of our findings must be more rigorously evaluated by studies that include infected patients and controls . a more substantive investigation into how age and season might affect polymicrobial infections the results of our current study indicate that associations can occur between some pathogens affecting the human gastrointestinal tract . the observation of selective positive associations among some gastrointestinal pathogens raises the question of how they interact in vivo ; e.g. , is the critical factor a modification of gastrointestinal tract microflora ? understanding the association(s ) among various co - infecting pathogens may help direct the development of treatment strategies .

intrinsically disordered proteins ( idps ) represent a new frontier in structural biology since the primary characteristic of idps is that structures need to be characterized as diverse ensembles of conformational substates . we compare two general but very different ways of combining nmr spectroscopy with theoretical methods to derive structural ensembles for the disease idps amyloid- 140 and amyloid- 142 , which are associated with alzheimer s disease . we analyze the performance of de novo molecular dynamics and knowledge - based approaches for generating structural ensembles by assessing their ability to reproduce a range of nmr experimental observables . in addition to the comparison of computational methods , we also evaluate the relative value of different types of nmr data for refining or validating the idp structural ensembles for these important disease peptides . during systematic active surveillance of the causes of diarrhea in patients admitted to the infectious diseases and beliaghata general hospital in kolkata , india , we looked for 26 known gastrointestinal pathogens in fecal samples from 2,748 patients . samples from about one - third ( 29% ) of the patients contained multiple pathogens . polymicrobial infections frequently contained vibrio cholerae o1 and rotavirus . when these agents were present , some co - infecting agents were found significantly less often ( p = 105 to 1033 ) , some were detected significantly more often ( p = 105 to 1026 ) , and others were detected equally as often as when v. cholerae o1 or rotavirus was absent . when data were stratified by patient age and season , many nonrandom associations remained statistically significant . the causes and effects of these nonrandom associations remain unknown .

many neuroscientists have been calling for a system for the mouse brain where digital atlases serve as the framework used to traverse the brain and information linked to it ( baldock et al . , 2003 ; bjaalie , 2002 ; boline et al . , 2007 ; mackenzie - graham et al . , 2003 ; martone et al . , 2004 ; toga , 2002 ) . in contrast to data repositories , which allow simple access to data through a single interface , sophisticated digital atlases backed by the appropriate technology can act as a neuroinformatics hub facilitating access to different databases , information sources , and related documents and annotations . they may act as the scaffold in which otherwise unrelated data may be housed and correlated , providing an intuitive interface to share , visualize , analyze , and mine data of multiple modalities , scales , and dimensions . the semantic and spatial information tied to an atlas can add a dimension to data in a manner that exponentially increases its potential use and reusability . semantic linking of data to the atlas requires the data provider to register it with an ontology or controlled vocabulary , while spatial registration requires alignment of an image to the atlas . this information is then used to place data into the context of the atlas , allowing it to inherit information tied to the spatial coordinates of the atlas . spatial information in this type of slice data is key to interpreting results , yet these images lose anatomical context during the data collection process . thus atlas - based tools for organizing and analyzing this and related types of data may be used to create a system ideal for sharing data . several projects offer access to gene expression image data with differing levels of spatial mapping in the mouse nervous system . these projects have been comprehensively reviewed ( brumwell and curran , 2006 ; koester and insel , 2007 ; sunkin , 2006 ; sunkin and hohmann , 2007 ) and among others , include the allen brain atlas ( aba , www.brain-map.org ) , bgem ( www.stjudebgem.org ) , gensat ( www.gensat.org ) , genepaint ( www.genepaint.org ) , eurexpress ( www.eurexpress.org ) , mgi ( http://www.informatics.jax.org/ ) and emap / emage ( http://genex.hgu.mrc.ac.uk ) . as illustrated by the images from gensat and mgi in figure 1 , the ability to examine spatial and temporal expression patterns is crucial for developing correlations between genotype and phenotype as well as for interpreting and comparing findings across experiments . also illustrated , is that the anatomical information tends to be sparse in these data sources , as is also the case with bgem , genepaint , and eurexpress ( sunkin and hohmann , 2007 ) . aba and emap / emage differ from these sources in that in addition to linking the images to semantic information , they have also linked their images to spatial information by registering their images to a reference atlas ( baldock et al . , 2003 ; while this extra step can be both difficult and time consuming , it adds the potential for a great deal of analytical power and the ability to generate spatial queries ( carson et al . , 2005 ; christiansen et al . , 2006 ; leergaard and bjaalie , 2007 ) . examples of databases which manage gene expression image without an anatomical framework . gensat and mgi both provide a rich repository of image data for gene expression . neither uses a standard framework for data organization , rather , both describe the pattern and location of gene expression revealed by the image . gensat ( a ) describes the expression pattern for major structures in each image , while the mgi ( b ) summarizes a batch of assays ( the mgi dataset shown here were used to derive the lef1 gene examples shown in lee et al . , 2007 ) . while these annotations aid interpretation , the ability to query or analyze these data in this format is very limited . as most resources are built for sharing a specific set of data , most discussed above are not yet set up to easily link their data to that offered by other groups , adding a barrier to analyzing data across experiments . also , with the exception of emage , mechanisms are not readily available for an individual to easily put data into a semantic and spatial framework that facilitates comparison of their own data to others . for these reasons , it still requires a great deal of research and work to compare data collected in different experiments , which is one of our communities greatest desires , but also most difficult tasks . these are some of the recent drivers for the call to create interoperability across data resources and offering access to this system to any scientist , within the context of a digital atlasing framework . integrating these and other data sources via such a framework would allow a researcher to easily query across these resources . one could look for studies of different collection modalities , strains , developmental stages , or disease models , or examine the expression patterns of genes or regions of interest across multiple studies . for example , a scientist investigating a disease model of parkinson 's with the microarray technique finds that an unexpected gene in the caudate / putamen area seems to have a reverse correlation with motor deficits . wanting to know more , he uses this system to find data from other experiments that have examined the same disease model . he finds a mri dataset illustrating a change in the shape and decrease in the volume of that area in later stages of the disease , and a high - resolution confocal dataset from this same region shows abnormal cell morphology with disease progression . in addition , he finds that expression of this gene in this region in normal animals decreases with age and that it is expressed at a higher level throughout life in a different mouse strain which shows resistance to parkinson 's . compiling and analyzing data from these different experiments , many of which he did not even realize were applicable to his situation , allows him to more fully examine the potential role of this gene and to better inform his next experiments . while individual researchers often do a similar type of information gathering on their own , it can be difficult to examine other datasets , or we miss a relevant dataset because the data producers published it in relation to a very different topic . as diverse data generation continues to grow at an accelerated rate , we are in dire need of systems that make it easy for our community to contribute , organize , and find relevant data . while we are a long way from a fully implemented system that could perform the previous example , different groups have already created many of its components . for atlases to be used to query and access spatially indexed data , they must be backed by databases , spatial and semantic registration methods , and a client that allows query and access to the data . building a digital atlas mapping framework to facilitate experimentation and as a result , atlas resources besides those discussed above , developed with the purpose of placing mouse gene image data within a context of an atlas and offering it to the public . these include a joint project between the mouse brain library ( mbl , www.mbl.org/ ) and neuroterrain ( www.neuroterrain.org ) , the mouse atlas project ( www.loni.ucla.edu/map/ ) , and the smart atlas ( https://portal.nbirn.net ) . the mbl consists of high - resolution histological images and associated databases of brains from many genetically characterized strains of mice ( rosen et al . , 2003 ) . neuroterrain offers a high - resolution 3d nissl atlas in macrovoxel format that may be accessed over the network and viewed in an arbitrary plane ( gustafson et al . , 2004 ) and has much of the mbl data registered to it . the mouse atlas project developed tools to bring together brain architecture , gene expression , and 2d and 3d imaging information into a single interface ( mackenzie - graham et al . , 2003 ) and the smart atlas integrates data using both semantic and spatial geographical information systems ( gis ) methods ( martone et al . , 2008 ) . it is based on coronal and sagittal plates from the paxinos and franklin ( 2001 ) atlas and includes functionality that allows a user to register image slices to the atlas plates and allows spatial query of these data ( zaslavsky et al . , 2004 ) . to go a step farther and build a system where atlases act as a point of access to data from multiple sources of different type and scale requires more than just connection to more databases . it requires a large community effort , and it is now a focus of collaboratives including the mouse biomedical informatics research network ( birn , www.nbirn.net and http://www.loni.ucla.edu/birn/projects/mouse ; martone et al . , 2004 ) and the international neuroinformatics coordinating facility ( incf , www.incf.org ; boline et al . , 2007 ) . the birn project has been focused on building infrastructure supporting data sharing through a federated model and the focus of mouse birn has been on developing and applying this infrastructure to promote data sharing through an atlasing framework . as a result , the mouse birn atlasing toolkit ( mbat , www.nbirn.net/tools/mbat_2.0 and http://cms.loni.ucla.edu/mbat/ ) has been built to offer easy access to this infrastructure to the public . the mbat project builds on the complementary strengths and functionality of the mbl , neuroterrain , map , and smart atlas projects and is shaped by experimental mouse models of neurodegenerative disease studied in the mouse birn project . if a system such as this is to grow in a flexible and extensible manner ( figure 2 ) , it requires support by a large set of integration resources , such as standard terminologies and/or ontologies , database integration resources ( such as the birn mediator , www.nbirn.net/research/data_integration ) , standard schemas for data exchange ( such as mage , www.mged.org/workgroups/mage/mage.html , or xcede , www.xcede.org ) , database webservices , and standard application programming interfaces ( apis ) . finally , bringing together resources that are registered to different atlases requires additional spatial and semantic integration resources that can facilitate interoperability between these atlases ( such as an atlas interoperability server and api , www.loni.ucla.edu/twiki/bin/view/mousebirn/atlasstateexchange ) . these integration resources give an entryway for groups other than birn to participate in creating interoperable data and tools which can allow linkage to data they offer in other projects . ideally , multiple methods may be employed for interested collaborators to share their data through this framework , whether it is via their own database , or through upload processes . gray elements are specialized servers , databases , tools , and standards built by the scientific community that facilitate integration of data . yellow cylinders represent data sources that offer a certain data type ( i.e. , genenetwork and the birn microarray database are both microarray databases with services that allow access to their data ) . standardized application program interfaces ( apis - blue arrows ) allow the creation of tools that access services offered by a source . however , expanding even a flexible system with a new data type requires its own community - driven approach to modify these existing resources to set standards , web - services , apis , and application of their own unique combination of integration methods . for instance , the preferred approach discussed for gene expression images has been spatial registration , but so far , the main approach for integrating microarray data is primarily semantic , with the exception of voxelized data registered to an atlas ( chin et al . , 2007 ) . moreover , bridging the gap between different developmental stages ( i.e. , embryonic and adult ) as well as that between species will require at least an initial reliance on semantic mapping due to the huge variations in spatial differences between species and some developmental stages . from the user 's point of view , the framework allows a simple , but complete cycle of data sharing . a user may enter this path by registering their data within the client or via other server resources . she manages and visualizes her data in the client , where she can compare her data to an atlas and some other sets of data . however , she also has the option to share her data set by uploading it through resources that take advantage of an integration infrastructure and stores the data and its associated information in the appropriate databases . this upload process requires semantic and spatial integration methods ( gray elements ) to give the appropriate context to the data . once the data are uploaded , the integration infrastructure allows other users to query and retrieve her dataset through a client that accesses the infrastructure through apis . the current version of mbat ( 2.0 beta ) integrates data using both semantic and spatial methods . it points a user to accessible mouse birn upload interfaces and even includes tools for a user to create their own atlas . it allows spatial - based queries , but its current focus is to allow semantic queries and access to data from multiple distributed sites ( figure 2 , yellow cylinders and table 1 ) . currently mbat does not completely fulfill the complete atlas - based framework outlined in figure 2 . however , we continue to develop , and offer access to tools and resources that aid in moving toward this goal . we recently reported the use of a high - resolution atlas as an anatomical framework for localizing gene expression data for the mouse brain at postnatal day 0 ( p0 ) ( lee et al . , 2007 ) along with new tools that help register , manage , and analyze gene expression image data ( figure 3 ) . local atlas - based data management facilitates data correlation via several atlas tools developed in the birn project . based on the intersection of the polygon and the anatomical delineation provided by the atlas , the anatomical composition within the roi can be determined , thus enabling the retrieval of information associated with the atlas coordinates or anatomical concepts . once the user 's data is brought into the atlas space , the contours outlining the regions of interest can be mapped to the brain anatomy . ( 2007 ) employed a similar vector organization as the polygon method used for spatial query in mbat ( inset ) . bringing the data into a common standard space not only facilitates data visualization , but also allows one to use the atlas as an interface for reusing and correlating other data derived from the same location . this publication shows that a management document specifying the linear data - to - atlas spatial transformation is sufficient for relating local gene expression image data to a predefined atlas space . to setup the atlas - based data management and interaction with atlas - associated resources , it only requires an application that reports the atlas transformation in an offline management document . since mbat uses a coordinate system and integration resources to associate with multiple resources ( i.e. , paxinos ' canonical templates , microarray data ) , atlas - associated data relating to the region of interest ( roi , area with enriched gene expression ) may be retrieved by a spatial query . this registration also maps the roi to the brain anatomy , thus data correlation may also be examined using anatomical structure names . it is important to note that the methods discussed in this publication are not focused on pushing forward registration methods . as the field of image registration is problematic with no simple solutions for all data types , the management tools need not be dependent on a single registration method . instead , the creators of registration tools may be able to take advantage of these management methods in order to interact with this framework . groups with components of this framework already in place may wish to contribute to , and shape the direction and specifics of the infrastructure . others may wish to make available and link tools to this framework that facilitate registration , query , visualization , or analysis . ideally the system is built in a model that allows tool - builders to easily add to or create new tools for the framework . data rich resource groups may wish to develop services for the type of data they offer so their resources may also be easily accessed by this sharing infrastructure . individuals may also wish to help shape the direction and specifics of the developing infrastructure and tools . as they begin to use these resources to share their data with the rest of the scientific community , they may find gaps that can only be filled with their expertise . finally , since the complexity of such a system must be hidden from those using it , feedback from both the research and programming community is essential to creating usable interfaces and points of integration . in addition to these technical issues it is obvious that conceptual differences related to anatomies , terminologies , and ontologies will exist as long as there are neuroscientists . however , an advantage of this framework is the flexibility offered by the integrate layer . this can be shaped by the community so scientists can map their own well - defined atlases , terms , and ontologies into this system and for a user to choose any of these to define their own data . in fact , a system such as this would be an ideal platform for facilitating these types of discussions and debates . as we begin to close in on such a framework and add accessible resources , we will find that we have developed powerful mapping tools that help us find and analyze diverse information about the brain of different scale and type from different laboratories across the world . if we succeed in building such a powerful , intuitive , and flexible framework , we may help spread access to data and speed discovery as never before possible . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . oral angiokeratomas are most commonly found as a component of the generalized systemic disorder in fabry 's disease or fucosidosis , where multiple angiokeratomas can be found on the skin and the oral mucosa . we report a rare case of a patient with multiple oral and scrotal angiokeratomas in the absence of a systemic disorder . a 60-year - old man presented with a 2-year history of painless red raised lesions involving the tongue . he complained of burning sensation in the mouth in the region of the papules . clinical examination revealed multiple erythematous to slightly bluish , shiny , vascular papules ranging from 1to3 mm size , discrete to grouped on the undersurface and lateral aspects of the tongue [ figure 1 a and b ] . biopsy from a vascular papule on the tongue showed acanthosis , and papillomatosis , with large , dilated spaces lined by normal - appearing endothelium which were filled with erythrocytes and organizing thrombi [ figure 1d ] . the patient was counseled about the benign nature of the disease after which he chose not to take any treatment . ( a - b ) multiple erythematous to bluish , shiny , vascular papules on the undersurface and lateral aspects of the tongue ; ( c ) similar vascular papules on the scrotum ; ( d ) acanthosis , and papillomatosis , with large , dilated spaces lined by normal - appearing endothelium which are filled with erythrocytes and organizing thrombi ( h and e , 200 ) angiokeratoma is an asymptomatic cutaneous vascular disorder of the papillary dermis characterized by dermal vasculsar ectasia with overlying hyperkeratosis of the epidermis . it is thought to be a telangiectatic lesion arising from local injury to papillary dermal capillaries , either from trauma or venous hypertension . they are ( 1 ) the generalized systemic type : angiokeratoma corporis diffusum of fabry characterized by multiple tiny red papules in a symmetrical distribution in the bathing area ; ( 2 ) angiokeratoma of mibelli , the bilateral form occurring on the dorsal areas of the fingers and toes ; ( 3 ) angiokeratoma of fordyce , the most common angiokeratoma which occurs on the scrotum ; ( 4 ) the solitary papular angiokeratoma which has a predilection for the legs ; and ( 5 ) angiokeratoma circumscriptum , the multiple papular and plaque- like form . oral mucosal angiokeratomas have also been associated , uncommonly , with angiokeratomas of the scrotum , gastrointestinal mucosa ( jejunum ) , or both . recently ranjan & mahajan have proposed a clinical classification for oral angiokeratomas , depending on the number of lesions , associated other muco - cutaneous involvement and systemic disease . oral angiokeratomas present as single / multiple , erythematous , shiny papules , which may be studded with keratotic tops . they are firm on palpation , nontender , show telangiectatic vessels on diascopy , and bleed occasionally . the tongue , with a predilection for the dorsal aspect , appears to be the most common site for isolated oral angiokeratoma . they may be mistaken for hemangiomas , lymph - hemangiomas , focal epithelial hyperplasia , or nevi . the histologic features are similar to their cutaneous counterparts ; hyperkeratosis , acanthosis and dilated capillaries in the papillary dermis , partly or completely enclosed by the papillomatous epidermis . organized or organizing thrombi are occasionally observed within the dilated capillaries . the only difference being the presence of parakeratosis in oral angiokeratomas . however , in fabry 's disease and fucosidosis , swollen and vacuolated ( lipid containing ) endothelial cells are characteristic differentiating features .

digital brain atlases are useful as references , analytical tools , and as a data integration framework . as a result , they and their supporting tools are being recognized as potentially useful resources in the movement toward data sharing . several projects are connecting infrastructure to these tools which facilitate sharing , managing , and retrieving data of different types , scale , and even location . with these in place , we have the ability to combine , analyze , and interpret these data in a manner not previously possible , opening the door to examine issues in new and exciting ways , and potentially leading to speedier discovery of answers as well as new questions about the brain . here we discuss recent efforts in the use of digital mouse atlases for data sharing . we report a 60-year - old man who presented with a 2-year history of painless red raised lesions involving the tongue and scrotum . histopathology was suggestive of angiokeratoma . oral angiokeratomas are most commonly found as a component of the generalized systemic disorder in fabry 's disease or fucosidosis . our patient had isolated mucosal angiokeratomas which is very rare .

there are approximately 287,000 preventable maternal deaths annually , of which 99% occur in developing countries . as a sentinel event , maternal death is also a prime indicator in evaluating the quality of a nation 's health care delivery systems [ 2 , 3 ] . mothers are pivotal to the social , economic , and cultural development of a community ; maintaining their health elevates the physical , psychological , and social well - being of their children and families and , by extension , society as a whole . for this reason , improving maternal health has been proposed by the world health organization ( who ) as one of their eight millennium development goals ( mdg ) . however , in countries with few maternal deaths , this approach fails to provide comprehensive information , leaving policy makers to react based on current rather than past statistics . to facilitate the development of precautionary measures and safer environments that minimize maternal deaths , it is essential that near miss data are recorded and analyzed . the who defines an individual having experienced severe acute maternal morbidity ( samm ) ( i.e. , near miss ) as a woman who nearly died but survived a complication that occurred during pregnancy , childbirth or within 42 days of termination of pregnancy . in fact , maternal near miss includes those cases in which a woman nearly died but survived during pregnancy or during 42 days after the delivery . using near miss data in maternal death prevention planning first , because the number of near miss cases exceeds maternal death cases , near miss is a better predicate for preventive planning . second , because the mother survives a near miss , she can provide valuable details on what she experienced . lastly , because near miss is one step removed from death , obtaining any information about the event could prove useful in preventing maternal death [ 810 ] . the prevalence of maternal near miss varies among different countries based on health care quality and availability . nevertheless , in a systematic review using disease - specific criteria , near miss rates have been reported to be between 0.6% and 14.98% . from a global perspective , iran has been notably successful in reducing maternal mortality . between 1990 and 2008 , iran has managed to decrease its maternal mortality rate by 80% , which , at present , translates to about 25 deaths in 100,000 . the availability of emergency obstetrical care , improvements in women 's education , and the expansion of family planning services have all contributed to the decrease in maternal mortality . in iran , a national maternal mortality surveillance system examines maternal death cases by reviewing files and interviewing key parties to determine the cause of death . although some studies have been carried out in iran and the middle east regarding the causes of maternal death and risk factors of pregnancy [ 1217 ] , the authors know of no published study on severe maternal morbidities and near miss occurrences from iran or other middle eastern countries . the present study aimed to establish a profile of severe maternal morbidities in iran and their relationship with other underlying factors this prospective study encompassed eight hospitals with maternity facilities located in the two large cities of kerman and jiroft in southeast iran . the study was performed in 2013 for a period of nine months . the study protocol was approved by the ethical committee of kerman university of medical sciences ( e.c./90/518 ) . after explaining the study 's nature and aims , oral consent was obtained from the participants who all were ensured that their information would remain confidential . first , a list of maternal near miss conditions was prepared based on say et al . 's recommendations for prospective surveillance of maternal near miss cases . this list included four major groups of haemorrhagic disorders , hypertensive disorders , severe management indicators , and other systemic disorders . moreover , a category including other conditions was also considered so as not to miss any other life - threatening illness . all women admitted during the nine - month study period for delivery or completion of pregnancy as well as women admitted within 42 days after the termination of pregnancy were enrolled into the study . after the participating hospitals ' maternity , labour , general icu , emergency , and admission departments were fully coordinated , our case survey was implemented . a check list was completed for cases with potentially life - threatening conditions by a team consisting of a midwife and gynaecologist . the first part included demographic and clinical data such as age , educational level , place of residency , gravidity , parity , type of delivery , and gestational age . next , a woman admitted to the delivery room at the same hospital was randomly selected from the list of patients as the control , and the first part of the check list was completed for her . in all stages , an experienced expert supervised completion of the check lists . according to the who definition maternal near miss ratio was defined as the number of maternal near - miss cases per 1000 live births . chi - square and independent t - tests were used to compare the qualitative and quantitative variables between near miss and control groups . a stepwise logistic regression model was used to determine the relationship between underlying variables and near miss ratio . the logistic regression model 's goodness of fit was evaluated by the hosmer - lemeshow test . during the study period , there were 501 cases of near miss in 19,908 live births ( a near miss ratio of 25.2 per 1000 live births ) . the highest near miss ratio ( 104.8 in 1000 ) was observed in the referral ( educational ) hospital ( table 1 ) . the mean age of near miss cases was 28.3 6.1 years versus the control group 's 26.0 5.8 years ( p < 0.001 ) . university degrees were seen more among near miss women ( table 2 ) . in the near miss group , 208 women ( 41.5% ) were primiparous , whereas , in the control group , the number was 225 ( 45.2% ; p = 0.243 ) . the frequency of abortion in the near miss and control groups was 18.6% and 1.6% , respectively ( p < 0.001 ) . the frequency of caesarean section in the near miss and control groups was 24.7% and 54.2% , respectively ( p < 0.001 ) . in our study group , there were two cases of maternal death . one was a 19-year - old woman diagnosed with intracerebral haemorrhage ( ich ) ; the other was a 28-year - old woman who had undergone curettage due to a failed abortion and died from sepsis because of perforations in the uterus and intestine . the rate of maternal death in near miss cases was 0.40% with a case : fatality ratio of 250 : 1 . as shown in table 3 , the most prevalent causes of near miss were severe preeclampsia ( 27.3% ) , ectopic pregnancy ( 18.4% ) , and abruptio placentae ( 16.2% ) . in all , 15.2% had at least one systemic disease , and 43 women in the near miss group were hospitalised in the icu ( table 3 ) . the majority of the near miss cases were observed in the haemorrhagic disorders group ( table 3 ) . logistic regression analysis showed that four variables had significant relationship with near miss occurrence ( table 4 ) . of the 377 near miss cases ( 75.2% ) , the major problem was discerned at or during the first six hours of admission . in the present study , the near miss ratio was 25.2 per 1000 live births . in the private setting , this rate was 7.5 per 1000 , and , in the tertiary care hospital , it was approximately 105 per 1000 . the main advantages of this study were its prospective nature and its use of standard criteria for determining near miss cases . because more than 97% of deliveries in iran occur in hospitals , the present report may be considered as a population - based study . because of the differences among patients and health care delivery systems , generalizing the results of this study on a countrywide basis should be done with caution . the literature shows that maternal near miss ratios vary greatly depending on the population studied , how near miss is defined , and how the study is conducted ( prospective versus retrospective ) [ 11 , 19 ] . near miss ratios have been reported as 44.3 per 1000 in brazil , 33 per 1000 in india , 3.83 per 1000 in scotland , and 34 per 1000 in a who survey . a recent systematic review using a unique definition for near miss showed samm rates in high - income countries to be significantly lower compared with those of low- and middle - income countries . in this study , like some other studies [ 19 , 20 ] , the rate of near miss was significantly higher in the tertiary care setting ( table 1 ) . the reason might be that , because of the limited facilities at private hospitals , women with complicated pregnancies are not usually referred to these centres . in the present study , we found a higher rate of near miss and consequently a lower fatality rate ( 0.4% ) . these findings may be derived from our broader definition for near miss events , which combined disease - specific criteria with management - based criteria . in netherland , with a near miss ratio of 7.1 per 1000 , the rate was 1.9% . in brazil , with a near miss ratio of 42 per 1000 , the rate was 1.6% . it should be mentioned that the maternal death rate shows a decreasing trend in iran . in our study , haemorrhagic disorders ( 46.1% ) and hypertensive disorders ( 31.9% ) these rates are similar to those reported from scotland and indonesia . in india , the two most common causes of near miss have been preeclampsia and haemorrhage . in a study on 64 cases of maternal death in kerman , iran , the same two factors were the most prevalent causes of maternal mortality . in our study , 43 cases required intensive care ; that is , for every 1000 live births , 2.2 mothers are hospitalized in an icu , which is nearly similar to the icu admission rate in netherland . according to our logistic regression model , four variables had a relationship with near miss ( table 4 ) . in older women with university degrees , in fact , most studies demonstrate a proportional relationship between higher near miss ratio and advancing age ( particularly over 35 years ) [ 22 , 23 ] . in the who 's global survey , this finding has been attributed to the tendency among women with higher educational levels to undergo caesarean section , which increases the probability of near miss events . the present study , as in previous studies , shows that being a primipara increases the probability of near miss ratio by 1.2%1.4% [ 22 , 23 ] . although pregnancy complications are to a great extent unpredictable and unpreventable , early awareness of near miss cases can prevent the progress of disease and maternal death . in this study , the feasibility of using who - recommended near miss criteria was recognized . however , because of inadequate health care services , it is necessary that the auditing of near miss cases be considered as important as the implementation of near miss surveillance systems [ 15 , 23 ] . the present study showed that using data related to near miss cases can provide more comprehensive information when reviewing maternal death cases ; therefore , establishing near miss surveillance systems in iranian hospitals is highly recommended . patients show increasing declines in cognition , and behavioral symptoms occur in all stages of ad . while all ad patients can present with depression , agitation , and aggressive behaviors , behavioral difficulties are most pronounced in the advanced stages of the disease.13 rivastigmine , inhibiting both acetylcholinesterase and butyrylcholinesterase , is the first transdermal treatment available for ad patients , and has been widely approved for the symptomatic treatment of ad.4,5 by providing continuous delivery of medication from the skin into the bloodstream , these patches achieve sustained , high drug concentration in the plasma over 24 hours.6 however , the investigation of transdermal exelon in alzheimer s disease ( ideal ) study revealed that ad patients receiving a 10 cm patch frequently suffered from nausea ( 7.2% ) and vomiting ( 6.2%).4 further , japanese ad patients receiving an 10 cm patch also showed similar adverse events , including nausea ( 7.0% ) and vomiting ( 8.0%).5 ad patients have been reported to stop the medication because of these adverse effects ; however , no technique is currently available to predict the occurrence of these adverse effects . the antiemetic domperidone is often administered in order to administer higher , more effective dosages of cholinesterase inhibitors ( cheis ) , and such a combination with domperidone has been shown to be effective in preventing rivastigmine - related gastrointestinal disturbances when administered orally in the rivastigmine capsule form.7 in the present study , we examined the occurrence of gastrointestinal disturbances related to the use of the rivastigmine patch using the frequency scale for the symptoms of gastroesophageal reflux disease ( fssg),8 bristol stool form scale,9 laboratory data , body weight , and amount of food intake . in addition , we compared the differences in gastrointestinal disturbances when using a rivastigmine patch with domperidone ( rwd ) and without domperidone ( rwod ) in order to examine the efficacy of domperidone orally . patients were recruited from february 2012 to january 2013 at the department of neurology , toho university omori medical center , tokyo , japan . subjects included outpatients diagnosed with ad who lived with or had regular daily visits from a responsible caregiver . the diagnosis of ad was based on the criteria from the national institute of neurological disorders and stroke and the alzheimer s association.10 the criteria have been established by clinical and neuropsychological examination . cognitive impairments have to be progressive and be present in two or more areas of cognition . the onset of the deficits has been between the ages of 40 and 90 years , and finally there must be an absence of other diseases capable of producing a dementia syndrome . a total of 30 patients with mild - to - moderate ad ( scores 1022 on the mini - mental state examination [ mmse]11 ) were enrolled and randomly allocated to two groups by the envelope method , ie , rwd and rwod ( n = 15 each ) . dose titrations of rivastigmine patches were performed every 4 weeks using 4.5 mg / day increments from 4.5 mg / day to 18 mg / day . domperidone was administered orally at a dose of 30 mg / day in the rwd group . patients received rivastigmine - patch treatment for 20 weeks , and were evaluated for gastrointestinal disturbances at baseline and at weeks 4 , 8 , 12 , 16 , and 20 . the fssg is a simplified questionnaire for evaluation of the symptoms of gastroesophageal reflux disease ( gerd ) , and has been demonstrated to be a useful tool for objectively evaluating symptoms in gerd patients.8 the questionnaire consists of twelve items that are scored as 04 based on the frequency of the symptom ( 0 = never , 1 = occasionally , 2 = sometimes , 3 = often , and 4 = always ) with total scores ranging from 0 to 48 . ( 2 ) does your stomach get bloated ? ( 3 ) does your stomach ever feel heavy after meals ? ( 4 ) do you sometimes subconsciously rub your chest with your hand ? ( 5 ) do you ever feel sick after meals ? ( 6 ) do you get heartburn after meals ? ( 7 ) ( 8) do you feel full while eating meals ? ( 9 ) do some things get stuck when you swallow ? ( 10 ) do you get bitter liquid ( acid ) coming up into your throat ? ( 11 ) do you burp a lot ? the bristol stool form scale consists of seven types ( 1 , separate hard lumps , like nuts ; 2 , sausage - shaped but lumpy ; 3 , like a sausage or snake , but with cracks on its surface ; 4 , like a sausage or snake , smooth and soft ; 5 , soft blobs with clear - cut edges ; 6 , fluffy pieces with ragged edges , a mushy stool ; and 7 , watery , no solid pieces ) . the fssg and bristol stool form scale scores were obtained not only from patients but also from their caregivers to obtain accurate information . if the scores differed between the patient and his / her caregiver , we usually took the caregiver s score , because of the patient s cognitive impairment . in addition , we evaluated the changes in laboratory data ( hemoglobin [ hb ] , albumin [ alb ] , and total cholesterol [ t - chol ] ) , body weight , and amount of food intake to evaluate the nutritional status of ad patients . the amount of food intake per day was set as a score of 10 at baseline , and any improvement or loss in appetite was reflected as increased or decreased scores , respectively , based on patient and caregiver feedback . statistical analysis was performed by statmate ( graph - pad software , la jolla , ca , usa ) , with the mann whitney u test and wilcoxon signed - rank test , with p < 0.05 used as the threshold for statistical significance . all patients and legally accepted caregivers participating in the trial were given an explanation of the trial by the investigators before the start of study , and informed consent to participate in the study was obtained . consent of patients was obtained orally or in writing , and the consent of legally accepted representatives was always obtained in writing . overall , three patients from the rwd group discontinued their participation due to skin irritation , and one patient from the rwod group withdrew because of nausea . no differences were observed between the rwd and rwod groups with respect to mean age , the sex ratio , mmse score , or body weight . the fssg scores for the rwod group showed significant increases after 12 weeks compared with baseline ( versus 12 weeks , p = 0.035 ; versus 16 weeks , p = 0.034 ; versus 20 weeks , p = 0.034 ) ; however , this was not observed in the rwd group ( figure 1 ) . before initiating rivastigmine - patch therapy , 23 of 30 patients ( rwd group , eleven of 15 ; rowd group , twelve of 15 patients ) showed the presence of some gastrointestinal symptoms based on the fssg questionnaire . the median value of the fssg at baseline was 3.8 in the rwd group and 3.1 in the rwod group ; therefore , we set the high - score threshold 4 points and low - score threshold 3 points . in the rwod group , the high - score subgroup showed increased fssg scores after 12 weeks compared with baseline scores ( versus 12 weeks , p = 0.038 ; versus 16 weeks , p = 0.035 ; versus 20 weeks , p = 0.034 ) ; however , no such changes were observed in the high - score subgroup in the rwd group ( figure 2 ) . in both rwd and rwod groups , the low - score subgroups showed no changes in fssg scores during the dose - escalation phase . the data for mmse , laboratory results ( hb , alb , and t - chol ) , body weight , amount of food intake , and bristol stool form scale scores are shown in table 2 ; no differences were noted between the baseline values and those at week 20 for either group . we compared the data for the rwd and rwod groups for all the parameters examined . no significant differences were observed between the two groups for the fssg ( figure 1 ) , mmse , laboratory data , body weight , amount of food intake , or bristol stool form scale scores ( table 2 ) . overall , three patients from the rwd group discontinued their participation due to skin irritation , and one patient from the rwod group withdrew because of nausea . no differences were observed between the rwd and rwod groups with respect to mean age , the sex ratio , mmse score , or body weight . the fssg scores for the rwod group showed significant increases after 12 weeks compared with baseline ( versus 12 weeks , p = 0.035 ; versus 16 weeks , p = 0.034 ; versus 20 weeks , p = 0.034 ) ; however , this was not observed in the rwd group ( figure 1 ) . before initiating rivastigmine - patch therapy , 23 of 30 patients ( rwd group , eleven of 15 ; rowd group , twelve of 15 patients ) showed the presence of some gastrointestinal symptoms based on the fssg questionnaire . the median value of the fssg at baseline was 3.8 in the rwd group and 3.1 in the rwod group ; therefore , we set the high - score threshold 4 points and low - score threshold 3 points . in the rwod group , the high - score subgroup showed increased fssg scores after 12 weeks compared with baseline scores ( versus 12 weeks , p = 0.038 ; versus 16 weeks , p = 0.035 ; versus 20 weeks , p = 0.034 ) ; however , no such changes were observed in the high - score subgroup in the rwd group ( figure 2 ) . in both rwd and rwod groups , the low - score subgroups showed no changes in fssg scores during the dose - escalation phase . the data for mmse , laboratory results ( hb , alb , and t - chol ) , body weight , amount of food intake , and bristol stool form scale scores are shown in table 2 ; no differences were noted between the baseline values and those at week 20 for either group . we compared the data for the rwd and rwod groups for all the parameters examined . no significant differences were observed between the two groups for the fssg ( figure 1 ) , mmse , laboratory data , body weight , amount of food intake , or bristol stool form scale scores ( table 2 ) . vomiting is a highly specific physical event , defined as the rapid , forceful evacuation of gastric contents in retrograde fashion from the stomach up to and out of the mouth . in contrast , nausea is an entirely subjective experience , defined as the sensation that immediately precedes vomiting , wherein patients feel that they are about to vomit . rivastigmine patch - related gastrointestinal disturbances have been reported in less than 10% of patients receiving this therapy.4,5 in our study , only one patient from the rwod group discontinued the therapy due to nausea . however , our fssg questionnaire findings revealed that 23 of 30 ad patients ( > 75% ) suffered from at least one gastrointestinal disturbance before the initiation of the rivastigmine patch . furthermore , ad patients with higher fssg were observed to show a further increase in the fssg score during the dose - escalation phase . our results may indicate that ad patients do not report gastrointestinal disturbances because of the memory deficits involved in ad . domperidone , a dopamine antagonist that acts by blocking dopamine receptors in the chemoreceptor trigger zone , has been previously shown to prevent gastrointestinal disturbances in rivastigmine therapy administered orally.7 in the present study , domperidone was effective in reducing or preventing gastrointestinal disturbances during the dose - escalation phase of rivastigmine - patch therapy in ad patients with higher fssg scores at baseline . the fssg is a convenient tool for evaluating the symptoms of gerd ; in our experience , patients / caregivers require approximately 1 minute to complete the questionnaire . the questionnaire consists of twelve detailed items ; therefore , the slightest gastrointestinal problem can be easily detected . in order to obtain accurate information in the present study , we confirmed the symptoms described by the ad patients using fssg questionnaires completed by their caregivers . at present , there is no technique for predicting the occurrence of gastrointestinal disturbances with rivastigmine - patch therapy . it has been suggested that pretreatment with domperidone may protect against the onset of centrally induced nausea and vomiting induced by cheis.12 however , our findings indicate that ad patients with low fssg scores at baseline may not require additional antiemetic and/or antiulcer medications . on the other hand , ad patients with initial high fssg scores showed elevations in the fssg scores during the dose - escalation phase , indicating an increased frequency of gastrointestinal disturbances . therefore , clinicians should pay careful attention to ad patients with high fssg scores at baseline during the dose - escalation phase to prevent gastrointestinal disturbances and to avoid patient discontinuation of rivastigmine - patch therapy . domperidone has , on rare occasions , been associated with acute extrapyramidal syndromes;13 however , our ad patients did not show such side effects . randomized , double - blind clinical trials conducted with other cheis , such as donepezil and galantamine , have provided evidence that these are safe and effective therapies against cognitive dysfunction and behavioral symptoms.1416 however , similar to the side effects of rivastigmine - patch therapy , gastrointestinal disturbances are the primary adverse effects associated with this therapy . we speculate that the fssg may be a useful tool for predicting and preventing chei - related gastrointestinal disturbances for these drugs . this study did not include a placebo for domperidone , and was limited by the small sample size ; therefore , improvements in cognitive dysfunction with rivastigmine - patch therapy could not be demonstrated fully . it is important to optimize the drug for ad in individuals to slow the rate of cognitive dysfunction and behavioral and psychological symptoms of dementia.35,1720 of note , behavior symptoms in patients with ad increase direct costs of care . for example , a 1-point increase in the neuropsychiatric inventory score21 was calculated to be equivalent to an increased cost of $ 247$409 in total annual direct costs.22 in addition , compared with those who never used cognitive enhancers , patients who used cheis had a significant delay in nursing home admission.23 in summary , the group receiving rivastigmine - patch therapy without domperidone showed significant increases in the fssg score after 12 weeks ( dose - escalation phase ) compared with baseline scores . domperidone was effective in preventing rivastigmine patch - related gastrointestinal disturbances in ad patients with higher fssg scores at baseline . further , the fssg questionnaire may be a useful tool for the prediction of gastrointestinal symptoms associated with rivastigminepatch therapy .

this prospective study aimed to estimate the incidence and associated factors of severe maternal morbidity in southeast iran . during a 9-month period in 2013 , all women referring to eight hospitals for termination of pregnancy as well as women admitted during 42 days after the termination of pregnancy were enrolled into the study . maternal near miss conditions were defined based on say et al . 's recommendations . five hundred and one cases of maternal near miss and 19,908 live births occurred in the study period , yielding a maternal near miss ratio of 25.2 per 1000 live births . this rate was 7.5 and 105 per 1000 in private and tertiary care settings , respectively . the rate of maternal death in near miss cases was 0.40% with a case : fatality ratio of 250 : 1 . the most prevalent causes of near miss were severe preeclampsia ( 27.3% ) , ectopic pregnancy ( 18.4% ) , and abruptio placentae ( 16.2% ) . higher age , higher education , and being primiparous were associated with a higher risk of near miss . considering the high rate of maternal near miss in referral hospitals , maternal near miss surveillance system should be set up in these hospitals to identify cases of severe maternal morbidity as soon as possible . objectivewhile acetylcholinesterase inhibitors , such as donepezil , galantamine , and rivastig - mine , are beneficial in treating behavioral symptoms of patients with alzheimer s disease ( ad ) , their dose - limiting effects include gastrointestinal disturbances , such as nausea , vomiting , and diarrhea . we aimed to predict the occurrence of these gastrointestinal disturbances with rivastigmine therapy for optimal drug choice and improved compliance.materials and methodsthirty patients with mild - to - moderate ad ( scores 1022 on the minimental state examination ) were administered a rivastigmine 18 mg patch with domperidone 30 mg ( rwd ) and without domperidone ( rwod ; n = 15 each ) for 20 weeks . gastrointestinal disturbances were evaluated using a frequency scale for symptoms of gastroesophageal reflux disease ( fssg ) , bristol stool form scale , laboratory data ( hemoglobin , albumin , total cholesterol ) , body weight , and amount of food intake.resultsafter 12 weeks , fssg scores were higher in the rwod group compared to baseline scores ; however , no significant differences were noted between the rwd and rwod groups . we then subdivided each group based on high and low baseline scores ; the rwod high - score ( 4 ) subgroup showed increased fssg after 12 weeks compared with the baseline score . in both rwd and rwod groups , the low - score ( 3 ) subgroups showed no changes during the dose - escalation phase.conclusionfor ad patients with higher fssg scores at baseline , domperidone was effective in preventing rivastigmine - related gastrointestinal disturbances .

low aqueous solubility of drug has always presented major obstacle towards the development of drug delivery systems which often compromises patient compliance . oral route is thought to be common and easy for drug administration . on oral administration of drug in its dosage form , it is expected to dissolve and release the drug into the gastrointestinal fluid before the absorption . poor solubility may limit the dissolution of drug in gastrointestinal tract resulting to low bioavailability that can pharmacologically affect the therapeutic efficacy of drug . the drugs belonging to bcs class ii and iv particularly fall in this category and have been extensively researched molecular optimization and development of novel efficacious dosage forms . cinnarizine , ( e)-1-(diphenylmethyl)-4-(3-phenylprop-2-enyl ) piperazine ( figure 1 ) , molecular formula : c26h28n2 and molecular weight : 368.51 g / mol , is white or almost white powder . originally obtained from woodreed roots ( cinna ) , cinnarizine was first synthesized by janssen pharmaceutica in 1955 and marketed in 1958 under the brand name stugeron . according to a paper the solubility of cinnarizine is highly ph dependent , that is , 0.29 mg / ml at ph 2 , 0.017 mg / ml at ph 5 , and 0.002 mg / ml at high ph of 6.5 ( all the results were at 37c ) . pharmaceutically it belongs to bcs class ii , as displays preferential absorption in stomach ( table 1 ) . after oral administration , cinnarizine is rapidly absorbed ( tmax = 24 h ) from gastrointestinal tract , with an absorption window in upper gastrointestinal tract . on repetitive dosing in healthy human , accumulation of cinnarizine occurs due to its pharmacokinetic properties . the drug can cross the blood - brain barrier by simple diffusion , due to weakly basic and lipophilic . cinnarizine is oxidized by cytochrome p450 into metabolites such as 1-(diphenylmethyl)piperazine(c1 ) , 1(diphenylmethyl)-4-[3-(4-hydroxyphenyl)-2-propenyl ] piperazine ( c2 ) , and benzophenone(c3 ) 1-[(4-hydroxyphenyl)-phenylmethyl]4-(3-phenyl-2-propenyl)piperazine(c4 ) . among the various cyp enzymes , only cyp2d6 can catalyze p - hydroxylation of the cinnamyl phenyl ring of cinnarizine ( c-2 formation ) and only cyp2b6 can catalyze p - hydroxylation of the diphenylmethyl group ( c-4 formation ) of cinnarizine . on the other hand , cyp2c9 with cyp1a1 , -1a2 , and/or -2a6 have affinity for n - desalkylation at the 1- and 4-positions of the piperazine ring that results in c-1 and c-3 formation . it has high protein binding capacity of 91% . the drug is predominantly excreted in urine as metabolites and in feces mainly as unchanged drug with elimination half - life of 36 h. the pharmacokinetic aspects are also compiled in table 1 . these agents do not show action on slow calcium channels in myocardium but have selective action on arteries . the inhibitory effects of cinnarizine on the noradrenaline and ca - induced contraction of the rabbit thoracic aorta and mesenteric arteries were compared to papaverine . cinnarizine was found to block the ca - evoked contraction of the depolarized vessels but was less effective against the noradrenaline - induced contraction of the mesenteric arteries and even failed to antagonize the response of the thoracic aorta to noradrenaline . cinnarizine was effective in mesenteric arteries exposed to noradrenaline by blocking the ca0 dependent response , while papaverine was less effective towards the ca0 dependent response but efficiently inhibited the initial fast response more . cinnarizine was less effective or even failed to inhibit the response produced by thoracic aorta to nonadrenaline . these results suggested reduction of membrane permeability to extracellular calcium was the mechanism by which cinnarizine acted . the report also suggested that papaverine inhibits the contraction to less extent than that of cinnarizine . so have wide application in treatment of cerebral apoplexy , cerebral arteriosclerosis , and posttraumatic cerebral symptoms . according to a recent paper , the effect against vestibular vertigo was due to inhibiting potassium ( k ) currents , activated by heightened hydrostatic pressure on the hair cells , instead of blocking calcium channels . reports on inhibitory action on 5 h serotonin uptake by platelets are also available . its moderate antagonistic properties at dopamine d2 receptors are known to resemble the mechanism of action of most antipsychotic drugs . the active metabolite of cinnarizine , [ 1-(diphenylmethyl)-4-[3-(4-hydroxyphenyl)-2-propenyl ] piperazine , has higher affinity for d2-receptors than the parent drug that indicates significant role in inducing parkinsonism as adverse effect on chronic medication . additionally the drug was suggested to be used in cosmeceuticals as skin whitening agents for treatment of skin hyperpigmentation . clinically , cinnarizine is used for treatment of vertigo and prevention of motion sickness , due to its calcium channel blocking activity . vertigo indicates a sensation of false movement ( generally described like rotation ) and vestibular vertigo may be induced by increase in the endolymphatic pressure that activates pressure - dependent k currents ( ik , p ) in vestibular hair cells ; this modulates transmitter release . cinnarizine acts by enhancing the transmitter release in vestibular type ii hair cells by inhibition of ( ik , p ) , thus enhancing depolarization and increasing the voltage - dependent activation of ca currents . the therapeutics of cinnarizine has been proved employing clinical trials and the reports are compiled in table 2 . additionally , cinnarizine has evoked clinical interest for other disorders . consequently , recently summarized reports suggest clinical application of drug for migraine prophylaxis . on the other hand , cinnarizine is known to aggravate parkinson 's disease . when used in average dose of 150 mg daily , it can aggravate motor function in patients with parkinson 's disease ; however , the effect was reversible and disappeared upon withdrawal of drug . commercial cinnarizine is available as tablets and capsules usually administered 2 - 3 times a day . high dose should be used with caution in patients with hypotension , owing to the possibility of further decrease in blood pressure , and is contraindicated in pregnancy due to teratogenic and embryocidal effects on the foetus . the drug can only be prescribed if the potential benefit justifies the potential risk to the foetus . cinnarizine is widely marketed as combination medication with other drugs . in the indian domestic pharmaceutical market , its combination with domperidone is predominant , whereas combination with other drugs such as dimenhydrinate , dihydroergocristine , chlorcyclizine , phenylpropanolamine , and piracetam are available with overseas pharmaceutical companies . india leads in terms of commercialization of different brands of cinnarizine comprising approximately more than 50 brands including both drug in alone as well as in combination with other drugs . among all brands stugeron it has been accounted in research that bioavailability of cinnarizine is remarkably influenced by gastric acidity , displaying its ph dependent solubility and in turn dissolution . the marketed dosage forms account for low and erratic bioavailability , mainly due to the above - mentioned pharmacokinetic reasons . also , high dosing frequency is needed because of its short elimination half - life . being a weak base it is very soluble in acidic solutions and clinical absorption of cinnarizine was found to depend on the gastric acidity of patients . cinnarizine showed rapid dissolution in individuals having high gastric acid content ( ph = 1.2 ) , providing good absorption characteristics , whereas , in case of low - gastric acid content ( ph = 6 ) , reduction was observed in cmax and auc approximately 75 to 85% . literature retrieval on enhancement in solubility and bioavailability of cinnarizine highlighted various effective approaches since 1984 till date . in 1984 , tokumura et al . , in his report , claimed 30 times improved dissolution for inclusion complex of cinnarizine with -cyclodextrin than the drug alone , whereas no difference in bioavailability was observed . further working with the cinnarizine -cyclodextrin complex , tokumura with other research devised a method for improvement of bioavailability on simultaneous administration of proposed -cyclodextrin with competing agent . it was found that the penetration rate constant of cinnarizine in complex decreased which was restored on addition of competing agents . these agents compete with -cyclodextrin in complex formation , thus keeping more available free drug to penetrate the lipid barrier . in the same content , two papers were projected . one was based on the investigation of the competing ability of dl - phenylalanine to improve the bioavailability of cinnarizine . on simultaneous administration of dl - phenylalanine with cinnarizine -cyclodextrin complex , it was concluded that a minimum effective dose of 1 g of dl - phenylalanine might be required for every 50 mg of cinnarizine in -cyclodextrin complex . on the similar lines , the ability of l - leucine and l - isoleucine was also investigated for competing effect in gastrointestinal tract . as a result the cmax was increased to 2.7 and 1.9 times as compared to that of drug alone and its -cyclodextrin , respectively . conclusively , l - isoleucine showed stronger competing effect than l - leucine coupled with significantly improved bioavailability of the drug . in the next year , in 1987 , enhancement of bioavailability of cinnarizine as its oleic acid solution in hard capsule for oral administration was reported . use of lipid vehicle for oral administration of drug was ascertained to improve the bioavailability when compared to cinnarizine tablet . when investigated in beagle dogs , the cmax and auc for oleic acid solution of cinnarizine were remarkably improved by 2.9 and 4 times than that of tablet , respectively . the results were encouraging and somewhat similar to those obtained with simultaneous administration of -cyclodextrin and competing agent discussed above . in yet another report the -cyclodextrin derivatives , sbe4-beta - cd and 2-hydroxypropyl--cyclodextrin ( hp - beta - cd ) , were used to form complex with cinnarizine to enhance the oral bioavailability . the absolute bioavailability of solution or capsule containing drug alone was compared to -cyclodextrin derivatives complex of drug as a solution and in capsule . to assess the bioavailability , cinnarizine was administered to beagle dogs in the dose of ( and an amount equivalent in case of -cyclodextrin derivatives complex ) 25 mg orally and 12.5 mg / ml intravenously . the bioavailability of cinnarizine was 8 4% for solution of drug and 0.8 0.4% for capsule filled with drug alone . this was observed to be significantly increased to 5560% and 38 12% in case of -cyclodextrin derivative complex of drug formulated as suspension and that filled in capsules , respectively . the higher bioavailability achieved in case of cinnarizine -cyclodextrin derivative complex formulated as suspension than that filled in capsules was attributed to rapid dissociation of drug from its inclusion complex , thus causing improved absorption . based on this concept , a simple technique of preparing solid dispersion of drug by fusion method was explored . the purpose was served by the use of two lipid carriers separately , gelucire 44/14 ( gastric acid soluble ) and other compritol 888ato ( gastric acid insoluble ) . in case of gelucire 44/14 , the results suggested initial rapid release of drug which then sustained for over 5 h giving a release of approximately 90% , whereas compritol 888ato at a maximum ratio of 1 : 1 ( drug : compritol888ato ) more prolonged the release when compared to pure drug and commercial tablet supported the efficiency of solid dispersion to enhance the bioavailability . the drug being a weak base exhibits higher solubility in acidic environment of stomach and hence dissolution . on the other hand it displays poor dissolution in basic environment of small intestine . the inhibited drug release of drug in basic environment can be raised by incorporation of acidifiers in the formulation that create acidic environment . incorporation of acidifiers like citric acid or fumaric acid in matrix tablet to raise the solubility and hence ph independent drug release was also suggested . the drug release under the influence of acidifiers was examined by preparing three combinations , formulation containing only citric acid ( 70 mg ) or fumaric acid ( 70 mg ) separately and in combination with citric acid and fumaric acid ( 35 : 35 mg ) too . the acidifiers are able to reduce the ph of the intestinal and maintaining it for extended period of time , thus providing sustained drug release . the drug release was 57.3% and 53.5% after 8 h which increases to 75.2% and 66.2% after 12 h for citric acid and fumaric acid , respectively , whereas it was only 26.5% after 12 h. this shows better efficiency of citric acid for maintaining low ph environment . the lack point is depletion of acidifier from the tablet matrix , the rate of which is dependent on type of acidifier . hence , the result suggested that proper selection of acidifiers may give ph independent release profiles that in turn would lead to higher bioavailability . conclusively , the problematic issue of low and erratic bioavailability of poorly aqueous soluble drug , cinnarizine , was a topic of interest to researchers . the efforts were dedicated from the use of well known -cyclodextrin complex to some recent use of liquid cubic phases and acidifiers like citric acid and fumaric acid . additional use of competing agents ( such as dl - phenylalanine and l - isoleucine ) in combination with -cyclodextrin complex and lipid vehicles like oleic acid was proved to be advantageous and further increased the bioavailability of drug . on establishment of comparison between these mentioned techniques to that of cinnarizine in alone or its marketed preparations , the results were encouraging with remarkable enhancement in bioavailability of cinnarizine . various dosage forms / delivery systems have been approached to solve the solubility and bioavailability issue . these include fast dissolving tablets , nanoemulsions , microparticles , and as gastroretentive systems . to the best of our knowledge , here is the compilation of research reports , till date , on delivery related approaches to overcome the pharmacokinetic constraints of cinnarizine . critical analysis based on in vitro results revealed improved bioavailability of cinnarizine formulated as various dosage forms , when compared to marketed preparation . in contrast to in vitro results , the in vivo data for improvement in bioavailability is limited and efforts are needed to be made to assess the bioavailability and performance of cinnarizine in vivo . cinnarizine could serve as suitable candidate for fast dissolving tablet that may improve the bioavailability and patient compliance . the in vitro dissolution rate of granules was increased ( 8090% , 60 min ) on comparison with drug alone ( less than 50% , 80 min ) , because of the higher hydrophilic character imparted by hydrophilic carriers and reduction in drug crystallinity in case of granules . the report suggested the use of fluidized hot melt granulation method as an advantageous means of producing granules of cinnarizine with peg 6000 as a melt binder , which does not need solvents or water . chitosan based orodispersible tablet of cinnarizine was reported with the aim of improving absorption of drug which steps towards improved oral bioavailability . the study guided the use of chitosan based orodispersible tablet to be a valuable alternative in respect to use of expensive superdisintegrant . the prepared tablets have advantage of disintegrating within few seconds without need of water , which lead to enhanced absorption of drug . the disintegration time of optimized formulation was 11 sec giving 90% of drug release in a time period of 5 min . according to this report , the researchers assumed that if 90% of drug is released in the dissolution media in vitro , then it will exert full effect in vivo . so based on the results , the sufficient dissolution was assumed to enhance the absorption of drug that leads to improvise the bioavailability when assessed in vivo providing effective therapy . this ultimately aims towards improving patient compliance with main focus on children and elderly patients . developed fast dissolving tablet of cinnarizine using combination of superdisintegrants sodium starch glycolate ( ssg ) and croscarmellose sodium ( ccs ) and camphor as a subliming material . ssg = 12 mg , ccs = 12 mg , and camphor = 10 mg ) when compared to widely used commercial tablet ( stugeron ) was reported to be more effective with drug release of 99% in 16 min . in another report , fast dissolving tablets of cinnarizine were prepared by sublimation technique and regarded as effective alternative approach compared to use of expensive excipients . the precompressional evaluation revealed good free flowing properties within the prescribed limit . considering the pharmacopoeial test , the drug was uniformly distributed throughout the tablet with high drug content ( 101.5% ) . the subliming agents have directly proportional effect on the disintegration of tablets which further affect the drug release . the optimized formulation was reported to have t50% of 6.25 min and t90% of 19.74 min . it was stated that upon storage the disintegration of tablet was significantly decreased ( p < 0.05 ) . the report concluded the effectiveness of sublimation method as an alternative approach for fast dissolving tablet of cinnarizine to reach the optimum point in shortest time with minimum efforts . in yet another report , oral dispersible tablets were prepared by three different methods : effervescent , superdisintegrant addition , and sublimation method . the tablets showed total release of drug in a period of 6 h with total disintegration in 25.3 sec . the report concluded that the superdisintegrant addition method with l - hydroxypropyl cellulose ( 10% w / v ) was best to fulfill the aim . recently heer et al . developed fast dissolving orodispersible film and fast dissolving tablet of cinnarizine and assessed the effect of superdisintegrants . for this purpose , three different superdisintegrants , sodium starch glycolate , crospovidone , and croscarmellose sodium , were used . the tablet was prepared by direct compression method and solvent casting method was employed for preparation of orodispersible film . the two preparations were evaluated for weight variation , thickness , drug content , folding endurance , and tensile strength ( for orodispersible film ) . as a result , among three superdisintegrants , crospovidone at 4% w / w was proved to have promising superdisintegrant property required to prepare fast dissolving formulations . fast dissolving orodispersible film was concluded to be better over fast dissolving tablet in terms of faster drug release with better flexibility suggestive to patient compliant dosage form . lipid based drug delivery systems were topic of interest to scientist as novel drug delivery platform and well known to improve the oral bioavailability of poorly water soluble drug that belongs to bcs class ii and iv . the most interesting mechanism of enhanced bioavailability was regarded as improved absorption due to enhancement of gastrointestinal solubilisation . the factors that relate to bioavailability from lipid formulations include digestion of lipid , the mean emulsion droplet diameter , the lipophilicity of drugs , and types of lipids . the lipids are preferred for oral administration as they can be formulated in solution , suspension emulsions , self - emulsifying , and microemulsions . reported lipid based formulation of drug and studied the effect of lipid on drug absorption . the in vivo absorption study revealed higher bioavailability with long chain triglyceride than medium chained triglyceride , giving a value of 32.1 2.3% and 16.6 2.3% , respectively , but was independent of quantity of lipid administered . however , the report concluded that the bioavailability was observed to increase with lipid dose of 125250 mg but reached static point at 500 mg . improvement in solubility of drug in self - nanoemulsified drug delivery system was reported [ 43 , 44 ] . snedds containing combination of brij 97 ( 17.6% w / w ) and peg 400 ( 8.8% w / w ) revealed good bioavailability in vivo with an area under curve ( auc048 h ) of 773 148 ng ml h. a double peak phenomenon was observed for plasma profile . this helped in keeping cinnarizine solubilized even at lower part of gastrointestinal tract ; thus it is available for absorption for longer duration . it was observed that the biorelevant media greatly affected the type and size of droplets formed . upon dispersion in media containing low bile acid content , vesicles were formed , whereas it was micelle formation in presence of higher bile acid content . the resulted nanostructured composition influenced the solubilization capacity of composition which potentially affected the absorption of drug from resultant media . this research report on snedds suggested the approach to be suitable for better absorption of drug . again working with snedds , the researcher published another paper this time to assess the effect of increasing loading level of cinnarizine . three formulations were developed , sneddshigh , sneddsmedium , and sneddslow with 50 , 25 , and 12.5 mg of drug , respectively . the in vitro lipolysis at ph 6.5 showed that snedds with high concentration of cinnarizine leads to precipitation of drug , whereas it decreased in drug loading . there was no significant difference in auc , tmax , and cmax for the three formulations . the tmax was 1.8 0.14 h , 23 0.64 h , and 3 0.71 h after 1 g sneddshigh , 2 g sneddsmedium , and 4 g sneddslow , respectively . the auc obtained was 355 74 , 485 68 , and 392 52 ng / mlh for the three formulations in order of their increased drug loading , respectively . the report presented that , on oral administration , the drug loading level in snedds and amount of snedds vehicle did not affect the bioavailability . a lipid based liquid crystalline matrix of cinnarizine was prepared for sustained release delivery of drug which ultimately enhances the bioavailability . the lipids used were glyceryl monooleate and oleyl glycerate . in vitro lipolysis study suggested less susceptibility of oleyl glycerate to hydrolysis by pancreatic lipase than that of glyceryl monooleate . in order to investigate the improvement in bioavailability , cinnarizine was orally administered in the form of suspension or solution in glyceryl monooleate or olely glycerate . the bioavailability was more enhanced by administering cinnarizine with oleyl glycerate and appeared to be 344% ( aqueous suspension formulation assigned a reference bioavailability of 100% ) . the report concluded the efficiency of lipids that have an ability to form liquid crystalline structures in excess water , to be set forth as an application for oral sustained delivery of drug to enhance bioavailability . the sedds with 30% w / w of capmul pg-12 when evaluated in phosphate buffer , ph 7.2 , provided remarkable enhancement in drug solubility in phosphate buffer(s ) with rapid drug release . the sedds showed least solubility of drug to be 1500 m in phosphate buffer , ph 7.2 . it was concluded that the sedds prepared by combination of capmul pg-12 , tween 20 , and cremophor rh 40 can be a suitable alternative dosage form to fulfill the aim of the research that is to improve solubility and dissolution of drug . snedds of the drug was also reported with the aim of exploring the effect of type of lipid on solubility and drug release . proper balance between rapid and efficient self - emulsifying ability , higher solubility of drug , maintaining high amount of drug in solution after aqueous dispersion ( at least 85% ) , and lower droplet size upon aqueous dilution were the selection criteria for optimal formulations . the length of lipid chain significantly influenced the solubility of drug and long chain lipids showed higher solubility than medium chained . in terms of glyceride composition furthermore , the unsaturated fatty acids with long chain lipids resulted in higher solubility than the above two . snedds showed significantly better release in vitro in simulated gastric fluid ( sgf ) ( to mimic the in vivo condition more accurately ) than marketed preparation , stugeron . the snedds showed a release of 8495% after 15 min which was only 66% in case of stugeron in simulated gastric fluid . after 15 min of shifting into simulated intestinal fluid , the marketed preparation showed comparatively much higher drug precipitation ( 83% ) than the snedds which causes only 723% of drug precipitation . a paper was also reported to enhance the oral bioavailability of cinnarizine from its medium chain lipid solution formulation . lipid based formulation of cinnarizine was administered intraduodenally and perorally to rats in order to assess the contribution of dispersion and digestion in the stomach towards bioavailability . the difference in bioavailability was also observed for medium chain and long chain triacylglycerides owing to difference in the dispersion and digestion in the gastric and intestinal compartments . the difference between the bioavailability was large in case of medium chain lipid when compared to long chain lipid in case of both peroral and intraduodenal administration . the result suggested that the passage of medium chain lipid formulation through stomach led to critical gastric digestion than long chain lipid formulations . the report concluded that rapid transfer of medium chain formulation to intestinal mixed micelles possibly was achieved by efficient dispersion and partial digestion in stomach . also absorption in the upper intestine prior to drug precipitation was preferred . in yet another report the high solubility and stability of cinnarizine loaded emulsion ( cle ) had been proved . egg lecithin was used with the aim of minimizing drug degradation from both water and oil . the drug was localized in the interfacial lecithin layer of emulsion so as not to contact with oil or water medium . on further working with the cle , in next year the researchers provided reports for pharmacokinetics and tissue distribution of drug . when assessed in vivo in rats , the novel formulation ( cle ) revealed significantly higher auc and lower clearance and distribution volume . on i.v . administration of 2 mg / kg of cinnarizine loaded emulsion , the auc was observed to be 1879.556 g / lh , whereas it was 865.725 g / lh for aqueous cinnarizine solution prepared using tween 80 ( 1 g ) and 1,2 propylene glycol ( 10 g ) . the result revealed lower clearance ( 0.947 l / h / kg ) for drug loaded emulsion . consequently , circulation in blood stream achieved for longer duration resulted in better therapeutic effects . contributing to bioavailability of drug , researchers explored self - microemulsifying system ( smedds ) . smedds is known to spread readily in the gastrointestinal tract , and the agitation provided by digestive motility of stomach and the intestine results in self - emulsification . smedds containing oleic acid ( 16.66% w / w ) , tween 80 ( 55.55% w / w ) , and transcutol p ( 27.77% w / w ) improved the solubility and drug release when compared to marketed formulation of cinnarizine . in the report , the authors have explained formation of small sized particles , leading to rapid drug release of 81.96% in 5 min which slightly increases to 87.67% in 60 min . this suggested the potential use of smedds to improve oral bioavailability by overcoming the solubility issue . in addition to this , smedds of cinnarizine was prepared to characterize a better understanding of the mechanisms behind the precipitation of cinnarizine during in vitro lipolysis of a self - microemulsifying drug delivery system . in the period of in vitro lipolysis of the smedds with or without cinnarizine , dissolution was performed in biorelevant media and initially the dissolution rate of cinnarizine from pellets containing precipitated cinnarizine was 10-fold higher than dissolution from blank pellet spiked with crystalline cinnarizine , which increased to more than 50% drug dissolved in the first minute . pellets were further characterized by x - ray powder diffraction ( xrpd ) and polarized light microscopy ( plm ) . results indicated that either amorphous form or a molecular dispersion of cinnarizine resulted in precipitation during in vitro lipolysis of smedds , thus improving dissolution profile . recently snedds was also investigated to eliminate the effect of food on cinnarizine absorption . a nutritional drink , fresubin energy ( 200 ml ) , the snedds capsules or tablets in fed state were assessed for bioavailability by administering snedds equivalent to 50 mg of drug orally to male beagle dog ( 1013.5 kg ) with one week wash - out period between dosing . the dogs were fasted overnight for about 2024 h. the nutritional drink was administered 30 min prior to dosing . pentagastrin ( 6 g / kg ) was administered to dogs in order to control the gastric ph . the result also established the effect of food to extend the tmax to 2.5 times than fasted state and increased bioavailability for conventional tablets ( ffasted = 20.9 5.7 and ffed = 53.8 30.1 ) . in contrast to this the food condition did not affect the absorption of drug from snedds capsules and tablets , while tmax was observed to extend the absorption and thus enhanced the bioavailability . the fed state showed 2.5 and 3.3 times longer tmax than fasted state for snedds tablets and snedds capsules , respectively . the bioavailability of snedds capsules was higher at both fed ( 79.3 14.7 ) and fasted state ( 58.1 16.7 ) than that of snedds tablet ( ffed = 43.7 6.7 and ffasted = 32.7 11.5 ) . snedds formulation was observed to diminish the variation in absorption at fasted and fed conditions , leading to overall increase in bioavailability compared to conventional tablets . at the end , the conclusive points were drawn that food affects the absorption of bcs class ii drug causing increased bioavailability in fed state which can be significantly reduced by the use of snedds capsules and tablets . in the same context , another report was published in the same year to predict the performance of two above discussed lipid formulations ( snedds capsules and snedds tablets ) . the two formulations were compared with conventional tablets on the basis of gastrointestinal in vitro lipolysis model in both fasted ( ph = 2 ) and fed conditions ( ph = 6 ) . the in vitro lipolysis model was supposed to simulate the digestion in the stomach and intestine . to mimic the lipolysis events in fed state conditions , fat milk ( 3.5% ) was administered with 0.4% mg / ml of pepsin . at gastric step during fasted state , a high concentration of drug was found in aqueous phase , whereas it was lower for snedds tablet which can be explained on the basis of presence of alkaline excipients that lead to increase of the ph to 3.64 and cause lower solubility . the solubilization of drug remained constant for snedds formulations upon initiation of duodenal step suggested the role of lipid to enhance the solubilization of cinnarizine . the total amount of cinnarizine recovered from fasted state lipolysis was almost similar for all formulation with an average of 84 12% . on examining fed state condition , approximately 1% of cinnarizine from snedds was shown in aqueous phase in gastric step , whereas it was only 0.1% for conventional tablet . on passing into duodenal step , snedds formulations showed an aqueous cinnarizine content of 40% after 83 min and maintained it throughout the lipolysis , while the conventional tablet lost solubilization capability decreasing to 25.4 1.7% at the end . when compared to in vivo data provided by paper discussed above , these in vitro results were in accordance with in vivo . the order was similar as snedds capsules > snedds tablet > conventional tablet for performance in vitro and in vivo . more conclusively , the in vitro fasted state model was able to predict the correlation with in vivo study , whereas the fed state model can not exactly establish a correlation but was able to predict the best formulation similar to that predicted by in vivo study . in the path of lipid based nanosized delivery of cinnarizine , a report was published to stabilize the nanosuspensions by the utilization of bead layering . the nanosuspension of drug was coated on sugar beads by the use of fluidized bed pellet coater with wurster insert . these nanosuspensions showed complete dissolution in 15 min , whereas it was only 11% in 1 h for unmilled powder . the coated nanocrystals resulted in slower release of cinnarizine than that of original nanosuspensions due to formation of reagglomeration upon release from coating . the conclusive point suggested influence of drug physicochemical properties on in vitro dissolution after bead layering . utilization of lipids for pharmaceutical and biomedical applications was known to overcome the issues related to biocompatibility and biodegradability . in this respect the unique mesophasic structure of lipids such as cubic , hexagonal , and sponge phase structures provides advantage of stability and production feasibility . the cubic structure has advantage of improved stability , bioadhesivity , and biocompatibility , whereas the hexagonal arrangement exhibited hexasomes accounts for encapsulation of different drugs with high stability . the similar reports on cinnarizine are discussed further . the contribution towards improved absorption of cinnarizine in addition to this , another highlight of cubic phase is its temperature sensitivity in the range of 2037c suggesting it to be a putative material for the purpose of in situ gelation . the in vivo absorption study was performed on anaesthetized male sprague - dawley rats ( 280320 g ) . the result illustrated large difference in the rate of cinnarizine absorption from cubic phase and suspension . the absorption profile was more sustained and continued beyond 8 h after dose period for cubic phase , thus supporting its application to improve bioavailability . the cmax obtained for cubic phase was 26.2 ng / ml after 4.7 h , whereas it was 99.9 ng / ml after 2.2 h for suspension proving sustained absorption for former case . the report was the first to reveal the ability of liquid crystalline nanostructured particles ( cubosomes ) to attain sustained absorption of cinnarizine . these cubosomes were developed using phytantriol ( an alcohol ) and glyceryl monooleate ( a lipid ) . phytantriol cubosomes showed improved results with sustained absorption beyond 48 h. the oral bioavailability was improved to 21% compared to suspension ( f = 9% ) and oleic acid emulsion ( f = 12% ) . in such cases of nondigestible phytantriol cubosomes , the slow release can be facilitated by stomach that acts as a nonsink reservoir . thus , the report suggested the potential use of nondigestible liquid crystalline nanostructured particles as a novel approach for sustained oral drug delivery systems leading to improvise the bioavailability of poorly water soluble drugs . in the same year hexagonal liquid crystals ( hexosomes ) of cinnarizine were prepared with the aim of sustaining the absorption for increment in bioavailability . the pharmacokinetics was evaluated on oral administration of bolus lipid solution of drug in selachyl alcohol to rats . the results revealed that , after oral administration , sustained plasma profile with 2040 ng / ml of drug maintained over the first 24 h was observed . when compared to suspension , the tmax was longer for hexosomes ( 1 h and 23.5 5.9 h resp . ) . the absolute oral bioavailability was improved for hexasomes ( 17% ) compared to that of suspension ( 9% ) . selachyl alcohol is not susceptible to digestion which attributed to prolong the absorption as similar to nondigestible phytantriol cubic system discussed above . the report concluded the potential use of nondigestible liquid crystalline systems with additional advantages of nanostructured particles as novel system for sustained drug delivery . the research reported two preparations : one was a buoyant tablet containing powdered soyabean , drug , and sodium bicarbonate and the other was laminated floating film comprising of a drug film , an effervescing film of sodium bicarbonate , and an outer drug release regulating film . the in vitro studies assured the suitable floating ability and sustained release property of the two formulations . linear dissolution was observed after 2 h in case of laminated film not similar to tablet due to structural difference . the absorption study was done by oral administration of buoyant tablet and film in beagle dogs . as a result , cinnarizine was observed in blood even 24 h after oral administration , concluding the efficiency of the two preparations as sustained delivery systems so as to upgrade the absorption and bioavailability of cinnarizine . floating microballoons were prepared by diffusion solvent evaporation technique using 2 factorial design to improve bioavailability . was performed in ph 1.2 for 8 h and then in ph 7.4 for 2 h. microballoon released approximately whole drug during 10 h period with sufficient buoyancy . the microballoons prepared with combination of eudragit s100 and eudragit rl in a ratio of 1 : 3 proved most effective results , suggesting the efficient use of microballoons for oral sustained delivery of cinnarizine . the sustained drug release was achieved due to rapid hydration of polymer on the surface of floating tablet , thus forming a gel layer surrounding the matrix . this controls penetration of water to the center of matrix tablet and retards the release . it was reported that , with increase in viscosity of polymer , drug release rate was increased . while studying the release rate with different polymers , it was also revealed that the drug release rate depends on the surface area as well as hydration capacity of polymer . based on these results , the optimized formulation containing hpmc k100lv ( 90 mg ) was compared ( in vivo ) to oral suspension and bioavailability was studied . the absorption phase was slow and prolonged in case of matrix tablet , thus indicating prolonged plasma concentration . the overall bioavailability of floating matrix tablet was 2.85 times greater than that of oral suspension . in 2012 , floating tablet of cinnarizine in combination of domperidone maleate was developed for gastroretentive delivery . the ph independent polymer , methocel polymer of different viscosity grades , was used at concentration of 2050% w / w to prepare floating tablets . the formulation was reported to remain buoyant for 12 h with lag time of 1015 sec in 0.1 n hcl . sodium bicarbonate ( 15 mg ) and sodium alginate ( 18 mg ) were used as gas generating agent necessary to impart sufficient buoyancy . so the report suggested improved retention of tablet of combination of two antiemetic drugs at gastric ph that was supposed to have improved therapeutic effects . multiparticulate systems reported as gastroretentive floating microspheres were developed to assess the effect of formulation variables on microsphere formation . microspheres were developed using acrycoat s100 , eudragit rs100 , and ethyl cellulose as gastroretentive polymers . when assessed in vitro in 0.1 n hcl , the microspheres remained buoyant for 12 h. among these , microspheres containing acrycoat s100 ( 100 mg ) showed best results for sustained drug release ( 98.14% ) , floatability ( 72% ) in 12 h , and stability . another effort to sustain the release of cinnarizine was attained by preparation of drug loaded chitosan methylcellulose interpenetrating polymer network ( cs / mc ipn ) microspheres intended for mucoadhesive gastroretentive application . the study explores the interpenetrating network of two biopolymers , chitosan and methylcellulose , to prolong the residence of microspheres in the stomach . the microspheres were prepared using central composite design at three different levels of polymeric ratio and glutaraldehyde . the formulations were evaluated for degree of mucoadhesiveness which was correlated to amount of polymer and interpenetrating network of polymers . for optimized formulation the percent of mucoadhesion was 73.85 2.78 at 4 h with t50 of 432.21 26.15 min . the other observed results were for mean diameter ( 61.32 1.38 m ) , swelling index ( 2.38 0.06 ) , and entrapment efficiency ( 84.13 1.32% ) . the report suggested the utilization of chitosan methylcellulose interpenetrating polymer network ( cs / mc ipn ) as potential approach to impart mucoadhesive property for gastroretentive delivery of weakly basic drugs . recently in the past year , patel et al . prepared mucoadhesive microparticles by supercritical fluid method using chitosan as mucoadhesive polymer . the efficiency of this physical coating process for producing microparticles was established by x - ray powder diffractometry and differential scanning calorimetry . the prepared microparticles were proposed for better bioavailability than the suspension in test animal due to gastroretention . the comparison was done in vivo by administering both suspension and mucoadhesive microparticles to rabbit in fasted state . the mucoadhesive microparticles showed sustained drug release for more than 20 h. good mucoadhesive properties and strong adherence of microparticles to gastric mucus layer were established both in vitro and in vivo , resulting in gastroretention for an extended period of time . these formulations accounts for sustained effect of drug reducing the dosing frequencies . in the same year mucoadhesive tablets of cinnarizine were developed using eudragit rlpo as polymer with the aim of investigating the effect of iron oxide in the formulation . the mucoadhesive tablets were prepared at different concentrations of eudragit rlpo , iron oxide , and pvp k 30 and evaluated for mucoadhesive strength , t50% , t90% , and mean dissolution time effect of iron oxide was investigated . the result clarifies the synergistic effect of combination of eudragit rlpo , iron oxide , and pvp k 30 to enhance the mucoadhesive strength when compared to their individual effect . as per the results the mucoadhesive tablets containing 8.58% w / w eudragit rlpo , 6.68% w / w iron oxide , and 7.86% w / w pvp k 30 were selected to be optimized and fulfilled maximum requirements with better mean dissolution time ( 266.44 3.82 min ) , t50% ( 124.94 5.40 min ) , t90% ( 755.53 4.29 min ) , and desirability of 0.727 . the incorporation of iron oxide was found to enhance the mucoadhesive strength due to induction of ionic interaction . retardation of drug release with mean dissolution time of 229.56 3.30 min at higher concentration of iron oxide was achieved due to reduction in polymeric chain disentanglement and polymer erosion caused by the entrapment of iron oxide in the polymeric chain network . a novel approach of gastroretention with controlled release was recently negotiated by preparing an unfolding type film of cinnarizine . the polymeric film was procured by solvent casting method using ethyl cellulose and hydroxyl propyl methyl cellulose k15 and folded in hard gelatin capsule . as obtained by in vitro drug release study , the film describes fast release of drug in the first hour due to initial leaching out of drug , which was followed by slow release by diffusion through hydroxyl propyl methyl cellulose and ethyl cellulose providing more steady controlled release giving total release of 81.93% in 13 h. the result also suggested more appropriate use of unfolding film in simulated gastric fluid when compared to roll folded pattern . the research strongly indicated the gastroretentive potential of prepared dosage form which can be explored further for in vivo studies . cinnarizine is known to precipitate at high ph on gastric emptying and show slow and erratic bioavailability . microparticles prepared using ph responsive polymer eudragit l by emulsion solvent evaporation method showed improved bioavailability . the microparticles inhibited the in vitro release of drug under gastric conditions due to high threshold ph 6 . at favorable intestinal conditions , this diminishes the impact of drug precipitation and improved the absorption . in vivo comparison with drug powder suspension was done by oral dosing in rats and eudragit l ( 3 g ) loaded microparticles showed twofold increase in bioavailability , although c(max ) and t(max ) were not significantly different . despite wide therapeutic applications and worldwide commercialization of cinnarizine , the marketed formulation accounts for low and erratic bioavailability , due to gastric acidity dependent absorption . earlier complexation techniques were practiced but now recently developed dosage forms such as snedds , gastroretentive systems , are in interest . lipid based systems emerged with improved solubility and dissolution in vitro ( 8095% ) and in vivo bioavailability ( auc approximately 4 times higher than tablets ) . the purpose to circumvent the obstacles associated with drug was also achieved by the preparations aimed to longer retention at gastric environment . these provided new achievements for sustained release of cinnarizine , so as to reduce dosing frequencies and attain patient compliance . use of nanosized liquid crystalline particles ( cubosomes ) can be proved as new field of interest for sustained absorption of cinnarizine . patient compliance is the ultimate aim for any pharmaceutical dosage form . for the control of drug release or sustained benefits for patients , the microsized particles , microballoons , microspheres , and microsponges alone or in combination with other approaches like use of bioadhesive polymers or coating materials to facilitate intimate adherence to gastric mucosa and incorporation of acidifiers in order to improve ph dependent solubility of cinnarizine , might result in the availability of new product with improved potential for the therapeutics of cinnarizine . cinnarizine solubility is highly dependent on gastric acidity , so ph controlled systems can be proposed to provide constant rate release , irrespective of location of dosage form in the git . release of cinnarizine from its dosage form independent of environmental conditions can also be achieved by designing osmotically controlled gastroretentive drug delivery systems which can further be modified by coating external surface with some erodible polymer to provide alternate chances of improvement and must be assessed further . use of lipid based systems as gastroretentive systems can set forth for the new era of overcoming the pharmacokinetic constraints of cinnarizine . tablets and capsules formulated using self - emulsifying drug delivery system containing droplets size extending from few nanometers to microns can be developed , while sustaining good flowability , cohesive property , and high content uniformity . methods by which a transiently supersaturated state can be generated and maintained in situin the gi tract can be set forth for development . in this context , novel formulation approach of use of polymeric precipitation inhibitors or retarders like hydroxyl propyl methyl cellulose incorporated into self - nanoemulsifying delivery systems to produce a supersaturated drug concentration and prolong such a drug concentration for an extended period of time for an optimal absorption , thereby improving oral bioavailability , can be set forth for assessment for future development of cinnarizine . the various techniques discussed herein can be better option for companies to focus on commercializing them .

the collected comments and pathologic diagnoses of several pathologists are summarized for 18 cases in which lesions were induced in the upper respiratory tract of rats and mice . specific neoplastic and nonneoplastic lesions of the nose and trachea are described and discussed , and opinions regarding pathogenesis and biologic significance of the lesions are presented . the anatomic and pathophysiologic complexities of the rodent nose in relation to lesion development following inhalation or systemic exposure to xenobiotics are important considerations in the genesis of pathologic changes in this organ.imagesplate 1.plate 2.plate 3.plate 4.plate 5.plate 6.plate 7.plate 8.plate 9.plate 10.plate 11.plate 12.plate 13.plate 14.plate 15.plate 16.plate 17plate 18plate 19plate 20.plate 21.plate 22.plate 23.plate 24.plate 25.plate 26.plate 27.plate 28.plate 29.plate 30.plate 31.plate 32.plate 33.plate 34.plate 35.plate 36.plate 37.plate 38.plate 39.plate 40.plate 41.plate 42.plate 43.plate 44.plate 45.plate 46.plate 47.plate 48.plate 49.plate 50.plate 51.plate 52.plate 53 . low solubility causing low dissolution in gastrointestinal tract is the major problem for drugs meant for systemic action after oral administration , like cinnarizine . pharmaceutical products of cinnarizine are commercialized globally as immediate release preparations presenting low absorption with low and erratic bioavailability . approaches to enhance bioavailability are widely cited in the literature . an attempt has been made to review the bioavailability complications and clinical therapeutics of poorly water soluble drug : cinnarizine . the interest of writing this paper is to summarize the pharmacokinetic limitations of drug with special focus on strategies to improvise bioavailability along with effectiveness of novel dosage forms to circumvent the obstacle . the paper provides insight to the approaches to overcome low and erratic bioavailability of cinnarizine by cyclodextrin complexes and novel dosage forms : self - nanoemulsifying systems and buoyant microparticulates . nanoformulations need to systematically explored in future , for their new clinical role in prophylaxis of migraine attacks in children . clinical reports have affirmed the role of cinnarizine in migraine prophylaxis . research needs to be dedicated to develop dosage forms for efficacious bioavailability and drug directly to brain .

health - care associated infections ( hcais ) are one among the major health related hazards and account for a major global disease burden . they pose a health threat toward patients and health care workers . these have a global impact and pose a disease burden to millions of people worldwide . these promote resistance to antibiotics , complicate the delivery of patient care and also lead to an additional monetary expenditure to the and also lead to additional monetary expenditure to the patients with underlying pathology . hospital - acquired infections account for more than 1.4 million people worldwide.1 health - care delivery exposes the hospital workers toward infections such as tuberculosis , needle stick injuries , and hiv . the practice of hand hygiene ( hh ) has long been considered as a gold standard and has passed the test of time as an infection control measure successfully preventing hcais . the single most important preventive measure against hcai is to practice proper hh technique by either washing the hands with soap or with a disinfectant , but this practice is usually given minimal priority by the hospital staff.2 in general , health care workers are not carriers , but vectors . centers for disease control and prevention defines the clinical contact surfaces as the surfaces that can be contaminated directly from patient materials either by direct spray or the generated splatter during dental procedures or with contact of dental health care provider s gloved hands . examples of such surfaces include light and door handles , switches , dental x - ray equipment dental chair , pens , telephone , door knobs , etc . contamination of these surfaces is frequently reported , but the risk of infection for patients and the dental staff is not yet , determined with high accuracy . dental healthcare workers skin could transiently carry potential pathogens coming from the environment and the use of gloves does not automatically eliminate the need for hand washing , because gloves can have unapparent defects or can be torn during use , due to sharp equipment , widespread in dental healthcare . in addition , the opportunistic pathogens may multiply at a very rapid rate in a moist environment that is usually present underneath the gloves . an aspect associated with microbial contamination of gloved hands is the use of finger rings . indeed , rings can make glove donning difficult with consequent damage and loss of integrity . a large number of studies are there establishing the association of hcais with improper hh , but only a handful of studies have demonstrated that the skin underneath finger rings is more enormously colonized than other comparable areas in the finger skin . however , whether wearing of the finger rings increases the likelihood of pathogen transfer is still unknown.3 a significance level of microbial contamination on the skin under ringed finger is also least examined . hence , the present study was done to investigate the occurrence of potential pathogenic bacteria and fungi on skin of gloved hands of dental health care workers who wore finger rings with those who did not wore finger rings under gloves . the sample consisted of 40 dentists working in the department of oral medicine diagnosis and radiology , career post graduate institute of dental sciences ( cpgids ) , lucknow . this study was conducted in compliance with the protocol ; written ethical approval was taken from the head of department of oral medicine and also from the principal of cpgids , lucknow . skin samples were collected in the middle of the working day from the dominant hand soon after glove removal and professional hand cleaning . sterile swabs previously moistened with tubes containing 1 ml of sterile saline solution ( 0.9% w / v nacl ) were gently rubbed over the complete ventral surface of the hand and around the periphery of finger ring sample . swabs were immediately transferred onto the plates containing mannitol salt agar and sabouraud dextrose agar plates were incubated aerobically at 28c and 37c for 48 h ( figures 1 and 2 ) . isolates recovered from cultures were preliminarily subjected to microbiological procedures and were identified using biomerieux equipment , uk . the laboratory procedures were made by courtesy of the department of microbiology , career medical college , lucknow . prevalence of potential pathogenic microorganisms with and without rings was assessed and differences were statistically analyzed . this study was conducted in compliance with the protocol ; written ethical approval was taken from the head of department of oral medicine and also from the principal of cpgids , lucknow . skin samples were collected in the middle of the working day from the dominant hand soon after glove removal and professional hand cleaning . sterile swabs previously moistened with tubes containing 1 ml of sterile saline solution ( 0.9% w / v nacl ) were gently rubbed over the complete ventral surface of the hand and around the periphery of finger ring sample . swabs were immediately transferred onto the plates containing mannitol salt agar and sabouraud dextrose agar plates were incubated aerobically at 28c and 37c for 48 h ( figures 1 and 2 ) . isolates recovered from cultures were preliminarily subjected to microbiological procedures and were identified using biomerieux equipment , uk . the laboratory procedures were made by courtesy of the department of microbiology , career medical college , lucknow . prevalence of potential pathogenic microorganisms with and without rings was assessed and differences were statistically analyzed . a cross - sectional study was conducted to measure and identify the microbial contamination isolated from the skin under rings and on skin without rings and to compare the results among volunteered dentists of cgpids and hospital , lucknow , india . all dentists were males aged between 21 and 33 years ( mean 27 years ) , equally divided into ( n = 20 ) with finger rings ( n = 20 ) without finger rings . all the estimated values of prevalence ratio ( pr ) were quite high , ranging between lower limit of 2.03 and the upper limit of 4.74 , but were statistically not significant at 95% of level excluding the pr value for fungi ( table 1 ) . occurrence of potential pathogens in skin samples from hands with and without finger rings of dentists . the potentially pathogenic bacteria , which were isolated were staphylococcus aureus , staphylococcus epidermidis , escherichia coli , enterococcus spp . , while fungi were candida albicans , aspergillus niger , and aspergillus flavus . excluding s. aureus which was isolated only in one non - ring wearing dentist , all these microorganisms were more frequent in ring wearing dentists ( table 2 ) . list of potential pathogenic bacteria and fungi isolated from skin samples of dentists hand with and without finger rings ( prevalence values between brackets ) . the statistical analysis was done by applying systat version 10 by cranes software , bangalore , india in the study . the prevalence of potentially pathogenic microorganisms with and without finger rings was assessed and differences were statistically analyzed using 2 test with yates correction for continuity . the pr , with 95% of confidence interval and a level of significance of 95% was selected to statistically analyze the data . bacteria were non - significantly more frequent in the dentist s with ringed finger ( 63% vs. 37% ; p = 0.06 ) . while fungi were significantly more frequent in the dentist s with a ringed finger ( 79% vs. 21% ; p = 0.0002 ) ( table 1 ) the statistical analysis was done by applying systat version 10 by cranes software , bangalore , india in the study . the prevalence of potentially pathogenic microorganisms with and without finger rings was assessed and differences were statistically analyzed using 2 test with yates correction for continuity . the pr , with 95% of confidence interval and a level of significance of 95% was selected to statistically analyze the data . bacteria were non - significantly more frequent in the dentist s with ringed finger ( 63% vs. 37% ; p = 0.06 ) . while fungi were significantly more frequent in the dentist s with a ringed finger ( 79% vs. 21% ; p = 0.0002 ) ( table 1 ) potentially pathogenic bacteria , which were isolated were s. aureus , e. coli , while fungi were c. albicans . excluding s. aureus which was isolated only in one non - ring wearing dentist , all these microorganisms were more frequent in ring wearing dentists . dentists who wore rings were nearly twice more likely to harbor potential pathogenic bacteria ( pr , 2.13 - table 1 ) and almost 5 times more likely to harbor fungi ( pr , 4.74 - table 1 ) than those dentists who did not wore finger rings . in a previous study on 84 nurses working in intensive care units in a rural indian those who wore rings showed more gram - positive and gram - negative bacteria than those who did not earrings , while no difference was found between nurses wearing plane wedding rings and those wearing rings with stones.4 in a sample of 20 veterinary medical students , 10 wearing plain rings , wearing rings , no difference in viable flora was detected between ring and non - ring hands.5 in a sample of 200 healthcare workers from an intensive care unit , ring wearers showed almost double prevalence of gram - negative bacteria than non - wearers , but no difference with respect of s. aureus.6 similar results have been reported from other studies conducted by hoffman et al.,7 field et al.8 in which bacterial significantly high in ringed hands compared with control hands . in an attempt to summarize these data , we could speculate that ring wearing is associable with a higher level of bacteria and fungi , including potential pathogens . such a high contamination level could be due to the lower effectiveness of hh in ring wearers than in non - wearers . in addition , the residual and relatively high level of contamination , due to inefficient hygiene , will increase during the working day , because of bacterial and fungal multiplication on the warm and moistened environment of skin beneath gloves . every study has some or the other kind of limitations , which need to be recognized . for the present study , a small sample size and all the samples from same department so some of the reported differences between dentists who wore and did not wear rings could be due to chance , which still leaves scope for further studies . clean care : safer care is the globally accepted slogan raised as a first line of defense in the global patient safety challenge , which is a core component of who s world alliance for patient safety launched in the academic year 2004.9,10 the results of the present study establish the association of ringed finger with increased microbial contamination . hence , it shows the need that a major focus must be there toward the improvement in hh to prevent hcai worldwide . the three recommended strategies to minimize hcais are as follows : generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infectionleadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . abrasions and cuts must be completely covered with a waterproof dressing . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practicedhigh - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infection leadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . abrasions and cuts must be completely covered with a waterproof dressing . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practiced high - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . clean care : safer care is the globally accepted slogan raised as a first line of defense in the global patient safety challenge , which is a core component of who s world alliance for patient safety launched in the academic year 2004.9,10 the results of the present study establish the association of ringed finger with increased microbial contamination . hence , it shows the need that a major focus must be there toward the improvement in hh to prevent hcai worldwide . the three recommended strategies to minimize hcais are as follows : generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infectionleadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practicedhigh - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . generation of awareness campaigns toward prevention of hcais like the staff involved in hospital hygiene activities must be included in education and training related to the prevention of hospital - acquired infection leadership , commitment and transparency of high standards must be practiced like the hands must be decontaminated immediately before and after every episode of direct patient contact / care . all wrist watches and hand jewelry must be removed at the beginning of each clinical shift beforehand decontamination begins . application of an alcohol - based hand rub or washing hands with a liquid soap and water to decontaminate hands between caring for different patients or between different caring activities for the same patient must be practiced high - level reinforcement of the proposed strategies must be examined by frequent surprise audits by the public health specialists at local / national levels . the challenges are enormous , but so are the reward : preventing illness , saving lives , improving patient safety , and providing an overall better quality of care to millions of patients and families . hcai are unintended , undesirable , and intolerable , but many are preventable . it is a time that hh promotion should be made a priority for public health and health care policymakers , medical and nursing schools , chief medical , and executive officers . the improvement in hh is feasible , affordable , and effective in a healthcare setting with limited resources . the who strategy represents evidence - based , ready - to - use solutions for planning and supporting hh promotion in healthcare facilities worldwide , including developing countries . however , the adoption of such a strategy on a national scale is the need of hour for patient s safety and control of hcai s . within the limitations of the present study , the general conclusion is that the potential pathogens are more likely detected in the hands of dentists who wore rings . although more such evidence is required before establishing guidelines regarding the wearing of finger rings during clinical procedures , but the results of this study adds to the evidence that wearing of finger ring can be a source of microbes and warrant more studies . resistance to fluoroquinolones ( flq ) and second - line injectable drugs ( slid ) is becoming more common , especially in eastern european countries , posing a serious threat to effective tuberculosis ( tb ) infection control . according to the most recent world health organization ( who ) report , extensively drug - resistant tb ( xdr - tb ) had been reported by 100 countries by the end of 2013 . on average , an estimated 9.0% ( 95% confidence interval [ ci [ , 6.5% to 11.5% ) of people with multidrug - resistant tb ( mdr - tb ) have xdr - tb ( 1 , 2 ) . to date , only 8 countries among the 36 with a high tb and/or mdr - tb burden have established a national surveillance system for resistance to second - line drugs among patients with mdr - tb ( 1 , 2 ) . efforts should be made to ensure that all patients diagnosed with mdr - tb undergo testing of susceptibility to flq and slid in order to initiate early effective treatment and appropriate measures of infection control . conventional phenotypic methods take weeks to months to fully define the drug resistance profile of mycobacterium tuberculosis isolates due to the low growth rate of the bacterium ( 3 , 4 ) . the development and implementation of rapid molecular assays for the detection of resistance to anti - tb drugs promise more - rapid drug resistance detection , which can be critical in areas with high rates of mdr - tb and xdr - tb and settings with limited conventional dst capacity . in contrast , the choice of molecular assays for second - line drugs is far more limited . to date , genotype mtbdrsl ( hain lifescience , nehren , germany ) is one of the few commercially available molecular tests for detection of resistance to the main second - line anti - tb drugs . it is a qualitative test for the identification of mycobacterium tuberculosis complex ( mtbc ) and its resistance to flq , to aminoglycosides and cyclic peptides ( ag / cp ) , and to ethambutol ( emb ) as either clinical isolates or pulmonary smear - positive clinical specimens ( 57 ) . diagnosis of flq resistance is enabled by the detection of mutations in the quinolone resistance - determining regions ( qrdr ) of the gyra gene ( coding for the a - subunit of the dna gyrase ) . resistance to ag / cp is detected by targeting a region of the 16s rrna gene ( encoded by the rrs gene ) , whereas identification of mutations in the embb gene enable detection of emb resistance . several studies have been conducted to evaluate the test performances in different settings and have demonstrated generally good performance characteristics and high confidence for resistance results ( 2 ) . however , sensitivities varied significantly among studies depending on the genetic background of the strains and were frequently suboptimal for kanamycin ( kan ) , in particular , with eastern european strains ( 810 ) . in eastern europe and russia , where the beijing subfamily is largely diffused , assays using the rrs gene alone failed to detect a large proportion of kan - resistant isolates ( 11 ) . in line with this observation , huang et al . showed that the low sensitivity of the genotype mtbdrsl assay was due to the presence of specific mutations not detected by the test , located in the eis gene promoter region ( 12 ) . polymorphisms in the eis promoter region lead to an increased synthesis of the enzyme and the subsequent inactivation of the drug ( 13 ) . these mutations are responsible for more than 80% of low - level kan - resistant cases ( 1315 ) , making this genetic region a valuable molecular marker of kan resistance . on the basis of published evidence , the who decided in 2013 not to endorse the second - line line probe assay ( lpa ) given that , with the test 's low pooled sensitivity , negative results can not be considered to reliably rule out resistance . the who expert group thus concluded that the assay can be used only as a method to rule in diagnosis of xdr - tb but not as a replacement for conventional phenotypic dst to rule out resistance ( 16 ) . to improve the overall performance of genotype mtbdrsl , and in particular its sensitivity for kan resistance , a new version of the assay ( i.e. genotype mtbdrsl v2.0 includes two new target genetic regions : the eis promoter region 10 to 14 ( in addition to rrs ) and the gyrb quinolone resistance - determining region ( qrdr ) ( in addition to gyra ) for the detection of mutations at codons 536 to 541 . mtbdrsl v2.0 thus includes a total of 27 probes for the detection of resistance exclusively to second - line drugs . we have conducted an international study to assess the performance of the new version of the genotype mtbdrsl assay for detection of resistance to flq and slid . the performance of the test was evaluated by comparison to dst and sequencing using a panel of 228 clinical isolates and 231 clinical specimens from subjects referred to the different study sites ( three tb supranational reference laboratories [ srl ] in europe : srl milan , italy ; srl solna , sweden ; and srl borstel , germany ) from countries with a high mdr - tb prevalence . a collection of mtbc clinical isolates and specimens obtained from the national and supranational reference laboratories involved in the fp7 project pan - european network for study and clinical management of drug - resistant tuberculosis ( tb pan - net ) ( funded by a european union grant ) were considered for the evaluation of the test performances . the collection includes strains isolated in italy and sweden and clinical specimens collected in italy , germany , and moldova . all tests were performed at a tb supranational reference laboratory ( srl ) ( srl milan , milan , italy ; srl solna , solna , sweden ; or srl borstel , borstel , germany ) . the study included 228 mycobacterial strains isolated on liquid ( bbl mgit ; bd , franklin lakes , nj , usa ) or solid ( bbl lwenstein - jensen ; bd , franklin lakes , nj , usa ) media and identified according to standard biochemical test and sequencing , as previously recommended ( 3 ) . all mtbc strains were phenotypically resistant to rifampin ( rif ) and isoniazid ( inh ) , harboring different patterns of mutations in rpob and katg or inha genes . the profiles of susceptibility to second - line drugs were determined by conventional phenotypic testing . two different panels were tested and analyzed independently , one that included 128 samples at srl milan , milan , italy , and another that included 100 samples at srl solna , solna , sweden . dna was obtained from 231 clinical specimens from patients with pulmonary mdr - tb during standard clinical management who were referred to the different study sites ( srl milan , milan , italy ; srl borstel , borstel , germany ; and nrl minsk , minsk , belarus ) for retesting . all respiratory specimens were classified as acid - fast bacillus ( afb ) positive by sputum smear microscopy . samples were decontaminated according to international guidelines using an n - acetyl - cysteine naoh procedure ( 17 ) . the drug susceptibility profile of the corresponding strains was obtained using a conventional phenotypic assay according to international guidelines . the development and validation of the mtbdrsl v2.0 assay were approved by the ethical committee of the san raffaele hospital given the role of srl milan as the coordinating institution . determinations of susceptibility to flq ( ofloxacin , levofloxacin , and moxifloxacin ) and slid ( amikacin , kanamycin , and capreomycin ) were performed either on a bactec mgit 960 system ( mgit ) ( bd bioscience , erebodegem , belgium ) or on lowenstein - jensen ( lj ) media using the proportional method according to international guidelines ( 18 ) . the dst critical concentrations used for mgit are as follows : for ofloxacin , 2 g / ml ; for levofloxacin , 1.5 g / ml ; for moxifloxacin , 0.5 g / ml ; for amikacin , 1 g / ml ; for kanamycin , 2.5 g / ml ; and for capreomycin , 2.5 g / ml . the dst critical concentrations used for solid lj media are as follows : for ofloxacin , 4 g / ml ; for amikacin , 30 g / ml ; for kanamycin , 30 g / ml ; and for capreomycin , 40 g / ml . genomic dna was extracted from isolates and decontaminated specimens by thermal lysis and sonication as described elsewhere ( 19 ) . alternatively , a commercially available kit , genolyse ( hain lifescience , nehren , germany ) , was used to extract dna from clinical specimens according to the manufacturer 's instructions . amplification and hybridization of dna extracted from isolates and clinical specimens were performed according to the manufacturer 's instructions . genomic regions harboring drug resistance mutations were amplified and sequenced ( 9 ) by the sanger method using the designed internal primers for each region . results were visualized as electropherograms and analyzed by clustalw application ( bioedit software ; ibis biosciences / abbott company , carlsbad , ca ) , aligning sequences with the corresponding reference strain ( m. tuberculosis h37rv ; genbank accession no . the performance of the mtbdrsl v2.0 assay was evaluated using conventional phenotypic dst and sanger sequencing as reference standards , following the standards for reporting of diagnostic accuracy ( stard ) recommendations ( 20 ) . sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , likelihood ratio , and diagnostic accuracy values were calculated according to the wilson score . all statistical analyses were carried out using open source epidemiologic statistics for public health version 3.03 ( 21 ) . the panel included a total of 73 strains phenotypically resistant to at least one flq ( ofloxacin or moxifloxacin ) and 155 susceptible ones ( table 1 ) . the genotype mtbdrsl v2.0 assay correctly identified 61 ( 83.6% ) flq - resistant isolates , missing 12 ( 16.4% ) phenotypically resistant strains . eleven phenotypically resistant strains showed a mix of wild - type ( wt ) and mutated bands at the level of the gyra gene , suggesting a mixed infection . phenotypic drug - susceptibility testing of 228 clinical isolates and 231 specimens used in the study fqs , fluoroquinolones ; slids , second - line injectable drugs ; r , resistant ; s , susceptible . the distribution of gene mutations in the 73 flq - resistant isolates is shown in table 2 . the predominant mutations detected by mtbdrsl v2.0 as conferring flq resistance are the gyra mut1 a90v and mut3c d94 g mutations , identified in 26 ( 35.6% ) and 15 ( 20.5% ) resistant strains , respectively . nine additional strains ( 12% ) showed a mix of wild - type sequence and a90v or d94 g mutations . gyra mutations mut3a ( d94a ) and mut3b ( d94n / d94y ) were detected in 4 ( 5.5% ) and 2 ( 2.7% ) resistant strains , respectively . the gyra mutation mut2 ( s91p ) was detected in 3 ( 4.1% ) samples either alone or together with other mutations as a mix of wild - type and mutated bands . in three cases , the lack of the gyra wild - type 3 ( wt3 ) probe was used to infer a mutation at the level of codon 94 . by sequencing the gyra gene , we confirmed the presence of mutations d94y and d94a in two and one resistant strains , respectively . fluoroquinolone resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay , phenotypic dst , and sequencing on 228 clinical isolates dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible ; np , not performed . mutations in the gyrb gene were very rare and were identified in only 1 out of 73 ( 1.4% ) resistant strains . in this unique case , the mutation could be inferred by the lack of the gyrb wild - type probe on the genotype mtbdrsl v2.0 strip . dna sequencing revealed the presence of the n538 t mutation , thus confirming the mtbdrsl test result . overall , the test sensitivity for flq resistance measured against culture - based dst was 83.6% ( 95% confidence interval [ ci ] , 73.4% to 90.3% ) whereas the test specificity was 100% ( 95% ci , 97.6% to 100% ) . the concordance between phenotypic dst and the mtbdrsl v2.0 test was 94.7% ( 95% ci , 91.0% to 97.0% ) ( table 3 ) . we also calculated mtbdrsl v2.0 test performance by looking separately at each single flq tested , namely , ofloxacin or moxifloxacin . for ofloxacin , we obtained a test sensitivity and specificity of 84.7% ( 95% ci , 74.7% to 91.2% ) and 100% ( 95% ci , 97.6% to 100% ) , respectively , whereas the test sensitivity and specificity for moxifloxacin were 90.0% ( 95% ci , 69.9% to 97.2% ) and 97.4% ( 95% ci , 91.0% to 99.3% ) , respectively . performance of genotypemtbdrsl v2.0 assay compared to phenotypic dst on 228 clinical isolates flq include ofloxacin and moxifloxacin . slid ci , confidence interval ; dst , drug susceptibility testing ; ppv , positive predictive value ; npv , negative predictive value . the comparison between the mtbdrsl v2.0 test and gyra qrdr sequencing showed an overall concordance of 98.3% ( 95% ci , 95.6% to 99.3% ) ( table 4 ) . among the 11 phenotypically resistant strains showing a mix of wild - type and mutated bands at the level of the gyra gene , sequencing showed a double - pattern profile in 9 out of 11 cases ; it identified only the mutated nucleotide in 1 case , whereas it completely missed the mutation ( d94 g codon substitutions ) in another case . sequencing also missed one fully mutated sample ( i.e. , mutation at the level of codon d94 and absence of the corresponding wild - type 3 [ wt3 ] probe ) in one case . in contrast , mtbdrsl v2.0 missed a d94y codon substitution and a d89n codon substitution in the gyra gene in two distinct isolates that were instead detected as mixed populations by sequencing ( see table 2 ) . overall , the test sensitivity for flq resistance compared to gyra gene sequencing was 96.7% ( 95% ci , 88.8% to 99.1% ) and the specificity was 98.8% ( 95% ci , 95.8% to 99.7% ) ( table 4 ) . performance of genotype mtbdrsl v2.0 assay compared to sequencing on 228 clinical isolates ci , confidence interval ; ppv , positive predictive value ; npv , negative predictive value . our panel included 147 isolates resistant to at least one slid ( amikacin , kanamycin , or capreomycin ) as determined by phenotypic dst and 81 strains susceptible to all the tested injectable drugs ( table 1 ) . the genotype mtbdrsl v2.0 assay correctly identified 127 out of 147 ( 86.4% ) slid - resistant isolates and 73 out of 81 ( 90.1% ) fully susceptible ones . the distribution of gene mutations in the 127 slid - resistant isolates identified by the mtbdrsl v2.0 test is shown in table 5 . the great majority of resistant strains carried mutations only at the level of the rrs gene , namely , 98 out of 127 ( 77.2% ) , including 12 ( 9.4% ) strains with a mixed mutated and wild - type profile . among these isolates , the most frequent mutation was rrs mut1 a1401 g , which was detected in 92 out of 98 ( 93.9% ) cases ; three ( 3% ) strains carried both a1401 g and g1484 t mutations , two ( 2% ) isolates carried the c1402 t mutation , and one isolate carried the mut2 g1484 t mutation . second - line injectable drug resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay , phenotypic dst , and sequencing on 228 clinical isolates dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible . two resistant strains carried mutations in both the rrs ( mut a1401 g ) and eis promoter regions , with one sample showing both mutated and wild - type bands . a total of 27 out of 127 ( 21.3% ) slid - resistant strains carried mutations only at the level of the eis promoter region . the most frequent mutation identified was the mut1 c-14 t mutation , which was detected in 12 out of 27 ( 44.4% ) isolates , including one strain with a mixed profile . the absence of the wild - type 2 ( wt2 ) probe in the eis region correlated with the presence of mutations g-10a , g-37 t , and c-12 t in 8 , 4 , and 3 cases , respectively . all strains with a mixed mutated and wild - type profile , in either the rrs or eis promoter region , were phenotypically resistant . a total of 20 strains phenotypically resistant to slid were detected as fully wild - type by the mtbdrsl v2.0 assay : the great majority ( 13 out of 20 [ 65% ] ) were resistant to capreomycin but susceptible to both amikacin and kanamycin , 4 isolates ( 20% ) were resistant uniquely to kanamycin , 2 isolates ( 10% ) were resistant to all slid , and one isolate was resistant uniquely to amikacin . the genotype mtbdrsl v2.0 test also detected mutations in the eis promoter region of 8 phenotypically susceptible strains . among those , 6 isolates were missing the eis wt2 probe corresponding to mutations g-10a and c-12 t , as detected by gene sequencing in 5 and 1 cases , respectively , 1 sample showed the eis c-14 t mutation , and 1 sample showed the g-37 t mutation ( table 5 ) . overall , the concordance between mtbdrsl v2.0 and phenotypic dst for slid resistance was 87.7% ( 200/228 ) , whereas the test sensitivity and specificity were 86.4% ( 95% ci , 79.9% to 91.0% ) and 90.1% ( 95% ci , 81.7% to 94.9% ) , respectively . as a test for detection of kanamycin resistance measured against culture - based dst , the diagnostic accuracy of genotype mtbdrsl v2.0 was 93.8% ( 95% ci , 89.9% to 96.3% ) , whereas the sensitivity and specificity were 95.5% ( 95% ci , 90.6% to 97.9% ) and 91.4% ( 95% ci , 83.9% to 95.6% ) , respectively ( table 3 ) . the comparison between the assay and rrs sequencing showed an overall concordance of 97.8% ( 95% ci , 95.1% to 99.1% ) , with the molecular assay correctly identifying all the mutations revealed by gene sequencing ( 100% sensitivity ) ( 95% ci , 96.2% to 100% ) . our panel included 13 strains with a mixed mutated and wild - type profile either in the rrs gene or in the eis promoter region . sanger sequencing confirmed the double - pattern profile in 7 out of 13 cases , whereas it missed the mutation in 2 cases and it recognized only the mutated nucleotide in the remaining 4 cases . we excluded from the analysis one isolate showing the lack of the rrs wt1 probe since we were not able to obtain a readable sequence , which was probably due to the low quality or partial degradation of the dna . the comparison between mtbdrsl v2.0 and sequencing of the eis gene promoter region showed an overall concordance of 98.1% ( 95% ci , 94.6% to 99.4% ) . the assay missed two mutations detected by sequencing at the level of codons 10 and 37 . in both cases , the sequencing profile showed a double population with the coexistence of both wild - type and mutated nucleotides . in one case , the lack of the eis wt2 probe did not correspond to any mutation in the corresponding genetic region of the eis promoter by sequencing . overall , the test sensitivity and specificity measured against eis sequencing were 94.9% ( 95% ci , 83.1% to 98.6% ) and 99.2% ( 95% ci , 95.4% to 99.9% ) , respectively ( table 4 ) . of the 232 respiratory specimens tested with mtbdrsl v2.0 , 231 showed an interpretable result , with only one sample not providing a valid hybridization profile for any of the 4 genomic regions . gene sequencing , either direct or using the corresponding isolate , was performed uniquely on samples with a discordant genotypic / phenotypic profile . performance of genotypemtbdrsl v2.0 assay compared to phenotypic dst on 231 clinical specimens flq include ofloxacin , levofloxacin , and moxifloxacin . slid ci , confidence interval ; dst , drug susceptibility testing ; ppv , positive predictive value ; npv , negative predictive value . our panel included a total of 57 clinical specimens phenotypically resistant to at least one flq ( ofloxacin , levofloxacin , or moxifloxacin ) and 174 fully susceptible ones ( table 1 ) . the test correctly identified 53 ( 92.9% ) of the flq - resistant clinical specimens , missing four resistant cases . in contrast , 3 specimens showed a flq - resistant profile by mtbdrsl v2.0 while being susceptible by phenotypic dst . note that 2 out of 3 of these samples showed a double - pattern profile at the level of the gyra gene , suggesting a mixed infection . the most frequent mutations detected by genotype mtbdrsl among the 53 flq - resistant strains were the gyra mut3c d94 g and the mut1 a90v mutations , each identified in 13 ( 22,8% ) and 9 ( 15,8% ) cases , respectively . in addition , 10 specimens ( 17.5% ) showed a mixed profile ( i.e. , wild - type sequence and d94 g and/or a90v mutations ) . other gyra mutations , including mut2 ( s91p ) and mut3a ( d94a ) , were detected either as fully mutant or as mixed populations in 6 and 5 flq - resistant specimens , respectively . mutations at the level of the gyrb gene were very rare ( only 2 cases ) and could be inferred by the lack of the wild - type probe . dna sequencing revealed the presence of gyrb mutations n538s and t539a , thus confirming the mtbdrsl test result . however , only one of the two samples carrying the mutations in gyrb was flq resistant by phenotypic testing . fluoroquinolone resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay and phenotypic dst on 231 clinical specimens dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible . overall , the diagnostic accuracy for flq resistance detection in clinical specimens was 97.0% ( 95% ci , 93.9% to 98.5% ) , with a sensitivity and specificity of 93.0% ( 95% ci , 83.3% to 97.2% ) and 98.3% ( 95% ci , 95.1% to 99.4% ) , respectively . we also calculated mtbdrsl v2.0 test performance by looking separately at each single flq tested , namely , ofloxacin , levofloxacin , or moxifloxacin . for ofloxacin , we obtained a test sensitivity and specificity of 92.9% ( 95% ci , 83.0% to 97.2% ) and 97.7% ( 95% ci , 94.3% to 99.1% ) , respectively ; for levofloxacin , the test sensitivity and specificity were 100.0% ( 95% ci , 67.6% to 100% ) and 91.1% ( 95% ci , 79.3% to 96.5% ) , respectively , whereas the test sensitivity and specificity for moxifloxacin were 100.0% ( 95% ci , 78.5% to 100% ) and 90.9% ( 95% ci , 62.3% to 98.4% ) , respectively . our panel included a total of 63 clinical specimens phenotypically resistant to at least one slid ( kanamycin , amikacin , or capreomycin ) and 168 fully susceptible ones ( table 1 ) . the test correctly identified 56 ( 88.9% ) of the slid - resistant samples and 154 out of 168 ( 91.7% ) of the fully susceptible ones . among the 7 slid - resistant specimens missed by the assay , 5 were susceptible to kanamycin and resistant to capreomycin or amikacin or both thirty - five out of 56 ( 62.5% ) samples classified as resistant by mtbdrsl v2.0 carried the a1401 g mutation at the level of the rrs gene , with an additional 10 samples ( 17.5% ) showing a mixed wild - type and mutated ( mut a1401 g ) profile , whereas only one sample carried the rrs c1402 t substitution . mutations at the level of the eis promoter region were detected in 9 slid - resistant specimens , with an additional sample showing a double mutation at the levels of both the rrs and eis regions . mtbdrsl v2.0 test detected mutations within the eis promoter region in 11 out of 168 ( 6.5% ) slid - susceptible samples and mutations in the rrs region in 3 out of 168 ( 1.8% ) slid - susceptible specimens . the lack of an eis wt2 probe was the most prevalent mutation , detected in 8 out of 14 susceptible specimens . direct gene sequencing of these 8 samples confirmed the presence of the c-12 t and g-10a mutations in 6 and 2 cases , respectively . all three phenotypically susceptible specimens with mutations in the rrs gene showed a double ( wild - type and mutation)-pattern profile by mtbdrsl v2.0 assay , but sequencing of the corresponding isolates resulted in a wild - type profile . the full distribution of mutations detected by the genotype mtbdrsl assay is summarized in table 8 . second - line injectable drug resistance and susceptibility results obtained by genotype mtbdrsl v2.0 assay and phenotypic dst on 231 clinical specimens dst , drug susceptibility testing ; wt , wild type ; r , resistant ; s , susceptible . overall , the diagnostic accuracy of the test for slid resistance detection in clinical specimens was 90.9% ( 95% ci , 86.5% to 94.0% ) , with a sensitivity and specificity of 88.9% ( 95% ci , 78.8% to 94.5% ) and 91.7% ( 95% ci , 86.5% to 95.0% ) , respectively . as a test for detection of kanamycin resistance , the diagnostic accuracy of genotype mtbdrsl v2.0 was 93.1% ( 95% ci , 88.9% to 95.8% ) , whereas the sensitivity and specificity were 96.0% ( 95% ci , 86.5% to 98.9% ) and 92.2% ( 95% ci , 87.1% to 95.4% ) , respectively ( table 6 ) . the genotype mtbdrsl v2.0 assay correctly identified 37 out of 46 ( 80.4% ) phenotypically identified xdr - tb isolates , thus missing 9 ( 19.6% ) cases . in particular , the test detected only slid resistance , missing flq resistance , in 7 out of 9 ( 77.8% ) xdr - tb isolates and detected only flq resistance , missing slid resistance , in the remaining two cases ( 22% ) . in contrast , the test gave a positive xdr - tb result for 8 isolates resistant uniquely to flq by detecting mutations at the level of the eis promoter region . overall , the test accuracy for detection of xdr - tb was 92.5% ( 95% ci , 88.4% to 95.3% ) , whereas the test sensitivity and specificity were 80.4% ( 95% ci , 66.8% to 89.4% ) and 95.6% ( 95% ci , 91.6% to 97.8% ) , respectively ( table 9 ) . performance of genotypemtbdrsl v2.0 assay for detection of xdr - tb measured against phenotypic dst on 228 clinical isolates and 231 clinical specimens ci , confidence interval ; ppv , positive predictive value ; npv , negative predictive value . the genotype mtbdrsl v2.0 assay correctly identified 18 out of 22 ( 81.8% ) phenotypically identified xdr - tb clinical specimens , while it missed flq resistance in 2 cases , slid resistance in 1 case , and both flq resistance and slid resistance in 1 case . however , the test also gave a positive xdr - tb result for 3 specimens that were flq resistant only by detecting mutations at the level of the eis promoter region and for one sample that was slid resistant only by failing to detect the gyrb wt probe . overall , mtbdrsl v2.0 diagnostic accuracy for detection of xdr - tb was 96.5% ( 95% ci , 93.3% to 98.2% ) , with a sensitivity and specificity of 81.8% ( 95% ci , 61.5% to 92.7% ) and 98.1% ( 95% ci , 95.2% to 99.3% ) , respectively ( table 9 ) . this was the first multicenter study to assess the diagnostic accuracy of the novel version ( v2.0 ) of the genotype mtbdrsl test for detection of resistance to flq ( ofloxacin , moxifloxacin , and levofloxacin ) and slid ( amikacin , kanamycin , and capreomycin ) . similarly to genotype mtbdrsl v1.0 , the concordance between mtbdrsl v2.0 and gene sequencing was > 97% for all the genomic regions analyzed . discrepancies were mainly observed in cases of isolates showing a double - pattern profile ( 15% for gyra and 8.2% for rrs ) , which could be explained by the known lower sensitivity of sanger sequencing in detecting heteroresistance ( 22 , 23 ) . all heteroresistant strains were phenotypically resistant , thus confirming the presence of the mutated strain . the inclusion of probes for the detection of mutations in the eis promoter region increased the overall sensitivity of mtbdrsl v2.0 for the detection of slid resistance to 86.4% ( 95% ci , 79.9% to 91.0% ) and the sensitivity for the detection of kanamycin resistance to 96% for both the isolates and the clinical specimens , which is higher than the pooled sensitivity values for mtbdrsl v1.0 ( 66.9% ; 95% ci , 44.1% to 83.8 ) ( 2 ) . notably , the assay primarily missed strains that are resistant uniquely to capreomycin , namely , 13 out of 20 ( 65% ) among isolates and 4 out of 7 ( 57% ) among clinical specimens , which is expected given that the mechanisms of resistance to capreomycin partly differ from those causing resistance to aminoglycosides ( 13 ) . if , on the one side , the inclusion of the eis promoter region increased the test sensitivity , on the other , it negatively affected its specificity , which decreased from 99.5% ( 95% ci , 97.1% to 99.9% ) in mtbdrsl v1.0 ( 2 ) to 91.1% ( 95% ci , 86.43% to 94.9% ) and 90.1% ( 95% ci , 81.7% to 94.9% ) in mtbdrsl v2.0 for direct and indirect testing , respectively . as expected , the discordance between the results from the slid - resistant samples as defined by this molecular test and phenotypic dst involved primarily samples carrying mutations in the eis promoter region : 8 out of 8 among isolates and 11 out of 14 ( 78.6% ) among clinical specimens . the majority of isolates with a discordant phenotypic / genotypic profile carried the g-10a mutation ( 62.5% ) , whereas the most frequent eis mutation among the discordant clinical specimens was c-12 t ( 6 of 11 ; 54.4% ) , followed by mutations c-14 t ( 27.3% ) and g-10a ( 18.2% ) . in line with published data , the c-12 t eis mutation was more often found in sensitive strains and thus appears to be a nonspecific marker of resistance to slid ( 24 , 25 ) . in contrast , the g-10a nucleotide substitution was reported to correlate well with kanamycin resistance in several studies ( 13 , 24 , 25 ) , whereas only engstrm et al . more evidence is thus required to univocally define the role of each mutation within the eis promoter region in the development of resistance to kanamycin and of cross - resistance to the other classes of injectable drugs . this also implies the necessity to include in the assay specific probes able to discriminate between eis mutations at the level of different nucleotides , in particular , in positions 12 and 10 . in addition , since resistance to capreomycin and amikacin can not be automatically inferred on the basis of the presence of eis mutations , results from mtbdrsl v2.0 testing for slid resistance should be reported accordingly . if , on the one hand , our understanding of the role of specific mutations within the eis promoter region and slid resistance is still limited , on the other , phenotypic dst might be a suboptimal gold standard for the assessment of the test performance . data from a recent publication by the alland group strongly suggest that eis promoter mutants should be considered to have low to moderate kanamycin resistance even if resistance is not detected by lj - based or even mgit - based susceptibility tests ( 27 ) . concerning the rrs gene , in line with previous observations ( 9 , 24 , 28 ) , we found a high correlation between mutations in this genetic region and slid resistance , with 100 out of 100 mutated isolates and 47 out of 50 ( 94% ) clinical specimens being phenotypically resistant to at least one slid . notably , the three specimens giving false slid resistance results were characterized by a double - pattern profile compatible with a mixed infection , which could explain the discrepancy with the phenotypic dst result . in such cases , the presence of a predominant susceptible strain might have in fact concealed the resistance ( 29 ) . also , in line with published data ( 9 , 24 , 28 , 30 ) , we found a high correlation between rrs mutations and cross - resistance to amikacin , kanamycin , and capreomycin , with 90% of mutated strains and 98% of mutated clinical specimens being resistant to both aminoglycosides and capreomycin . notably , the two strains and one clinical specimen carrying the rrs mutation c1402 t were resistant to all injectable drugs . overall , the incidence of the rrs c1402 t and g1484 t mutations in our panel was very low , with only 3 strains out of 228 and 3 specimens out of 231 carrying uniquely the g1484 t and c1402 t substitutions . given the low prevalence of these mutations ( 24 ) , rrs codons 1484 and 1402 have a low positive predictive value of slid resistance and are thus unlikely to provide strong clinical significance . the inclusion of probes for the detection of mutations in the qrdr of the gyrb gene did not significantly improve the overall sensitivity and specificity of the mtbdrsl v2.0 test compared to mtbdrsl v1.0 . indeed , the incidence of gyrb mutations , all inferred by the lack of the gyrb wt probe , was very low , with only 1 case among 228 strains tested and 2 cases among 231 clinical specimens . mutations found in gyrb , in the absence of gyra substitutions , correlated with phenotypic flq resistance in 2 out of 3 cases . given the very low prevalence of gyrb mutations ( 3133 ) and thus their low positive predictive value for flq resistance , the inclusion of gyrb probes in the new version of the test is unlikely to be clinically relevant . to date , genotype mtbdrsl has been one of the few rapid molecular assays available on the market for the detection of resistance to second - line drugs . given its high positive predictive value ( ppv ) and specificity , a positive result for flq resistance , slid resistance , or xdr - tb can be treated with confidence ( 2 ) . however , the low sensitivity , especially for detection of kanamycin resistance and its low negative predictive value ( npv ) , makes it necessary to confirm by conventional dst all cases classified as susceptible . the inclusion of probes for the detection of mutations at the level of the eis promoter region in the new version of the assay , mtbdrsl v2.0 , leads to a significant increase in test sensitivity for the detection of kanamycin resistance compared to the original version . importantly , these mutations allow the identification of strains with low or moderate kanamycin resistance which might be erroneously classified as susceptible by conventional phenotypic testing alone . if , on the one hand , this raises the problem of dealing with a suboptimal gold standard when assessing the performance of a new diagnostic test , on the other , it highlights the necessity to understand whether this low - level resistance has a real impact on the treatment efficacy . therefore , we need to further investigate the correlation between specific mutations and the level of drug responsiveness in vivo by routinely collecting clinical data on treatment outcomes . this will be essential for improving our capacity to correctly interpret the mutations identified by genotypic testing . summarizing , the mtbdrsl v2.0 assay has better performance characteristics for the detection of xdr - tb when performed either directly or indirectly on samples , by correctly identifying more than 80% of xdr - tb cases , than version 1.0 , which misses the xdr phenotype in between one in four and one in three cases ( 2 ) . on clinical specimens , mtbdrsl v2.0 showed a ppv and a npv of 81.8% ( 95% ci , 61.5% to 92.7% ) and 98.1% ( 95% ci , 95.2 to 99.2 ) , respectively . notably , the prevalence of xdr - tb cases in our panel of clinical specimens ( i.e. , a setting with high prevalence of mdr - tb ) is around 9.5% , which corresponds to the who - estimated prevalence of xdr - tb among mdr - tb cases ( 2 ) . therefore , our sample panel provides an evaluation of the diagnostic performance of mtbdrsl v2.0 in a real - world setting . indeed , the use of this rapid assay in countries with a high rate of drug resistance , especially kanamycin resistance , will further increase the test ppv and will reduce the number of xdr - tb cases missed by the test . despite some limitations , such as the fact that we tested smear - positive clinical samples only , the fact that flq resistance was measured primarily against ofloxacin phenotypic dst , and the lack of data on the mic for isolates carrying mutations in the eis promoter region , this report provides an accurate evaluation of the diagnostic performances of the new version of the genotype mtbdrsl assay based on standardized testing of a large set of samples from different settings . we show that the mtbdrsl v2.0 test has better performance characteristics in terms of increased sensitivity for detecting slid and kanamycin resistance and better accuracy for detecting xdr - tb than the original version of the test . mtbdrsl v2.0 thus represents a better screening tool for the rapid detection of resistance to second - line drugs , and it should be recommended in countries with a high burden of mdr / xdr - tb or in settings with high reported rates of resistance to ofloxacin in new and retreatment cases . in such contexts , the test can rapidly identify patients for whom mdr treatment regimens that include earlier - generation fluoroquinolones are likely to fail or who may not be good candidates for the shorter mdr treatment currently being tested in western and central africa ( 34 ) .

background : disease prevention is better than its cure . the role of healthcare worker s hand in the transmission and spread of an infectious disease to the patient is well acknowledged . indeed , the hands of a health care worker can easily pick potentially pathogenic bacteria and fungi from hand touch surfaces before wearing of gloves . for these microorganisms to multiply rapidly , a moist environment present underneath the gloves acts a good cultivating media . it is also reported that the multiplication rate also increases several folds with the duration of glove use.materials and methods : dentists 20 with rings and 20 without rings were considered . skin samples from the hand soon after professional hand cleaning and glove disposal were collected . the occurrence of potentially pathogenic fungi and bacteria were examined and investigated disposal were collected . the occurrence of potentially pathogenic fungi and bacteria were examined and investigated with biochemical and cultural laboratory tests.results:bacteria and fungi were significantly more frequent in dentist s hand with rings than those without rings . 63% versus 37% ( bacterial prevalence ) , among the isolated potentially pathogenic microorganisms were staphylococcus aureus , escherichia coli , and candida albicans.conclusion:in the present study potentially pathogenic microorganisms were more frequent in dentists who wore finger rings under gloves . resistance to fluoroquinolones ( flq ) and second - line injectable drugs ( slid ) is steadily increasing , especially in eastern european countries , posing a serious threat to effective tuberculosis ( tb ) infection control and adequate patient management . the availability of rapid molecular tests for the detection of extensively drug - resistant tb ( xdr - tb ) is critical in areas with high rates of multidrug - resistant tb ( mdr - tb ) and xdr - tb and limited conventional drug susceptibility testing ( dst ) capacity . we conducted a multicenter study to evaluate the performance of the new version ( v2.0 ) of the genotype mtbdrsl assay compared to phenotypic dst and sequencing on a panel of 228 mycobacterium tuberculosis isolates and 231 smear - positive clinical specimens . the inclusion of probes for the detection of mutations in the eis promoter region in the mtbdrsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing ( 96% and 95.4% , respectively ) than that seen with the original version of the assay , whereas the test sensitivities for detection of flq resistance remained unchanged ( 93% and 83.6% for direct and indirect testing , respectively ) . moreover , mtbdrsl v2.0 showed better performance characteristics than v1.0 for the detection of xdr - tb , with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing , respectively . mtbdrsl v2.0 thus represents a reliable test for the rapid detection of resistance to second - line drugs and a useful screening tool to guide the initiation of appropriate mdr - tb treatment .

psoas abscess is characterized by infrequent incidence , insidious onset , and nonspecific clinical features , which can lead to misinterpretation and delayed diagnosis ; thus , it is difficult to clinically diagnose psoas abscess1,5 ) . here , we present a rare case of tuberculous spondylitis and a large abscess in the left psoas muscle that occurred after spinal surgery for an acute burst fracture of the second lumbar ( l2 ) vertebral body caused by a traffic accident . moreover , the first radiological examination revealed some unusual findings that could have possibly indicated abscess before his spinal surgery . in may 2009 , a 44-year - old man was transferred to our hospital for lower back pain after he suffered from a blunt trauma in a pedestrian traffic accident . his medical and surgical histories were unremarkable . neurological examination and chest radiology revealed no abnormalities . laboratory evaluation revealed a erythrocyte sedimentation rate ( esr ) of 48mm / hr ( normal range , 0 - 10mm / hr ) and c - reactive protein ( crp ) of 239.52mg / l ( 0 - 5mg / l ) , and a normal wbc count of 7740/l ( 4000 - 10,000/l ) . lumbar computed tomography ( ct ) scan with bone window settings revealed a burst fracture of the l2 vertebral body with displacement of a posterior fragment into the spinal canal ( fig . lumbar magnetic resonance imaging ( mri ) revealed acute burst fracture with retropulsion of the posterior bone fragment at the l2 vertebral body and acute compression fracture of the l1 vertebral body ( fig . he underwent posterior reduction , transpedicular screw fixation , secondary anterior thoracoscopic decompression , and anterior reduction that was accomplished with a distractible cage and a modular anterior construct systemthoraco - lumbar ( macs - tl ) plate ( aesculap , tuttlingen , germany ) ( fig . the patient had no postoperative complications and was discharged on the 45 postoperative day . until july 2010 , the patient 's clinical condition was satisfactory ; thereafter , he exhibited a gradually enlarging swelling in the left flank , associated with pain extending up to the left thigh , and urinary incontinence . his esr was 120mm / h ; crp level , 59.94mg / l ; blood urea nitrogen ( bun ) level , 28.1mg / dl ; and serum creatinine level , 1.77mg / dl . abdominopelvic ct revealed the presence of a large abscess in the following sites : left psoas muscle , lower pole of the left kidney , and posterolateral abdominal wall . 2 ) . abscess drainage and irrigation and partial nephrectomy through the previous surgical route were performed under general anesthesia . the abscess and surrounding soft tissue samples yielded positive results in the acid - fast bacilli ( afb ) culture and polymerase chain reaction ( pcr ) for mycobacterium tuberculosis complex . the patient received antituberculous chemotherapy , consisting of isoniazid , rifampin , pyrazinamide , along with pyridoxine supplements , on a daily basis . in november 2010 the patient was transferred to the urological department for treatment of the left ureteral stricture . initially , the predominant pathogen was mycobacterium tuberculosis , which caused psoas abscess through either hematogenous spread or direct extension from vertebral osteomyelitis of the lumbar spine ( pott 's disease ) . with the decline of mycobacterium tuberculosis as a major pathogen of psoas abscess in developed countries , the condition was mostly detected secondary to diseases of the digestive tract , with other pathogens such as staphylococcus aureus gaining more dominance in the pathogenesis of this psoas abscess4,7 ) . however , in recent years , tuberculosis - induced psoas infections are again emerging with the recurrence of tuberculosis in industrial countries and increase in the number of human immunodeficiency virus ( hiv ) infections5 ) . mckley et al5 ) emphasized the role of the spine as the primary source of infection for secondary psoas abscess . according to the most case reports on psoas abscesses , tuberculous psoas abscess occurring secondary to direct extension from other adjacent structures or even hematogenous seeding from a distant site is less frequent8 ) . however , there have been hardly any reports on tuberculous psoas abscess presenting as a manifestation of tuberculosis , without evidence of an active infection elsewhere8 ) . even in the case of our patient , there was no evidence of a tubercle bacillus infection anywhere but except in the surgical portion . a retrospective review of the first mri of our patient revealed some unusual findings of the left psoas muscle , which had been overlooked earlier . axial mri of the l2 vertebra revealed that the left psoas muscle was irregular shaped , ill - defined , and heterogeneously enlarged compared to the right psoas muscle and both paraspinal muscles at the level of the vertebral burst fracture . in addition , the coronal t2-weighted mri revealed a thick line of high - signal intensity along the left psoas muscle ( fig . even though it is very difficult to determine whether asymmetric swelling of the left psoas muscle was due to trauma or infection , we consider that this unusual finding was a result of the infection . if it was caused by the trauma , there would have been more contusions of the paraspinal muscle than that of the psoas because the psoas is positioned anterior to the paraspinal muscle . moreover , because the abnormality was detected only in the left psoas and not in either paraspinal muscle or the right psoas , it is difficult to determine whether the condition was a result of the trauma . we also consider that the possibility of tuberculous spondylitis extending to the vertebral body before injury is low . we assume that vertebral fracture and subsequent spinal surgery can make the progress of the primary psoas abscess around surrounding tissues quicker . in the majority of earlier cases , the patients , as in our case , although fever , abdominal or back pain , and limited hip - joint mobility form the classical triad of psoas abscess , they can be inconsistently presented1 ) . retrospective examination revealed that our patient also had abdominal pain and intermittent limited hip - joint mobility , weeks before the traffic accident . therefore , suspicious clinical finding , radiological study , and microbial culture study of the pus , are crucial in the diagnosis of tuberculous psoas abscess . this report might be significant , considering the possibility of missed diagnosis of psoas abscess in patients with acute lumbar compression fracture caused by trauma , even though the unusual findings of the left psoas muscle in the first mri were not histologically examined . unfortunately , because we mainly focused on the traumatic injury , we did not conduct a medical examination through interrogation and radiology prior to surgery , and as a result , diagnosis and treatment of psoas abscess and tuberculous spondylitis were considerably delayed . therefore , despite the critical state of the patient in similar emergencies , surgeons should always pay close attention to the radiological findings and clinical symptoms of the patients , before considering a surgical intervention or biopsy . internet has become a ubiquitous feature at home , school and the work place as the world - wide population of internet users has grown rapidly in the recent years . internet user population world - wide has doubled from 1.15 billion to 2.27 billion and has grown from 418 million to 1 billion in the last 5 years in asia . rapid expansion and proliferation of the internet has provided better opportunities for communication , information and social interaction . however , the excessive undisciplined use by , some individuals has led to the emergence of the concept of internet addiction . the proposed diagnostic criteria for internet addiction are obsessive thoughts about internet and the use of the internet more than is intended or despite its negative consequences including withdrawal symptoms and tolerance . studies have shown that young internet users and males have a higher risk of addiction . this addiction has been linked to intermediate psychosocial variables such as avoidance , ( escapism ) male gender and applications such as online games . excessive use and pre - occupation about the internet has been shown to affect day - to - day life activities , time management , marriage , sexual life , productivity at work , education and academic work . studies have shown that excessive use of the internet is associated with such psychiatric conditions as anxiety , depression , social phobia , impulsiveness and substance abuse , as well as the induction of seizures , insufficient sleep and social isolation . an increasing number of young malaysians are resorting to the internet as the medium of choice for information and entertainment . a study by an international marketing firm taylor nelson sofres which observed the habits and behavior of internet users found that the largest number of social network partners belonged to malaysian internet users with the highest average number of 233 digital friends when compared with their peers from other countries . another survey by microsoft conducted among 2800 internet users in 11 countries in the asia - pacific region has shown that 71% of malaysian internet users spend more than 1 h / day on cyber social activities and that the largest number of internet friends on facebook belonged to malaysian internet users . studies from various countries have reported on internet addiction , its association with mental health , yet no reports have come from malaysia . therefore , an online survey was carried out of regular internet users to assess the frequency of the use of various internet applications and explore the association of compulsive internet use with mental health and socio - demographic factors . a cross - sectional online survey was carried out by university tunku abdul rahman ( utar ) . the utar opinion poll survey is an initiative undertaken by a committee with representatives from all faculties of the university . every month , the committee plans and executes opinion poll surveys on various topics of general interest about the youth in malaysia ( http://poll.utar.edu.my ) . the registered utar opinion poll survey participants are young adults from various parts of malaysia , most of whom are young university students with access to the internet . sampling method and sample size calculation were not used since this was an exploratory study . the survey also provided an option for participants to invite their friends to take part . the online survey questionnaire had sections on demographic information such as age , gender , marital status , monthly income , educational status , employment status , questions on availability of internet at the work place and the typical number of hours spent online per day and applications or the purpose for which the internet is used . frequency of using various internet applications such as gaming , social network , e - mails etc . , were assessed on a five - point likert scale as never , seldom , sometimes , often and always . the survey also contained a 12-item general health questionnaire ( ghq-12 ) and compulsive internet use scale ( cius ) . ghq-12 designed by goldberg , has reliability coefficients ranging from 0.78 to 0.95 in various studies with a well - established international validity . for example , the options given for each question in ghq-12 and the scores assigned ( as shown in parenthesis ) are better than usual - ( 0 ) , same as usual - ( 1 ) , less than usual - ( 2 ) and much less than usual - ( 3 ) . ghq-12 scores are calculated by summing up the scores of all 12 items giving a score range of 0 - 36 . a score > 15 suggests evidence of distress and a score > 20 suggests severe problems and psychological distress . ghq-12 and its scoring is available at http://www.workhealth.org/uci%202007/practicum%2008%20ghq%202007.pdf . cius is an easily administered , psychometrically sound , valid instrument used to assess the severity of compulsive internet use . cius originates from an analysis of criteria for dependence and obsessive - compulsive disorder as found in the dsm - iv , literature on behavioral addictions and from qualitative research on self - declared internet addicts . it has a high internal consistency and high correlations with concurrent and criterion variables demonstrating its good validity . for example , the options for each question and the scores assigned ( as shown in parenthesis ) are never - ( 0 ) , seldom - ( 1 ) , sometimes - ( 2 ) , often - ( 3 ) and always - ( 4 ) . scores are calculated by summing up the scores of all 14 items giving a score range of 0 - 36 . however , it has no cut - off point to categorize a participant as an internet addict . data extracted into microsoft excel was converted into statistical package for social sciences ( spss inc . the relationship of cius score with demographic factors and ghq was explored using bivariate statistics . correlation coefficients for ghq score and cius score with age , daily hours and years of internet use were calculated . mean ghq and cius scores were compared with categories of demographic variables and the statistical significance of the difference in mean scores was tested using independent samples t - test or anova as appropriate . the relationship of cius score with ghq , daily hours and years of internet use and socio - demographic variables was tested by linear regression analysis . a p < 0.05 was considered as significant . a cross - sectional online survey was carried out by university tunku abdul rahman ( utar ) . the utar opinion poll survey is an initiative undertaken by a committee with representatives from all faculties of the university . every month , the committee plans and executes opinion poll surveys on various topics of general interest about the youth in malaysia ( http://poll.utar.edu.my ) . the registered utar opinion poll survey participants are young adults from various parts of malaysia , most of whom are young university students with access to the internet . sampling method and sample size calculation were not used since this was an exploratory study . the survey also provided an option for participants to invite their friends to take part . the online survey questionnaire had sections on demographic information such as age , gender , marital status , monthly income , educational status , employment status , questions on availability of internet at the work place and the typical number of hours spent online per day and applications or the purpose for which the internet is used . frequency of using various internet applications such as gaming , social network , e - mails etc . , were assessed on a five - point likert scale as never , seldom , sometimes , often and always . the survey also contained a 12-item general health questionnaire ( ghq-12 ) and compulsive internet use scale ( cius ) . ghq-12 designed by goldberg , has reliability coefficients ranging from 0.78 to 0.95 in various studies with a well - established international validity . for example , the options given for each question in ghq-12 and the scores assigned ( as shown in parenthesis ) are better than usual - ( 0 ) , same as usual - ( 1 ) , less than usual - ( 2 ) and much less than usual - ( 3 ) . ghq-12 scores are calculated by summing up the scores of all 12 items giving a score range of 0 - 36 . a score > 15 suggests evidence of distress and a score > 20 suggests severe problems and psychological distress . ghq-12 and its scoring is available at http://www.workhealth.org/uci%202007/practicum%2008%20ghq%202007.pdf . cius is an easily administered , psychometrically sound , valid instrument used to assess the severity of compulsive internet use . cius originates from an analysis of criteria for dependence and obsessive - compulsive disorder as found in the dsm - iv , literature on behavioral addictions and from qualitative research on self - declared internet addicts . it has a high internal consistency and high correlations with concurrent and criterion variables demonstrating its good validity . for example , the options for each question and the scores assigned ( as shown in parenthesis ) are never - ( 0 ) , seldom - ( 1 ) , sometimes - ( 2 ) , often - ( 3 ) and always - ( 4 ) . scores are calculated by summing up the scores of all 14 items giving a score range of 0 - 36 . however , it has no cut - off point to categorize a participant as an internet addict . data extracted into microsoft excel was converted into statistical package for social sciences ( spss inc . the relationship of cius score with demographic factors and ghq was explored using bivariate statistics . correlation coefficients for ghq score and cius score with age , daily hours and years of internet use were calculated . mean ghq and cius scores were compared with categories of demographic variables and the statistical significance of the difference in mean scores was tested using independent samples t - test or anova as appropriate . the relationship of cius score with ghq , daily hours and years of internet use and socio - demographic variables was tested by linear regression analysis . a p < 0.05 was considered as significant . of the 330 respondents who completed the online survey , 185 ( 55.2% ) were females and 145 ( 44.8% ) were males with a mean age of 23.17 ( sd = 3.84 ) . two hundred and three ( 61.5% ) respondents were full - time students , 247 ( 74.8% ) were single ; 214 ( 64.8% ) had a bachelor 's degree and 204 ( 61.8% ) had monthly income of < 2000 malaysian ringgits ( 1 malaysian ringgit 0.32 us dollar ) [ table 1 ] . the average years of internet use was 8.7 ( sd = 3.85 ) and the mean hours of daily internet use was 6.56 ( sd = 3.73 ) [ table 2 ] . a majority of participants had an internet connection either at home ( 93.3% ) , or in college or at the work place ( 96.1% ) and 62.7% had an internet connection on their mobile phones ( data not shown ) . association of demographic factors with cius score and ghq score bivariate correlation coefficients of cius score and ghq scores with age , durations of internet use and ghq score the overall mean cius score was 19.85 ( sd = 10.57 ) and the mean ghq score was 15.47 ( sd = 6.29 ) . according to the recommended ghq-12 cut - off scores , 55.5% ( 183/330 ) respondents showed evidence of psychological distress and 28.2% ( 93/330 ) had severe distress . bivariate correlation between cius score and ghq score was 0.249 ( p < 0.001 ) . mean cius scores were compared with categories of socio - demographic variables of gender , education , income , marital status and job status . of these , the mean cius scores were significantly high among respondents who had a master 's or a higher degree [ table 1 ] . cius score was compared with age , years of internet use and the number of hours the internet is used daily , using bivariate correlations and the correlation coefficients were 0.118 , 0.014 and 0.242 respectively . all the correlations were weak , but statistically significant for age ( p = 0.03 ) and hours of internet use daily ( p < 0.001 ) [ table 1 ] . a comparison of the mean ghq scores among categories of gender , education , income , marital status and job status exhibited no statistically significant differences . a comparison of ghq score with age , years of internet use and daily hours of internet use by bivariate correlations gave correlation coefficients of 0.040 , 0.033 and 0.017 respectively suggesting that there was no correlation and no statistical significance ( p > 0.05 ) [ table 2 ] . a majority of the participants responded that they often / always used the internet for social networking ( 85.5% ) and personal e - mails ( 78.5% ) , followed by work - related surfing ( 67.2% ) and general information search ( 63.9% ) . the frequency of the use of the youtube and movie websites on the internet and to download ( software , movies , music etc . ) reported as often / always was 54.2% [ table 3 ] . frequency of using internet applications by multiple linear regression analysis age ( = 0.111 , p = 0.033 ) and marital status ( = 0.124 , p = 0.018 ) were negatively associated with cius score whereas daily hours of internet use ( = 0.269 , p = 0.001 ) and ghq score ( = 0.259 , p = 0.001 ) were positively associated with cius score after adjusting for the effect of socio - demographic variables [ table 4 ] . association of cius score with ghq-12 score , age , sex , marital status and hours of internet use per day social networking , personal e - mails and general information search were the most frequently used internet applications . mean cius score was not high , but its range was wide and weakly correlated with daily hours of internet use , age and marital status and ghq scores . by ghq case criterion , nearly a quarter of the respondents had severe psychological distress , but the proportion with internet addiction could not be reported since unlike the more recent internet addiction tests , cius does not have a cut - off score . however , studies using internet addiction test have reported the prevalence of internet addiction of adolescents from taiwan as 31 ( 19.8% ) , china 32 ( 6.44% ) , greece 33 ( 11.6% ) , japan 34 ( 8 - 10% ) and malaysia 35 ( 29% ) . the pattern of internet usage in our study was similar to the findings of a survey by accenture ( a multinational management consulting , technology services and outsourcing company ) which has reported that social network sites usage ( sns ) was the preferred method of communication among adolescents in malaysia , which ranks first as the most sns active country ( malaysian communications and multimedia commission ) . similar to the results of our study and those from usa and korea , the same study also showed that sns usage had a significant negative effect on psychological well - being . other studies about mental health and internet use have reported that mental health problems such as depression , low self - esteem , high level of stress , distorted view of body image , loneliness and prior addictions increased the risk of internet addiction . cius scores in the higher range are consistent with the frequency of internet addiction reported in studies from china and korea . research has suggested that the ease of access will tend to facilitate the use of internet from home , school / the work place , or mobile networks . this convenience has encouraged the use of the internet to the extent of addictive behavior . it is reasonable to argue that easy access to the internet may be another possible risk factor for addiction . the internet is an essential tool for learning in the modern education system , but a study from taiwan has reported availability of the internet as a possible risk factor for addiction among college students . nevertheless , it is not easy to determine the optimal use of internet or put restrictions on the use of various applications . in our study , the number of hours of daily internet use was associated with cius score , but years of internet use was not since most ( > 90% ) of our participants had an internet connection at home or place of work / study , increasing the likelihood of their using it for longer hours . our findings of positive correlation between cius and ghq scores are in agreement with the emerging concept of association between internet addiction and mental health . as cross - sectional design can only identify the views and reported behavior of respondents at a particular point in time . further research to observe actual behavior or a prospective study to follow - up the internet users might be helpful . despite online anonymous survey , some responses may have been biased leading to a reporting bias . the sample surveyed might have been relatively healthy , accounting for relatively low to moderate correlations found between the cius score and the ghq score . only about 30% of registered participants responded to the survey , but there was no information about those who did not complete the survey ( non - respondents ) for us to assess how non - response may have affected our results . future research directed at answering questions on the relationship between problematic or addictive use of the internet and mental health should include a more heterogeneous sample , in terms of age , occupation ( i.e. non - students ) and education . as cross - sectional design can only identify the views and reported behavior of respondents at a particular point in time . further research to observe actual behavior or a prospective study to follow - up the internet users might be helpful . despite online anonymous survey , some responses may have been biased leading to a reporting bias . the sample surveyed might have been relatively healthy , accounting for relatively low to moderate correlations found between the cius score and the ghq score . only about 30% of registered participants responded to the survey , but there was no information about those who did not complete the survey ( non - respondents ) for us to assess how non - response may have affected our results . future research directed at answering questions on the relationship between problematic or addictive use of the internet and mental health should include a more heterogeneous sample , in terms of age , occupation ( i.e. non - students ) and education . compulsive internet use was weakly correlated with ghq score and hours of internet use daily . internet users should be educated about the ill effects of over - use and ways of spending time away from internet .

the authors present a rare case of tuberculous spondylitis and a large abscess in the left psoas muscle that occurred after spinal surgery for an acute traumatic burst fracture of the l2 vertebral body . we retrospectively reviewed the patient 's first magnetic resonance imaging ( mri ) we found that some unusual findings , indicative of psoas abscess had been overlooked . as a result , diagnosis and treatment of tuberculous psoas abscess and spondylitis were considerably delayed . despite the critical condition of patients in a similar emergency , surgeons should always pay close attention to the radiological findings and clinical symptoms of the patient before considering a surgical intervention or biopsy . background : the last decade has seen the emergence of the internet as the prime communication medium changing the way people live and interact . studies from various countries have reported on internet addiction and its association with mental health , but none have come from malaysia.objectives:we aimed at assessing the frequency of the use of various internet applications and exploring the association of compulsive internet use with mental health and socio - demographic factors.materials and methods : a cross - sectional online survey was carried out among participants registered for the monthly opinion poll survey of university tunku abdul rahman , malaysia . the questionnaire contained socio - demographic information , the use of various internet applications on a five - point likert scale , compulsive internet use scale ( cius ) and 12 item general health questionnaire ( ghq-12 ) . correlations and linear regression analyzes were carried out.results:of the 330 respondents , 182 were females and 148 were males . the mean age was 23.17 ( sd = 3.84 ) . mean cius score was 19.85 ( sd = 10.57 ) and mean ghq score was 15.47 ( sd = 6.29 ) . correlation coefficients of cius score with age , years of use and daily hours of internet use were 0.118 ( p = 0.03 ) , 0.014 ( p = 0.81 ) and 0.242 ( p < 0.001 ) respectively . multiple linear regression analysis showed that age ( = 0.111 , p = 0.033 ) and marital status ( = 0.124 , p = 0.018 ) were negatively associated with cius scores whereas daily hours of internet use ( = 0.269 , p = 0.001 ) and ghq score ( = 0.259 , p = 0.001 ) were positively associated with the cius score.conclusions:compulsive internet use was correlated with ghq score . more research is needed to confirm our results . psychologists may consider assessing internet addiction when evaluating young psychiatric patients .

randomized controlled trials are primarily designed and conducted to meet the needs of regulatory bodies , so as to provide evidence on the efficacy and the safety of new drugs or other health care interventions . however , these studies are generally insufficient by themselves to meet the evidentiary needs of many health technology assessment agencies , that is , evidence on the drug s effectiveness . similarly , clinicians and payers desire evidence on comparative effectiveness of new drugs immediately after launch to take informed decisions.1 the sooner valid comparative effectiveness research results can be generated , the more useful they are to patients , clinicians , and payers . comparative effectiveness research using secondary health care data ( including electronic medical records , longitudinal claims data , and registries ) provides evidence on the benefits and risks of drugs in routine medical practice.1 however , channeling bias is a potential risk when relative effectiveness of a newly marketed drug compared to an established drug is investigated in observational data . channeling bias may occur when a newly marketed drug and an established drug , despite similar therapeutic indications , are prescribed to patients with different prognostics characteristics.2 over time , the prognostic characteristics of the patients who prescribed the two drugs may become more balanced as the newly marketed drug becomes more established . reasons for channeling bias could be a belief in extra advantages of the new drug compared to the established drug , or simply because doctors do not know how else to treat a subgroup of patients due to intolerance or low response to established drugs . it is possible that patients with a better prognostic are channeled to the newly marketed drug , but it is often theorized that a newly marketed drug is predominantly prescribed to patients with worse prognostics compared to the established drug being prescribed to patients with better prognostics.1,2 when channeling bias occurs , comparative effectiveness research becomes challenging because comparison with a drug will be confounded and knowledge of comparative effectiveness close to market entry may be biased or absent . the first glucagon - like peptide-1 analogs ( glp-1 ) , exenatide , was approved by the european medicines agency ( ema ) in november 2006 , and the first dipeptidyl peptidase-4 inhibitor ( dpp-4i ) , sitagliptin , was approved by the ema in march 2007 . a us study on prescription pattern of exenatide during the first half year after approval showed that a higher proportion of obese patients used exenatide compared to patients with other glucose - lowering drugs , indicating an awareness of weight - lowering effects of glp-1.3 three us studies46 compared the characteristics of sitagliptin users with patient receiving other oral glucose - lowering drugs ; two of the studies4,5 compared 20062007 versus 20082010 , and the third study6 investigated the first 2 years after approval of sitagliptin . the three studies46 found that dpp-4i users were older and have more comorbidities than patients receiving other oral glucose - lowering drugs , but with no change over time . these studies36 were all descriptive and did not investigate if the differences in characteristics could be taken into account in an outcome analysis . the objectives of this study were , first , to compare the characteristics of patients initiating glp-1 to those initiating basal insulin and of patients initiating dpp-4i to those initiating sulfonylurea stratified on calendar time and , second , to assess the relative effectiveness over time since the market entry of glp-1 and dpp-4i . the united kingdom clinical practice research datalink ( cprd ) was used for this study . the cprd is derived from uk primary care and holds longitudinal patient - level data such as diagnoses , mortality , laboratory results , and prescription data . it is the representative for the uk general population , and validation ensures high - quality data.710 anonymized data are available after protocol approval from the independent scientific advisory committee ( isac).11 for this study , data about patients aged 18 years with a diagnosis of type 2 diabetes and a prescription of glucose - lowering medication between january 1 , 2006 , and february 9 , 2015 , were extracted . validation studies have shown that diagnosis of type 2 diabetes alone is not always correct , so to ensure identification of patients with type 2 diabetes , patients were included if they fulfilled one of the following criteria:12 1 ) first diagnosis ( either type 1 or type 2 ) before age 35 years and no continual insulin treatment within six months of diagnosis , or 2 ) first diagnosis ( either type 1 or type 2 ) after age 35 years and not on continual insulin treatment from diagnosis . two comparison groups were used throughout the study ; initiators of glp-1 were compared with initiators of basal insulin , whereas initiators of dpp-4i were compared with initiators of sulfonylurea . the chosen comparator groups were to compare second - line ( dpp-4i and sulfonylurea ) and third - line ( glp-1 and insulin ) treatments , respectively.13 the glucose - lowering drugs were handled at the drug - class level , and combination products of metformin and dpp-4i and of rosiglitazone and sulfonylurea were considered as dpp-4i and sulfonylurea . prescriptions of drugs were used to identify episodes of treatment . for each patient , the end of a prescription was estimated from the daily dose and amount prescribed ( typically available for glp-1 , dpp-4i , and sulfonylurea ) , or as the patient s median duration of prescription of the drug ( typically the case for insulin ) . if there were < 120 days between the estimated end date of one prescription and the start date of the next prescription , the prescriptions were considered to make up a continuous episode of drug treatment . initiation was defined as patients with a prescription of one of the four glucose - lowering drugs after minimum 180 days without a prescription of the drug ( washout period ) . two cohorts were created , by analogy with the terms used in clinical trials : the intention - to - treat ( itt ) cohorts comprise subjects with a treatment duration of at least one day , whereas the per - protocol ( pp ) cohorts comprise subjects with a treatment duration of at least 180 days . if patients were identified as incident users more than one time for the same drug , only the first initiation was considered . if patients initiated glp-1 and insulin , or dpp-4i and sulfonylurea at the same time , this initiation was censored . within each of the four cohorts , time blocks of 365-day duration were defined with start date of the first initiation of glp-1 or dpp-4i , respectively . if patient initiated the comparison drugs within the same time block , only the first initiation was used and the second initiation was censored . measures of glycated hemoglobin ( hba1c ) and body weight were used to establish outcome measures . the goal was to observe change in hba1c and in body weight during a 180-day period . however , because measurements were not always available at the exact date of treatment initiation and 180 days after initiation , the following were deployed when measurements were not available : if a measurement was available within 90 days prior to initiation , this was used as the measurement at initiation , and if a measurement was available between 90 and 179 days after initiation , this was used as the measurement 180 days after initiation . if several measurements were available within these periods , the measurement closest to the initiation date and 180 days after initiation , respectively , was used . the following covariates were used : sex ; age at initiation ; weight at initiation ; body mass index ( bmi ) at initiation ; hba1c at initiation ; duration of diabetes at initiation ; number of oral glucose - lowering drugs at initiation ; any use of glucose - lowering drugs ( sulfonylurea , basal insulin , dpp-4i , metformin , and other oral glucose - lowering drugs ) during the washout period ; any use of antihypertensive , statins , and anticoagulants during the washout period ; charlson comorbidity index14 at initiation ; and any diagnosis of hypertension , end - stage renal disease , myocardial infarction , or stroke prior to initiation . the covariate number of oral glucose - lowering drugs at index was a count of prescriptions at index of any of the following glucose - lowering drugs ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone , guar gum and metformin in the comparison of dpp-4i and sulfonylurea , and in the comparison of glp-1 and basal insulin number of other oral glucose - lowering drugs at index also included dpp-4i and sulfonylurea . the covariate other oral glucose - lowering drugs during washout was a binary response if at least one of the following glucose lowering drugs were prescribed during the washout period ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone or guar gum . information on hba1c , bmi , weight , and diabetes duration was missing for some individuals . under the assumption that these missing data were missing at random , missing values were imputed . multiple imputation was applied , with 10 imputed datasets.15 in addition to imputation of missing measurements at initiation , missing measurements 180 days after initiation were also imputed.16 we used the listed covariates , treatment group and change in hba1c and weight for the imputation . the charlson comorbidity index was categorized into four groups : 01 , 2 , 3 , and more than 3 points . number of oads was categorized into four groups : 0 oads , 1 oads , 2 oads , 3 oads , and more than 3 oads . results for both the descriptive and the outcome analyses were pooled across the imputed datasets using rubin s rule.17 post hoc we noticed that a fraction of hba1c measurements was very low for glp-1 and insulin initiators , and that most of these measurements were located around the fourth year since glp-1 approval . as we suspected these measurements were incorrect , all hba1c measurements below 4% were treated as missing and were imputed as described above . propensity scores were estimated as the probability of initiating glp-1 in the comparison of glp-1 and insulin initiators and the probability of initiating dpp-4i in the comparison of dpp-4i and sulfonylurea initiators . the propensity score models included the covariates mentioned earlier , except the covariate describing use of the comparison drug during washout . standardized difference was used to assess balance on covariates and to select the appropriate propensity score model.18 the propensity score was estimated twice : in the whole cohort for the descriptive purpose of characteristics over time and in the subgroup that did not receive the comparison drug during washout to be used for matching in the outcome analyses . based on the propensity score , patients were matched in order to create treatment groups that were comparable with respect to the propensity score and thus the potential confounders . greedy matching was done in a 1:1 ratio , stratified by time block and within each imputed dataset . the greedy matching was done in up to eight iterations ; first the data were searched for matches based on eight digits of the propensity score , then seven digits , and continued to matches according to one digit ( see parson19 for details ) . to assess changes in patient characteristics over time , the covariates and propensity score were plotted stratified by time blocks and treatment groups . this shows both general differences between treatment groups and potential channeling , where characteristics change over time . the propensity score serves as a single variable to describe differences in characteristics across comparison groups . both the propensity score for all individuals and the propensity score for individuals suitable for matching ( without use of the comparison drug during washout ) were assessed . because of the many covariates available , only the propensity score over time will be shown in the result section , whereas graphs for each individual variable can be found in the supplementary material . in the subgroup of the pp cohort that did not receive the comparison drug during washout , linear regression analyses were used to estimate the relative effect regarding change in hba1c and change in body weight after 180 days of follow - up . crude analyses were performed based on all identified initiators , in addition to analyses of the propensity score - matched initiators , to take confounding into account . in the analyses of change in hba1c and body weight , baseline levels of hba1c and body weight , respectively , were included as covariates in the outcome model.20 the relative effect estimate and their 95% confidence intervals ( cis ) were plotted for graphical inspections of changes in effect estimates over time . data management was done using template programs developed by the department of epidemiology at novo nordisk for importing cprd data into sas format . access to cprd data was gained through approval of protocol by the isac ( https://www.cprd.com/isac/ ) . two comparison groups were used throughout the study ; initiators of glp-1 were compared with initiators of basal insulin , whereas initiators of dpp-4i were compared with initiators of sulfonylurea . the chosen comparator groups were to compare second - line ( dpp-4i and sulfonylurea ) and third - line ( glp-1 and insulin ) treatments , respectively.13 the glucose - lowering drugs were handled at the drug - class level , and combination products of metformin and dpp-4i and of rosiglitazone and sulfonylurea were considered as dpp-4i and sulfonylurea . the end of a prescription was estimated from the daily dose and amount prescribed ( typically available for glp-1 , dpp-4i , and sulfonylurea ) , or as the patient s median duration of prescription of the drug ( typically the case for insulin ) . if there were < 120 days between the estimated end date of one prescription and the start date of the next prescription , the prescriptions were considered to make up a continuous episode of drug treatment . initiation was defined as patients with a prescription of one of the four glucose - lowering drugs after minimum 180 days without a prescription of the drug ( washout period ) . two cohorts were created , by analogy with the terms used in clinical trials : the intention - to - treat ( itt ) cohorts comprise subjects with a treatment duration of at least one day , whereas the per - protocol ( pp ) cohorts comprise subjects with a treatment duration of at least 180 days . with the two comparisons , this makes up four cohorts in total . if patients were identified as incident users more than one time for the same drug , only the first initiation was considered . if patients initiated glp-1 and insulin , or dpp-4i and sulfonylurea at the same time , this initiation was censored . within each of the four cohorts , time blocks of 365-day duration were defined with start date of the first initiation of glp-1 or dpp-4i , respectively . if patient initiated the comparison drugs within the same time block , only the first initiation was used and the second initiation was censored . measures of glycated hemoglobin ( hba1c ) and body weight were used to establish outcome measures . the goal was to observe change in hba1c and in body weight during a 180-day period . however , because measurements were not always available at the exact date of treatment initiation and 180 days after initiation , the following were deployed when measurements were not available : if a measurement was available within 90 days prior to initiation , this was used as the measurement at initiation , and if a measurement was available between 90 and 179 days after initiation , this was used as the measurement 180 days after initiation . if several measurements were available within these periods , the measurement closest to the initiation date and 180 days after initiation , respectively , was used . the following covariates were used : sex ; age at initiation ; weight at initiation ; body mass index ( bmi ) at initiation ; hba1c at initiation ; duration of diabetes at initiation ; number of oral glucose - lowering drugs at initiation ; any use of glucose - lowering drugs ( sulfonylurea , basal insulin , dpp-4i , metformin , and other oral glucose - lowering drugs ) during the washout period ; any use of antihypertensive , statins , and anticoagulants during the washout period ; charlson comorbidity index14 at initiation ; and any diagnosis of hypertension , end - stage renal disease , myocardial infarction , or stroke prior to initiation . the covariate number of oral glucose - lowering drugs at index was a count of prescriptions at index of any of the following glucose - lowering drugs ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone , guar gum and metformin in the comparison of dpp-4i and sulfonylurea , and in the comparison of glp-1 and basal insulin number of other oral glucose - lowering drugs at index also included dpp-4i and sulfonylurea . the covariate other oral glucose - lowering drugs during washout was a binary response if at least one of the following glucose lowering drugs were prescribed during the washout period ; acarbose , canagliflozin , canagliflozin combined with metformin , dapagliflozin , dapagliflozin combined with metformin , empagliflozin , pioglitazone , pioglitazone combined with metformin , rosiglitazone , rosiglitazone combined with metformin , nateglinide , repaglinide , troglitazone or guar gum . information on hba1c , bmi , weight , and diabetes duration was missing for some individuals . under the assumption that these missing data were missing at random , missing values were imputed . multiple imputation was applied , with 10 imputed datasets.15 in addition to imputation of missing measurements at initiation , missing measurements 180 days after initiation were also imputed.16 we used the listed covariates , treatment group and change in hba1c and weight for the imputation . the charlson comorbidity index was categorized into four groups : 01 , 2 , 3 , and more than 3 points . number of oads was categorized into four groups : 0 oads , 1 oads , 2 oads , 3 oads , and more than 3 oads . results for both the descriptive and the outcome analyses were pooled across the imputed datasets using rubin s rule.17 post hoc we noticed that a fraction of hba1c measurements was very low for glp-1 and insulin initiators , and that most of these measurements were located around the fourth year since glp-1 approval . as we suspected these measurements were incorrect , all hba1c measurements below 4% were treated as missing and were imputed as described above . propensity scores were estimated as the probability of initiating glp-1 in the comparison of glp-1 and insulin initiators and the probability of initiating dpp-4i in the comparison of dpp-4i and sulfonylurea initiators . the propensity score models included the covariates mentioned earlier , except the covariate describing use of the comparison drug during washout . standardized difference was used to assess balance on covariates and to select the appropriate propensity score model.18 the propensity score was estimated twice : in the whole cohort for the descriptive purpose of characteristics over time and in the subgroup that did not receive the comparison drug during washout to be used for matching in the outcome analyses . based on the propensity score , patients were matched in order to create treatment groups that were comparable with respect to the propensity score and thus the potential confounders . greedy matching was done in a 1:1 ratio , stratified by time block and within each imputed dataset . the greedy matching was done in up to eight iterations ; first the data were searched for matches based on eight digits of the propensity score , then seven digits , and continued to matches according to one digit ( see parson19 for details ) . to assess changes in patient characteristics over time , the covariates and propensity score were plotted stratified by time blocks and treatment groups . this shows both general differences between treatment groups and potential channeling , where characteristics change over time . the propensity score serves as a single variable to describe differences in characteristics across comparison groups . both the propensity score for all individuals and the propensity score for individuals suitable for matching ( without use of the comparison drug during washout ) were assessed . because of the many covariates available , only the propensity score over time will be shown in the result section , whereas graphs for each individual variable can be found in the supplementary material . in the subgroup of the pp cohort that did not receive the comparison drug during washout , linear regression analyses were used to estimate the relative effect regarding change in hba1c and change in body weight after 180 days of follow - up . crude analyses were performed based on all identified initiators , in addition to analyses of the propensity score - matched initiators , to take confounding into account . in the analyses of change in hba1c and body weight , baseline levels of hba1c and body weight , respectively , were included as covariates in the outcome model.20 the relative effect estimate and their 95% confidence intervals ( cis ) were plotted for graphical inspections of changes in effect estimates over time . data management was done using template programs developed by the department of epidemiology at novo nordisk for importing cprd data into sas format . access to cprd data was gained through approval of protocol by the isac ( https://www.cprd.com/isac/ ) . in total , 304,953 patients with diabetes and a prescription of a glucose - lowering drug were identified . the first initiation of glp-1 was observed on june 14 , 2007 , whereas the first initiation of dpp-4i was observed on june 22 , 2007 . for the descriptive analyses of characteristics over time , 10,633 and 19,243 initiators of glp-1 and insulin , respectively , and 30,048 and 41,346 initiators of dpp-4i and sulfonylurea , respectively , were included in the itt cohorts . the numbers of individuals in the pp cohorts were a little smaller . for the outcome analyses , 8,398 and 13,286 initiators of glp-1 and insulin , respectively , and 10,847 and 30,643 initiators of dpp-4i and sulfonylurea , respectively , were identified . the exact number of propensity score - matched individuals varies within each imputed dataset , but within each time block nearly half of the initiators of glp-1 were matched with an initiator of insulin and nearly all initiators of dpp-4i were matched with an initiator of sulfonylurea . it was possible to create seven time blocks of 365 days in both the glp-1 and insulin , and the dpp-4i and sulfonylurea comparisons . the distributions of propensity score look similar for both the itt and pp cohorts of all identified initiators of glp-1 and insulin , and dpp-4i and sulfonylurea , respectively , and also for the pp cohort of initiators with no use of the comparison drug during washout , which is the subgroup used for propensity score matching . time blocks 17 are demonstrated as histograms of the propensity score of the latter in figures 1 and 2 . other propensity score plots are found in the supplementary material figure s2 . in general , the distribution of propensity score of the glp-1 and insulin initiators indicate an overlap in propensity score for all time blocks considered . the propensity score of dpp-4i and sulfonylurea initiators has an even greater overlap in all time blocks , and for both dpp-4i and sulfonylurea , the distribution moves from being centered on lower propensity scores in the early time blocks to being centered on 0.4 in later time blocks , which indicate comparable patients with a similar change in characteristics over time . figures of the individual covariates are found in figure s3s4 . in general , trends for the itt and pp cohort hence , there will be no distinction of the itt and pp cohort in the following text . for initiators of glp-1 and insulin , minor changes in hba1c at initiation both glp-1 and insulin initiators had an increase in use of dpp-4i during washout and a decrease in use of other oral glucose - lowering drugs during washout . for insulin initiators only , an increase in use of sulfonylurea , glp-1 , and metformin during washout was seen . plots of the individual covariates also show some general differences of the comparison groups across time . compared to initiators of insulin , the initiators of glp-1 tended to have higher bmi ; more number of glucose - lowering drugs at initiation ; increased use of metformin , other glucose - lowering drugs , dpp-4i , and statins during washout , increased proportion of with hypertension diagnoses ; lower age ; higher charlson comorbidity index , increased proportion of myocardial infarction and end - stage renal disease diagnoses ; and shorter duration of diabetes . for dpp-4i and sulfonylurea initiators , the plots of the individual covariates showed that initiators of both drugs had an increase in duration of diabetes and use of metformin during washout and a decrease in other oral glucose - lowering drugs during washout . for sulfonylurea initiators only , a minor increase in hba1c at initiation and an increase in use of dpp-4i and glp-1 during washout were seen . across the different time blocks , the initiators of dpp-4i , compared to initiators of sulfonylurea , tended to have higher bmi , more prevalent use of other glucose - lowering drugs and statins during washout , longer duration of diabetes , lower hba1c , and , in the late time blocks , higher charlson comorbidity index . the considerable overlap in propensity scores allowed for propensity score matching and thus propensity score - matched relative effectiveness estimation . figure 3a and 3b shows the difference in change in hba1c and body weight , respectively , for the comparison of glp-1 and insulin ; nonsignificant differences in change in hba1c , except from a peak in time block 4 , and a relative increase in body weight for the insulin initiators compared to glp-1 initiators in all time blocks were seen . across time blocks , the relative increase in body weight was 3.57 kg ( 95% ci : 3.21 , 3.92 ) for insulin initiators . similarly , figure 4a and 4b shows the difference in change in hba1c and body weight , respectively compared to dpp-4i and sulfonylurea ; a relative decrease in hba1c and a relative increase in body weight for sulfonylurea initiators compared to dpp-4i initiators in all time blocks were seen . across time blocks , the relative decrease in hba1c was 0.34% ( 95% ci : 0.38 , 0.30 ) and the relative increase in body weight was 1.58 kg ( 95% ci : 1.38 , 1.78 ) for sulfonylurea initiators . the distributions of propensity score look similar for both the itt and pp cohorts of all identified initiators of glp-1 and insulin , and dpp-4i and sulfonylurea , respectively , and also for the pp cohort of initiators with no use of the comparison drug during washout , which is the subgroup used for propensity score matching . time blocks 17 are demonstrated as histograms of the propensity score of the latter in figures 1 and 2 . other propensity score plots are found in the supplementary material figure s2 . in general , the distribution of propensity score of the glp-1 and insulin initiators indicate an overlap in propensity score for all time blocks considered . the propensity score of dpp-4i and sulfonylurea initiators has an even greater overlap in all time blocks , and for both dpp-4i and sulfonylurea , the distribution moves from being centered on lower propensity scores in the early time blocks to being centered on 0.4 in later time blocks , which indicate comparable patients with a similar change in characteristics over time . figures of the individual covariates are found in figure s3s4 . in general , trends for the itt and pp cohort were similar . hence , there will be no distinction of the itt and pp cohort in the following text . for initiators of glp-1 and insulin , minor changes in hba1c at initiation were observed ; however , all were within the range of 0.5% . both glp-1 and insulin initiators had an increase in use of dpp-4i during washout and a decrease in use of other oral glucose - lowering drugs during washout . for insulin initiators only , an increase in use of sulfonylurea , glp-1 , and metformin during washout was seen . plots of the individual covariates also show some general differences of the comparison groups across time . compared to initiators of insulin , the initiators of glp-1 tended to have higher bmi ; more number of glucose - lowering drugs at initiation ; increased use of metformin , other glucose - lowering drugs , dpp-4i , and statins during washout , increased proportion of with hypertension diagnoses ; lower age ; higher charlson comorbidity index , increased proportion of myocardial infarction and end - stage renal disease diagnoses ; and shorter duration of diabetes . for dpp-4i and sulfonylurea initiators , the plots of the individual covariates showed that initiators of both drugs had an increase in duration of diabetes and use of metformin during washout and a decrease in other oral glucose - lowering drugs during washout . for sulfonylurea initiators only , a minor increase in hba1c at initiation and an increase in use of dpp-4i and glp-1 during washout were seen . across the different time blocks , the initiators of dpp-4i , compared to initiators of sulfonylurea , tended to have higher bmi , more prevalent use of other glucose - lowering drugs and statins during washout , longer duration of diabetes , lower hba1c , and , in the late time blocks , higher charlson comorbidity index . the considerable overlap in propensity scores allowed for propensity score matching and thus propensity score - matched relative effectiveness estimation . figure 3a and 3b shows the difference in change in hba1c and body weight , respectively , for the comparison of glp-1 and insulin ; nonsignificant differences in change in hba1c , except from a peak in time block 4 , and a relative increase in body weight for the insulin initiators compared to glp-1 initiators in all time blocks were seen . across time blocks , the relative increase in body weight was 3.57 kg ( 95% ci : 3.21 , 3.92 ) for insulin initiators . similarly , figure 4a and 4b shows the difference in change in hba1c and body weight , respectively compared to dpp-4i and sulfonylurea ; a relative decrease in hba1c and a relative increase in body weight for sulfonylurea initiators compared to dpp-4i initiators in all time blocks were seen . across time blocks , the relative decrease in hba1c was 0.34% ( 95% ci : 0.38 , 0.30 ) and the relative increase in body weight was 1.58 kg ( 95% ci : 1.38 , 1.78 ) for sulfonylurea initiators . in this study , change in characteristics over time since beginning treatment with glp-1 and dpp-4i in comparison with basal insulin and sulfonylurea , respectively , was investigated with focus on potential channeling bias . propensity score matching was applied to assess the relative effect on change in hba1c and weight . channeling bias was not seen in the sense that the characteristics of glp-1 and dpp-4i initiators changed over time after market entry , whereas the characteristics of the initiators of the established drugs , insulin and sulfonylurea , were stable . distribution of propensity scores , used as an overall measure of differences in characteristics , showed that initiators of glp-1 and insulin were somewhat different , although with a substantial overlap , in all time blocks . initiators of dpp-4i and sulfonylurea were more comparable with an even greater overlap across time blocks and with a similar change in propensity scores over time . for the individual covariates , changes in characteristics were observed in both groups of glp-1 and insulin initiators , and among dpp-4i and sulfonylurea initiators , as well as among initiators of insulin or sulfonylurea only . there was sufficient overlap in distribution of characteristics so that propensity score matching was possible to conduct relative effectiveness analysis , even in the first year after launch . the outcome analyses suggest similar change in hba1c for glp-1 and insulin , except for the peak in time block 4 . overall , we consider this peak as a result of random variation given the absence of a difference in effects in the other time blocks . the outcome analyses suggest a greater reduction in hba1c and weight gain for sulfonylurea compared to dpp-4i . it is hypothesized that patients initiating a newly launched drug are those most ill and with more use of comedications , but such differences will disappear over time when the new drug becomes more established.1,2 in this study , also , it is hypothesized that patients initiating a newly launched drug have a history of suboptimal drug response or intolerance . direct information on drug response was not available in data , but hba1c level indicated how well - regulated patients with diabetes are . from the measurements of hba1c at initiation , there was not much difference over time between initiators of glp-1 and insulin , and dpp-4i initiators had lower hba1c at initiation compared to initiators of sulfonylurea . this indicates that patients initiating the newly launched drugs had similar response profiles as those who initiated the established drugs . the findings in this study are in line with us studies comparing sitagliptin ( merck & co. , inc . , whitehouse station , nj , usa ) with other oral glucose - lowering drugs where there were no changes in characteristics over time in the first years after launch.46 the use of real - world data is a necessity to investigate channeling bias , but it also brings along some general problems . the following potential limitations of the study were identified : real - world data are data of real - world patients , yet may suffer from missing information , which in this study was imputed by means of multiple imputation under the assumption that the missing data are missing at random . the quality of real - world data may be questioned as the data seldom are collected primarily for research purposes . this study concerns a period after implementation of quality outcome framework in cprd , through which the highest possible data quality is achieved . it was a concern that for the majority of patients in cprd , body weight was measured only once , and that measurement was carried forward . this was , however , not the case in the data available for this study . the risk of confounding is present in observational studies of the effects of medical treatments ; this study applied propensity score matching to take confounding into account . not all relevant potential confounding factors are captured in the cprd database ( eg , diet and ethnicity ) , but the propensity score model included a diverse set of covariates describing demographics , clinical measurements , comedication including glucose - lowering drugs , and comorbidities . immortal time bias , which is used to denote misclassification of observation time to exposure groups in survival analyses , was not an issue in this study since follow - up started at the first prescription made and was continued for the same duration for all subjects ( ie , 180 days ) . however , the risk of selection bias in the pp cohort in this study might be an issue because individuals who have survived or stayed in the dataset at least 180 days were selected . however , the analyses of a pp cohort are relevant because our outcome measure of hba1c is highly affected by exposure of the investigated drugs , which is more likely in the pp cohort . in this study , we looked at drug - class levels , and combination products of sulfonylurea and rosiglitazone , as well as products of dpp-4i and metformin , were classified as sulfonylurea and dpp-4i . change in characteristics over time may be affected by launch of specific brands and the use of monotherapy and combination products . it is possible that time blocks of shorter duration would reveal channeling in the very early period after launch , but the duration of 365 days was suitable as this will ensure some level of statistical power . studies in other medical areas have observed channeling bias in cprd data . in a study comparing new nonsteroidal anti - inflammatory drugs ( nsaids ) with old nsaids on the risk of gastrointestinal hemorrhage , baseline characteristics suggested that the new nsaid population had a higher baseline risk ( possibly explained by new nsaids having a safer profile with regard to gastrointestinal toxicity ) . outcome analyses seemed to adjust for confounding , including channeling bias , and showed a lower risk among patients treated with new nsaids.21 in the treatment of chronic obstructive pulmonary disease , corticosteroids have been compared with long - acting bronchodilators . the adjusted analyses showed higher risk of pneumonia among corticosteroid users , but it was not clear to the authors if this was related to the effect of the drug alone , or if channeling bias had occurred.22 the use of a sequential cohort design , where analyses are done at several time points as in this study , is generally proposed to take channeling bias into account.2327 despite the fact that no major channeling bias was observed in this study , some variation over time was seen . also , the characteristics of the initiators of the established drugs ( basal insulin and sulfo - nylurea ) changed over time , despite the time period observed ( 20062015 ) being more or less arbitrary for these drugs . the sequential cohort design in this study used the same propensity score model across time blocks , as standardized differences suggested that balance across treatment groups were established after matching . however , individual propensity score models may be needed within each time block in other settings . to conclude , we did not identify major channeling so that patients initiating the new glucose - lowering drugs differed importantly from patients initiating the established glucose - lowering drugs . in this study , the differences in patient characteristics and changes over time were at a magnitude where it was still possible to estimate propensity score and conduct matching to control for confounding in the outcome analyses , even in the first year after market entry . change in the characteristics of initiators of established drugs generally advocates for time - dependent analyses . amyotrophic lateral sclerosis ( als ) is a progressive and fatal neurodegenerative disease characterized by the loss of lower and upper motor neurons , leading to muscle atrophy , paralysis , and death . sleep disorders in patients with als are well - documented , and sleep - related complaints , such as insomnia , disturbed sleep , nightmare , and daytime sleepiness , have been frequently reported . moreover , several clinical studies on sleep disturbances in patients with als have been published , focusing on the frequency , characteristics , and severity of sleep problems . however , there are no animal or mechanistic studies on sleep disturbances in als . animal and mechanistic studies on sleep disturbances in other neurodegenerative diseases , such as alzheimer 's disease ( ad ) and parkinson disease ( pd ) , might give us insights into sleep disturbances in als . sleep problems in ad and pd involve disturbances in the neurotransmitter and hormone signaling , abnormal accumulations of neurotoxic proteins , and damage in the brain regions controlling the sleep / wake cycles , which could exist in als as well . precursor peptide prepro - orexin , which is produced in the hypothalamic neurons , matures into two peptides , orexin a and orexin b. these peptides promote wakefulness by activating wake - active neurons ( wan ) in the hypothalamus and brain stem . the actions of orexins are mediated by two receptors , orexin-1 ( ox1r ) and orexin-2 ( ox2r ) receptors . we hypothesized that there are disturbances of sleep and wakefulness in the als mouse models and that orexin is an important molecule responsible for those disturbances . in the present study with sod1-g93a transgenic mice , which are extensively used as animal model for mechanistic and therapeutic studies on als , we used sleep / wake activity recordings and molecular techniques to test our hypothesis . transgenic sod1-g93a mice used in this study were bred from male hemizygous sod1-g93 a mice ( b6sjl - tg [ sod1-g93a ] 1 gur / j ) to female b6sjl / f1 hybrids . the genotyping of sod1-g93a mice was performed by polymerase chain reaction ( pcr ) , as previously reported . male hemizygous sod1-g93a mice and female b6sjl / f1 hybrids were both purchased from the jackson laboratories ( bar harbor , me , usa ) . all mice were housed under a controlled temperature ( 22 1c ) and 12 hours : 12 hours light - dark cycle . all animal studies were approved by the institutional animal care and use committee of peking university third hospital , and conducted in accordance with the guide for the care and use of laboratory animals of peking university . to monitor disease progression , all mice were tested using the rota rod test apparatus ( ugo basile , varese , italy ) . the day on which a mouse first dropped off the rota rod within 600 seconds was designated as a day of disease onset for that mouse . using this testing criterion , we observed that all the sod1-g93a transgenic mice in this study had disease onset between 90 and 120 days of age . thus , we determined the two - time points in our study : 90-day representing the age before disease onset and 120-day representing the age after disease onset . twenty - eight mice were divided into four groups : sod1-g93a group at 90-day ( n = 8) , control group at 90-day ( n = 6 ) , sod1-g93a group at 120-day ( n = 6 ) , and control group at 120-day ( n = 8) . all mice received electroencephalogram / electromyogram ( eeg / emg ) recordings at 90 or 120 days of age . after performing the recordings , the cerebrospinal fluid ( csf ) and brain samples were collected . the hypothalamus and brain stem were isolated from the brain tissue for real - time reverse transcriptase ( rt)-pcr , western blotting , and enzyme - linked immunosorbent assay ( elisa ) . surgeries and eeg / emg recordings were performed as previously described . briefly , after anesthesia , 28 mice were implanted with eeg and emg electrodes . the eeg electrodes were placed epidurally on the cortex , and the emg electrodes were placed in the dorsal neck muscles . ten days after surgery , the electrodes were connected to recording cables attached to the mp150 system ( biopac , goleta , ca , usa ) . eeg and emg recordings were performed in this manner from the freely behaving mice for 24 hours . data were analyzed using sleep sign 2.0 software ( biopac , goleta , ca , usa ) . total rna from the hypothalamus and brainstem tissues was extracted using trizol reagent ( takara , dalian , china ) according to the manufacturer 's instructions . first - strand cdna was synthesized at 42c with fastquant rt kit ( tiangen , beijing , china ) using 1 g of total rna . the amplification was performed using a superreal premix plus kit ( tiangen ) and an abi 7500 rt - pcr system ( applied biosystems , foster city , usa ) . the cdna was amplified with an initial denaturation step ( 95c , 15 minutes ) , and then with 40 pcr cycles consisting of a denaturation step ( 95c , 10 seconds ) and an annealing / extension step ( 58c , 32 seconds ) . -actin was used as internal control to calculate the relative abundance of each mrna ( n = 46/group ) . the specific sets of primers were as follows : prepro - orexin : f : tgaactttccttctacaaaggttc , r : caacagttcgtagagacggca ; orexin1 receptor : f : cgccaaccctatcatctacaa , r : gctctgcaaggacaaggactt ; orexin2 receptor : f : gctcaccagcataagcacact , r : tatctctttgagcagacatggg ; -actin : f : cctagcaccatgaagatcaagat , r : actcatcgtactcctgcttgct . total protein was extracted from the hypothalamus samples using the total protein extraction kit ( applygen , beijing , china ) , consisting of radioimmunoprecipitation assay lysis buffer , phenylmethylsulfonyl fluoride , protease inhibitors , and phosphatase inhibitors , according to the manufacturer 's instructions . protein concentration was detected by the bicinchoninic acid protein assay using a protein assay kit ( applygen ) . samples containing equal amounts of total protein ( 20 g ) were separated by 12% sds - page and transferred to a polyvinylidene fluoride membrane ( millipore , billerica , ma , usa ) . the membrane was blocked with 3% bovine serum albumin ( bsa ) in tris buffered saline ( tbs ) containing 0.1% tween 20 ( tbs - t ) for one hour , and then incubated overnight at 4c with the primary orexin antibody ( anti - orexin - prepro , 1:500 , millipore , billerica , ma , usa ) or primary -actin antibody ( anti--actin , 1:5000 , earthox , san francisco , ca , usa ) . -actin was used as an internal loading control . the membrane was then incubated with secondary irdye 800cw goat anti - rabbit ( 1:10,000 , li - cor , lincoln , ne , usa ) or goat anti - mouse ( 1:10,000 , li - cor , lincoln , ne , usa ) antibodies at 37c for one hour . quantitation of immunoreactive bands was performed using an odyssey infrared imaging system ( li - cor , lincoln ) . levels of orexin a and orexin b in mouse csf and brain tissues were measured by orexin a / orexin b elisa kits ( bluegene biotech , shanghai , china ) . mol / l , ph 7.07.2 ) per 0.5 g tissue and centrifuged . samples ( csf or brain homogenates supernatant ) or standards were added to 96-well plates coated with anti - mouse orexin a / b antibody and elisa was performed according to manufacturer 's protocol . the independent - samples t - test and pearson correlation analyses were performed using spss 19.0 ( spss inc . , transgenic sod1-g93a mice used in this study were bred from male hemizygous sod1-g93 a mice ( b6sjl - tg [ sod1-g93a ] 1 gur / j ) to female b6sjl / f1 hybrids . the genotyping of sod1-g93a mice was performed by polymerase chain reaction ( pcr ) , as previously reported . male hemizygous sod1-g93a mice and female b6sjl / f1 hybrids were both purchased from the jackson laboratories ( bar harbor , me , usa ) . all mice were housed under a controlled temperature ( 22 1c ) and 12 hours : 12 hours light - dark cycle . all animal studies were approved by the institutional animal care and use committee of peking university third hospital , and conducted in accordance with the guide for the care and use of laboratory animals of peking university . to monitor disease progression , all mice were tested using the rota rod test apparatus ( ugo basile , varese , italy ) . the day on which a mouse first dropped off the rota rod within 600 seconds was designated as a day of disease onset for that mouse . using this testing criterion , we observed that all the sod1-g93a transgenic mice in this study had disease onset between 90 and 120 days of age . thus , we determined the two - time points in our study : 90-day representing the age before disease onset and 120-day representing the age after disease onset . twenty - eight mice were divided into four groups : sod1-g93a group at 90-day ( n = 8) , control group at 90-day ( n = 6 ) , sod1-g93a group at 120-day ( n = 6 ) , and control group at 120-day ( n = 8) . all mice received electroencephalogram / electromyogram ( eeg / emg ) recordings at 90 or 120 days of age . after performing the recordings , the cerebrospinal fluid ( csf ) and brain samples were collected . the hypothalamus and brain stem were isolated from the brain tissue for real - time reverse transcriptase ( rt)-pcr , western blotting , and enzyme - linked immunosorbent assay ( elisa ) . surgeries and eeg / emg recordings were performed as previously described . briefly , after anesthesia , 28 mice were implanted with eeg and emg electrodes . the eeg electrodes were placed epidurally on the cortex , and the emg electrodes were placed in the dorsal neck muscles . ten days after surgery , the electrodes were connected to recording cables attached to the mp150 system ( biopac , goleta , ca , usa ) . eeg and emg recordings were performed in this manner from the freely behaving mice for 24 hours . data were analyzed using sleep sign 2.0 software ( biopac , goleta , ca , usa ) . total rna from the hypothalamus and brainstem tissues was extracted using trizol reagent ( takara , dalian , china ) according to the manufacturer 's instructions . first - strand cdna was synthesized at 42c with fastquant rt kit ( tiangen , beijing , china ) using 1 g of total rna . the amplification was performed using a superreal premix plus kit ( tiangen ) and an abi 7500 rt - pcr system ( applied biosystems , foster city , usa ) . the cdna was amplified with an initial denaturation step ( 95c , 15 minutes ) , and then with 40 pcr cycles consisting of a denaturation step ( 95c , 10 seconds ) and an annealing / extension step ( 58c , 32 seconds ) . -actin was used as internal control to calculate the relative abundance of each mrna ( n = 46/group ) . the specific sets of primers were as follows : prepro - orexin : f : tgaactttccttctacaaaggttc , r : caacagttcgtagagacggca ; orexin1 receptor : f : cgccaaccctatcatctacaa , r : gctctgcaaggacaaggactt ; orexin2 receptor : f : gctcaccagcataagcacact , r : tatctctttgagcagacatggg ; -actin : f : cctagcaccatgaagatcaagat , r : actcatcgtactcctgcttgct . total protein was extracted from the hypothalamus samples using the total protein extraction kit ( applygen , beijing , china ) , consisting of radioimmunoprecipitation assay lysis buffer , phenylmethylsulfonyl fluoride , protease inhibitors , and phosphatase inhibitors , according to the manufacturer 's instructions . protein concentration was detected by the bicinchoninic acid protein assay using a protein assay kit ( applygen ) . samples containing equal amounts of total protein ( 20 g ) were separated by 12% sds - page and transferred to a polyvinylidene fluoride membrane ( millipore , billerica , ma , usa ) . the membrane was blocked with 3% bovine serum albumin ( bsa ) in tris buffered saline ( tbs ) containing 0.1% tween 20 ( tbs - t ) for one hour , and then incubated overnight at 4c with the primary orexin antibody ( anti - orexin - prepro , 1:500 , millipore , billerica , ma , usa ) or primary -actin antibody ( anti--actin , 1:5000 , earthox , san francisco , ca , usa ) . the membrane was then incubated with secondary irdye 800cw goat anti - rabbit ( 1:10,000 , li - cor , lincoln , ne , usa ) or goat anti - mouse ( 1:10,000 , li - cor , lincoln , ne , usa ) antibodies at 37c for one hour . quantitation of immunoreactive bands was performed using an odyssey infrared imaging system ( li - cor , lincoln ) . levels of orexin a and orexin b in mouse csf and brain tissues were measured by orexin a / orexin b elisa kits ( bluegene biotech , shanghai , china ) . brain tissues were homogenized in 1 ml pbs ( 0.02 mol / l , ph 7.07.2 ) per 0.5 g tissue and centrifuged . samples ( csf or brain homogenates supernatant ) or standards were added to 96-well plates coated with anti - mouse orexin a / b antibody and elisa was performed according to manufacturer 's protocol . the independent - samples t - test and pearson correlation analyses were performed using spss 19.0 ( spss inc . , sleep / wake recordings were performed in both sod1-g93a transgenic mice and their control groups . in the 90-day sod1-g93a transgenic mice , across a 24-hour recording period , the total sleep time ( tst ) was significantly decreased ( [ 443.23 40.42 minutes ] vs. [ 569.97 39.04 minutes ] , p < 0.05 ) and wakefulness was increased ( [ 996.78 40.42 minutes ] vs. [ 870.03 39.04 minutes ] , p < 0.05 ) , compared to the littermate controls . non - rapid eye movement ( nrem ) sleep ( [ 411.29 35.41 minutes ] vs. [ 533.48 37.01 minutes ] , p < 0.05 ) and deep sleep ( ds ) ( [ 47.35 9.55 minutes ] vs. [ 107.35 11.36 minutes ] , p < 0.01 ) were also reduced . no significant difference was found in rem sleep and light sleep between the two groups for the 24-hour period [ figure 1a ] . eeg / emg recordings for 24 hours from the sod1-g93a transgenic mice and littermate control mice at 90 days and 120 days of age . ( a ) tst , nrem , and ds are significantly decreased in the 90-day sod1-g93a transgenic mice . ( b ) in the 120-day sod1-g93a transgenic mice , wake is also significantly enhanced . tst : total sleep time ; wake : wakefulness ; rem : rapid eye movement sleep ; nrem : non - rapid eye movement sleep ; ls : light sleep ; ds : deep sleep ; eeg / emg : electroencephalogram / electromyogram . in the 120-day sod1-g93a transgenic mice , a remarkable increase in wakefulness ( [ 1110.33 38.16 minutes ] vs. [ 804.29 74.57 minutes ] , p < 0.01 ) , and a decrease in tst ( [ 329.67 38.16 minutes ] vs. [ 635.71 74.57 minutes ] , p < 0.01 ) , nrem ( [ 306.03 36.81 minutes ] vs. [ 534.68 82.68 minutes ] , p < 0.05 ) and ds ( [ 22.88 10.27 minutes ] vs. [ 107.90 25.21 minutes ] , p < 0.05 ) were observed . however , in contrast with the 90-day sod1-g93a transgenic mice , rem sleep was decreased ( [ 23.62 9.96 minutes ] vs. [ 101.08 13.93 minutes ] , p < 0.01 ) in the 120-day sod1-g93a transgenic mice compared to the 120-day littermate controls [ figure 1b ] . next , we evaluated the changes in the orexin system in the sod1-g93a transgenic mice . first , we tested the level of mrna and protein expression of prepro - orexin . q - pcr showed increased levels of prepro - orexin mrna in the hypothalamus of the 90-day ( 1.84 0.24 vs. 1.00 0.20 , p < 0.05 ) and 120-day ( 2.06 0.25 vs. 1.00 0.14 , p < 0.01 ) sod1-g93a transgenic mice , compared to controls [ figure 2a ] . the western blotting analysis also showed increased levels of prepro - orexin protein in the hypothalamus of both 90-day ( 2.33 0.24 vs. 1.00 0.18 , p < 0.01 ) and 120-day ( 2.30 0.14 vs. 1.00 0.19 , p < 0.01 ) sod1-g93a transgenic mice as compared to littermate controls [ figure 2b ] . prepro - orexin increases in sod1-g93a transgenic mice . ( a ) q - pcr was performed in the hypothalamic tissues from the sod1-g93a transgenic mice and control groups . prepro - orexin mrna is significantly elevated in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice , compared to control . ( b ) western blotting was performed with antibody anti - orexin - prepro in the sod1-g93a transgenic mice and control groups . prepro - orexin expression is increased in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice as compared to control . data are expressed as mean standard error of mean ( n = 46/group ) . we then tested the protein levels of orexin a and b , using elisa . in the hypothalamus , the level of orexin a was significantly enhanced in the 90-day ( [ 2126.47 70.65 pg / mg ] vs. [ 1591.52 61.25 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 2166.32 115.98 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice , compared to their respective littermate control mice [ figure 3a ] . the level of orexin a was also elevated significantly in the brain stem of the 90-day ( [ 1540.93 34.87 pg / mg ] vs. [ 1170.04 47.73 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 1583.96 21.64 pg / mg ] vs. [ 1224.87 51.62 pg / mg ] protein , p there was no significant difference in orexin a levels between the sod1-g93a transgenic mice and control groups in the csf , at either time points [ figure 3c ] . similarly , orexin b levels were also significantly increased in the hypothalamus of the 90-day ( [ 3485.35 74.62 pg / mg ] vs. [ 2352.45 142.56 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 3656.34 139.17 117.70 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice [ figure 3d ] . in the brain stem , orexin b was also significantly enhanced in both transgenic groups ( 90-day : [ 2726.79 34.06 pg / mg ] vs. [ 2411.70 64.18 pg / mg ] protein , p < 0.01 and 120-day : [ 2736.45 57.32 pg / mg ] vs. [ 2369.33 83.57 pg / mg ] protein , p < 0.05 ) as compared to littermate controls [ figure 3e ] . in the csf , no significant difference was found in orexin b levels between the respective transgenic and control groups [ figure 3f ] . orexin a and b increase in sod1-g93a transgenic mice . ( a - c ) elisa reveals that orexin a levels are enhanced in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice than controls , but there is no significant difference for orexin a in the csf . ( d - f ) elisa shows increased the level of orexin b in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice compared with control , but no significant difference is seen in the csf . ht : hypothalamus ; bs : brain stem ; csf : cerebrospinal fluid ; elisa : enzyme - linked immunosorbent assay . q - pcr analysis revealed no significant difference in the mrna levels of ox1r [ figure 4a and 4b ] and ox2r [ figure 4c and 4d ] in the hypothalamus or the brain stem between the sod1-g93a transgenic mice and controls . q - pcr of orexin-1 receptor mrna show no significant difference in the hypothalamus ( a ) and brain stem ( b ) of the 90 and 120 days sod1-g93a transgenic mice than controls . no significant difference is found in orexin-2 receptor mrna in the hypothalamus ( c ) and brain stem ( d ) of the 90 and 120 days sod1-g93a transgenic mice than controls . pearson correlation analyses were performed between sleep / wake time ( tst , wake , rem , nrem , and ds ) and expression levels of orexins ( prepro - orexin , orexin a and b ) . absolute values of the correlation coefficient confirmed high correlation between the sleep / wake time ( tst , wake , and nrem ) and expression levels of orexins . meanwhile , there was a moderate correlation between rem / ds and orexins [ table 1 ] . pearson correlation coefficient of sleep / wake time and orexins tst : total sleep time ; wake : wakefulness ; rem : rapid eyes movement sleep ; nrem : nonrapid eyes movement sleep ; ds : deep sleep ; ht : hypothalamus ; bs : brain stem . sleep / wake recordings were performed in both sod1-g93a transgenic mice and their control groups . in the 90-day sod1-g93a transgenic mice , across a 24-hour recording period , the total sleep time ( tst ) was significantly decreased ( [ 443.23 40.42 minutes ] vs. [ 569.97 39.04 minutes ] , p < 0.05 ) and wakefulness was increased ( [ 996.78 40.42 minutes ] vs. [ 870.03 39.04 minutes ] , p < 0.05 ) , compared to the littermate controls . non - rapid eye movement ( nrem ) sleep ( [ 411.29 35.41 minutes ] vs. [ 533.48 37.01 minutes ] , p < 0.05 ) and deep sleep ( ds ) ( [ 47.35 9.55 minutes ] vs. [ 107.35 11.36 minutes ] , p < 0.01 ) were also reduced . no significant difference was found in rem sleep and light sleep between the two groups for the 24-hour period [ figure 1a ] . eeg / emg recordings for 24 hours from the sod1-g93a transgenic mice and littermate control mice at 90 days and 120 days of age . ( a ) tst , nrem , and ds are significantly decreased in the 90-day sod1-g93a transgenic mice . ( b ) in the 120-day sod1-g93a transgenic mice , wake is also significantly enhanced . tst : total sleep time ; wake : wakefulness ; rem : rapid eye movement sleep ; nrem : non - rapid eye movement sleep ; ls : light sleep ; ds : deep sleep ; eeg / emg : electroencephalogram / electromyogram . in the 120-day sod1-g93a transgenic mice , a remarkable increase in wakefulness ( [ 1110.33 38.16 minutes ] vs. [ 804.29 74.57 minutes ] , p < 0.01 ) , and a decrease in tst ( [ 329.67 38.16 minutes ] vs. [ 635.71 74.57 minutes ] , p < 0.01 ) , nrem ( [ 306.03 36.81 minutes ] vs. [ 534.68 82.68 minutes ] , p < 0.05 ) and ds ( [ 22.88 10.27 minutes ] vs. [ 107.90 25.21 minutes ] , p < 0.05 ) were observed . however , in contrast with the 90-day sod1-g93a transgenic mice , rem sleep was decreased ( [ 23.62 9.96 minutes ] vs. [ 101.08 13.93 minutes ] , p < 0.01 ) in the 120-day sod1-g93a transgenic mice compared to the 120-day littermate controls [ figure 1b ] . next , we evaluated the changes in the orexin system in the sod1-g93a transgenic mice . first , we tested the level of mrna and protein expression of prepro - orexin . q - pcr showed increased levels of prepro - orexin mrna in the hypothalamus of the 90-day ( 1.84 0.24 vs. 1.00 0.20 , p < 0.05 ) and 120-day ( 2.06 0.25 vs. 1.00 0.14 , p < 0.01 ) sod1-g93a transgenic mice , compared to controls [ figure 2a ] . the western blotting analysis also showed increased levels of prepro - orexin protein in the hypothalamus of both 90-day ( 2.33 0.24 vs. 1.00 0.18 , p < 0.01 ) and 120-day ( 2.30 0.14 vs. 1.00 0.19 , p < 0.01 ) sod1-g93a transgenic mice as compared to littermate controls [ figure 2b ] . ( a ) q - pcr was performed in the hypothalamic tissues from the sod1-g93a transgenic mice and control groups . prepro - orexin mrna is significantly elevated in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice , compared to control . ( b ) western blotting was performed with antibody anti - orexin - prepro in the sod1-g93a transgenic mice and control groups . prepro - orexin expression is increased in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice as compared to control . data are expressed as mean standard error of mean ( n = 46/group ) . we then tested the protein levels of orexin a and b , using elisa . in the hypothalamus , the level of orexin a was significantly enhanced in the 90-day ( [ 2126.47 70.65 pg / mg ] vs. [ 1591.52 61.25 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 2166.32 115.98 pg / mg ] vs. [ 1446.18 41.31 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice , compared to their respective littermate control mice [ figure 3a ] . the level of orexin a was also elevated significantly in the brain stem of the 90-day ( [ 1540.93 34.87 pg / mg ] vs. [ 1170.04 47.73 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 1583.96 21.64 pg / mg ] vs. [ 1224.87 51.62 pg / mg ] protein , p < 0.01 ) transgenic groups [ figure 3b ] . there was no significant difference in orexin a levels between the sod1-g93a transgenic mice and control groups in the csf , at either time points [ figure 3c ] . similarly , orexin b levels were also significantly increased in the hypothalamus of the 90-day ( [ 3485.35 74.62 pg / mg ] vs. [ 2352.45 142.56 pg / mg ] protein , p < 0.01 ) and 120-day ( [ 3656.34 139.17 pg / mg ] vs. [ 2411.57 117.70 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice [ figure 3d ] . in the brain stem , orexin b was also significantly enhanced in both transgenic groups ( 90-day : [ 2726.79 34.06 64.18 pg / mg ] protein , p < 0.01 and 120-day : [ 2736.45 57.32 pg / mg ] vs. [ 2369.33 83.57 pg / mg ] protein , p < 0.05 ) as compared to littermate controls [ figure 3e ] . in the csf , no significant difference was found in orexin b levels between the respective transgenic and control groups [ figure 3f ] . orexin a and b increase in sod1-g93a transgenic mice . ( a - c ) elisa reveals that orexin a levels are enhanced in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice than controls , but there is no significant difference for orexin a in the csf . ( d - f ) elisa shows increased the level of orexin b in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice compared with control , but no significant difference is seen in the csf . ht : hypothalamus ; bs : brain stem ; csf : cerebrospinal fluid ; elisa : enzyme - linked immunosorbent assay . q - pcr analysis revealed no significant difference in the mrna levels of ox1r [ figure 4a and 4b ] and ox2r [ figure 4c and 4d ] in the hypothalamus or the brain stem between the sod1-g93a transgenic mice and controls . q - pcr of orexin-1 receptor mrna show no significant difference in the hypothalamus ( a ) and brain stem ( b ) of the 90 and 120 days sod1-g93a transgenic mice than controls . no significant difference is found in orexin-2 receptor mrna in the hypothalamus ( c ) and brain stem ( d ) of the 90 and 120 days sod1-g93a transgenic mice than controls . pearson correlation analyses were performed between sleep / wake time ( tst , wake , rem , nrem , and ds ) and expression levels of orexins ( prepro - orexin , orexin a and b ) . absolute values of the correlation coefficient confirmed high correlation between the sleep / wake time ( tst , wake , and nrem ) and expression levels of orexins . meanwhile , there was a moderate correlation between rem / ds and orexins [ table 1 ] . pearson correlation coefficient of sleep / wake time and orexins tst : total sleep time ; wake : wakefulness ; rem : rapid eyes movement sleep ; nrem : nonrapid eyes movement sleep ; ds : deep sleep ; ht : hypothalamus ; bs : brain stem . in summary , we observed marked sleep disturbances in the sod1-g93a mouse model of als , and demonstrated that the increase in expression of orexins correlated with these sleep disturbances in these mice . sleep / wake disturbances are often reported by patients with als , but no related animal studies have been published . the exact mechanism behind the sleep / wake disturbances in als remains to be determined . in the present study , for the first time , we observed enhanced wakefulness and reduced sleep time ( including tst , nrem , and ds ) in both 90-day and 120-day sod1-g93a transgenic mice . these results indicate that sleep / wake disturbances are early symptoms in these sod1-g93a transgenic mice , and these disturbances may be correlated with the onset of als . in addition , rem sleep time was significantly decreased in the 120-day sod1-g93a transgenic mice as compared to the 90-day sod1-g93a transgenic mice , suggesting an aggravation and the possible link of sleep disturbances with disease progression . therefore , studies on sleep disturbances in als are of significance to elucidate the etiology and pathogenesis of als . moreover , considering the early occurrence of sleep disorders in ad and pd , we speculate that the dysregulation of sleep might be common in the early stages of neurodegenerative diseases . to further investigate the mechanism of sleep / wake disturbances in als sleep is a complicated behavior regulated by wakefulness promoting systems , sleep promoting systems , and circadian rhythms . wakefulness is promoted by multiple groups of wan , which are mainly located in the brain stem and hypothalamus , including cholinergic neurons , orexinergic neurons , and dopaminergic neurons . neurotransmitters in these wan , including acetylcholine , dopamine , glutamate , histamine , norepinephrine , orexin , and serotonin , promote wakefulness . prepro - orexin , which is produced in hypothalamic neurons , matures into two neuropeptides , orexin a and orexin b. these two peptides activate monoaminergic and cholinergic neurons in the hypothalamus and brain stem to maintain a long , consolidated awake period . in our study , prepro - orexin was increased in the hypothalamus in the 90 and 120 days sod1-g93a transgenic mice . orexin a and b were significantly enhanced in the hypothalamus and brain stem of the 90 days and 120 days sod1-g93a transgenic mice compared with littermate controls . these results suggest a strong association between increased expression of orexins and the extension of wakefulness in als mice . pearson correlation analysis , which showed moderate to high correlations between sleep / wake time and expression of orexins , further confirmed this association . these results indicate that the extension of wakefulness and sleep / wake disturbances in the als mice may be caused , at least partly , by increase in expression of orexins . according to the absolute value of the correlation coefficient in table 1 , nrem had the highest correlation with expression of orexins among all stages of sleep . however , no differences were detected in the levels of orexin receptors in the hypothalamus and brain stem between the als mice and control groups , suggesting that enhanced wakefulness and other sleep disturbances in these transgenic mice were not promoted by upregulation of orexin receptors . in addition , the levels of orexin a and b were only elevated in the brain tissue other than the csf in the sod1-g93a transgenic mice . this is consistent with the clinical study by van rooij et al . , which demonstrated that csf orexin levels were normal in patients with als . sleep disturbances occur before disease onset in the als sod1-g93a transgenic mice . increased expression of orexins further experiments need to be carried out in the future to investigate the underlying mechanisms .

backgroundchanneling bias may occur when a newly marketed drug and an established drug , despite similar indications , are prescribed to patients with different prognostic characteristics ( ie , confounding).aimto investigate channeling bias and its impact on relative effectiveness of glucagon - like peptide-1 ( glp-1 ) analogs versus basal insulin and dipeptidyl peptidase-4 inhibitors ( dpp-4i ) versus sulfonylurea.methodsin the uk clinical practice research datalink , patients with type 2 diabetes initiating treatment between 2006 and 2015 were included . analyses were stratified by years since first prescription of glp-1 and dpp-4i , respectively . the characteristics of glp-1 versus insulin and dpp-4i versus sulfonylurea initiators were compared over time . after propensity score matching , the relative effectiveness regarding 6-month changes in glycated hemoglobin ( hba1c ) and body weight was estimated.resultsin total , 8,398 glp-1 , 14,807 insulin , 24,481 dpp-4i , and 33,505 sulfonylurea initiators were identified . no major channeling was observed . considerable overlap in distributions of characteristics allowed for propensity score - matched analyses . relative effectiveness was similar across time . the overall relative effect of glp-1 versus insulin showed no difference for hba1c and relative increase in body weight ( 3.57 kg [ 95% confidence interval { ci } : 3.21 , 3.92 ] ) for insulin . the overall relative effect of dpp-4i versus sulfonylurea showed relative decrease in hba1c ( 0.34% [ 95% ci : 0.38 , 0.30 ] ) and increase in body weight ( 1.58 kg [ 95% ci : 1.38 , 1.78 ] ) for sulfonylurea.conclusionno major channeling was identified in the investigated glucose - lowering drugs . relative effectiveness could be estimated already in the first year after launch and was consistent in the years thereafter . background : sleep / wake disturbances in patients with amyotrophic lateral sclerosis ( als ) are well - documented , however , no animal or mechanistic studies on these disturbances exist . orexin is a crucial neurotransmitter in promoting wakefulness in sleep / wake regulation , and may play an important role in sleep disturbances in als . in this study , we used sod1-g93a transgenic mice as an als mouse model to investigate the sleep / wake disturbances and their possible mechanisms in als.methods:electroencephalogram/electromyogram recordings were performed in sod1-g93a transgenic mice and their littermate control mice at the ages of 90 and 120 days , and the samples obtained from these groups were subjected to quantitative reverse transcriptase - polymerase chain reaction , western blotting , and enzyme - linked immunosorbent assay.results:for the first time in sod1-g93a transgenic mice , we observed significantly increased wakefulness , reduced sleep time , and up - regulated orexins ( prepro - orexin , orexin a and b ) at both 90 and 120 days . correlation analysis confirmed moderate to high correlations between sleep / wake time ( total sleep time , wakefulness time , rapid eye movement [ rem ] sleep time , non - rem sleep time , and deep sleep time ) and increase in orexins ( prepro - orexin , orexin a and b).conclusion : sleep / wake disturbances occur before disease onset in this als mouse model . increased orexins may promote wakefulness and result in these disturbances before and after disease onset , thus making them potential therapeutic targets for amelioration of sleep disturbances in als . further studies are required to elucidate the underlying mechanisms in the future .

subdural spinal hematoma is an extremely rare complication of spinal and epidural anesthesia and may lead to cauda equina syndrome , bowel bladder dysfunction or even complete permanent paraplegia . a 60-year - old female patient was diagnosed to have osteoarthritis of right knee joint and was advised knee arthroplasty . enoxaparin sodium 40 mg ( lmwh ) was started 9 h after surgery and was followed by once daily dose . foot pump and compression stockings were applied for deep vein thrombosis ( dvt ) prophylaxis . on day 2 , the epidural catheter was removed 13 h after last dose , and she was discharged on 3 postoperative day in stable condition . at the time of discharge , on 4 postoperative day , she developed sudden onset pain in the lower back which was radiating to left lower limb . the pain was followed by loss of sensation and weakness in the left lower limb . it showed a large subdural hematoma , which was compressing the thecal sac at l2-l3 level . laminectomy with decompression of spinal cord and evacuation of hematoma was done on the emergent basis . postoperatively patient recovered well with complete control over bowel and bladder functions , but foot drop on the left side persisted after 6 months of follow - up . combined spinal epidural anesthesia though safe has been associated with uncommon neurologic complications like subdural hematoma . venous stasis , endothelial injury and hypercoagulable conditions contribute to the postoperative development of dvt which can be a life - threatening complication . in surgeries as total knee and hip replacement , direct injury to the large vessels of lower limb predispose patients for increased chances of dvt especially in absence of postoperative anticoagulation prophylaxis . however , prophylaxis must be balanced against the risks for postoperative bleeding and epidural hematoma . spinal and epidural anesthetic procedures in combination with anti - coagulant therapy represent the fifth most common cause of spinal hematoma . three cases developed paraplegia due to subdural hematoma which was due to underlying coagulation disorder . and horlocker and wedel highlighted the risk of spinal hematoma which gets aggravated by concomitant administration of anticoagulants and anti - platelet drugs . fryburg et al . , reported two patients who suffered from spontaneous acute spontaneous subdural hematoma while taking fondaparinux for venous thromboembolism prophylaxis . it is recommended that lmwh should be used once daily and to be started 12 - 24 h postoperatively , especially when indwelling spinal catheter are present or when concomitant antiplatelet or oral anticoagulant medication is being given . patients who develop extra or subdural hematoma usually present with acute back or radicular pain radiating to limbs , followed by progressive sensory and motor involvement in either one or both the lower limbs along with saddle anesthesia and bowel and bladder dysfunction . in most of the cases , these symptoms usually develop shortly after the procedure with mean age of onset being 30 h after operation , but in our case , the patient developed sudden onset neurological symptoms suggestive of cauda equina syndrome on 4 postoperative day . urgent mri was done to confirm the diagnosis and patient was immediately taken to the operation theatre where laminectomy with decompression of spinal canal and evacuation of hematoma was done . , neurosurgical intervention must be sought immediately , because recovery is unlikely if surgery is postponed for more than 8 h. this case highlights that even in the absence of an existing spinal pathology and carefully administrated spinal - epidural anesthesia , cse may cause dreaded neurologic complication like subdural hematoma especially in the presence of anti - coagulant therapy . hence frequent 4 hourly neurological examination is essential for early detection of hematoma , which should be continued 24 hours postoperatively following a neuraxial block or upto 24 hours following catheter removal if patient is anticoagulated . in a case of suspected cauda equina syndrome , the patient should be investigated and urgent decompression should be done on emergent basis to prevent permanent neurological sequelae . hypophosphatasia is a rare enzymopathy that normally presents within the first few years of life and often has profound effects upon the periodontium . it is characterized by low or zero levels of serum and tissue non - specific alkaline phosphatase , which is necessary for normal bone mineralization . it is a heritable disorder characterised by defective mineralisation of the skeletal and dental structures of the body , and a deficiency in liver / bone / kidney isoenzyme of alkaline phosphatase ( alp ) . the symptoms are highly variable in their clinical expression , ranging from stillbirth without mineralized bone , to early loss of tooth without any bone symptoms . in 1957 , six clinical types of hypophosphatasia are currently recognized in the forms of perinatal lethal , benign prenatal , infantile , childhood , adult and odontohypophosphatasia . some infants can only live for a few days and have respiratory complications due to hypoplastic lungs and rachitic deformities of the chest . a few oral manifestations of hypophosphatasia can be given as follows : lack of cementum formationroot resorptionenamel hypoplasiadelayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . lack of cementum formation delayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . there appears to be no male / female preference , and mean age for the first tooth to be lost is 21 - 22 months . enlarged pulp spaces and roots either fail to develop fully or there is early resorption of the apices . long bones with elongated finger like processes of uncalcified tissue extending longitudinally into the poorly formed osseous tissue at the ends of long bones can be seen radiogpahically . beaten copper appearance multiple radiolucent areas on skull called gyral or convoluted markings which result due to increase in intra cranial pressure are seen . reduced enamel thickness , enlarged pulp chamber and alveolar bone atrophy are common dental findings along with generalised reduction in bone density in jaw bones . evidence of skeletal defects is found in nearly all patients and includes hypomineralization , rachitic changes , and incomplete vertebrate ossification and occasionally lateral bony spurs on the ulnae and fibulae . the most sensitive substrate marker for hypophosphatasia is an increased pyridoxal 5-phosphate ( plp ) plasma level , which often correlates with the disease severity . and , although it remains only a research technique , quantitation of urinary inorganic pyrophosphate ( ppi ) levels , which are elevated in most hypophosphatasia patients , has been reported to accurately detect carriers . in addition , increased urinary levels of phosphaoethanalamine ( pea ) are observed in most patients . differential diagnosis for this disease include osteogenesis imperfect , rickets and achondrogenesis this clinical report describes the prosthetic rehabilitation of a sixteen year old female patient with hypophosphatasia , who reported to our department with poor oral hygiene , missing teeth , unaaesthetic appearance and low self esteem . clinical examination revealed severely resorbed maxillary and mandibular alveolar ridges with partial anodontia and several root stumps . teeth present were right maxillary central incisor , maxillary right first and second molar , maxillary left first and second molar , mandibular left first and second molar , and mandibular right first and second molar . opg revealed severe alveolar bone loss with enlarged pulp chambers and stunted root growth of molars . there were horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible . [ figure 3 ] on examining the lateral cephalogram , a prognathic relation of mandible with maxilla was observed [ figure 4 ] . pre - operative intra - oral photograph showing missing teeth , root stumps and poor oral hygiene of the patient in the case report severely atrophied alveolar ridge and partial anodontia of the patient in the case report orthopantomogram showing horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible of the patient in the case report lateral cephalogram showing prognathic relation of the mandibular to the maxilla of the patient in the case report the patient was tested for alp levels , which were found to be 3 times lower than the normal values confirming the diagnosis of hypophosphatasia . treatment for this patient was planned keeping in mind her psychological aspect , young age and severe bone loss with partial anodontia . it was carried out step by step in various phases : rehabilitating the periodontal condition with non - surgical intervention that included complete oral prophylaxis and curettage ; extractions of the unwanted root stumps ; and , root canal treatment of 11 , 16 and 27 . patient was thus prepared for prosthetic intervention to replace missing teeth , improve the jaw relation and establish a comfortable working occlusion for the young girl . besides , her extraoral features like sunken cheeks , depressed mentolabial sulcus and decreased nasolabial angle were also kept in mind while fabricating the dentures . as the alveolar ridges were severely resorbed , conventional complete denture or partial denture fabrication implant retained prosthesis was also not an option as bone height was very less due to resportion , and the distance between the bone crest and the occlusal level was more , which would have led to an unfavourable crown : implant ratio . hence , it was decided to restore the missing teeth and occlusion with maxillary overdenture and mandibular cast partial denture , as this was the most favourable and conservative option for this young girl with severe bone loss and lost psychological confidence . after the pre - treatment procedures , the tentative aesthetic anterior try - in was performed [ figure 5 ] and improved aesthetics with adequate lip fullness and improved inter arch relationship was observed . the root canal treated teeth was carried out for the abutment teeth ( 11 , 16 and 27 ) , followed by the fabrication of post integrated metal copings [ figure 6 ] . mandibular cast partial metal framework was planned with a ring clasp placed on 37 and embrasure clasps on 46 and 47 . the regular procedures for overdenture and cast partial denture fabrication were carried out in the lab , followed by final denture insertion [ figure 8 ] . after placement of the dentures , we could appreciate the restored aesthetics , improved occlusion and an established vertical dimension for this young girl with a better functional occlusion that improved her speech , mastication and most importantly , her self confidence . the post operative frontal and lateral profile view depicted an improvement over the depressed mentolabial sulcus , and an increased and more acceptable nasolabial angle and cheek bulging , with an improved normal interjaw relationship . [ figures 9 10 ] patient was more confident with her improved aaesthetic smile and restored occlusion . tentative aesthetic anterior try - in of the patient in the case report post intergrated metal copings for the root canal treated abutment teeth ( 11 , 16 and 27 ) of the patient in the case report mesh framework of cast partial denture for the mandibular arch of the patient in the case report intra - oral post - operative view after maxillary overdenture and mandibular cast partial denture insertion in the mouth of the patient in the case report post - operative extra - oral frontal view with aesthetic smile , and adequate lip and cheek fullness in the patient in the case report post - operative extra - oral lateral profile view showing improved nasolabial angle , mentolabial sulcus and inter jaw relationship a few oral manifestations of hypophosphatasia can be given as follows : lack of cementum formationroot resorptionenamel hypoplasiadelayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . lack of cementum formation delayed appearance and premature decay of deciduous and permanent dentition and total tooth loss before 20 years of age . there appears to be no male / female preference , and mean age for the first tooth to be lost is 21 - 22 months . enlarged pulp spaces and reduced cortical bone thickness of the mandibular angle are one of the common characteristics . long bones with elongated finger like processes of uncalcified tissue extending longitudinally into the poorly formed osseous tissue at the ends of long bones can be seen radiogpahically . beaten copper appearance multiple radiolucent areas on skull called gyral or convoluted markings which result due to increase in intra cranial pressure are seen . reduced enamel thickness , enlarged pulp chamber and alveolar bone atrophy are common dental findings along with generalised reduction in bone density in jaw bones . evidence of skeletal defects is found in nearly all patients and includes hypomineralization , rachitic changes , and incomplete vertebrate ossification and occasionally lateral bony spurs on the ulnae and fibulae . the most sensitive substrate marker for hypophosphatasia is an increased pyridoxal 5-phosphate ( plp ) plasma level , which often correlates with the disease severity . and , although it remains only a research technique , quantitation of urinary inorganic pyrophosphate ( ppi ) levels , which are elevated in most hypophosphatasia patients , has been reported to accurately detect carriers . in addition , increased urinary levels of phosphaoethanalamine ( pea ) are observed in most patients . this clinical report describes the prosthetic rehabilitation of a sixteen year old female patient with hypophosphatasia , who reported to our department with poor oral hygiene , missing teeth , unaaesthetic appearance and low self esteem . clinical examination revealed severely resorbed maxillary and mandibular alveolar ridges with partial anodontia and several root stumps . teeth present were right maxillary central incisor , maxillary right first and second molar , maxillary left first and second molar , mandibular left first and second molar , and mandibular right first and second molar . opg revealed severe alveolar bone loss with enlarged pulp chambers and stunted root growth of molars . there were horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible . [ figure 3 ] on examining the lateral cephalogram , a prognathic relation of mandible with maxilla was observed [ figure 4 ] . pre - operative intra - oral photograph showing missing teeth , root stumps and poor oral hygiene of the patient in the case report severely atrophied alveolar ridge and partial anodontia of the patient in the case report orthopantomogram showing horizontally impacted third molars and reduced bone density with severe bone atrophy in both maxilla and mandible of the patient in the case report lateral cephalogram showing prognathic relation of the mandibular to the maxilla of the patient in the case report the patient was tested for alp levels , which were found to be 3 times lower than the normal values confirming the diagnosis of hypophosphatasia . treatment for this patient was planned keeping in mind her psychological aspect , young age and severe bone loss with partial anodontia . it was carried out step by step in various phases : rehabilitating the periodontal condition with non - surgical intervention that included complete oral prophylaxis and curettage ; extractions of the unwanted root stumps ; and , root canal treatment of 11 , 16 and 27 . patient was thus prepared for prosthetic intervention to replace missing teeth , improve the jaw relation and establish a comfortable working occlusion for the young girl . besides , her extraoral features like sunken cheeks , depressed mentolabial sulcus and decreased nasolabial angle were also kept in mind while fabricating the dentures . as the alveolar ridges were severely resorbed , conventional complete denture or partial denture fabrication was ruled out . implant retained prosthesis was also not an option as bone height was very less due to resportion , and the distance between the bone crest and the occlusal level was more , which would have led to an unfavourable crown : implant ratio . hence , it was decided to restore the missing teeth and occlusion with maxillary overdenture and mandibular cast partial denture , as this was the most favourable and conservative option for this young girl with severe bone loss and lost psychological confidence . after the pre - treatment procedures , the tentative aesthetic anterior try - in was performed [ figure 5 ] and improved aesthetics with adequate lip fullness and improved inter arch relationship was observed . the root canal treated teeth was carried out for the abutment teeth ( 11 , 16 and 27 ) , followed by the fabrication of post integrated metal copings [ figure 6 ] . mandibular cast partial metal framework was planned with a ring clasp placed on 37 and embrasure clasps on 46 and 47 . the regular procedures for overdenture and cast partial denture fabrication were carried out in the lab , followed by final denture insertion [ figure 8 ] . after placement of the dentures , we could appreciate the restored aesthetics , improved occlusion and an established vertical dimension for this young girl with a better functional occlusion that improved her speech , mastication and most importantly , her self confidence . the post operative frontal and lateral profile view depicted an improvement over the depressed mentolabial sulcus , and an increased and more acceptable nasolabial angle and cheek bulging , with an improved normal interjaw relationship . [ figures 9 10 ] patient was more confident with her improved aaesthetic smile and restored occlusion . tentative aesthetic anterior try - in of the patient in the case report post intergrated metal copings for the root canal treated abutment teeth ( 11 , 16 and 27 ) of the patient in the case report mesh framework of cast partial denture for the mandibular arch of the patient in the case report intra - oral post - operative view after maxillary overdenture and mandibular cast partial denture insertion in the mouth of the patient in the case report post - operative extra - oral frontal view with aesthetic smile , and adequate lip and cheek fullness in the patient in the case report post - operative extra - oral lateral profile view showing improved nasolabial angle , mentolabial sulcus and inter jaw relationship initially recognized by rathbun in 1948 , hypophosphatasia is a rare inborn error of metabolism caused by low activity of the tissue - nonspecific isoenzyme of alkaline phosphatase ( tnsalp ) . at least six clinical forms of hypophosphatasia have been reported , although form assignment is often challenging . two other forms include odontohypophosphatasia ( no clinical changes in long bones are present , only biochemical and dental manifestations ) and pseudohypophosphatasia . the latter is clinically indistinguishable from infantile hypophosphatasia , because serum alp activity is normal . the form od hypophosphatasia a patient is in , should be considered before making a dental treatment planning . there are less skeletal deformities ; however , more dental symptoms at the odontohypophosphatasia form as in our case several authors have reported that dentin structure is reduced in thickness and also mineral content of dentin is low in hypophosphatasia . also in this case as the number of teeth present were very few , it was difficult to obtain support to restore function by conventional prosthesis . the patient 's anxiety was also of concern during the treatment period , as the patient was young and apprehensive about how the treatment would proceed , and about the final outcome . however , constant motivation of the patient and her family led to the successful completion of treatment . a patient with hypophosphatasia , presenting with partial anodontia and highly resorbed ridges with severely constricted maxillary arch , was treated successfully with cast partial denture in mandibular arch , and an overdenture for the maxillary arch . the occlusion was restored to a functional form , and the patient was highly satisfied with the aaesthetic outcome of the treatment . thus , to conclude , a patient with hypophosphatasia poses a challenge for the clinician , especially when the patient is of young age as such patients have high expectations ; however , a thorough diagnosis , constant motivation and critical evaluation of all treatment options and correct treatment planning and selection can lead to a successful treatment for our patients . this case presentation is different as it presents a complete intra as well as extra oral rehabilitation of a young girl with hypophosphatasia , and with severely resorbed ridges , in whom conventional treatment planning was not possible . besides , managing the aesthetics , function and occlusion along with improving the extra - oral appearance of the patient posed a great challenge .

combined spinal - epidural anesthesia ( cse ) is considered safe in lower limb surgeries . we report a case of sudden neurological deterioration in a stable postoperative patient who was given cse for total knee replacement and low molecular weight heparin in postoperative period . on the 4th postoperative day , she developed sudden onset weakness in left lower limb along with bladder incontinence . magnetic resonance imaging spine revealed a subdural hematoma at l2-l3 level . immediate laminectomy along with cord decompression was done and patient recovered well except for a persistent foot drop on left side . hypophosphatasia is a congenital disease characterized by deficiency of serum and tissue non - specific alkaline phosphatase activity . the disease occurs due to mutations in the liver / bone / kidney alkaline phosphatase gene . six clinical forms of hypophosphatasia are recognized . systemic symptoms of the disease are respiratory complications , premature craniosynostosis , widespread demineralization and rachitic changes in the metaphases , stress fractures , chondrocalcinosis and osteoarthropathy . characteristic dental symptoms include premature loss of deciduous teeth , premature exfoliation of fully rooted permanent teeth , severe dental caries and alveolar bone loss . this clinical report describes the prosthetic rehabilitation of a sixteen year old female patient with hypophosphatasia with partial anodontia and no occlusion . patient was managed clinically by saving her remaining teeth , fabricating a maxillary overdenture and mandibular cast partial denture . this not only helped her functionally in biting and chewing food and achieving a good occlusion , but also restored her confidence and self image by improving the aesthetics .

chagas disease ( or american trypanosomiasis ) was recognized by world health organization as one of thirteen neglected tropical diseases in the world . it was estimated that over 10 million people are infected , and latin america was considered the area of the highest prevalence . this parasitic disease is caused by trypanosoma cruzi ( family , trypanosomatidae and order , kinetoplastida ) , a hemoflagellate protozoa , which can be found on several strains with different mechanisms of pathogenesis , immunogenicity , treatment response , and epidemiology . the development of infection includes an acute phase which lasts up to six months after infection , an indeterminate phase with no symptoms , and a chronic phase in which approximately 30% of patients show clinical evidence of heart disease or megavisceras . the current chemotherapy uses benznidazole , a nitroimidazole derivative , which is orally administered in acute phase and short - term chronic phase [ 6 , 7 ] . the usefulness of this drug is limited by its narrow therapeutic window and due to serious side effects , such as anorexia , nausea , headache , paresthesia , peripheral neuropathies , dermatitis , central nervous system ( cns ) depression , and maniac symptoms [ 7 , 8 ] . however , several parasite subpopulations with different host tissue 's tropism contribute to the low clinical efficacy of this drug . therefore , there are efforts for the discovery and design of new therapeutic compounds to treat chagas disease , due to the complexity of this disease . gallic acid ( 3,4,5-trihydroxybenzoic acid ) is a precursor of hydrolysable tannins in the biosynthesis that occurs in plants and its natural and semisynthetic derivatives have been associated with a wide variety of biological activities , such as antiproliferative [ 1113 ] , chemopreventive , antihemolytic , antioxidant , and anti - inflammatory activities . however , the main interest in gallic acid and in its derivatives has been associated with their antimicrobial properties . it has been shown that n - octyl gallate possesses fungicidal activity against saccharomyces cerevisiae and zygosaccharomyces bailii in any stage of their growth [ 18 , 19 ] . leal and coworkers reported the potent fungitoxicity of n - nonyl gallate against yeasts , dermatophytes , and hialohyphomycetes . lauryl gallate showed antibacterial activity against gram - positive bacteria , including methicillin - resistant staphylococcus aureus ( mrsa ) [ 21 , 22 ] . thus , the aim of this study was to investigate the antitrypanosomal activity of gallic acid and its esters against epimastigote forms of trypanosoma cruzi . further analysis involving interactions generated by combinations between alkyl gallates and benznidazole was also carried out . in addition , the apoptosis - inducing activity by alkyl gallates was studied through the parasite mitochondrial membrane potential assay . the compounds tested in this study were synthesized as described previously [ 14 , 15 ] . epimastigote forms of trypanosoma cruzi of y strain , described by silva and nussenzweig ( 1953 ) , which is considered a standard strain of type i , were grown in lit media ( liver infusion tryptose media ) at 28c . the cytotoxicity assay with epimastigotes t. cruzi was performed using the mtt colorimetric method as described by muelas - serrano et al . the parasites were treated with different concentrations of the substances for 72 hours and then , the parasite viability was estimated by measuring the absorbance at 595 nm . the influence of the treatments with the gallates in the mitochondrial membrane potential was assessed . the parasites were treated with the substances during 72 hours using , as standard concentration , the ic50 previously determined by the mtt assay . the percentage of parasite cells which suffered loss in the mitochondrial membrane potential due to the treatment with the tested compounds was measured by flow cytometry using jc-1 dye ( 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide , bd mitoscreen kit ) according to the manufacturer 's instruction [ 27 , 28 ] . a facscanto i cytometer was used , and the data were recorded and analyzed on the software of the equipment . the antitrypanosomal activity of a homologous series of alkyl gallates , gallic acid , and other analogues was evaluated against epimastigote forms of trypanosoma cruzi . this study was carried out in order to correlate chemical characteristics of the molecules with the observed activity , including the importance of free hydroxyl groups and side chain length . gallic acid ( 1 ) was found to be totally inactive against t. cruzi ( ic50 > 100 m ) and its derivatives up to hexyl gallate ( 27 ) exhibited similar ic50 values ( ic50 > 100 m ) . heptyl gallate ( 8 , ic50 = 37.3 0.9 ) showed weak activity which increased in the case of longer carbon chain derivatives up to undecyl gallate ( 12 , ic50 = 1.46 0.0 ) . however , the increase in the carbon chains , becoming bigger than undecyl gallate , led to lower activity , as shown for compounds 13 and 14 ( table 1 ) . such results indicate that the 3,4,5-trihydroxyphenyl moiety appeared to be necessary but not sufficient for antiprotozoal activity . on the other hand , kubo et al . ( 2001 ) verified that the length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity of the gallates . among the thirteen tested gallates ( 214 ) esterified with linear alcohols from c1 to c14 , best results were observed for esters with chain lengths ranging from 9 to 12 , which present clogp values of 4.115.30 , as reported previously by rosso et al . n - undecyl gallate ( 12 ) , the most active derivative , with ic50 of 1.46 m ( clogp = 4.90 ) , was twenty - three times more potent than benznidazole , used as positive control ( ic50 = 34.0 m ) . such results indicate that esterification led to appearance of antitrypanosomal activity , suggesting that a free carboxyl group was not crucial for protozoal death . in contrast , esters with clogp values lower than 3.32 or higher than 0.92 exhibited lower potency than the positive control . altogether , there is a clear and positive correlation among ic50 values , alkyl chain length and its contribution to the lipophilicity , which are in agreement with previous studies of this homologous series [ 19 , 29 ] . furthermore , the trypanocidal potential increased with the increase in the number of carbons found on the side chain until reaching the maximum activity , in this case at n - undecyl gallate ( 12 ) , and longer carbon chains showed lower trypanocidal activity , reaching a cutoff at n - tetradecyl gallate ( 14 ) . if the homologs longer than 14 might show antitrypanosomal activity , their ic50 values will be superior at 17.6 m , representing low potency , not corroborating for further chemical or biological investments . in previous studies , the cutoff phenomenon was observed for alkanols , which was correlated with their amphipathic properties . in the case of alkyl gallates , their amphiphilicity appeared to be dependent on the presence of two features : hydrophilic phenolic hydroxyls and hydrophobic alkyl chain . fujita and kubo ( 2002 ) reported the antifungal activity of gallates , suggesting that these compounds possessed amphiphilic capacity and surfactant properties and they might act disrupting the fluidic bilayer membrane , leading to fungal death . the synergistic effects of the combination using gallic acid and its esters with benznidazole , the antichagasic drug , were also evaluated . the definitions and mathematical determination of the formula for synergism were used as described by hellmann et al . briefly , the fractional inhibitory concentration ( fic ) index was calculated by using the formula fic index = a / b + a / c . in this formula , a , b , and c were ic50 of gallate with benznidazole , ic50 of gallate alone , and ic50 of benznidazole alone , respectively . from this calculation , a fic index 0.5 was considered evidence of synergistic effect , as this value can be generated only if the concentrations of both compounds in combination do not exceed one - quarter of the ic50 of either drug tested alone . a fic index > 4.0 indicates antagonism . when tested alone , some gallates ( 27 ) have not led to a significant number of epimastigotes toxicity , even at the highest concentration tested ( 100 m ) . therefore , to calculated fic index for these compounds , the ic50 was arbitrarily defined as 50 m ( table 2 ) . the interactions between compounds 114 and benznidazole were highly variable , indicating dependence on the gallates chain length . association between gallic acid ( 1 ) and short esters ( 24 ) with benznidazole was indifferent . additive interaction was observed for inactive short esters ( 5 and 6 ) and most potent long esters , which exhibited fic index values ranging from 0.560 to 0.765 . interestingly , medium chain length gallates ( 79 ) and the long chain length ester 14 with fic index values bellow 0.257 exhibited synergism . the most significant synergistic effect was observed for heptyl gallate ( 8 , fic = 0.084 ) , which reduced by 10-fold the concentration of benznidazole necessary to inhibit cell growth in 50% ( ic50 of 37.3 0.93 m ) . synergism was observed at m level , evidencing a strong antitrypanosomal activity and the potential of medium and long chain alkyl gallates as prototypes for further studies and development of novel therapeutic agents . it is also worth mentioning that the significant results from the association of benznidazole and alkyl gallates , which enabled the use of smaller doses of benznidazole , extended dosing intervals between consecutive administrations and short - term treatment leading to fewer and less intense side effects . urbina and coauthors ( 1988 , 1995 , 2002 , 2009 ) [ 3134 ] described systematic studies involving antifungal drugs and their associations as trypanocidal combined agents for treatment of chagas disease , such as azoles ( ketoconazole and posaconazole ) with mevinolin , terbinafine , aspirin , and amiodarone . recently , the interaction between benznidazole and parthenolide was investigated against t. cruzi , which evidenced synergistic and additive activities against epimastigotes and trypomastigotes , respectively . additionally , trypanocidal gallates ( 814 ) were investigated for their influence on the mitochondrial membrane potential . considering that the loss of mitochondrial membrane potential is a typical characteristic of apoptotic cells , it was used to elucidate a possible mode of parasite death . in living cells , the membrane of healthy mitochondria is polarized and jc-1 is rapidly internalized , increasing the concentration gradient , which may lead to its aggregation . in the flow cytometer , the jc-1 monomers were detected in the fl-1 channel ( fitcs ) , while its aggregates were observed in the red channel fl-2 ( pe ) . on the other hand , injured mitochondria exhibits depolarized membrane and jc-1 remains in the cytoplasm as monomers . then , we suggest that the cells which loss their mitochondrial membrane potential , which could be apoptotic cells , lose their fluorescence and could be captured in q2 quadrant , whereas normal cells are detected in q4 quadrant . figure 1 presents the flow cytometry analysis of n - undecyl gallate ( 12 ) on t. cruzi . among the tested gallates , compounds 8 , 9 , and 14 promoted important loss in the mitochondrial membrane potential , which maybe an apoptosis - inducing activity , with percentage of cells with reduction of mitochondrial membrane potential of 46.6 , 43.6 , and 53.2 , respectively . figure 1(a ) shows that untreated epimastigotes ( control ) did not lose their mitochondrial membrane potential which can be observed due to the high fluorescence detected in fl-2 channel ( pe ) . similarly , it is observed that n - undecyl gallate ( 12 ) ( figure 1(b ) ) did not stimulate this influence in the treated cells since it did not induce a reduction of fluorescence as shown by the control , suggesting the maintenance of the membrane potential . in contrast , the cells treated with pentamidine ( figure 1(c ) ) show intense decrease in the fluorescence represented by pe , suggesting a loss of mitochondrial membrane potential which may indicate apoptosis of the parasites analyzed . the results of flow cytometry for n - undecyl gallate ( 12 ) are presented on table 3 . the results showed that the gallate esters used in this study induced loss of the mitochondrial membrane potential and maybe apoptosis in the parasites . this indicates that one of the mechanisms of cell death induced by the gallates activity could be similar to the apoptosis . this can be visualized by comparing the results with pentamidine , an apoptosis - inducing drug , which led to different percentages of mitochondrial membrane potential relatively to the control . the treatment with some gallates , such as the substances 8 , 9 , and 14 , showed a percentage of loss of mitochondrial membrane potential similar to the pentamidine treatment . pentamidine is a dicationic drug which has been used in the last 50 years for the treatment of african trypanosomiasis and antimony - resistant leishmaniasis and is known to cause changes in mitochondrial metabolism . the drug that can cause a collapse in mitochondrial membrane potential leads to an imbalance in intracellular ca . electronic microscopy transmission studies with cells treated with pentamidine showed condensation and disruption of kinetoplast dna core and collapse of mitochondrial membrane [ 39 , 40 ] . based on the results obtained by the flow cytometry and cytotoxicity analysis , it is possible to infer that other mechanisms of action , different from apoptosis , might be associated with the death of the parasites , since the substances that caused more significant dysfunction in the mitochondrial membrane potential ( 8 , 9 , and 14 ) are not the same substances that according to the cytotoxicity assay cause higher rates of parasites death ( 10 , 11 , 12 , and 13 ) . in addition , maintenance of cell viability might be associated with low affinity or steric hindrance of the molecular target due to its physical - chemical properties . another possible mechanism of action of the gallic acid esters would be similar to the results found by abe et al . these authors showed that some gallates are potent inhibitors of the enzyme squalene epoxidase , which is involved in the biosynthesis of ergosterol and , therefore , is essential for the survival of the microorganism . ergosterol is essential to the parasite because it is the most abundant sterol found in the membrane of lower eukaryotes and is therefore found in fungi and parasitic protozoa such as leishmania and trypanosoma [ 4346 ] . the inhibition of sterol biosynthesis is associated with the loss of cell membrane fluidity , which might lead to the parasite death through the same mechanism of action of commercial drugs , such as terbinafine . mitochondria are one of the first organelles to be affected after treatment of t. cruzi with ergosterol biosynthesis inhibitors , and even stronger effect may occur when an inhibitor of squalene epoxidase is used [ 3647 ] . due to the results of our experiments that demonstrated that there are changes in the mitochondrial membrane potential of the parasites when treated with gallates , we suggest that these compounds cause the parasite death by more than one pathway , which probably includes loss of mitochondrial membrane potential , correlated with the induction of apoptosis and , also , interfering in the biosynthesis of ergosterol in the parasites . the phenol moiety is often associated with cytotoxic effects due to the possible free radical or reactive oxygen species formation , which might be deleterious to the parasite metabolism . however its esterification yielding gallate esters with different side chain lengths afforded compounds with strong trypanocidal potential , which is probably also due to a change in the mitochondrial membrane potential and an interference in the biosynthesis of ergosterol in the parasites . it is important to clarify that previous studies demonstrate a correlation between epimastigote and trypomastigote forms of t. cruzi , the last one the infective form . thus , this research encourages the development of new research aiming to apply the gallates as an alternative to current therapy against chagas disease . the prevalence and the severity of posture and gait disorders increase with ageing ( salthouse and somberg 1982 ; maki et al 1994 ; mourey et al 1998 ; rankin et al 2000 ) . indeed , balance controlis a complex sensori - motor function which requires the integration of vestibular , visual , and somesthetic information into the central nervous system ( pyramidal , extrapyramidal , and cerebellar systems ) in order to maintain antigravitar postures and to produce a suitable response to any balance perturbation ( horak and mcpherson 1996 ) . in addition , the quality of muscle and joints is important in the balance function ( alencar et al 2007 ) . increased age is associated with impaired performance in vestibular , visual and somesthesic systems , a decline in the speed of information transmission and changes in the mode of information processing in the brain , allof which lead to balance and postural disorders ( brocklehurst et al 1982 ; aniansson et al 1986 ; woollacott 1993 ; choy et al 2003 ; yordanova et al 2004 ) . concerning the vestibule , it has been reported that the number of vestibular hair cells falls by 20% to 40% in healthy subjects aged 70 years and older , when compared to young subjects , and the number of vestibular nerve fibers has also been found to decrease with age ( alpini et al 2004 ) . regarding vision , decreased visual acuity , restriction of visual field , poor depth perception , losses in contrast sensitivity at the intermediate and high spatial frequencies have been found in elderly subjects ( alpini et al 2004 ) . at the somesthesic level , various alterations in the morphology and functions of the peripheral nervous system are related to ageing . elderly subjects also suffer from a loss of myelinated and unmyelinated nerve fibers associated with axonal atrophy . aging also induces a decline in nerve conduction velocity and sensory discrimination ( verdu et al 2000 ) . moreover , the age - related decline in muscle and joint quality contributes to the proprioceptive abnormalities and also explains effector disorders found in elderly subjects . indeed , with advancing age , the frequency of sarcopenia increases and the decrease in muscle mass is associated with the replacement of muscle fibers by fat and connective tissue ( lexell 1995 ; alpini et al 2004 ; crepaldi and maggi 2005 ) . moreover , there is an increase in degenerative osteoarticular lesions and osteoporosis ( crepaldi and maggi 2005 ) . independently of sensorimotor system , postural controlis also enabled by spatial references such as the subjective vertical ( massion 1992 ) . the subjective vertical ( which can be haptic , postural , or visual ) corresponds to an individual s perception of the vertical and is therefore different from the true or gravitational vertical ( manckoundia et al 2007a ) . concerning gait , physiological ageing is associated with a reduction in the length and the height of step , an increase in dual foot contact time , and a resulting reduction in gait speed ( craik et al 1992 ; alexander 1996 ) . the negative effects of certain chronic afflictions on posture and gait will compound those of physiological ageing . these chronic diseases may be neurological ( parkinson s disease or syndrome , the after - effects of stroke , neuropathy , normal pressure hydrocephalus and amyotrophic lateral sclerosis , for example ) or osteoarticular ( osteoarthritis of the lower limbs ) , muscular , visual ( cataract , age - related macular degeneration ) , vestibular and can be associated with certain geriatric syndromes such as psychomotor disadaptation syndrome ( knutsson 1972 ; fisher 1982 , pfitzenmeyer et al 1999 ; hausdorff 2000 ; lee and scudds 2003 ; corriveau et al 2004 ; manckoundia et al 2008 ) . these balance and postural disorders make elderly people vulnerable , accelerate functional decline and contribute to the loss of autonomy ( rankin et al 2000 ) . backward disequilibrium ( bd ) is a postural disorder which is characterized by a posterior position of the centre of mass with respect to the base of support in the standing and sitting position predisposing subjects to backward falls . this postural disorder is defined by a posterior body tilt ( mourey et al 2004 ) . the pathophysiological mechanisms leading to bd behavior , however , have not yet been clearly identified . nevertheless , as observed in patients with stroke , deterioration in the subjective vertical could be associated with the appearance of bd . indeed , the literature reports that the pusher behavior due to hemiplegia is associated with deterioration in the representation of the subjective verticalin the frontal plane ( lopez et al 1997 ; prennou et al 1998 ) . a similar patho - physiological hypothesis may be involved in bd . to test this hypothesis we conducted a pilot study and found a direct correlation between the subjective vertical and the bd ( manckoundia et al 2007a ) . in this pilot study , the subjective vertical was assessed by measuring the postural vertical , which corresponds to the self - perception of the body s orientation in space and in the sagittal plane ( manckoundia et al 2007a ) . moreover , bd could be related to accentuated postural tone of the extensor muscles due to a disorder in the excitability of the corresponding descending pathways ( porter and lemon 1993 ) , or to the fear of the void in front of the subject linked to the fear of falling in the context of post - fall syndrome ( murphy and isaacs 1982 ; pfitzenmeyer et al 1999 ; mourey et al 2004 ) . although the geriatrician can recognize bd , which is not uncommon in daily clinical practice , there are no tools to evaluate the severity . moreover , only a few authors have examined this postural disorder in elderly subjects ( mourey et al 2004 ) and these studies concern mainly parkinsonian syndromes in the anglo - saxon literature ( visser et al 2003 ) . finally , there are no epidemiological data on bd . thus , in this paper , we review bd , a postural disorder that has severe consequences and can compromise functional autonomy in elderly subjects . indeed , the autonomy and functional well - being of elderly subjects suffering from bd could be improved by early diagnosis and management using basic interventions ( manckoundia et al 2007b ) . cortical and subcortical lesions of the central nervous system are the main abnormalities leading to bd . there are reports of a positive correlation between cortical and subcortical cerebral lesions , identified on magnetic resonance imaging , and bd ( pfitzenmeyer et al 2002 ) . ischemic , degenerative and traumatic lesions of the cerebral trunk , the white matter , the frontal lobes , the gray matter and the cerebellum could induce bd in elderly subjects ( van wegen et al 2002 ) . ischemic and hemorrhagic strokes are the most frequent etiologies of bd as they are in nonidiopathic parkinsonian syndromes ( visser et al 2003 ) . in addition , the literature reports other subcortical abnormalities such as normal pressure hydrocephalus or amyotrophic lateral sclerosis , which could also be associated with bd ( desai and swash 1999 ; blomsterwall et al 2000 ) . backward disequilibrium could also be due to an imbalance between the ankle extensor muscles and the ankle flexor muscles , because of hypertonia of extensor muscles , which could be caused by a peripheral neurological lesion ( porter and lemon 1993 ) . the main psychosomatic disorder associated with bd is psychomotor disadaptation syndrome and its acute form , the post - fall syndrome ( pfitzenmeyer et al 2001 ) . psychomotor disadaptation syndrome , which was first described by gaudet and colleagues in 1986 , is characterized by postural impairments , including bd , neurologic signs and psychological disorders ( mourey et al 2004 ) . several chronic diseases such as parkinsonian syndromes , multiple system atrophy , progressive supranuclear palsy , lewy body disease , binswanger s disease , subcortical vascular lesions , normal pressure hydrocephalus , lymphomas of nervous system , meningoencephalitis , hiv infection , and depression may be associated with psychomotor disadaptation syndrome ( pfitzenmeyer 1999 ; mourey et al 2004 ) . finally , other situations such as confinement to bed and/ or depression could be associated with motor and postural disorders including bd ( mobily and skemp kelley 1991 ; mourey et al 2004 ; turcu et al 2004 ) . elderly subjects suffering from bd have a high risk of falling backwards ( pfitzenmeyer et al 1999 ) . falls are associated with an increase in morbi - mortality in the elderly ( wild et al 1981 ) . hip fracture is the more dreaded injury ( rose and maffulli 1999 ) , and there is a risk of posttraumatic subdural hematoma ( noltie and denham 1981 ) . fallers can also be affected by many other injuries ( tinetti et al 1995 ) . these traumatic consequences are associated with a high risk of mortality ( tinetti et al 1995 ) . among the psychological consequences of falls , there is the fear of standing , walking , and falling ( gomez and curcio 2007 ) , allof which can exacerbate bd behavior and result in a vicious circle . the social consequences of falls include the loss of autonomy , institutionalization and the risk of becoming bedridden ( tinetti and williams 1997 ) . in addition , the loss of autonomy is associated with a decrease in physical activity leading to reduced muscle mass and functional decline which predispose the elderly to falls . falls therefore have economic repercussions and represent a major public health problem ( tinetti et al 1994 ) . regarding the consequences of falls , it is necessary for all health workers , especially physicians , involved in the management of elderly subjects to know how to recognize and manage syndromes and afflictions that could cause falls . in this context , bd , which is one of main risk factors of falls , must be detected . however , elderly subjects suffering from mild form of bd show postural instability that does not necessarily result in falls . this , nevertheless , limits their daily activities , induces hypoactivity and may lead to the use of walking aids . backward disequilibrium could have severe psychological consequences in elderly subjects even in the absence of falls . indeed , bd is often associated with a loss of self - confidence during standing and walking . this could cause fear of standing , walking and falling as described in psychomotor disadaptation syndrome ( mourey et al 2004 ; manckoundia et al 2007b ) . this fear of standing , walking and falling can appear even in the absence of a history of falls ( legters 2002 ) . the progressive loss of autonomy is the logicaloutcome of both loss of self - confidence during standing / walking , and fear of standing . bd and its motor and psychological consequences lead to postural disadaptation which in turn leads to dependence . finally , bd can bring about depression mostly because of the fear of walking and falling , loss of independence and decreased of quality of life . indeed , elderly people who become aware of the deterioration in their physical capacities will lose self - confidence and self - esteem , develop a sense of insecurity and become withdrawn . as a result elderly subjects suffering from bd have a high risk of falling backwards ( pfitzenmeyer et al 1999 ) . falls are associated with an increase in morbi - mortality in the elderly ( wild et al 1981 ) . hip fracture is the more dreaded injury ( rose and maffulli 1999 ) , and there is a risk of posttraumatic subdural hematoma ( noltie and denham 1981 ) . fallers can also be affected by many other injuries ( tinetti et al 1995 ) . these traumatic consequences are associated with a high risk of mortality ( tinetti et al 1995 ) . among the psychological consequences of falls , there is the fear of standing , walking , and falling ( gomez and curcio 2007 ) , allof which can exacerbate bd behavior and result in a vicious circle . the social consequences of falls include the loss of autonomy , institutionalization and the risk of becoming bedridden ( tinetti and williams 1997 ) . in addition , the loss of autonomy is associated with a decrease in physical activity leading to reduced muscle mass and functional decline which predispose the elderly to falls . falls therefore have economic repercussions and represent a major public health problem ( tinetti et al 1994 ) . regarding the consequences of falls , it is necessary for all health workers , especially physicians , involved in the management of elderly subjects to know how to recognize and manage syndromes and afflictions that could cause falls . in this context , bd , which is one of main risk factors of falls , must be detected . however , elderly subjects suffering from mild form of bd show postural instability that does not necessarily result in falls . this , nevertheless , limits their daily activities , induces hypoactivity and may lead to the use of walking aids . backward disequilibrium could have severe psychological consequences in elderly subjects even in the absence of falls . indeed , bd is often associated with a loss of self - confidence during standing and walking . this could cause fear of standing , walking and falling as described in psychomotor disadaptation syndrome ( mourey et al 2004 ; manckoundia et al 2007b ) . this fear of standing , walking and falling can appear even in the absence of a history of falls ( legters 2002 ) . the progressive loss of autonomy is the logicaloutcome of both loss of self - confidence during standing / walking , and fear of standing . bd and its motor and psychological consequences lead to postural disadaptation which in turn leads to dependence . finally , bd can bring about depression mostly because of the fear of walking and falling , loss of independence and decreased of quality of life . indeed , elderly people who become aware of the deterioration in their physical capacities will lose self - confidence and self - esteem , develop a sense of insecurity and become withdrawn . as a result among the tests to assess balance and gait in elderly subjects , only the minimum motor test includes items for a qualitative analysis of bd ( mourey et al 2005 ) . the other usual tests , such as the tinettitest ( tinetti 1986 ) , the timed up and go test ( podsiadlo and richardson 1991 ) or the berg balance scale ( berg et al 1992 ) , are not able to diagnose bd . indeed , these tests are only suitable to evaluate the consequences of bd on certain daily tasks such as sit - to - stand , back - to - sit , maintaining a standing position or walking . given the absence of a specific tool to assess bd , diagnosis remains only qualitative , based on observations of elderly subjects in the sagittal plane , in the sitting position as well as in the static and dynamic standing position . however , a quality assessment of this kind of postural disorder requires a quantitative scale . the management of elderly subjects suffering from bd has to be multidisciplinary , associating different categories of health professionals including physicians , physiotherapists , psychologists , nurses , and nurse s aides . in addition , the management of bd must begin quickly , immediately after making the diagnosis , which is based on clinical observation . the medical management consists of the search for and the treatment of possible causes of the bd . the physician must therefore make a detailed examination , which could be completed by targeted biological analyses or radiological examinations . there are few reports concerning the rehabilitation of frail patients showing major limitations in motor performance . the association of strength training and progressive functional training improves the physical capacities in frail geriatric patients ( hauer et al 2001 ) . a recent review of the literature shows that progressive resistance training alone is not an effective therapy for balance control ( orr et al 2008 ) . improving the dynamic and static control of the centre of mass is the main objective of physiotherapy . practicing turning over in bed is introduced very early in the rehabilitation program in order to restore autonomy in bed and the capacity to achieve transfers . then , balance training is implemented in static and dynamic positions . it is advisable to start balance rehabilitation using intrinsic exercises first , followed by extrinsic exercises in the sitting position as well as in the standing position . the forward projection of the trunk and the stabilization of the head in space are developed in order to make the sit - to - stand easier . indeed , the sit - to - stand is a determinant stage of functional rehabilitation . in the standing position , the use of a removable heelpiece could help to reduce bd at the beginning of the physiotherapy . in addition , subjects suffering from bd have to be trained to rise from the floor . the physiotherapist , therefore has to control the heart rate and the breathing frequency ( brondel et al 2005 ) . the choice of exercise must be personalized according to the postural abilities of the subject . in parallel with rehabilitation by the physiotherapist , nurses and nurse s aides have to stimulate elderly patients suffering from bd through the activities of everyday life , but in doing so they must avoid pulling on the patient s upper limbs during transfers . finally , the psychologist must reassure and motivate elderly subjects suffering from bd and not overdramatize the situation to help them regain self - confidence . besides , the psychologist must help elderly subjects suffering from bd to overcome disafferentation , fear of standing and walking as well as fear of falling ( manckoundia et al 2007b ) . although it has been known by physicians for several decades , bd , which could have serious physical , psychologicalor social consequences , has rarely been studied in the literature . the causes of bd are often mixed and associate somatic and psychological pathologies among which there is post - fall syndrome . the management of bd must be interdisciplinary ( involving physicians , physiotherapists , psychologists , nurses , nurses aides ) . physiotherapy , which is a major component of the management strategy , must be started early and adjusted to allof the situations of everyday life .

american trypanosomiasis or chagas disease is a debilitating disease representing an important social problem that affects , approximately , 10 million people in the world . the main aggravating factor of this situation is the lack of an effective drug to treat the different stages of this disease . in this context , the search for trypanocidal substances isolated from plants , synthetic or semi synthetic molecules , is an important strategy . here , the trypanocidal potential of gallates was assayed in epimastigotes forms of t. cruzi and also , the interference of these substances on the mitochondrial membrane potential of the parasites was assessed , allowing the study of the mechanism of action of the gallates in the t. cruzi organisms . regarding the preliminary structure - activity relationships , the side chain length of gallates plays crucial role for activity . nonyl , decyl , undecyl , and dodecyl gallates showed potent antitrypanosomal effect ( ic50 from 1.46 to 2.90 m ) in contrast with benznidazole ( ic50 = 34.0 m ) . heptyl gallate showed a strong synergistic activity with benznidazole , reducing by 105-fold the ic50 of benznidazole . loss of mitochondrial membrane potential induced by these esters was revealed . tetradecyl gallate induced a loss of 53% of the mitochondrial membrane potential , at ic50 value . backward disequilibrium is observed frequently in daily clinical practice . however , there are no epidemiological data concerning this postural disorder . defined by a posterior position of the centre of mass with respect to the base of support , backward disequilibrium is abnormal postural behavior , usually characterized by a posterior trunk tilt in standing and sitting positions , which predisposes subjects to backward falls . many afflictions whether they are somatic ( degenerative , ischemic and traumatic brain lesions ) , psychosomatic ( psychomotor disadaptation syndrome , confinement to bed , nonuse situations ) or psychological ( depression ) can cause backward disequilibrium . a vicious circle of falls , and loss of autonomy can arise and this is the main consequence of backward disequilibrium . thus , in this paper , we review backward disequilibrium in elderly subjects with regard to the causes , consequences , assessment , and management .

conjunctival tick attachment is a rare occurrence , with only 7 cases reported in the literature . the tick species ixodes holocyclus is found in australia and belongs to the family of hard ticks , ixodidae , which are a group of blood - sucking acarine ectoparasites [ 1 , 2 ] . it is also known as the paralysis tick because it produces a toxin that can cause motor paralysis , which is mainly a problem seen in veterinary medicine and rarely in humans , although children under 5 years of age are at greatest risk . ticks are known vectors of infectious diseases and are associated with a range of ocular and systemic conditions . we describe a rarely seen ophthalmic finding : conjunctival attachment of a live paralysis tick . we also discuss the literature regarding ocular tick attachment , clinical features , and management . a 10-year - old boy presented to the clinic with a 2-day history of right - eye redness and itch . the patient was visiting a property surrounded by bush land in the northern suburbs of sydney , n.s.w . his visual acuity was 6/6 in the right eye and 6/5 in the left eye . slit lamp examination revealed a live translucent - bodied organism with pulsatile fluid movement ( at a different rate to the patient 's heart ) , firmly attached to his right temporal bulbar conjunctiva ( fig . there was conjunctival vessel dilatation surrounding the organism , but otherwise , ocular examination was unremarkable . under general anaesthesia , the conjunctival tissue and the organism onto which it was attached ( see arrow in fig . 1b ) were elevated and excised en bloc . the excised area ( approximately 3 mm in diameter ) the patient was started on a 1-week course of chloramphenicol ophthalmic ointment and oral cephalexin 4 times a day . the recovery of our patient , after 3 weeks , has so far been uneventful . the organism was examined and identified as the larval stage of the tick species i. holocyclus from the family of hard ticks , ixodidae . histopathological examination revealed deep embedding of the larval tick 's mouthparts , with extension into the conjunctival stroma ( fig . conjunctival attachment of a live tick is a rare occurrence with only 7 cases ( 6 reports ) found in the literature [ 4 , 5 , 6 , 7 , 8 , 9 ] . the paralysis tick , i. holocyclus , is one of 7 species in the subgenus sternalixodes , all of which are exclusive to australia . the paralysis tick is distributed predominantly along the east coast from north queensland to southern victoria , and possibly into tasmania ( fig . hard ticks , which include the paralysis tick , go through 4 life stages from the egg to larva , to nymph , and finally to the adult stage . from the vegetation where they quest for a host , female ticks attach onto passing vertebrate hosts , including humans , and feed on the blood . paralysis ticks , including the larval form , have long mouthparts to enable a strong attachment to the host [ 1 , 10 ] . the paralysis tick tends to show seasonality in life stages , and the larval stage is most active at this time of the year ( summer ) in australia . during blood feeding , ticks transfer saliva , which contains a cocktail of chemicals including toxins , various allergy - inducing proteins , and potentially disease - causing pathogens [ 10 , 11 , 12 , 13 ] . tick bites to the eye may cause ocular irritation at the attachment site , conjunctivitis , uveitis , keratitis , and vasculitis . ticks can become embedded in the meibomian gland orifice and cause eyelid swelling and pain . although rarely reported in humans , toxins transmitted from the paralysis tick 's saliva have been associated with ascending motor paralysis , diplopia , blurred vision , and photophobia especially in children . the paralysis tick , however , needs several days of engorgement on its host before these signs of tick paralysis are manifested . this is because adult ticks go through various phases of feeding , including a slow phase , where the production of toxins in the tick 's salivary glands is very low on day 1 and 2 of the blood feed , and a rapid phase , where salivary production peaks on day 5 [ 3 , 13 ] . the timing of these phases varies according to life stage and , typically , larvae feed for a shorter duration . our patient had a larval tick attached to his conjunctiva for 2 days and has so far not developed any visual or neurological symptoms . paralysis ticks are vectors of infectious diseases such as queensland tick typhus and flinders island spotted fever . tick bites have been associated with lyme borreliosis , although more conclusive evidence is required to determine that the borrelia bacteria is present in ticks in australia . regarding the larval stage of hard ticks , the infectious diseases society of america ( idsa ) does not recommend routine antibiotic prophylaxis due to the low risk of disease transmission . this is because the larval form typically has not previously fed on another host from which they may have acquired a pathogen . however , antibiotic prophylaxis is often given when the tick bite has occurred in a location where infection would have more detrimental consequences , such as the eye . our patient was started on a 1-week course of oral cephalexin and chloramphenicol ophthalmic ointment . tick allergy has been well documented and ranges from mild urticaria , allergic conjunctivitis , to anaphylaxis . it was recently recognised that patients previously bitten by the paralysis tick may subsequently develop anaphylaxis from the consumption of mammalian meat and meat by - products such as gelatine , although this condition has not yet been associated with the larval stage . the mechanism of this is beyond the scope of this paper , but clinicians may consider close monitoring of the patient for hypersensitivity reactions . five out of the 7 cases of conjunctival tick attachment had the removal by conjunctival excision ( table 1 ) . the reasons for surgical removal were either a firmly attached tick [ 5 , 6 , 7 ] or simply that the conjunctiva was involved . one case report proposed that blunt , medium - tipped , angled forceps offered the best result for removing ticks not deeply seated in the conjunctiva . another case successfully used blunt force with a cotton applicator but admitted difficulty with this approach for embedded ticks . in all 7 cases , recovery of the patient was uneventful , and no parts of the tick remained in the conjunctiva . the australasian society of clinical immunology and allergy ( ascia ) recommends first killing an embedded tick with ether - containing agents or permethrin - based products and then letting it drop off the host or removing it with fine - tipped forceps to avoid squeezing saliva into the host 's vasculature . however , ascia recognises that this method is not suitable for all patients , especially when the tick is embedded in sensitive areas of the body such as the eye . despite the different tick removal methods postulated , there is consensus in the literature that the goal of treatment should be complete removal of the tick from the host to prevent further transmission of pathogens , allergens , and toxins [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ] . in our patient , the larval tick was too firmly attached to the bulbar conjunctiva , so complete and safe removal of it using forceps was not possible in a conscious child . it is not uncommon for ticks to inject further toxins into the host if disturbed . the tick was removed completely and our patient has so far not experienced any post - operative complications or systemic illness . the goal of treatment for conjunctival tick attachment should be to completely and safely remove the tick as soon as possible to prevent further transmission of pathogens , toxins , and allergens to the patient . surgical removal of the tick and surrounding conjunctiva ( with general anaesthetic if necessary ) proved simple and effective with no adverse sequelae , a decision supported by the histopathological examination revealing deep embedding of the tick 's mouthparts into the conjunctiva . twenty - three patients were administered a high - dose and low - dose atd mixture ( 100 vs. 25 g amino acids ) in a double - blind randomized crossover design with two sessions , separated by at least 4 days ( booij et al . the 100 g and 25 g atd mixtures have previously been shown to lower plasma trp levels by approximately 90% and 50% , respectively , in this sample ( booij et al . , 2005b ) as well as in other samples ( e.g. , booij , van der does , haffmans , & riedel , 2005a ) . the study was approved by an independent medical ethics committee ( metigg , utrecht ) and was performed according to their guidelines and regulations . all patients were informed about the study by their clinician and in detail by one of the investigators ( l.b . ) , and provided written informed consent . the sample has been described in detail previously ( booij et al . , 2005b ) . inclusion criteria were : age between 18 and 65 years ; ongoing treatment with an ssri or a serotonin noradrenaline reuptake inhibitor for at least 4 weeks , meeting dsm - iv criteria for depression in full or partial remission , and a hamilton depression rating scale ( hrsd , 17 items ) ( hamilton , 1960 ) lower than 15 ( frank et al . , 1991 ) . exclusion criteria were : substance abuse within the past 3 months , psychosis ( lifetime ) , major physical illness , lactation , and pregnancy . after excluding two drop - outs and two statistical outliers , 19 participants remained for statistical analyses ( cf . booij et al . , 2005b ) . as described in the original article ( booij et al . , 2005b ) , depressive symptoms were assessed with the 10-item montgomery asberg depression rating scale ( madrs ) ( montgomery & asberg , 1979 ) . the sleep items were omitted , since this could not change within an atd session . diagnoses , and demographic and clinical background variables were verified with the structured clinical interview for dsm - iv ( scid - i ) ( first , spitzer , gibbon , & williams , 2005 ) . the simon task consisted of 64 trials presented in two consecutive blocks in which the stimulus interval differed ( 2,250 ms fixed vs. 2,2505,500 ms variable ; originally included to investigate a possible effect of task complexity ) right was presented in randomized order either at the left or the right side of the screen . participants were instructed to respond to the meaning of the word ( target)that is , making a left - hand response to the word left and a right - hand response to the word the same number of compatible ( distracter location matches the target word ) and incompatible ( distracter location conflicts with the target word ) stimuli was used . 1 venous blood ( 10 ml ) was taken in the morning , 6 hr after atd and the next day ( t + 24 ) and was analyzed for both total plasma trp and the other large neutral amino acids ( fekkes , vandalen , edelman , & voskuilen , 1995 ) . mood was assessed 1 hr before atd ( t 1 ) , 6.5 hr later ( t + 6.5 ) , and the next morning ( t + 24 ) . the simon task ( left / right task ) was administered ( booij et al . , 2005b ) approximately 5.5 hr after administration of the atd mixture . the order of the low - dose session and the high - dose session was counterbalanced . cognitive performance and mood repeated measures anovas were used for sequential analysis of simon performance , separately for correct reaction time ( rt ) and percent accuracy . in order to measure sequential adjustments in simon task performance , we not only included the factor compatibility of the current trial as is usually done , but we also added the factor compatibility of the previous trial . standard conflict adaptation that is , the modulation of the compatibility effect as a function of previous - trial compatibility ( cf . 1a)should yield an interaction effect between current and previous trial compatibility ( egner , 2007 ; gratton et al . , 1992 ) . to analyze direct effects of the atd manipulation on conflict adaptation , intervention ( baseline vs. low - dose vs. high - dose atd ) the effect of mood state on conflict adaptation , for the low - dose atd and high - dose atd sessions separately , was analyzed by using mood scores ( measured at + 6.5 hr ) as a covariate . additionally , we also ran separate anovas in which we included the binary between - subjects factors ongoing treatment ( ssri vs. snri ) and mood response ( absence vs. presence of depressed mood increase from t-1 to t + 6.5 in the high - dose atd session).fig . 1a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation to visualize the hypothesized association between mood and conflict adaptation , we calculated standard individual conflict - adaptation scores by subtracting the interference effect following a correct conflict ( incompatible ) trial from the interference effect following a correct nonconflict ( compatible ) trial ( cf . 1a ) . before averaging sequential conditions for each individual , the first trial of each block , trials following an error , and trials with rts not fitting the outlier criterion ( deviating more than 2.5 sds from the individual condition - specific mean ) were excluded from analyses . the sample has been described in detail previously ( booij et al . , 2005b ) . inclusion criteria were : age between 18 and 65 years ; ongoing treatment with an ssri or a serotonin noradrenaline reuptake inhibitor for at least 4 weeks , meeting dsm - iv criteria for depression in full or partial remission , and a hamilton depression rating scale ( hrsd , 17 items ) ( hamilton , 1960 ) lower than 15 ( frank et al . , 1991 ) . exclusion criteria were : substance abuse within the past 3 months , psychosis ( lifetime ) , major physical illness , lactation , and pregnancy . after excluding two drop - outs and two statistical outliers , 19 participants remained for statistical analyses ( cf . as described in the original article ( booij et al . , 2005b ) , depressive symptoms were assessed with the 10-item montgomery asberg depression rating scale ( madrs ) ( montgomery & asberg , 1979 ) . the sleep items were omitted , since this could not change within an atd session . diagnoses , and demographic and clinical background variables were verified with the structured clinical interview for dsm - iv ( scid - i ) ( first , spitzer , gibbon , & williams , 2005 ) . the simon task consisted of 64 trials presented in two consecutive blocks in which the stimulus interval differed ( 2,250 ms fixed vs. 2,2505,500 ms variable ; originally included to investigate a possible effect of task complexity ) . the word left or right was presented in randomized order either at the left or the right side of the screen . participants were instructed to respond to the meaning of the word ( target)that is , making a left - hand response to the word left and a right - hand response to the word right,and to ignore its location ( distracter ) , as fast as possible . the same number of compatible ( distracter location matches the target word ) and incompatible ( distracter location conflicts with the target word ) stimuli was used . 1 venous blood ( 10 ml ) was taken in the morning , 6 hr after atd and the next day ( t + 24 ) and was analyzed for both total plasma trp and the other large neutral amino acids ( fekkes , vandalen , edelman , & voskuilen , 1995 ) . mood was assessed 1 hr before atd ( t 1 ) , 6.5 hr later ( t + 6.5 ) , and the next morning ( t + 24 ) . the simon task ( left / right task ) was administered ( booij et al . , 2005b ) approximately 5.5 hr after administration of the atd mixture . the order of the low - dose session and the high - dose session was counterbalanced . cognitive performance and mood repeated measures anovas were used for sequential analysis of simon performance , separately for correct reaction time ( rt ) and percent accuracy . in order to measure sequential adjustments in simon task performance , we not only included the factor compatibility of the current trial as is usually done , but we also added the factor compatibility of the previous trial . standard conflict adaptation that is , the modulation of the compatibility effect as a function of previous - trial compatibility ( cf . 1a)should yield an interaction effect between current and previous trial compatibility ( egner , 2007 ; gratton et al . , 1992 ) . to analyze direct effects of the atd manipulation on conflict adaptation , intervention ( baseline vs. low - dose vs. high - dose atd ) the effect of mood state on conflict adaptation , for the low - dose atd and high - dose atd sessions separately , was analyzed by using mood scores ( measured at + 6.5 hr ) as a covariate . additionally , we also ran separate anovas in which we included the binary between - subjects factors ongoing treatment ( ssri vs. snri ) and mood response ( absence vs. presence of depressed mood increase from t-1 to t + 6.5 in the high - dose atd session).fig . 1a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation a conflict adaptation in the simon task as evidenced by a reduced compatibility effect after conflict trials versus nonconflict trials . b more depressive symptoms ( madrs score ) after the high - dose atd intervention are associated with increased conflict adaptation to visualize the hypothesized association between mood and conflict adaptation , we calculated standard individual conflict - adaptation scores by subtracting the interference effect following a correct conflict ( incompatible ) trial from the interference effect following a correct nonconflict ( compatible ) trial ( cf . 1a ) . before averaging sequential conditions for each individual , the first trial of each block , trials following an error , and trials with rts not fitting the outlier criterion ( deviating more than 2.5 sds from the individual condition - specific mean ) were excluded from analyses . as described in detail elsewhere ( booij et al . , 2005b ) , high - dose atd but not low - dose atd led to a both statistically and clinically significant induction of depressive symptoms as measured by madrs scores 6.5 hr after depletion [ 7.9 7.8 vs. 3.7 3.8 , mean standard deviation ; t(18 ) = 3.34 , p < .005 ] . the simon task produced a standard interference effect : incompatible trials produced longer rts than compatible trials , f(1 , 18 ) = 23.47 , p < .001 , mse = 1933.65 . the analysis also revealed a robust conflict - adaptation effect as indicated by an interaction between current- and previous - trial compatibility , f(1 , 18 ) = 38.27 , p < .001 , mse = 1143.45 . 1a shows , the interference effect was eliminated after conflict ( incompatible ) trials but not after nonconflict ( compatible ) trials , indicating standard conflict adaptation that is , reduced interference after conflict trials . this reduction in interference was driven by both postconflict speeding of incompatible trials [ illustrating that increased conflict - driven control reduces interference ; t(18 ) = -4.2 , p < .001 ] and by postconflict slowing of compatible trials [ illustrating that increased conflict - driven control reduces facilitation ; t(18 ) = 4.5 , p < .001 ] . although participants made only a few errors in general , error rate analyses still showed standard interference , f(1 , 18 ) = 5.38 , p < .05 , mse = 0.001 , and conflict adaptation , f(1 , 18 ) = 6.30 , p < .05 , mse = 0.001 , effects . in addition , they revealed a main effect of previous compatibility , f(1 , 18 ) = 4.46 , p < .05 , mse = .002 , indicating improved accuracy after conflict . notably , no main effects or interactions with congruency or conflict adaptation in rt or accuracy were observed for atd intervention . thus , atd did not have an effect on interference ( as reported earlier by booij et al . ongoing treatment did not have main or interactive effects.table 1behavioral data for all sessions and conditionsreaction time ( in milliseconds)error rate ( in percentages)msemsebaselinecompatible trial following a compatible trial65514.40.60.3incompatible trial following a compatible trial70512.33.61.2compatible trial following an incompatible trial67612.10.90.4incompatible trial following an incompatible trial67511.61.00.5low - dose sessioncompatible trial following a compatible trial65016.81.40.9incompatible trial following a compatible trial70618.22.91.0compatible trial following an incompatible trial68816.70.90.9incompatible trial following an incompatible trial68014.40.80.8high - dose sessioncompatible trial following a compatible trial64918.31.40.8incompatible trial following a compatible trial71119.92.30.9compatible trial following an incompatible trial66717.30.90.5incompatible trial following an incompatible trial67816.41.10.8 behavioral data for all sessions and conditions an ancova using mood score as a covariate confirmed our hypothesis : depressed - mood scores during the high - dose atd condition predicted increased conflict adaptation in rt as indicated by a significant three - way interaction between mood , current - trial compatibility , and previous - trial compatibility , f(1 , 17 ) = 5.30 , p < .05 , mse = 396.75 . as fig . 1 shows , individuals with more depressive symptoms after the atd intervention showed more conflict adaptation in the simon task . as is typically observed ( chepenik et al . , 2007 ; van steenbergen et al . , 2010 2 moreover , no mood effects were found for accuracy , thus showing that the effect on conflict adaptation could not be attributed to a speed accuracy tradeoff . because the low - dose atd session did not lead to any mood changes ( booij et al . , 2005b ) , data from this session were used for a control analysis : no association between mood and performance emerged.3 we report the first evidence for a link between low tryptophan concentrations , depressed mood , and conflict adaptation in remitted depressed patients : individuals with higher levels of depressive symptoms following high - dose atd showed increased conflict adaptation . this finding is in line with predictions derived from mbm theory ( gendolla , 2000 ) , with earlier behavioral and physiological observations from mood - induction studies in healthy populations ( e.g. , gendolla , abele , & krusken , 2001 ; gendolla & krusken , 2002 ; van steenbergen et al . , 2010 ) , and with neural evidence ( e.g. , evers et al . , 2009 ) our present study has important implications for understanding how depressive affect regulates cognitive control . in line with mbm theory ( gendolla , 2000 ) , our data illustrate that depressed mood per se does not have motivational implications ( as would be indicated by a modulation in attentional interference effects ) , but may facilitate increased cognitive control after a behavioral challenge . interestingly , this effect was observed in a relatively low - demanding simon task in which people were merely instructed to do their best ( see also brinkmann & gendolla , 2007 ) , and in the context of depression scores that were mainly below the cut - off value for a depression diagnosis , but were still clinically relevant and much larger than the effect of mood inductions in healthy participants . note that our findings suggest that depressive symptoms as induced by atd did not simply mimic the effect of depressive symptoms in currently depressed patients . that is , whereas mdd has been associated with reduced postconflict adaptation ( meiran et al . , 2011 ; pizzagalli , 2011 ) mbm theory ( gendolla , 2000 ) has proposed that the association between mood and effort mobilization is nonlinear , because it is mediated by task difficulty appraisal . accordingly , negative mood will facilitate demand - driven adjustments in effort only up to a certain level : if demands become perceived as too high to actively cope with , negative mood may actually trigger demand - driven disengagement ( cf . evidence for this effect has been reported in mood - induction studies and can also be shown in dysphoric participants when they perform tasks with extremely high fixed demands ( brinkmann & gendolla , 2008 ) . taken collectively , these observations suggest an inverted - u relationship between depressive symptoms and conflict adaptation ( see brehm & self , 1989 ) . it is an important aim for future studies to understand the generalizability of these findings and to disentangle the effects of increased negative affect and putative reduced availability of resources ( e.g. , due to rumination ) in mdd ( cf . mbm theory assumes that the interaction between both factors determines the actual appraisal of the demand , which in turn modulates effort mobilization . this hypothesis is now ripe for further testing in other studies using sequential analyses of conflict - task performance . at the neural level , the joint impact of depressed mood and demand evaluation on subsequent effort mobilization and cognitive control may be associated with ( hyper)activation of the anterior cingulate cortex ( acc ) , a region important for signaling the need for more cognitive effort to the dorsolateral prefrontal cortex ( dlpfc ) ( botvinick et al . , 2001 ; davidson , pizzagalli , nitschke , & putnam , 2002 ; olvet & hajcak , 2008 ; pizzagalli et al . , dlpfc circuit in unipolar depression also produces catastrophic reactions to errors ( for a review , see pizzagalli , 2011 ) . further study is needed to understand the exact neuromodulating role that 5-ht may play in this affective regulation ( cf . future studies that combine neuroimaging methods with effort - related physiological and behavioral measures will advance our understanding of the functional role of the acc in the affective ( dys)regulation of adaptive control to fluctuating task demands .

we describe a rare clinical finding of conjunctival tick attachment in a child . a 10-year - old boy presented to the clinic with right - eye itch . he was found to have a live tick firmly attached to his right temporal conjunctiva . the tick was identified as the larval stage of the paralysis tick , ixodes holocyclus . the tick was removed completely by conjunctival excision . although various methods of removing a tick have been described in the literature , the goal of treatment is the safe and complete removal of the tick to prevent further transmission of pathogens , allergens , and toxins to the patient . negative affect in healthy populations regulates the appraisal of demanding situations , which tunes subsequent effort mobilization and adjustments in cognitive control . in the present study , we hypothesized that dysphoria in depressed individuals similarly modulates this adaptation , possibly through a neural mechanism involving serotonergic regulation . we tested the effect of dysphoria induced by acute tryptophan depletion ( atd ) in remitted depressed patients on conflict adaptation in a simon task . atd temporarily lowers the availability of the serotonin precursor l - tryptophan and is known to increase depressive symptoms in approximately half of remitted depressed participants . we found that depressive symptoms induced by atd were associated with increased conflict adaptation . our finding extends recent observations implying an important role of affect in regulating conflict - driven cognitive control .

metal - ceramic restorations have been successfully applied in restorative dentistry due to high fracture strength . however , achieving a natural translucency is more difficult with metal - ceramic restorations than all - ceramic restorations because metal substructures prevent light transmission in metal - ceramic restorations . this has led to an increase in use of all - ceramic restorations . among all - ceramic restorations , however , high translucency of a restoration is not always an advantage , for instance in cases with discolored teeth , colored core materials and cast metal post and cores . in these clinical conditions , a restorative material with optimal masking ability of the underlying substructures a method to evaluate the masking ability of restorative materials is to measure color differences in cie lab color system . in this color system , the l * , a * and b * color parameters and e refer to lightness , red - green value , yellow - blue value , and color difference , respectively , which can be measured by a spectrophotometer . the e color difference is calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 which is the most commonly used formula for e . this formula can detect even a small amount of color difference between two objects [ 9 , 10 ] . as spectrophotometers can detect even the slightest amount of color difference not perceptible by the human eye , perceptual and clinically acceptable thresholds have been defined based on e values . it has been reported that the clinically acceptable threshold is more than the perceptual threshold , because of optical conditions of oral environment . if the e ( color difference ) between two objects is more than the perceptual threshold , a color mismatch will be detected by the human eye . the perceptual threshold has been reported from e=1 to e=5.5 in different studies [ 1214 ] . if a restorative material has an ideal masking ability , e will be equal to zero when placing the material on white and black substrates , namely that the material has a similar color on different substrates , and the substrates can not affect the color of the material . from a clinical point of view , if e of a material on different backgrounds is less than the threshold , it can thoroughly mask the background color . several studies have surveyed the optical properties of different ceramic materials [ 8 , 1518 ] . some studies have evaluated various factors affecting the final color of zirconia restorations including dental substrates [ 1922 ] , luting agents [ 21 , 23 ] , ceramic veneers [ 2426 ] , glaze , and firing process . suputtamongkol et al , reported that the background color could affect the overall color of posterior zirconia restorations on a metal post and core or a prefabricated post and a composite core ; however , color changes did not exceed the perceptual threshold . choi and razzoog assessed the masking ability of a zirconia ceramic ( zrc ) with and without the veneering porcelain and revealed that the zirconium oxide coping material alone had some degree of masking ability . oh and kim disclosed that abutment shade , ceramic thickness and coping type affected the resulting color of zirconia restorations . vichi et al , evaluated the effects of different substrates with different colors on the final color of all - ceramic ips - empress glass - ceramic restorations , and advised to take the color of substrate into consideration in cases with ceramic thickness of less than 1 mm . masking ability of a zrc is related to its color coverage over underlying substructures including cement and dental substrates . it may not be feasible to mask a substrate with cements , because different shades do not exist for all cements . thus , it seems that the color masking ability of a zrc on different substrates may be an important factor affecting the esthetic results of zirconia - based restorations . on the other hand , posts and cores , which are widely applied in restorative dentistry to restore severely damaged teeth the effects of these core materials ( as substrates ) on masking ability of zrcs ( as substructures in zirconia - based restorations ) have to be clearly determined . therefore , this in vitro study aimed to evaluate the effect of three different core materials including a nickel - chromium alloy ( nca ) , a non - precious gold alloy ( npga ) , and a zrc on masking ability of a zrc . the null hypothesis was that the zrc would mask the three aforementioned core materials with no significant difference . the discs were placed over four substrates including a white ( w ) substrate ( as the control ) , nca , npga and zrc . the procedure was conducted as follows : specimen preparation : a computer - aided design / computer - aided manufacturing system ( coritec 250i , imes - icore gmbh , eiterfeld , germany ) milled zirconia blanks ( luminesse high strength / low translucency , 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate zirconia discs . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . a digital micrometer ( 293 mdc - mx lite ; mitutoyo corporation , tokyo , japan ) with the accuracy of 0.002 mm an adjustment / polishing kit ( bruxzir ; glidewell direct , irvine , ca , usa ) was used to reduce the thicknesses according to the afore - mentioned acceptable range . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . 1).substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . then , two cylindrical acrylic resin ( duralay , reliance dental mgf co. , worth , il , usa ) patterns were formed according to the afore - mentioned dimensions . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . an adjusting / polishing kit ( np alloy adjustment kit ; shofu inc . , kyoto , japan ) was employed to polish the alloy substrates . in order to fabricate the zrc substrate , a cylindrical pattern was virtually designed by a software program ( solidworks 2015 , solidworks corp . , the same computer - aided design / computer - aided manufacturing system milled a zirconia blank ( luminesse high strength 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate a zirconia substrate based on the above - mentioned virtual design . the zirconia substrate was dipped in a2 shade color liquid ( dd bio zx2 monolithic zero lzdd ; dental direkt gmbh , spenge , germany ) for 10 seconds . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . all the substrates were cleaned in the same ultrasonic bath containing 98% ethanol for 15 minutes ( fig . 2 ) color measurement : a spectrophotometer ( spectroshade micro , mht , verona , italy ) was employed for spectrometric measurements . a putty silicone material ( speedex ; coltene , altstatten , switzerland ) was adapted to the mouth piece of the spectrophotometer to match the conditions of spectrophotometry for all specimens and to prevent external lights . the specimens were located at the center of this putty mold . before each measurement , the spectro - photometer was calibrated by the white and green calibration plates , respectively . the discs were placed over the substrates with a water drop in - between to prevent the refraction of light . each disc was placed on each of the substrates , and the color measurements were made . all the color measurements were made at the center of specimens marked by a pen on the monitor screen of spectrophotometer . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 specimen preparation : a computer - aided design / computer - aided manufacturing system ( coritec 250i , imes - icore gmbh , eiterfeld , germany ) milled zirconia blanks ( luminesse high strength / low translucency , 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate zirconia discs . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . a digital micrometer ( 293 mdc - mx lite ; mitutoyo corporation , tokyo , japan ) with the accuracy of 0.002 mm an adjustment / polishing kit ( bruxzir ; glidewell direct , irvine , ca , usa ) was used to reduce the thicknesses according to the afore - mentioned acceptable range . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . then , two cylindrical acrylic resin ( duralay , reliance dental mgf co. , worth , il , usa ) patterns were formed according to the afore - mentioned dimensions . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . an adjusting / polishing kit ( np alloy adjustment kit ; shofu inc . , kyoto , japan ) was employed to polish the alloy substrates . in order to fabricate the zrc substrate , a cylindrical pattern was virtually designed by a software program ( solidworks 2015 , solidworks corp . , the same computer - aided design / computer - aided manufacturing system milled a zirconia blank ( luminesse high strength 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate a zirconia substrate based on the above - mentioned virtual design . the zirconia substrate was dipped in a2 shade color liquid ( dd bio zx2 monolithic zero lzdd ; dental direkt gmbh , spenge , germany ) for 10 seconds . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . all the substrates were cleaned in the same ultrasonic bath containing 98% ethanol for 15 minutes ( fig . 2 ) color measurement : a spectrophotometer ( spectroshade micro , mht , verona , italy ) was employed for spectrometric measurements . a putty silicone material ( speedex ; coltene , altstatten , switzerland ) was adapted to the mouth piece of the spectrophotometer to match the conditions of spectrophotometry for all specimens and to prevent external lights . the specimens were located at the center of this putty mold . before each measurement , the spectro - photometer was calibrated by the white and green calibration plates , respectively . the discs were placed over the substrates with a water drop in - between to prevent the refraction of light . each disc was placed on each of the substrates , and the color measurements were made . all the color measurements were made at the center of specimens marked by a pen on the monitor screen of spectrophotometer . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 zirconia disc specimens tested substrates ( from left to right : zirconia ceramic , non - precious gold alloy , nickel - chromium alloy and white substrate ) the perceptual threshold of e=2.6 was considered in this study [ 1214 ] . normal distribution of the data was accepted in all groups by the kolmogorov - smirnov test ( p>0.05 ) . the values of l * , a*and b * for each disc in npga , nca and zrc groups were subtracted from l*white , a*white and b*white values , which were the corresponding values for w substrate . in order to compare the standard l * , a * , b * ( from which , the values of w substrate were subtracted ) and e values among the groups , randomized block anova was employed . stata software ( statacorp lp , lakeway , tx , usa ) compared the e values with the predetermined perceptual threshold of e=2.6 using one - sample t - test . the results were explained according to the measured variables of l * , a * , b * , and e in four sections . l * parameter ( lightness ) : the mean and standard deviation of the l * values for the w , nca , npga and zrc groups were 88.351.46 , 78.241.31 , 78.981.26 , and 82.161.16 units , respectively ( table 1 , fig . 3 ) . statistical analysis showed a significant difference among the groups in this regard ( p<0.0001 ) . pairwise comparisons of the groups using bonferroni test revealed significant differences between the nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.0001 ) . the color parameter values in the four groups of white substrate ( w ) , nickel - chromium alloy ( nca ) , non - precious gold alloy ( npga ) and zirconia ceramic ( zrc ) the mean l * values of the groups a * parameter ( red - green value ) : the mean and standard deviation of the a * values for the w , nca , npga and zrc groups were 0.400.42 , 0.040.52 , 0.600.42 , and 1.520.38 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test revealed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) and npga and zrc ( p<0.0001 ) . the a * values increased in all the groups compared to the control . the increase in the a the mean a * values of the groups b * parameter ( yellow - blue value ) : the mean and standard deviation of the b * values for the w , nca , npga , and zrc groups were 3.380.36 , 1.921.01 , 3.860.49 , and 4.880.57 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.001 ) . zrc had the highest b * value , and nca had the lowest b * value among the groups . the mean b * values of the groups e ( color difference ) : the mean and standard deviation of the e values for the nca , npga and zrc groups were 10.262.43 , 9.451.74 , and 6.701.91 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and zrc ( p<0.0001 ) , and npga and zrc ( p=0.001 ) . the difference between nca and npga was not statistically significant ( p=0.65 ) . in order to compare the e values of the groups with the predetermined perceptual threshold of e=2.6 , the null hypothesis of e 2.6 was rejected for all the groups ( p<0.0001 ) . the present study evaluated the color parameters of l * , a * , b * , and e for zirconia disc specimens on four different substrates including w , nca , npga and zrc . statistical analysis indicated significant differences among the groups in the l * , a * , b * and e values . the examined zrc on the tested substrates showed perceptible color change namely that the examined zrc could not thoroughly mask the tested substrates . the l * ( lightness ) values decreased in all the groups compared with the control . the change in l * value shows the impact of the substrates on the zirconia specimens . this can be explained by the optical properties of zrc , which is a semi - translucent material . according to the results , metal core materials such as ncas and npgas can decrease the lightness of zrc more than a zirconia core material the a * ( red - green ) values increased in all the groups compared to the control . a reason for this may be the natural a * value of the zrc as a substrate . the tested zirconia substrate ( a2 shade ) positively shifted the a * value towards red . the b * ( yellow - blue ) values changed in the groups depending on the substrates . the nickel - chromium substrate decreased the b * value while the non - precious gold and zirconia substrates increased the b * value compared to the control . this may be due to the yellow color tendency of a2 shade zirconia substrate and npga , which impact on the color of zrc and shift it towards yellow . on the contrary , nca shifted the color of zrc towards blue , and negatively affected the b * value . the e values of all groups were more than the predetermined perceptual threshold of e=2.6 . assessment of the l * , a * and b * values indicated their changes in the substrate groups . thus , color changes were derived from all three color parameters . the l * , a * , and b * values manifested significant differences between the substrate groups . this demonstrated that the quality of the color change was different depending on the substrate . the e values manifested no significant difference between the metal substrates ; however , significant differences existed between the zirconia substrate and the metal substrates , and the least color change belonged to the zirconia substrate . this demonstrated that the quantity of the color change was different depending on the substrate as well . hence , both quality and quantity of the color of zrc were altered by the substrates . the used metal substrates did not differ in terms of the quantity of color change , and both affected the quantity of the color change more than the zirconia substrate . suputtamongkol et al , reported that the color of an underlying substructure could affect the overall color of posterior zirconia - based restorations on a metal post and core or a prefabricated post and a composite core , ranging from e = 1.2 to 3.1 . additionally , slight color changes of zirconia crowns were detected by measuring the e . although some differences exist between the afore - mentioned study and the current research such as zrc brands , thicknesses of ceramics , veneered versus non - veneered zrcs , hypothesized perceptual thresholds and substrate types , both studies showed that zrc could not thoroughly mask the underlying materials . oh and kim in an in vitro study of color masking ability assessed the effects of abutment shade , ceramic thickness and coping type on the final color of zirconia restorations . the abutments were fabricated of gold alloy , base metal ( nickel - chromium ) alloy , and four different shades of composite resins . in their study , the average e value of lava specimens between the a2 shade composite resin and gold alloy abutments was higher than that between the a2 shade composite resin and other abutments , and was close to e=5.5 . a similar result was obtained in the current study , although the substrates and zrcs used in the two studies were different . according to the study by oh and kim , gold alloy substrate yielded the highest e among the tested substrates ; however , the npga and the nca yielded close e values in the current study . this dissimilarity in the results may be caused by the color difference of the precious gold alloy used by oh and kim and the npga used in the current study . choi and razzoog evaluated the masking ability of zrc with and without a veneering porcelain . in their study , color differences caused by zrc and the veneering porcelain were compared with substrates alone . they concluded that the non - veneered zrc had some degree of masking ability over different tested substrates ( white , black , gray , and a3 shade tooth - colored substrates ) . however , the current study compared substrate - induced color differences with a zrc over w substrate and concluded that the tested zrc could not mask the examined substrates . choi and razzoog measured the e values between the substrate alone ( as control ) and the zrc over the substrate , while the current study measured the e values between the zrc over w substrate ( as control ) and zrc over the tested substrates . based on the results of our study , the three core materials including nca , npga and zrc can change the color of tested zrc . the color of zrc may be closer to the final color of a restoration than the metal cores . therefore , it is esthetically advised to preferably choose a zrc post and core instead of nca or npga post and cores in zirconia - based restorations and to reduce the core material - induced color change by further core reduction , applying sufficient thickness of the veneering porcelain , and use of proper luting agents in zirconia - based restorations . it should be noted that given that the physical and mechanical properties of zirconia post and core are confirmed in further studies , zirconia post and core should be clinically recommended . this needs future investigations . considering the optical properties of zrc and the thickness of zirconia sub - structures , which is approximately 0.5 mm in a normal case , light transmission through the zirconia structure can be expected in zirconia - based restorations . the present study evaluated the effects of three core materials in this respect . on the other hand , the zirconia core overlaying materials such as porcelain veneers and glaze may influence the final color of zirconia - based restorations , which were not assessed in this study . therefore , evaluation of the effect of other factors in this respect is recommended in future studies . the present study had some limitations such as using a specific brand of zrc , an uncolored zrc , and a specific brand of composite resin . within the limitations of this study , it was concluded that the tested zrc could not thoroughly mask the three core materials of nca , npga , and zrc . however , it has not been reported with fluoroscopically - guided endovascular procedures.the rapidly proliferating keratinocytes in the hair matrix are most sensitive to radiation . the insult damages the keratinocytes , which results in thin , weakened hair shaft that is susceptible to trauma.complete hair re - growth occurs in a time period between two to four months following the insult . the insult damages the keratinocytes , which results in thin , weakened hair shaft that is susceptible to trauma . complete hair re - growth occurs in a time period between two to four months following the insult . fluoroscopy - guided neuro - radiological endovascular procedures for treatment of arteriovenous malformations are getting commonplace in modern health care facilities of india . temporary alopecia of the scalp is an uncommon but documented side - effect of these procedures , especially when a certain threshold of radiation is exceeded . to the best of our knowledge , this is the first case of temporary alopecia after fluoroscopically - guided coiling procedure for a ruptured cerebral aneurysm being described in a dark haired woman from india . a 47-year - old apparently healthy woman was referred to this clinic ( sv ) for a dramatic non - scarring hair loss of occipito - vertical region since a month . it became known upon eliciting history that three weeks ago , she developed acute excruciating headache of the nape of the neck , profuse perspiration , vomiting , and lack of consciousness with incontinence . an urgent ct scan showed a ruptured anterior communicating artery aneurysm . a fluoroscopically - guided guglielmi detachable coil ( gdc ) coiling procedure as a non - invasive treatment was suggested . a preliminary angiography lasting for two and half hours was done followed by an attempt to perform coiling that lasted unsuccessfully for four hours . shoots had to be taken due to severe vasospasm . after waiting for two days to let the dye drain out , the procedure was attempted again , and coiling was done successfully in two and a half hours . a week later , the patient developed headache , for which another ct scan was done . the headache was attributed to cerbritis , and 24 mg of methylprednisolone was instituted on a slow taper . two weeks later , she noticed clumps of hair falling out initially during combing and then even while spontaneously running her fingers through her hair . there was a 6 4 inch - sized , asymptomatic , skin - colored alopecic patch in the occipital area with no erythema , scarring or scaling [ figure 1 ] . hair was sent for trichogram to an academic hospital in coimbatore where they demonstrated 80% anagen hair showing dystrophy , the so called bayonet hair. [ figure 2 ] . she had no exclamation mark hairs , poliosis , hair shaft disorders or positive history of similar affliction or atopy in her family . a vascular cause for the hair loss was ruled out by loud arterial pulse palpable in the temporal and occipital arteries . pressure alopecia due to prolonged supine position was ruled out by absence of fractured hair shafts . occipito - vertical alopecia after radiation exposure anagen hair with the pointed distal end to the left and telogen hair to the right diagnosis of fluoroscopic endovascular catheterization - induced alopecia was made . after a period of 14 weeks of observation and no active treatment , she showed significant re - growth of dark hair [ figure 3 ] . flouroscopically - guided endovascular procedures are becoming increasingly common all over the globe . while dermatologists have since long been consulted for hair loss following radiotherapy of tumors , there are only few reports of temporary hair loss following fluoroscopic endovascular procedures in dermatologic literature . other than radiation oncology , the maximum radiation exposure comes from equipment used for non - invasive interventions such as these . the two commonest temporary side - effects of such procedures , erythema , and temporary hair loss are termed deterministic and are indicative of a certain threshold of irradiation , below which the phenomenon would not be seen . the probability of encountering such a side - effect would increase as the dose exceeds the threshold . the time between the exposure and appearance of clinical symptoms depend on the cell turnover rate . in tissues such as hair follicle with its rapid turnover rate , it is thought that irradiation doses exceeding 3 gy are followed by such a hair loss during fluoroscopic procedures . an estimated 7% of such radiological interventions are said to reach the threshold set for temporary hair loss by the international commission on radiological protection . according to the interventionalist , it would have grossly exceeded 3 gy because the procedure turned out to be a very long one with prolonged exposure to the radiation owing to the difficulty in negotiating the loop through the site of pathology . it is not unusual for the patient to skip the history of radiation exposure because they are unaware of the fact that the procedure they have undergone involves radiation exposure and have not been forewarned by the interventionalist . severity of radiation - induced temporary alopecia is largely dependent on the dose , total duration of irradiation , interval between irradiations ( dose fractionation where applicable ) , the size of the skin area irradiated , the angle of irradiation , all affect the expression and severity of the injury . one of the commonest working positions during gdc coiling procedure is posteroanterior , which explains the occipito - vertical hair loss in this patient . loss of scalp hair can occur at relatively lower doses when compared with doses at which other body hair is lost . our patient with jet black hair developing this dramatic hair loss is suggestive of gross excess of radiation . the radiation insult damages these keratinocytes and diminishes the growth of the growing hair shaft . this results in thin , weakened hair shaft that is susceptible to trauma when it reaches the surface of scalp . hair breakage in anagen effluvium occurs within days to weeks ( 1 - 3 weeks ) following insult to the follicle . microscopic hair examination shows that the distal hair shaft is dystrophic with a rapidly tapering configuration called the bayonet hair. tapered hair can arise in any process that inhibits the cell division in the hair matrix . since 90% of hair on the scalp is in growing phase , smoking , malnutrition , pre - existing autoimmune and connective tissue disorders like diabetes , hyperthyroidism , scleroderma , rheumatoid arthritis , and systemic lupus erythematosus predispose patients to the development of severe radiation effects in an unpredictable manner . drugs that increase radiosensitivity include actinomycin d , doxorubicin , bleomycin , 5-fluorouracil , and methotrexate , mitoxantrone , 5-fluorourcil , cyclophosphamide , paclitaxel , docetaxel , and possibly tamoxifen . , it was literally adding insult to injury considering she had just recovered from a life - threatening situation . fortunately , unassisted re - population of hair in 2 - 4 months is the most likely outcome and , therefore , a gentle handholding and confidence building are all that is required . interventionalists should explain this possibility to patients in whom such a deterministic dose has been exceeded to avoid distress . fortunately , at such doses , there are very few chances of a serious or irreversible long - term consequence . however , all dermatologists should be aware of the association of hair loss with prolonged fluoroscopic endovascular procedures where the deterministic dose has been exceeded . the absorbed skin dose should be recorded on the patient 's charts or medical record . it is recommended that this dose be taken into account if the patient requires a future procedure involving exposure to ionizing radiation . minimizing the effective dose to achieve the therapeutic goal is the aim , and efforts are on to reach that goal . fluoroscopically - guided interventional procedures are now performed fairly commonly in advanced tertiary care centers in india . dermatologists who saw radiation - induced dermatoses until recently will now increasingly start seeing side - effects to the skin and hair due to these procedures , which is the second commonest cause of radiation exposure after radiotherapy.temporary alopecia of the scalp is a well - known early side - effect of ionizing radiation , which is emitted during the fluoroscopic - guided interventional procedures and is seen when a dose of 3 gy is exceeded . other factors that play a role include total duration of irradiation , interval between irradiations , size of the skin area irradiated , and the angle of irradiation.the resultant alopecia is due to anagen effluvium , and there is complete spontaneous re - growth in about 2 - 4 months . fluoroscopically - guided interventional procedures are now performed fairly commonly in advanced tertiary care centers in india . dermatologists who saw radiation - induced dermatoses until recently will now increasingly start seeing side - effects to the skin and hair due to these procedures , which is the second commonest cause of radiation exposure after radiotherapy . temporary alopecia of the scalp is a well - known early side - effect of ionizing radiation , which is emitted during the fluoroscopic - guided interventional procedures and is seen when a dose of 3 gy is exceeded . other factors that play a role include total duration of irradiation , interval between irradiations , size of the skin area irradiated , and the angle of irradiation . the resultant alopecia is due to anagen effluvium , and there is complete spontaneous re - growth in about 2 - 4 months .

objectives : masking ability of a restorative material plays a role in hiding colored substructures ; however , the masking ability of zirconia ceramic ( zrc ) has not yet been clearly understood in zirconia - based restorations . this study evaluated the effect of three different core materials on masking ability of a zrc.materials and methods : ten zirconia disc samples , 0.5 mm in thickness and 10 mm in diameter , were fabricated . a white ( w ) substrate ( control ) and three substrates of nickel - chromium alloy ( nca ) , non - precious gold alloy ( npga ) , and zrc were prepared . the zirconia discs were placed on the four types of substrates for spectrophotometry . the l * , a * , and b * values of the specimens were measured by a spectrophotometer and color change ( e ) values were calculated to determine color differences between the test and control groups and were then compared with the perceptual threshold . randomized block anova and bonferroni test analyzed the data . a significance level of 0.05 was considered.results:the mean and standard deviation values of e for nca , npga , and zrc groups were 10.262.43 , 9.451.74 , and 6.701.91 units , respectively . significant differences were found in the e values between zrc and the other two experimental groups ( nca and npga ; p<0.0001 and p=0.001 , respectively ) . the e values for the groups were more than the predetermined perceptual threshold.conclusions:within the limitations of this study , it was concluded that the tested zrc could not well mask the examined core materials . a 47-year - old indian woman with dark hair developed dramatic alopecia of the occipito - vertical area two weeks after a fluoroscopically - guided endovascular procedure for treatment of cerebral aneurysm . there was spontaneous repopulation of hair in 14 weeks . neuro - radiological intervention procedures are becoming commoner by the day in india , and the rare but documented possibility of such reactions occurring in patients should be kept in mind by the treating surgeon as well as dermatologist . necessary counseling regarding this uncommon side - effect is of essence , especially when the radiation dose exceeds 3 gy . we believe this is the first case ever reported in indian dermatology literature .

hemicrania continua ( hc ) is an uncommon primary headache disorder characterized by a continuous , mild to moderate intensity , unilateral headache [ 14 ] . most patients will experience superimposed exacerbations of more severe pain , often associated with ipsilateral autonomic symptoms . headache for greater than 3 months fulfilling the following criteria : all of the following : unilateral pain without side shift , daily and continuous , without pain - free periods , moderate intensity , but with exacerbations of severe pain.at least one of the following autonomic features occurs during exacerbations and ipsilateral to the side of pain : conjunctival injection and/or lacrimation , nasal congestion and/or rhinorrhea , ptosis and/or miosis.a complete response to therapeutic doses of indomethacin.finally , not attributed to another disorder . unilateral pain without side shift , daily and continuous , without pain - free periods , moderate intensity , but with exacerbations of severe pain . at least one of the following autonomic features occurs during exacerbations and ipsilateral to the side of pain : conjunctival injection and/or lacrimation , nasal congestion and/or rhinorrhea , ptosis and/or miosis . a complete response to therapeutic doses of indomethacin . finally , not attributed to another disorder . there is a group of patients that meets the above criteria for hc , but has to discontinue indomethacin secondary to adverse effects , primarily involving the gastrointestinal ( gi ) tract . the side effects can range from gastritis to severe bleeding , patients may also develop an allergy to indomethacin , see an elevation of blood pressure , or an exacerbation of asthma symptoms . gabapentin is often used in an off label fashion for the treatment of neuropathic pain and is well tolerated from a side effect profile . marinano da silva , alcantara , bordini , and speciali reported a case of a unilateral headache similar to hc that was responsive to gabapentin . institutional review board approval was obtained for the study . a retrospective chart review of nine patients with hc between october 2006 and february 1 , 2008 . inclusion criteria included men and women age 18 or above presenting to the headache center with a headache that meets ihs criteria for hc , including a response to indomethacin , but were unable to continue on indomethacin secondary to adverse side effects . exclusion criteria included anyone under the age of 18 and anyone not meeting ihs criteria for hc including an adequate trial at therapeutic doses of indomethacin and response to indomethacin . patients with concomitant disease , specifically involving pain were not excluded , although every effort was made to enroll patients where hc was their primary complaint . this was decided since the purpose of this study was to examine gabapentin as an alternative therapy , indomethacin remains the gold standard . points of measurement included the duration of disease which is considered to be from onset to completion of the study , temporal profile , duration of therapy , range of dose , and concurrent use of other medications for pain , concomitant disease , especially related to pain , reduction of pain , and the side effects of gabapentin . the duration of therapy on gabapentin ranged from 3 months to 13 months with eight out of nine patients remaining on gabapentin at the end of the study . one patient discontinued after reaching 3,600 mg a day due to lack of efficacy . the other doses ranged from 600 mg to 3,600 mg per day of gabapentin . one patient also on fentanyl patch 50 mcg every 72 h reported an 80% reduction in pain after reaching 2,400 mg of gabapentin . the second patient was taking topiramate and became pain free upon reaching 600 mg of gabapentin.table 1treatment of hc with gabapentinpgardi / day ( mg)arirdg dose / day ( mg)rpdd ( months)tpdt ( months)cdcmse#1f41150gastritis1,5008/100/1013c5nonenonenone#2f5575gastritis2,4006/101/106c4lumbar radiculopathyfentanyl patch 50 mcg/72 hsedation#3f44300g.i . bleed 2 episodes3,6009/109/1025c6nonenonecognitive impairment#4f25150fatique9004/101/1036c12nonenonenone#5f46150gastritis9005/104/107c4nonenonenone#6f33150gastritis1,8008/100/1034c7nonenonevertigo#7m6375rectal bleed3,60010/104/1017c13nonenonenone#8f45300excerbation of asthma60010/100/108c5migrainetopiramate 50 mg / daynone#9f42250gastritis9005/100/103ep3atypical facial painnoneweight gainp patient , g gender , f female , m male , a age , rdi responsive dose of indomethacin , ari adverse reaction to indomethacin , rdg responsive dose of gabapentin , rp reduction of pain , dd disease duration , tp temporal profile , c chronic , ep episodic , dt duration of treatment , cd concomitant disease , cm concomitant medication , se side effects on gabapentin treatment of hc with gabapentin p patient , g gender , f female , m male , a age , rdi responsive dose of indomethacin , ari adverse reaction to indomethacin , rdg responsive dose of gabapentin , rp reduction of pain , dd disease duration , tp temporal profile , c chronic , ep episodic , dt duration of treatment , cd concomitant disease , cm concomitant medication , se side effects on gabapentin the seven patients that achieved the primary end point and reported a 5080% reduction of pain had doses ranging from 900 to 3,600 mg per day . the one man reported a 50% reduction of pain and reached the maximum dose of 3,600 mg of gabapentin per day . four patients achieved the secondary end point and reported being completely pain free on gabapentin . the dose ranges for these patients were , 600 mg , 900 mg , 1,500 mg , and 1,800 mg / day respectively . it should be noted that one of the four patients was also taking topiramate , which has been reported to be efficacious for hc [ 7 , 8 ] . the patient , who reported no benefit , responded to indomethacin on two separate occasions but developed gi bleeding as a result of indomethacin . the major side effects reported on gabapentin include sedation , cognitive impairment , depression , and weight gain . none of the patients chose to discontinue gabapentin as a result of the above side effects . hc was first described in 1981 and officially named by sjaastad and spierings in 1984 [ 9 , 10 ] . hc was originally believed to be a rare primary headache disorder but more recent evidence suggests that hc is not rare , just under recognized . in the clinical setting , hc is characterized by a unilateral headache that is continuous and of mild to moderate severity . the pain is usually reported as dull , aching , or pressure - like pain without associated symptoms . exacerbations or more severe pain , lasting anywhere from 20 min to several days , are experienced in the majority of those with hc , and when present are associated with one or more autonomic symptoms on the ipsilateral side . migrainous features , such as photophobia , phonophobia , nausea , and vomiting commonly occur during exacerbations of pain . many patients will experience a sensation of a foreign body in their eye similar to sand or an eyelash on the affected side . the pathophysiology of hc is not known , some scientist believe it is a subtype of migraine , while others believe it is more closely related to the trigeminal autonomic cephalalgias ( tac ) . it was hoped that functional brain imaging would help to clarify our understanding of hc , but it revealed that hc is a distinct headache syndrome , not belonging to the migraine or tac group . the scans revealed activation of the contralateral posterior hypothalamus and ipsilateral dorsal rostral pons , as well as activation of the ipsilateral ventrolateral midbrain , extending over the red nucleus and substania nigra and the bilateral pontomedullary junction . these areas have been previously demonstrated to be the sites of activation in the tacs and migraine , ipsilateral activation hypothalamic activation in short - lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing ( sunct ) and cluster ; in hc the findings are reversed . hc was not included in the first edition of the ichd - i , but is included within the other primary headaches section of the second edition of the ichd - ii . hemicrania continua presents as two temporal profiles : an episodic form with distinct headache phases separated by periods of pain - free remissions , and a chronic form in which the headache persists without remission for years . hc is chronic from onset in 53% of patients ; the disorder began in the episodic form and evolved into the chronic form in 35% , and it begins and remains episodic in 12% of patients . this can occur by focusing exclusively on the ipsilateral autonomic features that accompany the painful exacerbations of hc . similarly , focusing on the associated photophobia , phonophobia , nausea , and vomiting that may occur during exacerbations of hc may lead to misdiagnosing hc as migraine . autonomic features on the ipsilateral side , which are present during exacerbations of hc , are at times also present during acute attacks of migraine . hc is also distinguished from cluster headache and migraine by the presence of a continuous baseline headache that is usually unilateral and mild to moderate in severity . the associated features of photophobia , phonophobia , nausea , and vomiting , as well as the ipsilateral autonomic features of cluster are absent with the continuous baseline pain of hc . some patients are unable to tolerate indomethacin due to gi distress , renal disease , or concurrent anticoagulation therapy , and alternative types of therapy must be considered . peres and silberstein demonstrated in a study using cyclooxygenase-2 inhibitors ( cox-2 ) , efficacy of celecoxib and rofecoxib , the latter of which has since been removed from the market . they found 60% of patients who received celecoxib and 33% who received rofecoxib experienced a complete response . there is one case in the literature of a unilateral headache similar to hc that did not respond to indomethacin , and many other standard headache agents , but was responsive to gabapentin at 1,200 mg per day . there have also been two separate reports of melatonin being efficacious for hc , one case reported by spears , and three cases by rozen . there have also been four cases in the literature of patients responding to topiramate , one by camarda et al . , one by matharu et al . , and two cases by brighina et al . . gabapentin is an antiepileptic drug that is approved for use in the adjunctive treatment of partial seizures with and without secondary generalization in patients over 12 years of age . gabapentin is also indicated as adjunctive therapy in the treatment of partial seizures in pediatric patients age 312 years of age . benefit has been reported in gabapentin treatment for hypobaric hypoxia - induced headache , high altitude headache , nummular headache , sunct syndrome , chronic cluster headache , post - dural puncture headache , and primary thunderclap headache just to mention headaches disorders [ 1826 ] . the mechanism by which gabapentin exerts its analgesic action is unknown , but in animal models of analgesia , gabapentin prevents allodynia ( pain related behavior in response to a normally innocuous stimulus ) and hyperalgesia ( exaggerated response to painful stimuli ) . the mechanism by which gabapentin exerts its anticonvulsant action is unknown , but in animal test systems designed to detect anticonvulsant activity , gabapentin prevents seizures . gabapentin is structurally related to the neurotransmitter gamma - aminobutyric acid ( gaba ) but does not modify gaba a or gaba b radioligand binding , it is not converted metabolically into gaba or a gaba agonist , and it is not an inhibitor of gaba uptake or degradation . gabapentin was tested in radioligand binding assays and did not exhibit affinity for a number of other common receptor sites including benzodiazepine , glutamate , n - methyl - d - aspartate ( nmda ) , quisqualate , kainate , strychnin - insensitive glycine , alpha 1 , alpha 2 , or beta adrenergic , adenosine a1 or a2 , cholinergic muscarinic or nicotinic , dopamine d1 or d2 , histamine h1 , serotonin s1 or s2 , opiate mu , delta or kappa , cannabinoid 1 , voltage sensitive calcium channel sites labeled with nitrendipine or diltiazem , or at voltage sensitive sodium channel sites labeled with batrachotoxinin a 20 alpha benzoate . one patient reported an 80% reduction of pain on 2,400 mg of gabapentin but was also on the fentanyl patch . there have been reports in the literature of enhancement of pain control on the combination of opioids and gabapentin [ 28 , 29 ] . this patient met the primary endpoint , but the concomitant use of an opioid is a confounding factor . this study demonstrates that gabapentin is a good choice for alternative therapy in the treatment of hc . seven out of nine patients demonstrated at least a 50% reduction of pain on gabapentin , four patients becoming completely pain free . the results of the study are confounded in two patients that were taking concomitant medications specifically fentanyl patch and topiramate . another potential confounding factor is that a proton pump inhibitor was not initiated when patients had difficulty tolerating indomethacin . clinically it may be prudent to first try initiating therapy with a proton pump inhibitor while continuing indomethacin prior to changing to gabapentin . hc is a particularly difficult headache disorder to treat if the patient is not able to tolerate indomethacin , therefore alternative therapies are being sought to provide relief to this patient population . the results of this study should be validated in a double - blinded randomized control trial . the study population consisted of 85 caucasian , northern spanish girls representing a continuum of female subjects with hyperinsulinemic androgen excess that commences in prepuberty in those girls with a combined history of lbw and precocious pubarche , is followed by a preclinical phase starting around menarche of biochemical ovarian hyperandrogenism and yields to an overt clinical period of ovarian hyperandrogenism 23 years after menarche . all subjects were included in similar clinical trials investigating the safety and efficacy of metformin treatment on their hyperinsulinemic androgen excess . the prepubertal girls ( n = 18 ; tanner stage i ; aged 7.9 0.2 years ; bmi 19.3 0.6 kg / m ) had a combined a history of lbw ( 2.3 0.1 kg at term birth ) and precocious pubarche ( appearance of pubic hair before age 8 years ) attributable to an exaggerated adrenarche , based on high serum levels of dehydroepiandrosterone sulfate or androstenedione at diagnosis of precocious pubarche ( 8) . the girls were , in addition , hyperinsulinemic , as judged by the insulin response after an oral glucose tolerance test ( 16 ) . all of these subjects participated in a controlled long - term study exploring the effects of early metformin treatment ( isrctn84749320 ) ; more specifically , they form together the study subgroup that was initially randomly assigned to receive metformin for at least 1 year ( 425 mg , daily at dinner time ) ( 9 ) . the early postmenarcheal girls ( n = 31 ; aged 12.2 0.1 years ; bmi 21.5 0.5 kg / m ) manifested biochemical androgen excess characterized as the ensemble of 1 ) absence of clinical signs or symptoms of androgen excess ( 17 ) and 2 ) hyperinsulinemia ( oral glucose tolerance test ) and 3 ) ovarian androgen excess suggested by increased testosterone levels and confirmed by 17-hydroxyprogesterone hyperresponse to gonadotropin - releasing hormone agonist ( 17,18 ) . the girls in the present analysis were enrolled in clinical studies that were initiated before 2003 , thus before compulsory trial registration ; as described , all these girls had a history of lbw - precocious pubarche and were already in the early postmenarche phase ( 612 months beyond menarche ) at start of metformin intervention ( 8,19 ) . the adolescent girls ( n = 36 ; aged 15.5 0.3 years ; bmi 21.9 0.5 kg / m ) were consecutive patients with pcos who were at least 2 years beyond menarche and who were followed in the outpatient clinic according to standard procedures , including yearly blood sampling in the fasting state ( once with dna extraction ) and yearly assessment of body composition . the inclusion criteria were the same as those for the early postmenarcheal girls , except that the presence of clinical markers of androgen excess was now required : hirsutism ( ferriman - gallwey score > 8) plus either amenorrhea ( menses absent for > 3 months ) or oligomenorrhea ( menstrual cycle > 45 days ) . in all postmenarcheal girls , baseline assessments were performed in the follicular phase ( day 37 ) or after 2 months of amenorrhea . all postmenarcheal girls received metformin for at least 1 year in monotherapy ( 850 mg , daily at dinner time ) . at the start of metformin treatment , none of the 85 girls presented evidence for thyroid dysfunction , cushing syndrome , hyperprolactinemia , glucose intolerance , or late - onset adrenal hyperplasia ; for at least 6 months before the start of metformin start , none of the girls received an estro - progestagen or other medication known to affect gonadal function or carbohydrate metabolism . height and weight were measured , and bmi was calculated ( weight in kilograms divided by the square of height in meters ) . bmi standard deviation scores ( sdss ) were derived from regional normative data ( 20 ) . fasting blood glucose , serum insulin , igf - i , sex hormone binding globulin ( shbg ) , and testosterone were measured as described previously ( 9 ) . genomic dna was purified from whole blood samples using commercial reagents ( gentra puragene cell kit ; qiagen iberia , madrid , spain ) . the stk11 rs8111699 snp was genotyped by a fluorescent genotyping system ( kaspar snp genotyping system ; kbiosciences , hoddesdon , u.k . ) . in brief , 10 pg genomic dna was incubated in a 8-l solution of a combined mix containing 100 mol / l allele specific primer 1 ( fam , x - axis , allele g ) , 5-gaaggtgaccaagttcatgctccactgactgttcaggaaagcac-3 , 100 mol / l allele - specific primer 2 ( vic , y - axis , allele c ) , 5-gaaggtcggagtcaacggattccactgactgttcaggaaagcag-3 , 100 mol / l common reverse primer 5-ggtggaagcctgactgtgagagt-3 , 10 mol / l tris - hcl , ph 8.3 , ktaq polymerase , 1.8 mmol / l mgcl2 , and rox as a passive reference . the pcr mix was incubated at 94c for 15 min , followed by 20 cycles of 94c for 10 s , 57c for 5 s , and 72c for 10 s. thereafter , the pcr mix was incubated for 18 cycles at 94c for 10 s , 57c for 20 s , and 72c for 40 s. samples were then read in a fluorescence plate reader . the kaspar system uses the fluors fam and vic for distinguishing among genotypes and rox as a passive reference . the corresponding genotypes for this snp ( cc vs. cg vs. gg ) did not differ among prepuberty ( 33% vs. 44% vs. 22% ) , early postmenarche ( 26% vs. 49% vs. 26% ) , and pcos ( 25% vs. 42% vs. 33% ) subjects in our study ( p = 0.89 ) . the distributions were also tested for hardy - weinberg equilibrium , and none deviated significantly . body composition was assessed by dual - energy x - ray absorptiometry with a lunar prodigy and lunar software ( version 3.4/3.5 ; lunar , madison , wi ) ( 9 ) . total irradiation dose per assessment was 0.1 msevert ; coefficients of variation for scanning precision are 2.2 and 2.6% for fat and lean mass ( 8,9,19 ) . the study protocol was approved by the institutional review board of hospital of sant joan de du . informed consent was obtained from the patients and/or from parents , and assent was obtained from girls , as appropriate . the clinical , biochemical , and body composition data from prepubertal and early postmenarcheal subjects have been reported separately ( 8,9,19 ) . statistical analyses were performed using spss ( version 12.0 ; spss , chicago , il ) . quantitative phenotypic data were compared across genotypes by a repeated - measures general linear model , computing the genotype as the between - subjects effect and both baseline and 1-year values as the within - subjects effect . differences in 1-year changes across genotypes were tested by the interaction term among the between- and within - subjects effects . height and weight were measured , and bmi was calculated ( weight in kilograms divided by the square of height in meters ) . bmi standard deviation scores ( sdss ) were derived from regional normative data ( 20 ) . fasting blood glucose , serum insulin , igf - i , sex hormone binding globulin ( shbg ) , and testosterone were measured as described previously ( 9 ) . genomic dna was purified from whole blood samples using commercial reagents ( gentra puragene cell kit ; qiagen iberia , madrid , spain ) . the stk11 rs8111699 snp was genotyped by a fluorescent genotyping system ( kaspar snp genotyping system ; kbiosciences , hoddesdon , u.k . ) . in brief , 10 pg genomic dna was incubated in a 8-l solution of a combined mix containing 100 mol / l allele specific primer 1 ( fam , x - axis , allele g ) , 5-gaaggtgaccaagttcatgctccactgactgttcaggaaagcac-3 , 100 mol / l allele - specific primer 2 ( vic , y - axis , allele c ) , 5-gaaggtcggagtcaacggattccactgactgttcaggaaagcag-3 , 100 mol / l common reverse primer 5-ggtggaagcctgactgtgagagt-3 , 10 mol / l tris - hcl , ph 8.3 , ktaq polymerase , 1.8 mmol / l mgcl2 , and rox as a passive reference . the pcr mix was incubated at 94c for 15 min , followed by 20 cycles of 94c for 10 s , 57c for 5 s , and 72c for 10 s. thereafter , the pcr mix was incubated for 18 cycles at 94c for 10 s , 57c for 20 s , and 72c for 40 s. samples were then read in a fluorescence plate reader . the kaspar system uses the fluors fam and vic for distinguishing among genotypes and rox as a passive reference . the corresponding genotypes for this snp ( cc vs. cg vs. gg ) did not differ among prepuberty ( 33% vs. 44% vs. 22% ) , early postmenarche ( 26% vs. 49% vs. 26% ) , and pcos ( 25% vs. 42% vs. 33% ) subjects in our study ( p = 0.89 ) . the distributions were also tested for hardy - weinberg equilibrium , and none deviated significantly . body composition was assessed by dual - energy x - ray absorptiometry with a lunar prodigy and lunar software ( version 3.4/3.5 ; lunar , madison , wi ) ( 9 ) . total irradiation dose per assessment was 0.1 msevert ; coefficients of variation for scanning precision are 2.2 and 2.6% for fat and lean mass ( 8,9,19 ) . the study protocol was approved by the institutional review board of hospital of sant joan de du . informed consent was obtained from the patients and/or from parents , and assent was obtained from girls , as appropriate . the clinical , biochemical , and body composition data from prepubertal and early postmenarcheal subjects have been reported separately ( 8,9,19 ) . statistical analyses were performed using spss ( version 12.0 ; spss , chicago , il ) . quantitative phenotypic data were compared across genotypes by a repeated - measures general linear model , computing the genotype as the between - subjects effect and both baseline and 1-year values as the within - subjects effect . differences in 1-year changes across genotypes were tested by the interaction term among the between- and within - subjects effects . genotype effects were similar in prepubertal , early postmenarcheal , and pcos subjects , and therefore genotype - phenotype associations are presented for all 85 girls studied as a single group ( adjusted for group allocation ) to maximize the statistical power . clinical , biochemical , and body composition variables before and after 1 year of metformin in the study subjects values are means sem . values in healthy girls matched for bmi : prepubertal ( n = 24 ; aged 8.3 0.3 years ) : insulin 6.5 0.5 miu / l , igf - i 200 15 ng / ml , shbg 3.6 0.1 g / dl , testosterone 16 2 ng / dl ; pubertal ( n = 24 ; age 15.3 0.2 years ) : insulin 8.8 0.4 miu / l , igf - i 384 26 ng / ml , shbg 1.9 0.1 g / dl , testosterone 31 3 ng / dl . lipid profile : triglycerides 55 4 mg / dl ; ldl 83 3 mg / dl ; hdl 65 2 mg / dl ( 8,23 and ibez et al . ; jcem 2004 ; 89:4,331337 ) . the g allele in stk11 rs8111699 was associated with a poorer metabolic profile , specifically with higher insulin and igf - i ( both p < 0.005 ) ( table 2 ) . the presence of this allele was at baseline unrelated to age , weight , body composition parameters , and serum androgens and lipids ( table 2 ) . clinical , biochemical , and body composition variables before and after 1 year on metformin according to stk11 rs8111699 genotype in the study subjects values are means sem . p values for comparisons of baseline , 1-year data , and 1 year changes across genotypes are from a repeated - measures general linear model . results are adjusted for group allocation ( i.e. , prepubertal , early postmenarcheal , or pcos ) ; those for 1 year of metformin and for 01 year are also adjusted for baseline insulin levels . p 0.005 is considered the acceptable threshold of significance in these data because of the multiple tests performed ; these values appear in bold in the table . values in healthy girls matched for bmi : prepubertal ( n = 24 ; aged 8.3 0.3 years ) : insulin 6.5 0.5 miu / l ; igf - i 200 15 ng / ml ; shbg 3.6 0.1 g / dl ; testosterone 16 2 ng / dl ; pubertal ( n = 24 ; aged 15.3 0.2 years ) : insulin 8.8 0.4 miu / l ; igf - i 384 26 ng / ml ; shbg 1.9 0.1 g / dl ; testosterone 31 3 ng / dl . lipid profile : triglycerides 55 4 mg / dl ; ldl 83 3 mg / dl ; hdl 65 2 mg / dl ( 8,23 ) . after 1 year of therapy , there were pronounced differences in the response to metformin according to the stk11 rs8111699 genotype , as reflected by a robust response in the main endocrine - metabolic and body composition parameters in gg homozygotes , with an intermediate response in cg heterozygotes and almost no response in cc homozygotes ( table 2 ) . such differences were found for 1-year changes in body composition , circulating insulin , igf - i , free androgen index , and lipids ( all p < 0.005 ; table 2 ) . our results indicate that the stk11 rs8111699 snp influences both insulin sensitivity and metformin efficacy in hyperinsulinemic girls with androgen excess . despite the fact that neither stk11 nor ampk is a direct target of metformin ( 14 ) , both are necessary for metformin actions ( 14 ) . it has been proposed that stk11 is constitutively active and that metformin exerts its hypoglycemic effects through a modification in ampk , rendering it a better substrate for stk11 ( 10 ) . although the role of the studied intronic snp in stk11 has yet to be defined , it is known that intronic snps may regulate gene expression and alternative splicing ( 21 ) . the stk11 rs8111699 snp may also be in linkage disequilibrium with an active snp in the kinase gene region . the stk11 rs8111699 snp was associated with baseline insulin sensitivity in girls with hyperinsulinemic androgen excess . bearing in mind that stk11 may be constitutively active , we postulate that carrying the g allele in the stk11 rs8111699 snp can result in lower stk11 activity and therefore lower efficiency of ampk phosphorylation by stk11 . the same g allele in the stk11 rs8111699 snp was associated with pronounced changes in endocrine - metabolic and body composition parameters upon metformin treatment , effects that were nearly absent in c homozygous subjects . we suggest that metformin restores the efficiency of ampk phosphorylation by stk11 in carriers of the g allele and has less or no effect in the absence of such allele . ( 15 ) , who found a stepwise increase in ovulation rate from c / c to c / g to g / g genotype in women with pcos treated with metformin . the stk11 rs8111699 snp may thus play a broader role in metabolic and cardiovascular diseases by modulating the baseline condition as well as the response to treatment with the above - mentioned pharmacological agents . a limitation of our study is the insufficient power to analyze the effect of the stk11 snp in each of the three subgroups within the total study population . such an analysis would have been of interest because each of the consecutive subgroups is thought to represent a developmental stage of hyperinsulinemic androgen excess . it should be noted , however , that the results remained essentially the same with and without adjustment for subgroup allocation in the multivariate model . the identification of genetic polymorphisms that contribute to predict the response to commonly prescribed medications should assist clinicians in their selection of available drugs to individualize treatment , thereby improving the therapeutic response , both on the efficacy and on the safety side .

the objective of this study is to examine the efficacy of gabapentin for the treatment of hemicrania continua ( hc ) in cases where patients had difficulty tolerating indomethacin due to adverse effects . a retrospective chart review of nine patients with hc between october 2006 and february 2008 . inclusion criteria included men and women age 18 or above presenting to the headache center with a headache that meets international headache society criteria for hc including a response to indomethacin , but were not able to continue on indomethacin secondary to adverse effects . four patients report being pain free , three patients report a 5080% reduction of pain , one patient reports a 10% reduction of pain , and one patient reports no change in pain . seven out of nine patients demonstrated at least a 50% reduction of pain on gabapentin , four patients becoming completely pain free . objectiveserine - threonine kinase stk11 catalyzes the amp - activated protein kinase complex . we tested the hypothesis that a gene variant in stk11 contributes to variation in insulin sensitivity and metformin efficacy.research design and methodswe studied the effects of a single nucleotide polymorphism ( snp ) ( rs8111699 ) in stk11 on endocrine - metabolic and body composition indexes before and after 1 year of metformin in 85 hyperinsulinemic girls with androgen excess , representing a continuum from prepuberal girls with a combined history of low birth weight and precocious pubarche over to postmenarchial girls with hyperinsulinemic ovarian hyperandrogenism . metformin was dosed at 425 mg / day in younger girls and 850 mg / day in older girls . stk11 rs8111699 was genotyped . endocrine - metabolic features were assessed in the fasting state ; body composition was estimated by absorptiometry.resultsgenotype effects were similar in younger and older girls . at baseline , the mutated g allele in stk11 rs8111699 was associated with higher insulin and igf - i levels ( both p < 0.005 ) . the response to metformin differed by stk11 genotype : gg homozygotes ( n = 24 ) had robust metabolic improvements , gc heterozygotes ( n = 38 ) had intermediate responses , and cc homozygotes ( n = 23 ) had almost no response . such differences were found for 1-year changes in body composition , circulating insulin , igf - i , free androgen index , and lipids ( all p < 0.005).conclusionsin hyperinsulinemic girls with androgen excess , the stk11 rs8111699 snp influences insulin sensitivity and metformin efficacy , so that the girls with the least favorable endocrine - metabolic profile improve most with metformin therapy .

the centers for disease control and prevention report that autism spectrum disorder ( asd ) now affects 1 in 88 children in the usa and 1 in 47 in utah . asd is 5 times more likely in boys with 1 in 54 boys identified nationally . more children will be diagnosed with asd this year than with aids , diabetes , and cancer combined . a diagnosis of autism is based on observed behavior and educational and psychological testing . currently , there is no single definitive treatment for autism , nor is there presently a cure . traditional treatments may be biomedical , sensory , behavioral , developmental , sports - related , or arts - based . the objective was to conduct qualitative research using light hands - on touch energy work with children diagnosed with asd with focus on the following behaviors : eye contact , appropriate social behavior , verbal tics , motor control and coordination , neural impulses related to the sensory organs , immune system , and ability to learn . six participants between 9 and 16 years of age were treated by teams of two practitioners in 10 sessions using light touch energy work protocol focusing on boosting the immune system and strengthening organs of the body and brain functioning . over a 15-month period , participants had an overall improvement of 70% in 5 categories evaluated . specifically , there was an improvement of 81% in eye contact , 72% in social behavior , 61% in motor control / coordination , 75% in sensory neural reorganization , and 65% in learning evolution . energy work may positively impact people living with asd and can be safely integrated with other healthcare modalities . further research needs to be conducted , and more reliable research instruments for complementary and alternative healthcare need to be developed . the optimal time of catheter removal following radical prostatectomy ( rp ) is not defined . from a patient 's perspective , early removal of the urinary catheter is desirable due to catheter - related discomfort , penile pain , bladder irritation / spasm , pericatheter urine leaks , etc . , however , the surgeon 's concerns with early catheter removal are the possibility of anastomotic site leak and acute urinary retention . with experience and improvement in techniques , the duration of catheterization has decreased from an average of 21 days in open rp era to 710 days in cases where robot - assisted rp ( rarp ) is performed . in fact , there are numerous reports about the feasibility and safety of even earlier ( i.e. on the postoperative day 35 ) catheter removal after radical retropubic prostatectomy or rarp . with early catheter removal , some concerns have been raised about increased risk of acute urinary retention . therefore , in practice , the majority of the institutions perform catheter removal after 7 days of surgery . however , there is a variation in the practice of performing cystograms before catheter removal which is done primarily to rule out anastomotic site leak . with the improvement in anastomotic technique , it is rare to find anastomotic site leak after rarp , and it may not always be necessary to obtain a routine check cystogram . we , therefore , evaluated our 230 consecutive rarp patients and the outcomes of their cystogram findings to determine the indications for cystogram before catheter removal . we reviewed our prospectively collected rarp database of 230 consecutive patients who underwent robot - assisted rp by 2 surgeons from april 2010 to january 2015 . all the patients underwent rarp through transperitoneal approach using 4 arm da vinci surgical system . clinical characteristics included patient 's age , biopsy gleason score , clinical stage , and history of transurethral resection of prostate ( turp ) or other urethral / bladder neck surgery . intraoperative data recorded included : estimated blood loss , operative time , bladder neck reconstruction , visible leak upon bladder irrigation at completion of anastomosis , and placement of pelvic drains . bladder neck sparing was done whenever possible . in patients with a wide bladder neck , bladder neck reconstruction was performed by placing sutures at the 3 and 9 oclock positions . urethrovesical anastomosis was performed in 2 layers ( described earlier ) . at the end of anastomosis , a bladder fill test was performed ( using 120 ml of saline ) to assess intraoperative anastomotic leak . patients were called for office cystography and catheter removal , usually a week after surgery . cystography was performed at low pressure by gravity instillation of 150200 ml of diluted contrast through the catheter placed during rarp . patients were observed under fluoroscopy in lateral oblique position for any contrast leak at the site of anastomosis . in the absence of extravasation , the catheter was left in place until a subsequent cystogram revealed resolution , typically 1 week later . in patients with retention of urine after catheter removal , placement of a 16fr foley catheter all patients were followed for a minimum of 6 months , and the longest follow - up was 5 years . postoperative data included pathologic gleason score and stage , hospitalization time , and catheterization time . postoperative complications included urinary retention , urinary tract infection , bladder neck contracture , and urinary incontinence . continence status was defined as the use of either no pad or just a security liner . continence rate was assessed with the self - administered questionnaire during a visit in the outpatient department or by e - mail / postal mail at 6 week and at 3 , 6 , 9 , 12 , 18 , and 24 months and then once a year . continuous parametric variables were reported as the mean standard deviations or as the median values and interquartile range . a total of 207 patients ( 90% patients ) underwent catheter removal on the 7 postoperative day while 10% ( 23 patients ) had catheter removal on the 10 day or later . a decision about delay in catheter removal was usually made by the operating surgeon , based upon satisfaction with bladder neck reconstruction or observation of minimal leak on bladder fill test during surgery . two of these patients had prior bladder neck reconstruction for the wide neck , and other two underwent standard anastomosis . as a routine , patients with bladder neck reconstruction and/or intraoperative leak ( on bladder fill test ) had their catheter removed on the day 10 . only one patient out of four with an intraoperative leak on bladder fill test had leak on postoperative cystogram . demographic details of patients total of nine patients ( 3.9% ) had extravasation on initial cystogram . among the patient with anastomotic site leak , two patients had a history of turp and seven other had wide bladder neck ( resulting from wide margin due to basal tumor ) . out of the total nine patients with leak , 3 patients had a minimal amount of contrast leak ( which cleared on post void film ) ; therefore , catheter was still not replaced [ flow chart 1 ] . in rest of the 6 patient showing leak , continued catheter drainage was done for another week and repeat cystogram was done [ figure 1 ] . four out of nine patient showing leak needed catheter for a total of 14 days while the two patients needed the catheter for a total of 21 days . no significant difference in the intraoperative variables , blood loss , duration of drain , length of hospital stay , and continence outcomes was noted between the patients with anastomotic leak ( noted on cystogram ) when compared to rest of the patients . flow chart of outcome of cystogram and their management ( a ) cystogram showing minimal anastomotic leak on the postoperative day 7 . ( b ) resolution of leak after 7 days of indwelling catheter ( day 14 ) acute urinary retention after catheter removal was noted in 3 patients . only 1 out of 3 patients who had retention after catheter removal had bladder neck sparing surgery . none of the patient experienced immediate retention of urine . out of those who developed retention ( 3 cases ) , one occurred after a week of catheter removal , and the other two developed after 48 h. the catheter was easily replaced in an emergency without any problem . these patients were started on alpha - blocker and catheter removed after 3 days with successful voiding . no pad status at 3 months , 6 months , and 1 year was 76% , 86% , and 94% , respectively . all but one patient with urine leak was continent at 6 months ( one patient became continent by 9 months of follow - up ) . no significant difference in the intraoperative variables , blood loss , and duration of drain , length of hospital stay , prostate volume , grade or stage of the tumor , and continence outcomes was noted between the patients with leak compared to rest of the patients . cystogram at the time of catheter removal is an important tool that can help in documentation of healing at anastomotic site and identifying leak , if any . however , as noted in our study and also studied by other authors , the incidence of urine leak noted on cystogram done at day 7 is very low ( 25% ) . therefore , instead of using cystogram in all the patients , selective use of cystogram can prove to be cost - effective . compared to studies published from developed countries , our patients had a higher clinical stage and more cases of post - turp status , thus requiring wide bladder neck dissection and subsequent reconstruction . we found that there is specific utility of cystogram in the patients who have had a history of turp , wide bladder neck with intraoperative bladder neck reconstruction , or minimal leak noted on intraoperative bladder fill test . although not supported by any evidence , it is our practice to delay catheter removal to postoperative day 10 ( instead of day 7 ) , in patients who undergo bladder neck reconstruction . the optimal time for catheter removal ( or indwelling catheter drainage ) is still a point of scrutiny . understandably , catheter - related symptoms and discomfort are less with shorter duration of indwelling catheter . however , the risk of anastomotic leak , acute urinary retention , and the impact of urine leak on long - term continence and bladder neck contracture are some of the concerns with early catheter removal . most of the authors consider catheter removal after 7 days minimizes the risk of anastomotic leak as well as the chance of acute retention . interestingly , there are studies that have suggested even earlier catheter removal ( at 34 days ) without any significant increase in complication . have reported 1% cystogram leak rate after catheter removal at 24 days , but with a higher rate of acute retention . it is considered that anastomotic site urine leak can potentially delay the time to continence , and adversely impact the overall continence rates . several authors have studied the outcomes in their patients who had a varying degree of anastomotic site urine leak . all the studies based on robotic prostatectomy have concluded that urinary extravasation had no adverse impact on the long - term continence status . patil et al . reported that those patients who had a leak , 70% were dry at 3 months , compared to 90% in those with no leaks . at the 12-month follow - up , 95.2% of the patients with no leak were dry , compared with 94% of those with a leak , thus concluding that at 12 months there was no difference . in our study , the number of patient with anastomotic site leak was too small to make a comparative statement about the impact on recovery pattern of continence . however , all but one patient with urine leak had zero pad status at 6 months ( one patient became continent by 9 months ) . it is also postulated that urine leak ( especially prolonged duration ) will lead to a sustained inflammatory reaction , hampering wound healing , thus increasing the risk of delayed complication such as bladder neck contracture . in a large series reporting urine leak eight of 287 ( 2.8% ) patients it was further clarified that bladder neck contracture developed in patients with higher grade leaks . only three of our patient had retention of urine after few hour of catheter removal . it is likely that the cause of acute retention is mucosal edema or hematoma or increase bladder neck smooth muscle tone . the reported risk of acute retention is reportedly higher ( 6.721% ) with catheter removal at 4 days or less . therefore , results may not be generalizable . however , at the same time , constancy of anastomotic technique , operating surgeons and the follow - up methodology is the strength of this study . the results of our study suggest that in usual circumstances , urinary catheter can be safely removed on the 7 postoperative day after rarp without a routine cystography . selective use of check cystogram can be done in cases where bladder neck reconstruction is performed or those who have a history of turp with wide bladder neck and when an intraoperative leak noted on the bladder fill test .

focus area : pediatricsintroduction : the centers for disease control and prevention report that autism spectrum disorder ( asd ) now affects 1 in 88 children in the usa and 1 in 47 in utah . asd is 5 times more likely in boys with 1 in 54 boys identified nationally . more children will be diagnosed with asd this year than with aids , diabetes , and cancer combined . a diagnosis of autism is based on observed behavior and educational and psychological testing . currently , there is no single definitive treatment for autism , nor is there presently a cure . traditional treatments may be biomedical , sensory , behavioral , developmental , sports - related , or arts-based.objectives:the objective was to conduct qualitative research using light hands - on touch energy work with children diagnosed with asd with focus on the following behaviors : eye contact , appropriate social behavior , verbal tics , motor control and coordination , neural impulses related to the sensory organs , immune system , and ability to learn.methods or target groups : six participants between 9 and 16 years of age were treated by teams of two practitioners in 10 sessions using light touch energy work protocol focusing on boosting the immune system and strengthening organs of the body and brain functioning . assessments included parental and practitioner observations using verified and unverified research instruments.results or activities : over a 15-month period , participants had an overall improvement of 70% in 5 categories evaluated . specifically , there was an improvement of 81% in eye contact , 72% in social behavior , 61% in motor control / coordination , 75% in sensory neural reorganization , and 65% in learning evolution.conclusion or deliverables : energy work may positively impact people living with asd and can be safely integrated with other healthcare modalities . further research needs to be conducted , and more reliable research instruments for complementary and alternative healthcare need to be developed . introduction : with the improvement in anastomotic technique , it is rare to find anastomotic site leak after robot - assisted radical prostatectomy ( rarp ) . it may not always be necessary to do regular check cystogram before catheter removal . we evaluated our 230 consecutive rarp patients and their cystograms to determine the indications for selective use of cystogram before catheter removal.materials and methods : we reviewed our prospectively collected rarp database of 230 consecutive patients . cystography was performed at low pressure by gravity instillation of diluted contrast through the catheter . patients were observed under fluoroscopy in lateral oblique position for any contrast leak at the site of anastomosis . all patients were followed for a minimum of 6 months , and the longest follow - up was 5 years.results:a total of 207 patients ( 90% ) underwent catheter removal on postoperative day 7 . nine patients ( 3.9% ) had extravasation on initial cystogram . two patients with leak had a history of transurethral resection of prostate ( turp ) and seven other had bladder neck reconstruction for wide bladder neck . three patients with minimal leak did not require catheter replacement . in rest of the 6 patient with leak , continued catheter drainage was done . no significant difference in the intraoperative variables , blood loss , duration of drain , length of hospital stay , and continence outcomes was noted between the patients with leak compared to rest of the patients . none of the patient needed any procedure / intervention related to the surgery and none developed bladder neck stenosis.conclusion:in usual circumstances , catheter removal can be done safely on a postoperative day 7 without routine cystography . selective use of check cystogram can be done in the case where bladder neck reconstruction is performed or those had a prior turp and a wide bladder neck .

successful laparoscopic treatment has been described previously in only a few cases . rarely the diagnosis is suspected preoperatively when using only ultrasound . other diagnostic tests like magnetic resonance cholangiopancreatography ( mrcp ) , endoscopic retrograde cholangiopancreatography ( ercp ) and intraoperative cholangiogram can be more accurate in the diagnosis of this condition . intraoperative cholangiogram represents a powerful tool for the surgeon when dealing with double gallbladder , it help to delineate the anatomy and confirms the diagnosis . ( 1,2 ) we present a case report of a patient with symptomatic cholelithiasis with double gallbladder treated laparoscopically using intraoperative cholangiogram . a 21 year old male otherwise healthy with no previous surgical history , presented to the emergency room with the chief complaint of several hours of right upper quadrant pain that started after a large meal . the patient described a history of several weeks of right upper quadrant pain after meals but normally it would subside without any intervention . on admission , the patient had normal and stable vital signs and on physical exam there was tenderness to palpation on the right upper quadrant but no peritoneal signs . his laboratory exams were unremarkable with a normal white blood cell count and normal liver function test . the patient was admitted to the hospital and he was taken to the operating room where he underwent a laparoscopic cholecystectomy using the standard 4 port technique with intraoperative cholangiogram ( fig 1 ) . also appreciated in the image : the common bile duct , the common hepatic duct and the left and right hepatic ducts . during the operation ( fig 23 ) the intraoperative cholangiogram helped to identify each cystic duct and the operation was otherwise uneventful . he was started on clear liquid diet postoperatively and he was discharged home on postoperative day one . gallbladder duplication is a very rare diagnosis . even though the true incidence reported during autopsy is about 1:40005000 the gallbladder primordium can split and form a gallbladder duplication but this only represents a congenital anomaly of the gallbladder . true gallbladder duplication has two cystic ducts and two cystic arteries . the true double gallbladder forms from an extra primordium during the embryonic development . the symptomatic patient with two gallbladder represents a diagnostic and treatment challenge to the general surgeon.(13 ) to aid in the diagnosis the surgeon can use ultrasound , mrcp , ercp and ultrasound . none of these tools are 100% sensitive and only in little more than 50% of the cases the diagnosis is made preoperatively.(2,3 ) in our case the ultrasound was questionable but certainly prompted the use of intraoperative cholangiogram to help define the anatomy . in figure 2 two cystic ducts can clearly be seen which makes our case a true gallbladder duplication treated successfully with standard laparoscopic technique . the differential diagnosis of a gb duplication seen in us is large , but the most common diagnosis are a phrygian cap , gallbladder diverticulum or a choledochal cyst.(3 ) once the diagnosis has been made by seen two cystic ducts in the symptomatic patient , the surgical removal of both gallbladders is indicated . there are only a few case reports of double gb successfully managed laparoscopically in the literature.(3 - 7 ) our case shows that the use of intraoperative cholangiogram confirms the diagnosis and helps to identify both cystic ducts . previous literature have shown conflicting and controversial results when trying to use intraoperative cholangiogram to prevent bile duct injuries , but there no large series or randomized studies evaluating the use of intraoperative cholangiogram in patients with double gallbladder . ( 8 - 10 ) we recommend routine use of ioc when dealing with abnormal anatomy or congenital anomalies of the gallbladder . the evidence is anecdotal but as shown on this case report clearly seen two cystic ducts accurately diagnosed a gallbladder duplication and prevented the surgeon from leaving an unidentified gallbladder which later can become a diagnostic enigma . phantom limb pain ( plp ) is classified as a kind of neuropathic pain that is induced after nerve injury . it is an after effect of amputation occurring in up to 50 - 85% of the patients,[13 ] and often leads to significant impairment in occupational and social functioning . daily pain is reported by 48% of patients with plp and it is supposed that these symptoms potentially affect their quality of life . more importantly , emotional factors are influential in patients experience of prolonged pain in a phantom limb after amputation . on the one hand , it is likely that phantom pain develops poor health - related quality of life such as emotional problems including anxiety and depression . on the other hand , it is also possible that poor quality of life due to emotional problems induces phantom pain and some conditions , such as anxiety and depression , may themselves worsen plp.[1810 ] although some psychological investigations have been performed in patients suffering from plp , controversial findings have been reported . although multiple psychological modalities have been attempted in managing plp[1119 ] and psychotherapy was reported to yield good results , katz and melzack found no significant difference in standardized tests of psychological dysfunction between patients who experienced phantom pain and those who did not . they concluded that the pain is more likely to vary with the experience of pre - amputation pain , even retaining many of its characteristics . a review of the literature on measures used to diagnose psychopathology found that many measures include items that confound emotional distress with the physical disorder and it is estimated that since the period 1996 - 2002 , the number of hads papers that have been published has increased almost fourfold . a recent review of the literature on the validity of hads clearly indicates that it is a well - performed questionnaire in assessing the symptom severity and caseness of anxiety disorders and depression in somatic , psychiatric , and primary care patients and even in the general population . however , the prevalence and severity of these psychological dysfunctions have not been compared in plp patients with other chronic pain sufferers . previous comparative studies have focused on the comparison of psychiatric disorders in plp patients with non - pain amputees in which the effect of pain itself was assessed . therefore , the aim of our study was to evaluate anxiety and depression in patients with amputated limbs suffering from phantom pain and to compare these psychological disorders with those of patients with non - phantom chronic pain using the hads questionnaire . thus , in contrast to previous investigations , it could be possible to evaluate the effect of amputation instead of the pain itself . this prospective analytical cross - sectional study was conducted in khatam - al - anbia pain clinic , tehran , iran , between 2008 and 2010 . a total number of 16 male amputees consecutively participated in the study as the plp group . for this purpose , patients with upper and lower limb amputations between the ages of 25 years and 65 years with traumatic amputations and with plp ( at least five points on a visual analogue scale [ vas ] for at least 3 months ) were included . plp was defined as pain perceived at parts of an extremity that were no longer present . therefore , the presence of plp and phantom sensation was based on the answers to the following questions : ( i ) do you feel the amputated part of your limb ? ( ii ) do you feel pain in the amputated part of your limb ? if the answer to the second question was yes the severity of the pain was investigated using the vas ( 0 - 10 cm ) . as a control group , 24 male age - matched patients who were experiencing non - phantom chronic pain were recruited and evaluated at the same period of time . those eligible were men aged 25 years or older with neuropathic pain of at least 3 months in duration caused by trauma or surgery , with allodynia or hyperalgesia , and with an average weekly pain intensity score greater than five on a 10 cm vas . it must be noted that all of the patients were administered the same analgesic protocol , had a stable analgesic regimen , and were followed in the khatam - al - anbia pain clinic . the study was approved by the medical ethics committee of khatam - al - anbia hospital . patients were told that their data and the results of their assessment were being using for research purposes , and each patient provided written informed consent . after each patient fulfilled the eligible criteria , a list of demographic , baseline , and pain characteristics were asked , which were recorded at the time of enrollment . these variables consisted of age , marital status , educational level , body mass index ( bmi ) , and pain characteristics including duration , average , highest and lowest intensity , and aggravating factors ( e.g. , somatic , psychotic , prolonged fix position , changed position , prolonged rest ) . pain intensity measurements were taken at baseline for all of the 40 recruited patients using a 10 cm vas . the patients were instructed to record the highest , the lowest , and the average intensity of their pain within the last 4 weeks prior to the study enrollment . they were asked to point out each intensity on a 10 cm vas , which theoretically ranged between 0 and 10 , where 0 indicates no pain and 10 corresponds to the worst pain imaginable . the hads was used to assess two psychological dysfunctions anxiety and depression in two groups of the patients . it has been shown that hads provides clinically meaningful results as a psychological screening tool , in clinical group comparisons and in studies with several aspects of disease and quality of life . as mentioned previously , the validity of this questionnaire was previously evaluated and it was indicated that hads is a well - performed questionnaire in assessing the symptom severity and caseness of anxiety disorders and depression in chronic pain sufferers . the persian version of the hads questionnaire was first validated by montazeri et al . in 2003 . they showed that the iranian version of the hads questionnaire was generally acceptable to almost all patients . checking the reliability of the hads , cronbach 's alpha coefficient was found to be 0.78 for the anxiety subscale and 0.86 for the depression subscale . using the known groups comparison and convergent analysis , the persian version of the hads questionnaire was shown to be valid . moreover , both anxiety and depression subscales discriminated well between subgroups of their sampled patients with different statuses . the hads questionnaire contains 14 items and consists of two subscales : anxiety and depression . each subscale consists of seven items , where the odd - numbered questions belong to the anxiety subscale ( hads - a ) and the even items belong to the depression subscale ( hads - d ) . each item is rated on a four - point scale ( scored between 0 and 3 ) , giving maximum scores of 21 for anxiety and depression . in addition , the total score of each subscale could be categorized using some cutoff points . scores of 11 or more on either subscale are considered to be a significant case of psychological morbidity , whereas scores of 8 - 10 represent borderline and 0 - 7 represent normal. all the 40 participants were asked to complete the hads questionnaire , following which the scores were calculated for each subscale for each patient . a sample size of 16 patients in each group was calculated to detect a difference of two units in the total score of the hads questionnaire in each subscale with an estimation of 5% for type i error and 20% for type ii error based on a two - sided independent samples t - test . descriptive statistics were used to determine frequency and percentage for categorical variables , and mean and standard deviation ( sd ) for continuous measurements . after checking the normal distribution with the kolmogorov - smirnov test , the differences in patient characteristics and hads scores ( including each item and total scores ) between the plp group and non - phantom chronic pain patients were analyzed with the mann whitney u test , independent samples t - test , fisher 's exact test , and the chi - square test , as appropriate . analyses were performed with the statistical package for the social sciences , version 16 ( spss inc . , this prospective analytical cross - sectional study was conducted in khatam - al - anbia pain clinic , tehran , iran , between 2008 and 2010 . a total number of 16 male amputees consecutively participated in the study as the plp group . for this purpose , patients with upper and lower limb amputations between the ages of 25 years and 65 years with traumatic amputations and with plp ( at least five points on a visual analogue scale [ vas ] for at least 3 months ) were included . plp was defined as pain perceived at parts of an extremity that were no longer present . therefore , the presence of plp and phantom sensation was based on the answers to the following questions : ( i ) do you feel the amputated part of your limb ? ( ii ) do you feel pain in the amputated part of your limb ? if the answer to the second question was yes the severity of the pain was investigated using the vas ( 0 - 10 cm ) . as a control group , 24 male age - matched patients who were experiencing non - phantom chronic pain were recruited and evaluated at the same period of time . those eligible were men aged 25 years or older with neuropathic pain of at least 3 months in duration caused by trauma or surgery , with allodynia or hyperalgesia , and with an average weekly pain intensity score greater than five on a 10 cm vas . it must be noted that all of the patients were administered the same analgesic protocol , had a stable analgesic regimen , and were followed in the khatam - al - anbia pain clinic . the study was approved by the medical ethics committee of khatam - al - anbia hospital . patients were told that their data and the results of their assessment were being using for research purposes , and each patient provided written informed consent . after each patient fulfilled the eligible criteria , a list of demographic , baseline , and pain characteristics were asked , which were recorded at the time of enrollment . these variables consisted of age , marital status , educational level , body mass index ( bmi ) , and pain characteristics including duration , average , highest and lowest intensity , and aggravating factors ( e.g. , somatic , psychotic , prolonged fix position , changed position , prolonged rest ) . pain intensity measurements were taken at baseline for all of the 40 recruited patients using a 10 cm vas . the patients were instructed to record the highest , the lowest , and the average intensity of their pain within the last 4 weeks prior to the study enrollment . they were asked to point out each intensity on a 10 cm vas , which theoretically ranged between 0 and 10 , where 0 indicates no pain and 10 corresponds to the worst pain imaginable . the hads was used to assess two psychological dysfunctions anxiety and depression in two groups of the patients . it has been shown that hads provides clinically meaningful results as a psychological screening tool , in clinical group comparisons and in studies with several aspects of disease and quality of life . as mentioned previously , the validity of this questionnaire was previously evaluated and it was indicated that hads is a well - performed questionnaire in assessing the symptom severity and caseness of anxiety disorders and depression in chronic pain sufferers . the persian version of the hads questionnaire was first validated by montazeri et al . in 2003 . they showed that the iranian version of the hads questionnaire was generally acceptable to almost all patients . checking the reliability of the hads , cronbach 's alpha coefficient was found to be 0.78 for the anxiety subscale and 0.86 for the depression subscale . using the known groups comparison and convergent analysis , the persian version of the hads questionnaire was shown to be valid . moreover , both anxiety and depression subscales discriminated well between subgroups of their sampled patients with different statuses . the hads questionnaire contains 14 items and consists of two subscales : anxiety and depression . each subscale consists of seven items , where the odd - numbered questions belong to the anxiety subscale ( hads - a ) and the even items belong to the depression subscale ( hads - d ) . each item is rated on a four - point scale ( scored between 0 and 3 ) , giving maximum scores of 21 for anxiety and depression . in addition , the total score of each subscale could be categorized using some cutoff points . scores of 11 or more on either subscale are considered to be a significant case of psychological morbidity , whereas scores of 8 - 10 represent borderline and 0 - 7 represent normal. all the 40 participants were asked to complete the hads questionnaire , following which the scores were calculated for each subscale for each patient . a sample size of 16 patients in each group was calculated to detect a difference of two units in the total score of the hads questionnaire in each subscale with an estimation of 5% for type i error and 20% for type ii error based on a two - sided independent samples t - test . descriptive statistics were used to determine frequency and percentage for categorical variables , and mean and standard deviation ( sd ) for continuous measurements . after checking the normal distribution with the kolmogorov - smirnov test , the differences in patient characteristics and hads scores ( including each item and total scores ) between the plp group and non - phantom chronic pain patients were analyzed with the mann whitney u test , independent samples t - test , fisher 's exact test , and the chi - square test , as appropriate . analyses were performed with the statistical package for the social sciences , version 16 ( spss inc . , sixteen male patients with plp and 24 age - matched males who suffered from non - phantom chronic pain were recruited in this study . a complete list of demographic , baseline , and pain characteristics of the patients are shown in table 1 . the mean age of the patients with plp was 48.88 7.47 year , which was similar to that of chronic pain sufferers ( 48.63 9.42 year , p = 0.930 ) . comparison of the baseline and pain characteristics between the two groups of the study regarding pain characteristics , the mean duration of pain was 12.00 10.36 year in plp patients , which was not significantly different from the pain duration in the other group ( 10.35 11.54 year , p = 0.315 ) . using vas , the mean of the average severity of pain was found to be 8.12 1.86 and 7.82 1.94 in phantom pain and non - plp patients , respectively ( p = 0.627 ) . although psychotic factors were the most involved pain - aggravating factors in the plp group ( 87.5% ) , prolonged fix position was more responsible for worsening of pain in patients of group b ( 95.2% ) . the results of hads scores in each item and overall anxiety and depression status are shown and compared between the two groups of the study in table 2 . as is seen , the mean of hads scores for each of the seven items of anxiety ( hads - a ) is higher in patients of group b who suffered from non - plp chronic pain , which is in favor of a more severe anxiety . consequently , the mean of total anxiety score was significantly lower in patients with plp ( 8.00 3.93 vs. 11.25 5.23 ; p = 0.041 ) . comparison of the hospital anxiety and depression scale scores in each item and overall anxiety and depression status between two groups of the study moreover , when the total anxiety score is categorized using the proposed cutoff points of previous studies , the prevalence of anxiety caseness ( hads - a score 11 ) was found to be higher in chronic pain sufferers ( 58.3% vs. 25% ; p = 0.112 , power = 31.7% ) . as listed in table 2 , except for item number 14 , the other hads scores of six items in the depression subcategory ( hads - d ) were also higher in patients with non - phantom chronic pain , which was in favor of a more severe depressed mood . the mean of total depression score was 7.69 5.51 and 9.38 6.11 in patients of the plp and chronic pain groups , respectively . however , this difference was not statistically significant ( p = 0.340 , calculated power = 15% ) . when the total depression score is categorized using the proposed cutoff points of previous studies , the prevalence of depression caseness ( hads - d score 11 ) was also found to be lower in patients with phantom pain ( 37.5% vs. 50% ; p = 0.710 , power = 8% ) . sixteen male patients with plp and 24 age - matched males who suffered from non - phantom chronic pain were recruited in this study . a complete list of demographic , baseline , and pain characteristics of the patients are shown in table 1 . the mean age of the patients with plp was 48.88 7.47 year , which was similar to that of chronic pain sufferers ( 48.63 9.42 year , p = 0.930 ) . comparison of the baseline and pain characteristics between the two groups of the study regarding pain characteristics , the mean duration of pain was 12.00 10.36 year in plp patients , which was not significantly different from the pain duration in the other group ( 10.35 11.54 year , p = 0.315 ) . using vas , the mean of the average severity of pain was found to be 8.12 1.86 and 7.82 1.94 in phantom pain and non - plp patients , respectively ( p = 0.627 ) . although psychotic factors were the most involved pain - aggravating factors in the plp group ( 87.5% ) , prolonged fix position was more responsible for worsening of pain in patients of group b ( 95.2% ) . the results of hads scores in each item and overall anxiety and depression status are shown and compared between the two groups of the study in table 2 . as is seen , the mean of hads scores for each of the seven items of anxiety ( hads - a ) is higher in patients of group b who suffered from non - plp chronic pain , which is in favor of a more severe anxiety . consequently , the mean of total anxiety score was significantly lower in patients with plp ( 8.00 3.93 vs. 11.25 5.23 ; p = 0.041 ) . comparison of the hospital anxiety and depression scale scores in each item and overall anxiety and depression status between two groups of the study moreover , when the total anxiety score is categorized using the proposed cutoff points of previous studies , the prevalence of anxiety caseness ( hads - a score 11 ) was found to be higher in chronic pain sufferers ( 58.3% vs. 25% ; p = 0.112 , power = 31.7% ) . as listed in table 2 , except for item number 14 , the other hads scores of six items in the depression subcategory ( hads - d ) were also higher in patients with non - phantom chronic pain , which was in favor of a more severe depressed mood . the mean of total depression score was 7.69 5.51 and 9.38 6.11 in patients of the plp and chronic pain groups , respectively . however , this difference was not statistically significant ( p = 0.340 , calculated power = 15% ) . when the total depression score is categorized using the proposed cutoff points of previous studies , the prevalence of depression caseness ( hads - d score 11 ) was also found to be lower in patients with phantom pain ( 37.5% vs. 50% ; p = 0.710 , power = 8% ) . as an approximately common condition after limb amputation , plp serves as a disabling chronic pain syndrome for which regular pain treatment is seldom effective . this painful condition may be the result of inappropriately stored or chronically activated pain memories that continue to disturb the subject even after the disease or injury has been successfully treated or the limb has been amputated.[2831 ] on the other hand , it seems that both the amputation of a limb and the consequent phantom pain may affect quality of life , especially with regard to psychological functions . the incidence of anxiety and depression symptoms after amputation has been reported to be 37% and 20% , respectively , and even as high as 41%[3336 ] and much higher than the rates in the community . alongside these considerable prevalence rates , there are some other evidences that show these psychological dysfunctions are not more prevalent among those amputees reporting phantom pain . moreover , research in this field is difficult because of the different measurement tools used for anxiety and depression as well as the lack of prospective studies . in the present study , it was hypothesized that the prevalence rates and scores of anxiety and depression could be more common in patients with plp compared with non - phantom chronic pain sufferers . regarding the proposed cutoff points for the caseness of anxiety and depression in the hads questionnaire , 25% and 37.5% of our patients with plp fulfilled the criteria , respectively . nevertheless , our results indicate that depression and anxiety are more common in plp patients , and these conditions are more prevalent in non - phantom chronic pain sufferers . in 1973 , parkes suggested that emotional and psychological factors are dominant in patients with prolonged pain due to phantom limb after amputation . in another study using the hads scale , badura - brzoza et al . declared that patients after limb amputation achieved a higher score in hads - a and hads - d compared with the control group and concluded that higher levels of anxiety and depression are noticed in patients suffering from plp . in contrast , katz and melzack found no significant difference in psychological dysfunction between patients who experienced phantom pain and those who did not . in another study by fisher and hanspal , the hads score was compared between 29 plp patients with 64 non - phantom pain amputees with the mean time of post - amputation of 9.7 years . the mean reported hads scores in their study were 4.66 and 3.45 for anxiety and depression subscales , respectively . they concluded that only a few patients experienced emotional distress after amputation , and anxiety is being reported more often than depression . in agreement with the studies of katz and melzack and fisher and hanspal , our results also confirmed the lower prevalence of depression and anxiety in patients with plp compared with the control group . however , it should be noted that in the study of katz and melzack and fisher and hanspal , the control group consisted of amputees who did not experience any plp , whereas in our study the patients with non - phantom chronic pain were considered as the control subjects . moreover , compared to their results , the mean score of anxiety and depression was higher in our plp patients ( 8.00 and 7.69 , respectively ) though it was considerably lower when compared with that of chronic pain sufferers ( 11.25 and 9.38 , respectively ) . a longitudinal study by singh et al . even showed that there is a dramatic drop in the incidence of psychological symptoms in individuals after amputation by the time of discharge from a rehabilitation ward . they have concluded that it may be due to adjustment after the emotional stress of limb loss as well as the learning of new skills to adjust to life after amputation . this explanation could be also indicated in our patients , as they received different special cares at our pain clinic , too . however , in another more recent study by singh et al . , it was shown that after an initial drop in incidence of depression and anxiety symptoms after amputation , there is then an increase again after 2 - 3 years post amputation . in contrast , our evaluated plp patients had a mean duration of 12 years post amputation and still showed a lower prevalence of anxiety and depression . to the best of our knowledge , our study is the first one to compare the prevalence of anxiety and depression in plp patients with a sample of non - phantom chronic pain sufferers . in addition to the lesser mean scores of these psychological dysfunctions by the hads questionnaire , it was shown that the caseness of anxiety ( 25% vs. 58.3% ) and depression ( 37.5% vs. 50% ) were also lower in plp patients compared with subjects with chronic pain . a review of the literature reveals that the considerable prevalence of depression in chronic pain sufferers has been previously found in some investigations such as that of magni et al . in subjects with chronic musculoskeletal pain where the authors suggest a two - way relationship similar to most of the previous studies , one of the important limitations of our study was the cross - sectional design and lack of longitudinal assessment . in addition , the low statistical power in the comparison of depression score emphasizes the need for a higher sample size in further investigations . the results of our study suggest that although the prevalence rates of anxiety and depression are considerable in patients with plp , they were not as frequent as what is observed in non - phantom chronic pain sufferers . these lower levels of anxiety and depression in plp compared to chronic pain is a new finding , which needs to be further evaluated and may lead to new insights into the pathogenesis of phantom pain in further studies .

double gallbladder is a rare finding in patients with symptomatic cholelithiasis or acute cholecystitis . the incidence has been described as 1 in every 4000 - 5000 patients during autopsy . to identify the gallbladder ( gb ) duplication prior to surgical removal of the gb is of upmost importance . it is not unusual to identify this diagnosis intraoperatively , but by using us , ercp or mrcp more than 50% of the cases are diagnosed preoperatively . the use of intraoperative cholangiogram helps to identify the anatomy and confirm the diagnosis during laparoscopic cholecystectomy in patients with gallbladder duplication . background : phantom limb pain ( plp ) is approximately a common condition after limb amputation , which potentially affects the quality of life . we aimed to evaluate anxiety and depression in patients with amputated limbs suffering from plp and to compare these psychological dysfunctions with that of patients with non - phantom chronic pain.methods:a total number of 16 male amputees with plp and 24 male age - matched patients with non - phantom chronic pain were recruited in this study , which was performed at khatam - al - anbia pain clinic , tehran , iran . a validated persian version of the hospital anxiety and depression scale ( hads ) was used to compare two psychological dysfunctions anxiety and depression between the two groups of study.results:the mean of total anxiety score was significantly lower in patients with plp ( 8.00 3.93 vs. 11.25 5.23 ; p = 0.041 ) and the prevalence of anxiety caseness ( hads - a score 11 ) was also lower in the plp group ( 25% vs. 58.3% ; p = 0.112 , power = 31.7% ) . the mean of total depression score was 7.69 5.51 and 9.38 6.11 in patients of plp and chronic pain groups , respectively ( p = 0.340 , power = 15% ) . consequently , the prevalence of depression caseness ( hads - d score 11 ) was lower in plp patients ( 37.5% vs. 50% ; p = 0.710 , power = 8%).conclusion : our results indicate that depression and anxiety are not more common in plp patients , whereas they are more prevalent in subjects with non - phantom chronic pain . these lower levels of anxiety and depression in plp compared with chronic pain is a new finding that needs to be evaluated further , which may lead to new insights into the pathogenesis of phantom pain in further studies .

isolated double - orifice mitral valve ( domv ) is an extremely rare congenital anomaly whose exact incidence has not been conclusively determined . interestingly , this cardiac abnormality rarely presents as an isolated finding , preferentially associated with valvular regurgitation rather than stenosis . the etiology of domv has been recognized in the abnormal fusion of the endocardial cushions and abnormal development of the mitral valve during the delamination process . transthoracic echocardiography is the modality of choice for the characterization of this anomaly ; in this respect , the parasternal short - axis view allows a full morphologic characterization of the anomaly and should always be performed in the case of a meaningful clinical suspect . we present the case of an asymptomatic patient with isolated domv who was referred for cardiac evaluation to achieve a certificate of fitness for competitive sports . a 39-year - old asymptomatic and healthy man was referred to our outpatients clinic for cardiac evaluation to achieve a certificate of fitness for competitive sports . he had no previous medical history , but presented a strong family history of early coronary artery disease . accordingly , a complete transthoracic two - dimensional echocardiography with color doppler examination was performed with a philips ie33 ultrasound machine ( philips healthcare , leiden , the netherlands ) . the echocardiographic examination in the parasternal short - axis view showed a distinctive morphology of an isolated domv [ figure 1 ] . the two mitral orifices were located in the posteromedial and posterolateral positions and their estimated areas were 2.20 cm and 1.98 cm , respectively [ figure 2 ] . there was a single mitral annulus with the two valvular orifices separated by a central fibrous bridge [ figure 1 ] . the echocardiographic examination seemed to show separated subvalvular structures for each of the two orifices [ figure 3 ] . a normal function of both the anterior and posterior leaflets of each orifice was present without evidence of either systolic mitral regurgitation jets or diastolic turbulence [ figure 4 ] . moreover , the two peak and the mean transmitral gradients were normal . similarly , a normally functioning mitral valve ( i.e. without stenosis or insufficiency ) was evident also in the four - chamber view . parasternal short - axis view at the level of the mitral valve in diastole showing the bridge of tissue dividing the mitral valve into two orifices parasternal short - axis view at the level of the mitral valve in diastole showing the areas of the orifices of the double - orifice mitral valve apical four - chamber view seems to show the two separate subvalvular structures of the double - orifice mitral valve apical four - chamber view with color doppler demonstrating the absence of mitral regurgitation the resting echocardiographic examination excluded the presence of any sign of either congenital or acquired morphological cardiac abnormalities . to definitively exclude the presence of any other cardiac or vascular anomaly , a cardiac magnetic resonance imaging evaluation was performed , which confirmed the findings of two - dimensional echocardiography as it showed domv and each of the two orifices with its own chordal apparatus [ figures 57 ] . the patient is now followed up on a yearly basis for the early recognition of any valvular alteration . cardiac magnetic resonance imaging demonstrating normal left ventricular volumes , apex hypertrabeculation , and absence of atrioventricular / ventricular septal defects cardiac magnetic resonance imaging demonstrating the two orifices of the double - orifice mitral valve in the absence of other cardiac abnormalities cardiac magnetic resonance imaging demonstrating the two orifices of the double - orifice mitral valve in the absence of other cardiac abnormalities this anomaly is characterized by the presence of a single mitral annulus with two orifices , each having independent subvalvular apparatus and papillary muscles . domv is an extremely rare condition , usually associated with other congenital heart anomalies ( above all , atrioventricular septal defect , coarctation of aorta , interrupted aortic arch , patent ductus arteriosus , and ventricular septal defect ) . it generally presents with symptoms related to mitral regurgitation or stenosis , but it can also present as an incidental finding . three specific types of domv can be described at two - dimensional echocardiography : ( 1 ) complete bridge type ( about 15% of domvs ) , in which both openings are visible from the leaflet edge and both orifices appear circular ( equal or unequal in size ) with normal subvalvular apparatus and papillary muscles ; ( 2 ) incomplete bridge type , in which the connection is seen only at the leaflet edge ; and ( 3 ) hole type ( the most frequent ) characterized by a small accessory orifice situated at either the posteromedial or anterolateral commissure and identifiable only at the mid - leaflet level . accordingly , our patient presented the rarest form of the anomaly , as represented by the complete bridge type domv . transesophageal echocardiography ( tee ) defines the anatomy of mitral valve better than transthoracic echocardiography and may help to differentiate domv from other conditions such as lesions of valvular leaflets provoked by endocarditis . however , despite these theoretical advantages , tee was not performed in the diagnostic work - up of the patient because of its invasiveness . on the other hand , once domv is nearly assured by two - dimensional echocardiography , cardiac magnetic resonance imaging evaluation becomes a mandatory step to confirm the clinical suspicion , and above all , to exclude any concurrent cardiac and vascular abnormalities . moreover , three - dimensional echocardiography is generally useful to add anatomical details to that obtained by two - dimensional echocardiography . however , it should be performed only in case of uncertain findings at the two - dimensional echocardiography , which is considered the screening test for domv . the management of domv is related to the type and severity of mitral valve dysfunction . asymptomatic domv usually requires no active intervention ; in case of severe mitral regurgitation , stenosis , or associated cardiac anomalies , surgical repair is needed . in all cases of domv , a long - term follow - up is required for the early detection of complications . thus , domv is a very rare anomaly which is usually detected by transthoracic two - dimensional echocardiographic examination in short - axis parasternal views . nevertheless , it can be rarely diagnosed in asymptomatic middle - aged and elderly patients . in any case , the presence of other coexistent cardiovascular abnormalities should be excluded . the largest mercury processing in the western hemisphere was in huancavelica in the peruvian andes , now a town of 30,000 people situated at 3676 m altitude . the extensive mercury deposits have been mined since pre - hispanic times , they are just above the present town at ~4000 m. although the mine closed 30 years ago , mining continues on small scales . the legacy of 500 years of mercury mining is now found in the city trenches and waterways , which are lined with mercury but this legacy is less evident in today 's inhabitants of huancavelica . mining is a dangerous , unhealthy occupation but nowhere was it more health threatening than in colonial south america , especially in huancavelica . the widespread ecological impacts of mining in the andes such as landscape destruction and pollution through the introduction of mercury and other toxicants into the biosphere were overshadowed by the population shifts to the then sparsely inhabited highlands . there , the challenges of survival in the ambient hypoxia of altitude , at which mining generally occurs in the andes , were added to the deleterious health effects of mining . in 1928 , carlos monge of lima , peru , described chronic mountain sickness ( cms ) in a native miner from cerro de pasco , a mining town in peru situated at 4338 m above sea level . it occurs in high altitude natives ( hypoxia adapted individuals ) who for , as yet , unknown reasons lose their adaptation to their hypoxic homeland . recently , cms was found to be associated with deranged expression of hypoxia - related genes . in colonial times , less so now , mining required heavy physical labor which predisposes to cms . the toxicity of mercury , when added to the stress of inescapable hypoxia at high altitude and the dangers to health and predisposition to injury of mining in colonial huancavelica , earned the mine the infamous reputation as the mina de la muerte ( the mine of death ) . because of its importance to the spanish economy , mercury killed thousands of andeans . prior to the conquest , the locals used the huancavelica cinnabar ( mercury sulfide , hgs ) deposits for body paint and cosmetics for their women . but , after the spanish arrival , the lord of the local angaraes people betrayed the cinnabar deposits to amador de cabrera in 1563 . the riches of the deposits enticed newly arriving colonists to stake claims begin mining cinnabar , and distill mercury which they then sold to mexico 's silver refineries . demand for mercury increased when the silver mines at potosi began using mercury to amalgamate the ores in 1570 . the subsoil now belonged to the crown which granted its use to miners upon payment of taxes . under the new rules the huancavelica treasury contracted with former claim holders to keep the mine operating , setting the amount of mercury to be produced and the price to be paid . a change in the production of silver which required mercury for amalgamation of the ore increased the demand for labor in the mines . toledo committed the colonial government to provide adequate manpower for the mercury producers . to do this , he copied inca rules which required adult males to take turns on public projects called a mit'a ( turn ) and ordered neighboring provinces to send 900 mitayos . the population in the surrounding areas decreased because of disease and the demands of the mita . this forced the government to reduce the size of the mita to 620 by 1645 and expand the area from which mitayos were committed into the labor force . mitayos received a low wage but served only for 2 months at a time , taken here as evidence for governmental awareness of the devastating health effects of mercury mining . huancavelica became notorious as a dangerous workplace by the late 16th century and the spanish crown received reports that the mita was abusive , unchristian , and unhealthy . the high altitude of the mine added the stresses of ambient hypoxia , extreme temperatures and harsh weather to the hazards of mining , such as landslides and tunnel collapse . by 1600 the reputation of the huancavelica mine as the mine of death was well deserved . the franciscan miguel agia , in favor of the continuation of the mita , admitted that work in the mine was equivalent to sending [ indians ] to die . and the protector of indians damin de jeria likened the tunnel of the mine to a luis de velasco , another viceroy , wrote to the king about the health aspects of the huancavelica mine in 1600:these quicksilver ores , when they extract them in the mines , they give out a dust that enters itself into the indians as they breath and settles in the chest , of such evil quality , that it causes them a dry cough and light fever and at the end death without repair , because the doctors have it for an incurable evil . these quicksilver ores , when they extract them in the mines , they give out a dust that enters itself into the indians as they breath and settles in the chest , of such evil quality , that it causes them a dry cough and light fever and at the end death without repair , because the doctors have it for an incurable evil . he wanted to close the mine but because of its economic importance , he pleaded for guidance from the crown [ 8 , 9 ] . de jeria wrote to the viceroy that although the mitayos had committed no crimes they were forced to endure conditions worse than those imposed on criminals in prisons and galleys . he argued that huancavelica mining will eventually cause economic collapse because its continuance , costing so many lives of so many indians that which may be lost from not producing mercury is gained in the conservation of the indians , for without them there will be neither quicksilver , nor silver , nor the common good , nor peru . by 1630 , the count of chinchn , then viceroy , wrote to the king about the mita : it is a most terrible matter because a free , innocent , defenseless , poor and afflicted people are condemned to a notorious risk of death their fear of the mita was such that trustworthy people who have seen it have reported to me that they take these miserable indians by force against all their will from their houses and take them in iron collars and chains more than 100 leagues to put them in this risk and from this has resulted their own mothers maiming and crippling their sons to preserve them . it is a most terrible matter because a free , innocent , defenseless , poor and afflicted people are condemned to a notorious risk of death their fear of the mita was such that trustworthy people who have seen it have reported to me that they take these miserable indians by force against all their will from their houses and take them in iron collars and chains more than 100 leagues to put them in this risk and from this has resulted their own mothers maiming and crippling their sons to preserve them . pick - men and ore carriers developed silicosis ( deposition of dust and silica particles in the lungs ) rapidly because of enormous dust levels in the mine , but this also affected work in other mines . in huancavelica , additionally , mercury killed by its inhalation . after returning to their huts , they transferred the dust to their living quarters and contaminated their families by prolonging exposure and mercury absorption through the skin and food . the symptoms of mercury poisoning were known to the workers who called it the huancavelica sickness or the evil of huancavelica . although the usual treatment for poisoned miners was bloodletting to put their bodily humors back into balance , this increased their susceptibility to intercurrent illnesses . eventually the graves of mitayos were said to contain puddles of mercury after their bodies decayed [ 4 , 7 ] . the threat of mercury poisoning was even greater to those who worked the ovens to heat the ore and extract the mercury as the vapors cooled . and indians were made to open the ovens before they had completely cooled to increase mercury extraction by reloading them with fresh ore . those rendered unable to work because of mercury poisoning were known as azogados ( azogue = quicksilver ) . weight loss , tremors , excessive salivation , ulcers of the mouth , restlessness , anemia , and eventually severe depression which often lead to alcoholism . many miners had to be fed by their relatives because of the violent tremors which could not be controlled to bring food to their mouths . the prince of santo buono ( viceroy 17161720 ) urged the abolition of the mita . although initially rejected by the crown and the council of the indies it was finally approved on humanitarian grounds . by 1740 skilled miners were now able to stand and swing sledge hammers in the mining shafts which , by now , had acquired improved ventilation . ulloa , governor of huancavelica , wrote in 1760 that miners were now free of mercury intoxication because the ore contained so little quicksilver but poisoning still occurred at the ovens when workers failed to let them cool completely before reloading them . ulloa reported that azogados descended to warmer lowlands , drank huge amounts of chicha , a beer made from maize , worked very hard in the fields , and sweated out the mercury . the documented improvement was , likely , due to the diuretic effect of the beer which expelled the mercury concentrated in the kidneys . after some weeks of heavy drinking and hard physical labor the shakes , excessive salivation and mouth ulcers disappeared and some miners returned to work in the huancavelica mine . although the mine was especially deadly because of its mercury , most spaniards at that time thought that the indians were destined to die anyway if this was necessary to obtain the metal . camargo wrote to the king in 1595 : they are barbarous people and without knowledge of god ; in their lands they are only occupied in idolatries and drunkenness and other vices of great filthiness these people going to work the mines first seems to me a service to god and to your holy catholic royal majesty and for the good of the natural indians themselves , because the mines they teach them doctrine and make them hear mass and they deal with spaniards by which they become ladinos [ hispanicized] . losing their barbarity , and they have no place for the idolatries of their lands by being outside of those sites where they do them and being occupied , which is the principal thing . they are barbarous people and without knowledge of god ; in their lands they are only occupied in idolatries and drunkenness and other vices of great filthiness these people going to work the mines first seems to me a service to god and to your holy catholic royal majesty and for the good of the natural indians themselves , because the mines they teach them doctrine and make them hear mass and they deal with spaniards by which they become ladinos [ hispanicized] . losing their barbarity , and they have no place for the idolatries of their lands by being outside of those sites where they do them and being occupied , which is the principal thing . camargo affirmed his great desire to [ also ] die in the service of your holy catholic royal majesty . some improvements in health occurred with new mining methods but these failed to take into account the huge pollution of the biosphere of huancavelica which persists to the present . the refining ovens were along the river where the escaping vapors settled on the ground , in the water , and blew across town depositing mercury on everything . these vapors were converted into methyl mercury by bacterial activity which then easily entered the food chain and which is especially injurious to the nervous system . the best - described mass poisoning occurred in minamata bay , japan , in the 1950s . but intoxications restricted to single families due to ingestion of mercury with longterm consequences have also been reported . mercury poisoning in infants ( pink disease , also known as acrodynia ) associated with the use of teething powders containing mercury , especially in england and australia , has been eliminated by the removal of the metal from these remedies . pain , paresthesias , and hypotonia allowed the assumption of characteristic postures by the affected infants . these symptoms reflect the especially injurious effects of mercury on the autonomic nervous system ( ans ) . the syndrome has not been found in recent epidemics of mercury poisoning in japan , but it reemerged in an epidemic of methyl mercury ( used as a fungicide ) poisoning employed for the sterilization of diapers in argentinean babies ( for review see ) . mercury compounds occur in the environment and their levels are rising due to anthropogenic activities , especially the burning of fossil fuels . coal contains large amounts of mercury , which is released into the atmosphere upon burning and it is then transported , possibly , around the globe . even low concentrations of mercury can cause subclinical effects , and slightly increased levels of mercury in hair are associated with decreased academic performance . the world health organization ( who ) places the level at which toxicity occurs at 50 ppm of mercury in hair and advises that healthy people should have no more than 5 ppm of mercury in their hair . however , normal mercury levels in hair are difficult to come by because they are dependent on the amount and type of fish eaten , on location ( especially where the environmental mercury burden may be high ) on the sampling methods , and on analytical methods . thus , in populations that have been exposed to high burdens of mercury for centuries , like those in huancavelica , the effects of such exposure are difficult to assess . hair is renewed throughout life ; it has a well - defined variability in growth influenced by diet , age , blood flow to the skin , and hormonal and metabolic signals which are , ultimately , regulated by the ans . but hair also reflects biologic rhythms because of clock - like signals that affect its growth through the intermediary of the ans . recurrent circa - annual periods of slow and fast rhythms in the hydrogen isotope ratios of human and animal hair can be recognized . using power spectral analysis of the ratios along the length of the hairs can reveal the effects of life at altitude and toxin accumulation on biologic rhythms . here we report a study of hydrogen isotope ratios in the hair and of heart rate variability in the same residents of huancavelica , peru , to assess their biologic rhythms and ans function . we carried out this study to determine whether centuries of ancestral and present exposure to increased mercury in the biosphere could affect ans function and give evidence for continuing deleterious effects on the health in modern huancavelica residents . the demographics of the 5 subjects and mercury and copper levels in their hair are given in table 1 . because the length of the hair reflects the duration of the stored information on biologic rhythms , our study was confined to women who generally have longer hair . we confirmed these very low levels in the hair from two huancavelica subjects using the x26a hard x - ray microprobe at the brookhaven synchrotron . no detectable mercury in single hairs from each subject was reported by the x - ray microprobe . to strengthen our conclusions about the surprisingly low mercury levels in the hair in huancavelica residents we used a control from the usa . in table 1 cold vapor atomic absorption spectrometry was used to measure total mercury ( organic + inorganic ) and inductively coupled plasma - atomic emission spectrometry for the other element ( one sample was misplaced by the analytical company performing the assays no values for hg are available ; here labeled lost ) . to assess the impact of individual metals on the hydrogen isotope ratios of low frequency / high frequency ( lf / hf ) power bands , a measure of ans control of biologic rhythms these power bands were affected by both copper and mercury levels in the hair clearly separating the lf and hf power according to mercury and copper levels . the annual growth rate reported for normal human hair is ~16 cm / year . in our controls ( n = 4 ) as determined from the sinusoidal variation in hydrogen isotope ratios in our samples from the usa and europe , it was 14.2 3.7 sd cm / year . the growth rate of hair from huancavelica ( n = 5 ) was 6.4 1.7 sd cm / year ( p < 0.001 ) ( figure 2 ) . the standard deviation of the variance of the power spectra of the hydrogen isotope ratios between controls from other parts of the world and that from huancavelica is shown in figure 3 . remarkably , this variance is significantly less in huancavelica residents ( p = 0.01 ) . pooling of hydrogen isotope power spectra from huancavelica and control hair is shown in figure 4 . the only statistically significant difference between these spectra is in the low - power frequencies ( p < 0.05 ) , though the power of high frequencies appears also to be less ( ns ) . the annual growth rates of hair can be shown to occur in fast and slow periods of weekly cycles ( figure 5 ) . the shorter fast period , and very prolonged slow period which correspond to the rest phase of hair growth , are consistent with the reduced annual hair growth in huancavelica residents . we compared the power spectra derived from heart rate variability with those obtained from the hydrogen isotope ratios in the hair , in the same subjects ( figure 6 ) . the low- and high - power frequency bands are of comparable length but , not surprisingly , the spectral power is different because of the different time scales heart rate variability in milliseconds , hydrogen isotope ratios in days . because of the complexity of the interactions between mercury and biologic rhythms as derived from hydrogen isotope ratios along the length of the hairs we used fractal analysis to illustrate the differences between those living in a mercury - polluted environment and a control ( figure 7 ) . these results are consistent with severe dysfunction of the ans evidenced also by the reduced annual hair growth in huancavelica residents . the most famous mercury poisoned character was the mad hatter , the one from the mad tea party in alice in wonderland . he was partner to the march hare , mad as only a march hare can be ( though the hare was not poisoned by mercury ) . however , the phrases mad as a hatter and mad as a march hare were in common use in 1837 , well before the publication of alice in wonderland in 1865 . hatters did go mad because felt used for making hats was cured by mercurious nitrate and the mercury vapors inhaled by hatters gave them the characteristic symptoms reported also by azogados ( quicksilver poisoned miners from huancavelica ) centuries earlier who noted the shakes , hallucinations , and psychosis as found in hatters centuries later . mercury was used in europe throughout medieval times for medicinal purposes or as mercury sulphide , an ingredient of the bright red ink used by scribes in monasteries . increased levels were found in medieval bones of those who had either employed it for the treatment of leprosy or syphilis , or with increased occupational exposure such as pharmacists . mercury is a neurotoxin but its damaging effects on humans are unpredictable and dependent , in part , on its chemical speciation and interactions with other metals in the environment and in the diet . organic mercury which is the primary source of mercury exposure is principally derived from the diet . huancavelica is listed amongst the ten most polluted places in the world and despite its listing , our measurement of mercury levels in the hair of current huancavelica residents was noticeably low ( table 1 ) and , clinically , huancavelica residents showed no signs of mercury poisoning as recorded in hatters and azogados centuries earlier . however , measurements of mercury levels in the hair are not good indicators of mercury 's toxicity . while levels of mercury can be accurately measured in the blood , the hair has no blood supply and the levels derived from hair represent metabolism over time and reflect the chronobiology of the individuals rather than the blood levels of this metal . in modern times , hair is also frequently washed with shampoos and dried with hair driers ; these manipulations can add or remove mercury and thus compound the difficulties of assaying the mercury levels in the hair . here we show that people living in a heavily mercury - polluted environment , in huancavelica , may have acceptable mercury levels , by international standards , in their hair and no overt clinical symptoms of intoxication . nevertheless , the effects of mercury exposure are evident from the reduced rate of hair growth and disturbed biologic rhythms in these people . taken together these findings give unequivocal evidence of the toxicity of mercury to the human autonomic nervous system the most vulnerable part of the nervous system to mercury 's damage , in modern huancavelica , peru . the high - frequency / low - frequency ( hf / lf ) ratios are measures of the control by the autonomic nervous system ( ans ) of biologic rhythms . we find that hg and cu both affected the power bands of the hf and lf . mercury does interact with other metals to produce its effect on biologic rhythms in huancavelica and such interactions might account for the unpredictable effects of hg on individual physiology in other parts of the world too ( figure 1 ) . the growth rate of human scalp hair is variable but on average ~16 cm / year . we suggest that the altered biologic rhythms in modern huancavelica residents may have contributed to this failure to renew the hair at an appropriate rate ( figure 2 ) . the standard deviation of the variance of the power spectra of the hydrogen isotope ratios along the length of the hairs between our controls from other parts of the world and that from huancavelica is shown in figure 3 . normal rhythmic oscillations in biology are marked by high variability ( large variances ) ; less variability degrades performance of the system . the reduction in variance we observed is yet another expression of the deranged biologic rhythms in our huancavelica subjects . the low - frequency peak of the power spectra reflects primarily the sympathetic nervous system 's control of autonomic variability . a decrease or absence of this peak in heart rate variability and in the power spectra derived from hydrogen isotope ratios in the hair in the same individuals is seen in ans diseases such as pure autonomic failure ( paf ) , manifested by widespread failure of sympathetic nervous system function . our identification of significant reduction in low - frequency power in the hydrogen isotope ratios in huancavelica subjects ( figure 4 ) further supports ans impairment in these individuals . long - term rhythms recurring at weekly , monthly , or yearly intervals are well known in animal and human physiology . the recurring annual fast periods reflect growth while the slow periods reflect the rest stages in hair growth . the shortened fast period rhythms in huancavelica residents ' hair ( figure 5 ) are in keeping with the reduced hair growth rate and further support the altered ans function ( and biologic rhythms ) in these people . to confirm our findings from hydrogen isotope derived biologic rhythms , we measured heart rate variability using 2 minutes of recording of cardiac action ( ~150 heart beats ) in the same subjects ( figure 6 ) . averaged power spectra showed comparable durations of low- and high - frequency bands and spectral power did not differ significantly . we have previously shown that spectra obtained from human heart rate variability were comparable to those derived from power spectra obtained from hydrogen isotope ratios in the hair and from growth lines from teeth . our results confirmed that biologic rhythms as gleaned from power spectral analysis reflected the pulsing of life , the varying rhtymicity of physiologic functions even when the ans has been affected by disease or mercury as in huancavelica residents . . they can show complex natural phenomena in graphic form such as ice crystals on a window pane and the intricate contours of mountains and ridges , and river branching . in biology they illustrate the 3/4 scaling of metabolism with body size , the complexity of heart rate control , of blood circulation , growth , development , and lifespan . here we used mandelbrot fractal sets to show the complex relationships between hg , its effects on the ans 's control of human biologic rhythms , and the variability of hydrogen isotope ratios along the length of the hair in our subjects ( figure 7 ) . the fractals derived from all huancavelica subjects clearly reflect derangement in biologic rhtymicity and the lack of variance which characterizes their exposure to hg , supporting our contention of continuing influence of this metal on human life in modern huancavelica . taken together our results show that survival in a heavily , mercury , polluted environment is possible without overt signs of mercury intoxications such as those found during mercury epidemics of the past or in miners during the spanish exploitation of peru 's mineral riches . nevertheless , careful analyses of the function of the most mercury vulnerable part of the nervous system gives evidence of continued effects in contemporaneous huancavelica residents . how can we reconcile the clear evidence of hg 's effects on biologic rhythms and ans function with the unexpected low levels of the metal in the hair of these same individuals and their apparent lack of overt signs of intoxication ? first , there is the fact that hg in hair is a poor indicator of its possible toxic effects . secondly , we propose a parallel between how human height and life expectancy increase in a short span of ~300 years after the industrial revolution . this well - documented phenomenon has been explained by several contributing factors such as technological advances and physiological adaptation . the history of mining in the high andes is replete with anecdotes of how technical advances in the extraction of ores improved miners ' survival and at the same time , in huancavelica , increased the yield of hg extraction . while the cause of the shortened lifespan in huancavelica miners remained a mystery for long , nevertheless , the miners themselves devised useful adaptations which allowed them to return to work after total incapacitation , extending their lifespan . just like in the early years of the industrial revolution in europe , improved health , better nutrition through obtaining the means of continuing to work , increased productivity of the mines and of the miners themselves , implied greater resilience , increase in longevity , and improved fertility in the high andes . human genetic potentials that could produce drastic changes in the relatively short span of 1215 generations in europeans during the industrial revolution may also have been operative in the mining towns of the high andes for more than 500 years . the andean miners had the opportunity to adapt to enormous environmental pollution and now their descendants tolerate , relatively , but not entirely , unscathed , the persistent hg in the huancavelica biosphere . nevertheless , our multilevel analyses clearly showed that even 21 generations is insufficient for complete adaptation to the horrendous mercury pollution . the microbiome , the symbiotic bacteria in the gut of the flies , profoundly affected their physiology . the molecular mechanisms that underlie the interactions of gut bacteria with their host are beginning to be unraveled in the flies . in humans since one route of entry of mercury in humans is through the gut , the microbiome might be the first to encounter the toxin and the bacteria adapt to increasing levels of ingested mercury . the survival of a functioning microbiome in the human gut like in the flies is , in turn , closely linked to the survival of the host . adaptation over a period of some 500 years to enormous environmental mercury loads in huancavelica may have ultimately been the results of bacterial ( microbiome ) adaptation to mercury . for this proposal , the nutritional and smoking habits of paternal grandparents could influence the health of their grandchildren and in australia the mercury sensitivity of paternal grandparents who had pink disease as infants , affected their grandchildren . a third possible explanation is found in the theory of tolerance to disease or to environmental stressors such as toxins . this theory has the most support from infectious diseases , where tolerance to bacterial invaders has been shown to offer evolutionary advantages by avoiding the costly means to eliminate the infectious agents . in this setting , tolerance to invaders offers a defense strategy that favors survival in heavily contaminated surroundings . similarly , cellular responses to stress can offer survival advantages in stressful environments such as the hypoxia of altitude or excessive heat by inducing reactive oxygen species ( ros ) or activating numerous heat - shock proteins , respectively . resistance and tolerance are thought to be complimentary strategies for living also in heavily polluted environments such as in huancavelica . analyzing the molecular pathways which allow relatively normal survival in a heavily mercury , polluted environment after five hundred years of mercury exposure may offer insights into strategies of coping with the climate and environmental changes wrought by the anthropocene , the man - made world , we now live in . the ethics committee of the new mexico health enhancement and marathon clinics ( nmhemc ) research foundation reviewed and approved the study . informed written consent was obtained from all participants . this was a field study , and as such suffers from limitations common to such studies such as small number of subjects , poor adherence to study protocols , and low number of controls . nevertheless , wherever statistical analyses were possible these were performed and gave clear and meaningful results . microfocused synchrotron x - ray fluorescence analyses were performed at beamline x26a at the national synchrotron light source , brookhaven national laboratory , upton , ny , usa . beamline x26a utilizes a bending magnet source on the nsls 2.8 gev electron storage ring , which operates at a current of 300 ma . for these experiments a monochromatic x - ray beam was used , monochromatized using a si(111 ) channel - cut monochromator and tuned to an incident beam energy of 12.5 kev . the beam was focused to a spot size of 5 m in the vertical x 8 m in the horizontal using a pair of dynamically bent , grazing - incidence mirrors , each 100 mm long , arranged in a kirkpatrick - baez ( kb ) geometry . x - ray fluorescence from the human hairs was measured using a combined set of three energy dispersive detectors ; a canberra 9-element hpge array detector placed 90 to the incident beam within the plane of the storage ring to minimize backgrounds from compton scattering . the other two detectors are single element radiant vortex - ex silicon drift diode detectors also at 90 to the incident beam , but each sitting 45 above and below the plane of the storage ring , respectively . the 9-element hpge array provides an active area of 900 mm and each silicon drift diode detector has an active area of 50 mm . all eleven detector elements are integrated simultaneously using the xmap series of compact pci - based digital spectrometers produced by x - ray instrumentation associates ( xia ) interfaced through epics and controlled through in - house client software written in idl . incident beam intensity was monitored using an ion - chamber upstream of the focusing optics and all images were corrected for changes in incident beam flux through normalization to the change in ion - chamber counts over time . hydrogen isotope ratios were determined using the continuous - flow high - temperature - reduction technique . hair was wrapped in silver foil and placed into the combustion chamber of a mass spectrometer using a carlo erba as 200-ls autosampler . the stable isotopic compositions of low - mass elements such as hydrogen are reported as delta ( ) values in parts per thousand ( ) . values are calculated as follows : in ( ) = ( r sample / r standard 1 ) 1000 , where r is the ratio of the heavy to the light isotope in the sample . the stable isotope standard for hydrogen is reported relative to the standard mean ocean water ( smow ) . isotope composition is reported in relation to this standard which has been defined as 0 . approximately 150 rr intervals were measured for each subject . the statistical analysis was similar to that used for the hydrogen isotope power spectra analysis ( see below ) and the frequency bands were identical to those chosen for the hydrogen isotope bands of the power spectra derived from the hair of the same subject . data derived from each hair were entered into the mandelbrot set - online generator by dawid makiela ( http://www.mandelbrot.ovh.org/ ) . x 1 : mercury content ; y1 : low - frequency / high - frequency ratio of power spectrum ; x2 : elemental content ( cu ) ; y2 : total power of low , mid- and high frequencies ; x : total low power ; y : total high power ; r : sum of low , mid , and high frequencies of the power spectrum obtained from the hydrogen isotope ratios along the length of each hair . maximum number of iterations was 100 . to strengthen our conclusions about biologic rhythms derived from the hair in huancavelica residents we used an aged control from the usa . the variance of biologic rhythms decreases with advancing age thus an old control subject , us resident , should serve better as a standard for comparison to the biologic rhythms derived from the hair of young mercury exposed subjects from huancavelica . in order to determine the relationship between metals in hair and the hydrogen isotope spectra in hair , we computed pearson and spearman correlations of each metal with measures of the spectra . the log - transformed quantities of hg and cu were found to be related to the spectral power in the high - frequency band we identify two clusters of high - frequency power ( low and high power ) in the hg - cu scatter plot of the 5 huancavelica subjects ( see figure 1 ) . the fact that periodicity of this sinusoid is 52 weeks allows us to compute annual growth rates in centimeters . fitting the annual sinusoid for huancavelica subject # 3 by nonlinear regression yielded the function of length along the hair in cm : ( 1)predicted dd=91.1315 + 1.5832sin ( .8850cm+1.3243 ) . one can use the frequency of the fitted sinusoid to estimate the growth rate that matches the 0.0193 cycles / week = 1 cycle/52 weeks , whose periodicity is 52 weeks . so ( freq/2)*(growth / year)/(52 weeks / year ) set equal to 1 cycle/52 weeks implies the following : ( 2)growthyear=2(frequency of fitted annual sinusoid ) . here the average growth rate for the 5 huancavelica subjects was 6.4 1.7 sd cm / year . this is compared to the average growth rate of ~16 cm / year in human hair by a one - sample t - test ( see figure 2 ) . all power spectra were computed using the finite fourier transform , decomposes time series into a sum of sine and cosine waves of different amplitudes and wavelengths ( proc spectra from sas ) . the spectral variance for a given time series of hydrogen isotope ratios in hair is the total power ( area under the periodogram ) . the comparison of these standard deviations ( square root of the variance ) for the 5 huancavelica subjects to our sample of controls are made by a two - sample t - test ( see figure 3 ) . the spectral power in the low - frequency band and their standard errors are computed as described in priestly . these are then compared by t - test ( see figure 4 ) . fast and slow periods of hair growth in weekly cycles is computed from the frequencies of higher power observed in the periodogram . the form of the calculation is as follows : ( 3)period = dt(freq)(growth ) , where dt is the increment of the series ( see figure 5 ) . for figure 6 , the power spectra for both heart rate interval ( rr ) and hydrogen isotope ratio time series measured in the same 5 huancavelica subjects ( the 5 series

double - orifice mitral valve ( domv ) is a very rare congenital anomaly that usually presents as mitral regurgitation . we present the case of a 39-year - old asymptomatic , healthy man with no previous medical history who was affected by isolated complete bridge type domv , incidentally detected by two - dimensional echocardiographic examination in the parasternal short - axis view . the mitral valve of the patient was normally functioning without any other coexistent cardiac abnormalities . isolated domv was also confirmed by cardiac magnetic resonance imaging . the patient is now followed up to detect possible complications . mercury is added to the biosphere by anthropogenic activities raising the question of whether changes in the human chromatin , induced by mercury , in a parental generation could allow adaptation of their descendants to mercury . we review the history of andean mining since pre - hispanic times in huancavelica , peru . despite the persistent degradation of the biosphere today , no overt signs of mercury toxicity could be discerned in present day inhabitants . however , mercury is especially toxic to the autonomic nervous system ( ans ) . we , therefore , tested ans function and biologic rhythms , under the control of the ans , in 5 huancavelicans and examined the metal content in their hair . mercury levels varied from none to 1.014 ppm , significantly less than accepted standards . this was confirmed by microfocused synchrotron x - ray fluorescence analysis . biologic rhythms were abnormal and hair growth rate per year , also under ans control , was reduced ( p < 0.001 ) . thus , evidence of mercury 's toxicity in ans function was found without other signs of intoxication . our findings are consistent with the hypothesis of partial transgenerational inheritance of tolerance to mercury in huancavelica , peru . this would generally benefit survival in the anthropocene , the man - made world , we now live in .

in the present issue of critical care , linder and colleagues present a new study in which they assess the clinical importance of serial measurements of heparin - binding protein ( hbp ) plasma levels in critically ill septic and nonseptic patients . they found that hbp plasma levels are significantly higher in patients with severe sepsis and septic shock in comparison with patients with a nonseptic critical condition . the authors also demonstrated that hbp plasma levels obtained at admission to the icu and during the last individual sampling are higher in nonsurvivors as compared with survivors in both the septic group and the whole study group . moreover , the high baseline hbp plasma levels in septic patients were associated with an increased 28-day mortality rate . altogether , these results indicate that serial hbp measurements might be very helpful in stratification of icu patients . however , there are some issues that should be raised before the study results can be translated into the daily routine . the study was designed to compare hbp plasma levels in patients with severe sepsis and septic shock with levels in patients with noninfectious critical illness . however , these two groups of patients were not equal in size . also , a significant proportion of patients from the nonseptic group developed infection and was treated with antibiotics . this raises the question of whether the comparison between septic and nonseptic icu patients is hindered by this treatment . nevertheless , the study design is logical because finding a biomarker that would predict development of any shock state is highly desirable . previous studies have demonstrated significant predictive values of elevated levels of lactate , cortisol and il-6 in the blood of patients with different etiologies of shock [ 2 - 4 ] . however , these biomarkers have some limitations : lactate levels are less influenced by arterial sampling , endogenous cortisol levels are downregulated by corticosteroids used in the treatment of septic shock or by relative adrenal insufficiency , and il-6 analysis is not generally available in regular hospital laboratories . other routinely measured biomarkers - such as procalcitonin , c - reactive protein , neutrophil and lymphocyte counts - have only a limited value in prognostic scoring of the critically ill patients and are mostly used in the early diagnostics of bacterial etiology of critical illness [ 6 - 8 ] . notably , hbp has antibacterial activity , which includes a direct microbicidal effect , and also helps neutrophils to migrate into the focus of infection . similarly to hbp , c - reactive protein and il-6 play an active role during the immune responses against infections : c - reactive protein is an inflammation opsonin , and the major function of il-6 is amplification as well as downregulation of inflammatory reactions , depending on the concentrations . regarding procalcitonin , there are only limited data from animal studies - which demonstrate that immunoneutralization of procalcitonin improved survival in experimental porcine sepsis . additionally , elevated cortisol levels in peripheral blood during sepsis are considered an integral part of compensatory anti - inflammatory response syndrome , leading to downregulation of exaggerated systemic immune responses . from the functional point of view , in comparison with the abovementioned biomarkers , hbp therefore plays the most complex role in severe sepsis and septic shock - highlighting its potential for clinical use . in conclusion , the serial measurements of hbp plasma levels can be a useful tool for close monitoring of critically ill septic patients . however , availability of a routine diagnostic method for hbp analysis is essential to confirm these interesting data . the authors thank dr e david mcintosh ( imperial college , london , uk ) for help with the manuscript . the information gathered during pre - marketing drug development is inevitably incomplete with regard to possible adverse reactions . this is mainly due to : animal testing is insufficient to predict human safety;patients enrolled in clinical trials are selected and limited in number;the conditions of use are different from those in clinical practice;the period of testing is limited . animal testing is insufficient to predict human safety ; patients enrolled in clinical trials are selected and limited in number ; the conditions of use are different from those in clinical practice ; the period of testing is limited . clinical trials often lack important information about rare but serious adverse reactions , chronic toxicity , use in special groups ( such as children , women , elderly or pregnant ) or interactions with other drugs . therefore , post - marketing surveillance activities are important to allow the early detection of unexpected and/or serious adverse reactions . it is estimated that only 6 - 10% of all adrs are reported : this underestimation is a major problem . although the extent of under - reporting is widely variable depending on the estimates , it is certain that the number of reported adrs represents only a small percentage of the total number of occurring adrs . adrs have a great impact on public health and represent a significant economic burden on both healthcare systems and society . in fact , it is estimated that in the united states , the total cost of adrs may be comparable to that of diabetes , as well as it represents the fourth leading cause of death by disease . in the european union ( eu ) , it is estimated that 5% of all hospital admissions are due to adrs , which are the fifth leading cause of hospital death : approximately 197,000 deaths per year in the eu are caused by adrs , and the total cost to the society of adrs in the eu is about 79 billion . based on these alarming data , on july 21 , 2012 , a new eu pharmacovigilance legislation came into force in order to strengthen and rationalize the eu pharmacovigilance system , with the overall objectives of better protection of public health , ensuring proper functioning of the internal market and simplifying the existing procedures . pharmacovigilance was officially born in december 1961 with the publication of a letter ( case report ) in the lancet by w. mcbride , the australian doctor who first suspected a causal link between serious fetal deformities ( phocomelia ) and thalidomide drug used during pregnancy : thalidomide was used as an antiemetic and sedative agent in pregnant women . in 1968 , programme for international drug monitoring , a pilot project aimed to the centralization of world data on adrs . in particular , the main aim of the who programme is to identify the earliest possible pharmacovigilance signals . as of april 2013 , 112 countries have joined the who programme for international drug monitoring , and in addition 32 countries participating in the who programme for international drug monitoring , with year of joining * all member countries contribute sending at least quarterly individual case safety reports ( icsrs ) to the who global icsr database system , named vigibase . it is updated with incoming icsrs on a continuous basis . the who worldwide database of adrs is located in uppsala in sweden , and it is developed and maintained by the uppsala monitoring center for pharmacovigilance ( umc ) on behalf of the who.by april 2013 over 8 million reports were contained in the database . the term pharmacovigilance was proposed in the mid-70s by a french group of pharmacologists and toxicologists to define the activities promoting the assessment of the risk of side effects potentially associated with drug treatment . pharmacovigilance is defined by the who as the science and activities relating to the detection , assessment , understanding and prevention of adverse effects , or any other problem in the field of medicine . the monitoring of spontaneous suspected adrs reports represents the key component of the integrated systems of pharmacovigilance . pharmacovigilance has four main objectives : recognize , as quickly as possible , new adrs;improve and increase the information about already known or suspected adrs;assess the benefits of a drug compared to other ones or other therapies;communicate the information in order to improve the therapeutic practice . recognize , as quickly as possible , new adrs ; improve and increase the information about already known or suspected adrs ; assess the benefits of a drug compared to other ones or other therapies ; communicate the information in order to improve the therapeutic practice . the main aim of pharmacovigilance is to provide an alarm signal through the early detection of new adrs . in pharmacovigilance a signal of suspected causality is defined as follows : information that arises from one or multiple sources , including observations and experiments , which suggests a new potentially causal association , or a new aspect of a known association , between an intervention and an event or set of related events , either adverse or beneficial , which would command regulatory , societal or clinical attention , and is judged to be of sufficient likelihood to justify verifiable and , when necessary , remedial actions . ad hoc studies will be performed to test this hypothesis and possibly provide additional information , not achievable through spontaneous reporting systems , such as the incidence and relative risk for evaluated adrs . . the main limitations of spontaneous reporting are related to under - reporting , variable quality of the reported data and lack of information on drug exposure . there are differences between countries ( and also between regions within countries ) in the occurrence of adrs and other drug - related problems . this may be due to many factors such as : disease and prescribing practices;genetics , diet , traditions of the communities;processes of drugs production influencing the quality of the pharmaceutical composition;drugs distribution and use including indications , doses and availability;the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . disease and prescribing practices ; genetics , diet , traditions of the communities ; processes of drugs production influencing the quality of the pharmaceutical composition ; drugs distribution and use including indications , doses and availability ; the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . according to the circumstances , the information obtained in a country ( for example , the country of origin of the drug ) may not be relevant in other ones . at european level , government agencies responsible for pharmacovigilance in member states are in contact with each other and with the european medicines agency ( ema ) . ema has established a web - based european network ( eudravigilance ) for reporting andexchangingsuspected adrs reports , during the development ( pre - authorization phase ) and following the marketing a - authorization ( post - authorization phase ) of medicinal products in the european economic area . eudravigilance is an european database , which supports : the early detection of possible safety signals associated with medicinal products for human use.the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions.the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . the early detection of possible safety signals associated with medicinal products for human use . the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions . the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . in the early 80 's , the italian rules on the safety of marketed drugs identified the manufacturers as responsible for the communication to the ministry of health about possible drug - related adverse effects . in fact , in 1980 , in italy , the marketing - authorization holders were required to transmit to the ministry of health a periodical report in which they must specify the nature and frequency of any toxic effects and secondary consequences or correlated with drugs use . according to the ministerial decree ( dm ) of june 23 , 1981 , ( article 8) and the dm of july 28 , 1984 , the data collection forms on drugs use doctors could also decide to send the form directly to the ministry of health . in 1987 , two major changes occurred : the term pharmacovigilance appeared and the local health units were involved . since then , the doctors were required to notify the local health units , which , in turn , communicated to the ministry information about the most serious cases and deaths . in 1997 , the national system of pharmacovigilance was established . by the legislative decree of april 8 , 2003 , n. 95 was clarified that the detectors can be doctors and healthcare professionals , who may report suspected serious or unexpected adverse reactions to vaccines and drugs undergoing intensive monitoring . in december 2003 , a new pharmacovigilance form was approved and is still currently in use [ form 1 ] . with the legislative decree no . 129 - 134 regions began to provide , within its competences , the dissemination of information to healthcare personnel and the training of operators in the field of pharmacovigilance . the healthcaresettings must nominate a qualified person responsible for pharmacovigilance ( qppv ) for adrs reports management . physicians and other healthcare professionals are required to report all suspected serious and/or unexpected adrs , which they learn as part of their activity . they are in duty bound to report all suspected adverse reactions , serious , not serious , expected and unexpected from all vaccines and medicines under intensive monitoring . all healthcare professionals are required to send reports of suspected adverse reactions by means of the appropriate form , promptly , to qppv . upon verifying completeness and consistency of the reporting form data , the rafv must store the report , no later than seven days from the date of receipt , in the database of the national pharmacovigilance network . the national pharmacovigilance system is headed by the italian medicines agency ( agenzia italiana del farmaco , aifa ) , acting in accordance with the rules laid down at eu level by the ema . it is a public body that operates autonomously , transparently and according to cost - effectiveness criteria , under the direction of the ministry of health and under the supervision of the ministry of health and the ministry of economy . aifa collaborates with the regions , the national institute of health , research institutes , patients associations , healthcare professionals , scientific societies , the pharmaceutical industry and the distributors . specifically , the agency : guarantees access to the drugs and their safe and appropriate use in order to protect public health;provides an unified national pharmaceutical system in agreement with the regions;provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry;ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases;strengthens the relationships with the agencies of other member countries , with the ema and other international organizations;encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation;interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors;promotes pharmaceutical knowledge and culture . guarantees access to the drugs and their safe and appropriate use in order to protect public health ; provides an unified national pharmaceutical system in agreement with the regions ; provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry ; ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases ; strengthens the relationships with the agencies of other member countries , with the ema and other international organizations ; encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation ; interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors ; promotes pharmaceutical knowledge and culture . the mission of the aifa in pharmacovigilance is to ensure a positive risk / benefit ratio for all authorized drugs through the continuous monitoring of all safety information and adrs . these data arise from different sources : spontaneous reports of suspected adrs , clinical trials , scientific literature , reports submitted by the pharmaceutical companies , and other sources . in particular , all data of spontaneous adrs reporting are managed and coordinated through the national network of pharmacovigilance ( rete nazionale di farmacovigilanza , rnf ) , a database that allows the collection , management and analysis of spontaneous reports of suspected adrs . it is active since november 2001 . the rnfisan aifa - managed , extensive network throughout the national territory that includes the regional authorities and the autonomous provinces of trento and bolzano , the regional centers of pharmacovigilance , 204 local health authorities , 112 hospitals , 43 research institutes , more than eight hundreds pharmaceutical companies and obviously aifa . through this network , periodicallyt he rnf data are transmitted to eudra vigilance database ( european database of suspected adrs reports ) , managed by the ema . eudravigilance collects all data provided at national level by the eu countries in a single database , which , in turn , communicates with vigibase , the who global icsr database . in december 2010 , european legislation on pharmacovigilance was changed with the adoption of the eu regulation 1235/2010 and directive 2010/84/eu amending , as regards pharmacovigilance , regulation ( ec ) 726/2004 and directive 2001/83/ec , respectively . this new legislation was the biggest change to the regulation of human medicines in the eu since 1995 . ema is responsible for implementing much of the new legislation , which has been effective since july 2012 . in october 2012 , the eu 's new pharmacovigilance legislation was further amended by : regulation ( eu ) no 1027/2012 ( applies from 5 june 2013);directive 2012/26/eu ( applies from 28 october 2013 ) . regulation ( eu ) no 1027/2012 ( applies from 5 june 2013 ) ; directive 2012/26/eu ( applies from 28 october 2013 ) . new legislation will greatly improve public - health protection and , ultimately , save lives by enhancing the overall efficiency and effectiveness of the pharmacovigilance system . basically , changes tend to increase the efficiency , speed and transparency of pharmacovigilance tasks through rules that aim to : strengthening pharmacovigilance systems ( more clearly defined roles and responsibilitiesof the many actors involved);streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort;increasing engagement of the patients and healthcare professionals;improving the communication systems of decision - making processes , giving proper justification to the public;increasing transparency . strengthening pharmacovigilance systems ( more clearly defined roles and responsibilities of the many actors involved ) ; streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort ; increasing engagement of the patients and healthcare professionals ; improving the communication systems of decision - making processes , giving proper justification to the public ; increasing transparency . first of all , the definition of adverse reaction has been changed and broadened to ensure that it now covers noxious and unintended effects resulting not only from the authorized use of a medicine at normal doses , but also from medication errors and uses outside the terms of the marketing authorization ( off - label use ) , including overdose , misuse and abuse of the product , and from occupational exposure . as a consequence , therefore , there will be an increase of reports and resulting a greater monitoring activity . in all eu countries , the patients will be able to report directly suspected adverse reactions . in italy , this possibility has already been considered since some years back by a paper form , however , from now on , also in accordance with the new directive , reports may also be sent by the web . all reports of adverse reactions will merge into the european database eudravigilance , but with different timing depending on the severity of the reaction ( within 15 days for serious reports and within 90 days for non - serious ones ) . reports from pharmaceutical companies will also be transmitted to the eudravigilance database . regarding some medicinal products authorized through the centralized procedure , it is already possible to consult the european database of adverse reactions at the following website http://www.adrreports.eu/. in summary , directive 2010/84/eu and regulation ( eu ) 1235/2010 aim to maintain , reinforce and further develop the eudravigilance database as the single or at least the main- point of receipt , storage and exchange of information concerning the safety of medicinal products for human use authorized in the eu . the data collected in the eudravigilance database will be monitored by the ema in cooperation with the member states , while monitoring of the data originated at the national level will be carried out by the member state involved ; these activities are aimed at the identification of changes in risk or new risks through the signals analysis . the methodology for the identification and management process of the signal were defined in the implementing regulation ( eu ) n. 520/2012 of 19 june 2012 , concerning the performance of pharmacovigilance activities provided for in regulation ( ec ) n.726/2004 of the european parliament and of the council and directive 2001/83/ec of the european parliament and of the council . as afore mentioned , the new legislation aims to guarantee greater transparency and improve communication . in fact , on its web portal , aifa makes available to the public at least the following information : public assessment reports , together with a summary there of;summaries of product characteristics and package leaflets;summaries of risk management plans;list of medicinal products that are subject to additional monitoring;information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . public assessment reports , together with a summary there of ; summaries of product characteristics and package leaflets ; summaries of risk management plans ; list of medicinal products that are subject to additional monitoring ; information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . medicines subject to additional monitoring will be the products containing new active substances not included in the authorized medicines in the eu before the january 1 2011 ; in particular , biologics and biosimilars , but the list may also include products whose license is subject to particular conditions or products authorized in exceptional circumstances ; e.g. , products subject to safety studies after the granting of the marketing authorization ( post - authorisation safety studies ) . these medicinal products subject to additional monitoring will be identifiable from the package leaflet that will bear the statement this medicinal product is subject to additional monitoring . this statement will be preceded by a black symbol and followed by an explanatory sentence explaining the concept of additional monitoring . the additional monitoring list will be regularly kept up to date by the ema in collaboration with member states . the new legislation has empowered the relevant authorities to impose on the marketing authorization holders the obligation to perform post - authorization safety and/or efficacy studies . these studies would be required at the time of the granting of the marketing authorization or later . according to another key point of the 2010 reform , each member state and each marketing authorization holder is obliged to adopt a pharmacovigilance system with the aim of analyzing suspected adrs and implementing provisions in order to prevent them . at last , the new eu pharmacovigilance legislation has established within the ema a new scientific committee , the pharmacovigilance risk assessment committee ( prac ) in which all member states are represented . the role of the prac in protecting public health requires close interaction with the agency 's committee for medicinal products for human use ( chmp ) and the coordination group for mutual recognition and decentralized procedures - human ( cmdh ) . its members include experts from regulatory authorities in eu member states and independent scientific experts who provide additional expertise in particular scientific areas . members from the patient organizations and healthcare professionals will be included in the near future further to appointment from the european commission . in its role in monitoring the safety of human medicines , the prac assesses all aspects of risk management , including the detection , assessment , minimization and communication of the risk of adrs , giving due consideration to the benefits of the medicine . the prac is also involved in designing and evaluating post - authorization safety studies and in conducting pharmacovigilance audits . when the prac has concluded an evaluation related to the safety of medicines , it prepares a recommendation that is sent either to the chmp ( if it concerns at least one centrally authorized medicine ) or to the cmdh ( if it only concerns medicines that are not centrally authorized in the eu ) . the regional authorities are responsible for the development and promotion of active pharmacovigilance programs , which agreed with aifa and subsequently , allow the regions to access to funds . in this context , aifa has promoted several projects and studies in italy , with the aim of increasing knowledge on drugs and the culture of pharmacovigilance the funding of pharmacovigilance projects by aifa has contributed to the improvement of spontaneous reporting . in italy , the situation of spontaneous adrs reports in 2010 showed an increase of 39% in comparison to 2009 . this result is in line with the average annual increase of 30% observed in the last five years , and it also demonstrates the crucial role of active pharmacovigilance programs , funded by aifa , in increasing knowledge on drugs , to better define their safety profile and to improve their safe use in clinical practice . pharmacovigilance was officially born in december 1961 with the publication of a letter ( case report ) in the lancet by w. mcbride , the australian doctor who first suspected a causal link between serious fetal deformities ( phocomelia ) and thalidomide drug used during pregnancy : thalidomide was used as an antiemetic and sedative agent in pregnant women . in 1968 , the world health organization ( who ) promoted the programme for international drug monitoring , a pilot project aimed to the centralization of world data on adrs . in particular , the main aim of the who programme is to identify the earliest possible pharmacovigilance signals . as of april 2013 , 112 countries have joined the who programme for international drug monitoring , and in addition 32 countries participating in the who programme for international drug monitoring , with year of joining * all member countries contribute sending at least quarterly individual case safety reports ( icsrs ) to the who global icsr database system , named vigibase . it is updated with incoming icsrs on a continuous basis . the who worldwide database of adrs is located in uppsala in sweden , and it is developed and maintained by the uppsala monitoring center for pharmacovigilance ( umc ) on behalf of the who.by april 2013 over 8 million reports were contained in the database . the term pharmacovigilance was proposed in the mid-70s by a french group of pharmacologists and toxicologists to define the activities promoting the assessment of the risk of side effects potentially associated with drug treatment . pharmacovigilance is defined by the who as the science and activities relating to the detection , assessment , understanding and prevention of adverse effects , or any other problem in the field of medicine . the monitoring of spontaneous suspected adrs reports represents the key component of the integrated systems of pharmacovigilance . pharmacovigilance has four main objectives : recognize , as quickly as possible , new adrs;improve and increase the information about already known or suspected adrs;assess the benefits of a drug compared to other ones or other therapies;communicate the information in order to improve the therapeutic practice . recognize , as quickly as possible , new adrs ; improve and increase the information about already known or suspected adrs ; assess the benefits of a drug compared to other ones or other therapies ; communicate the information in order to improve the therapeutic practice . the main aim of pharmacovigilance is to provide an alarm signal through the early detection of new adrs . in pharmacovigilance a signal of suspected causality is defined as follows : information that arises from one or multiple sources , including observations and experiments , which suggests a new potentially causal association , or a new aspect of a known association , between an intervention and an event or set of related events , either adverse or beneficial , which would command regulatory , societal or clinical attention , and is judged to be of sufficient likelihood to justify verifiable and , when necessary , remedial actions . following signal , subsequent ad hoc studies will be performed to test this hypothesis and possibly provide additional information , not achievable through spontaneous reporting systems , such as the incidence and relative risk for evaluated adrs . . the main limitations of spontaneous reporting are related to under - reporting , variable quality of the reported data and lack of information on drug exposure . there are differences between countries ( and also between regions within countries ) in the occurrence of adrs and other drug - related problems . this may be due to many factors such as : disease and prescribing practices;genetics , diet , traditions of the communities;processes of drugs production influencing the quality of the pharmaceutical composition;drugs distribution and use including indications , doses and availability;the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . disease and prescribing practices ; genetics , diet , traditions of the communities ; processes of drugs production influencing the quality of the pharmaceutical composition ; drugs distribution and use including indications , doses and availability ; the use of traditional medicines and supplements ( for example , herbal remedies ) , which may constitute specific toxicological problems . the data resulting from the whole country or single region may have greater relevance and educational value and may affect the national regulations . according to the circumstances , the information obtained in a country ( for example , the country of origin of the drug ) may not be relevant in other ones . at european level , government agencies responsible for pharmacovigilance in member states are in contact with each other and with the european medicines agency ( ema ) . ema has established a web - based european network ( eudravigilance ) for reporting andexchangingsuspected adrs reports , during the development ( pre - authorization phase ) and following the marketing a - authorization ( post - authorization phase ) of medicinal products in the european economic area . eudravigilance is an european database , which supports : the early detection of possible safety signals associated with medicinal products for human use.the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions.the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . the early detection of possible safety signals associated with medicinal products for human use . the continual monitoring and evaluation of potential safety issues in relation to reported adverse reactions . the decision making process , based on a broader knowledge of the adverse reaction profile of medicinal products . in the early 80 's , the italian rules on the safety of marketed drugs identified the manufacturers as responsible for the communication to the ministry of health about possible drug - related adverse effects . in fact , in 1980 , in italy , the marketing - authorization holders were required to transmit to the ministry of health a periodical report in which they must specify the nature and frequency of any toxic effects and secondary consequences or correlated with drugs use . according to the ministerial decree ( dm ) of june 23 , 1981 , ( article 8) and the dm of july 28 , 1984 , the data collection forms on drugs use doctors could also decide to send the form directly to the ministry of health . in 1987 , two major changes occurred : the term pharmacovigilance appeared and the local health units were involved . since then , the doctors were required to notify the local health units , which , in turn , communicated to the ministry information about the most serious cases and deaths . in 1997 , the national system of pharmacovigilance was established . by the legislative decree of april 8 , 2003 , n. 95 was clarified that the detectors can be doctors and healthcare professionals , who may report suspected serious or unexpected adverse reactions to vaccines and drugs undergoing intensive monitoring . in december 2003 , a new pharmacovigilance form was approved and is still currently in use [ form 1 ] . with the legislative decree no . 129 - 134 regions began to provide , within its competences , the dissemination of information to healthcare personnel and the training of operators in the field of pharmacovigilance . the healthcaresettings must nominate a qualified person responsible for pharmacovigilance ( qppv ) for adrs reports management . physicians and other healthcare professionals are required to report all suspected serious and/or unexpected adrs , which they learn as part of their activity . they are in duty bound to report all suspected adverse reactions , serious , not serious , expected and unexpected from all vaccines and medicines under intensive monitoring . all healthcare professionals are required to send reports of suspected adverse reactions by means of the appropriate form , promptly , to qppv . upon verifying completeness and consistency of the reporting form data , the rafv must store the report , no later than seven days from the date of receipt , in the database of the national pharmacovigilance network . the national pharmacovigilance system is headed by the italian medicines agency ( agenzia italiana del farmaco , aifa ) , acting in accordance with the rules laid down at eu level by the ema . it is a public body that operates autonomously , transparently and according to cost - effectiveness criteria , under the direction of the ministry of health and under the supervision of the ministry of health and the ministry of economy . aifa collaborates with the regions , the national institute of health , research institutes , patients associations , healthcare professionals , scientific societies , the pharmaceutical industry and the distributors . specifically , the agency : guarantees access to the drugs and their safe and appropriate use in order to protect public health;provides an unified national pharmaceutical system in agreement with the regions;provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry;ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases;strengthens the relationships with the agencies of other member countries , with the ema and other international organizations;encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation;interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors;promotes pharmaceutical knowledge and culture . guarantees access to the drugs and their safe and appropriate use in order to protect public health ; provides an unified national pharmaceutical system in agreement with the regions ; provides pharmaceutical expenditure governance in the context of economic and financial compatibility and competitiveness of the pharmaceutical industry ; ensures innovation , efficiency and simplification of the marketing authorization procedures , in order to allow rapid access to innovative drugs and drugs used for rare diseases ; strengthens the relationships with the agencies of other member countries , with the ema and other international organizations ; encourages and rewards investments in research and development ( r and d ) in italy , promoting and rewarding innovation ; interacts with the community of patients associations , the medical / scientific world , pharmaceutical companies and distributors ; promotes pharmaceutical knowledge and culture . the mission of the aifa in pharmacovigilance is to ensure a positive risk / benefit ratio for all authorized drugs through the continuous monitoring of all safety information and adrs . these data arise from different sources : spontaneous reports of suspected adrs , clinical trials , scientific literature , reports submitted by the pharmaceutical companies , and other sources . in particular , all data of spontaneous adrs reporting are managed and coordinated through the national network of pharmacovigilance ( rete nazionale di farmacovigilanza , rnf ) , a database that allows the collection , management and analysis of spontaneous reports of suspected adrs . it is active since november 2001 . the rnfisan aifa - managed , extensive network throughout the national territory that includes the regional authorities and the autonomous provinces of trento and bolzano , the regional centers of pharmacovigilance , 204 local health authorities , 112 hospitals , 43 research institutes , more than eight hundreds pharmaceutical companies and obviously aifa . through this network , periodicallyt he rnf data are transmitted to eudra vigilance database ( european database of suspected adrs reports ) , managed by the ema . eudravigilance collects all data provided at national level by the eu countries in a single database , which , in turn , communicates with vigibase , the who global icsr database . in december 2010 , european legislation on pharmacovigilance was changed with the adoption of the eu regulation 1235/2010 and directive 2010/84/eu amending , as regards pharmacovigilance , regulation ( ec ) 726/2004 and directive 2001/83/ec , respectively . this new legislation was the biggest change to the regulation of human medicines in the eu since 1995 . ema is responsible for implementing much of the new legislation , which has been effective since july 2012 . in october 2012 , the eu 's new pharmacovigilance legislation was further amended by : regulation ( eu ) no 1027/2012 ( applies from 5 june 2013);directive 2012/26/eu ( applies from 28 october 2013 ) . regulation ( eu ) no 1027/2012 ( applies from 5 june 2013 ) ; directive 2012/26/eu ( applies from 28 october 2013 ) . new legislation will greatly improve public - health protection and , ultimately , save lives by enhancing the overall efficiency and effectiveness of the pharmacovigilance system . basically , changes tend to increase the efficiency , speed and transparency of pharmacovigilance tasks through rules that aim to : strengthening pharmacovigilance systems ( more clearly defined roles and responsibilitiesof the many actors involved);streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort;increasing engagement of the patients and healthcare professionals;improving the communication systems of decision - making processes , giving proper justification to the public;increasing transparency . strengthening pharmacovigilance systems ( more clearly defined roles and responsibilities of the many actors involved ) ; streamline the activities among the member states ( distribution of tasks and work sharing , with less duplication of effort ; increasing engagement of the patients and healthcare professionals ; improving the communication systems of decision - making processes , giving proper justification to the public ; increasing transparency . first of all , the definition of adverse reaction has been changed and broadened to ensure that it now covers noxious and unintended effects resulting not only from the authorized use of a medicine at normal doses , but also from medication errors and uses outside the terms of the marketing authorization ( off - label use ) , including overdose , misuse and abuse of the product , and from occupational exposure . as a consequence , therefore , there will be an increase of reports and resulting a greater monitoring activity . in all eu countries , the patients will be able to report directly suspected adverse reactions . in italy , this possibility has already been considered since some years back by a paper form , however , from now on , also in accordance with the new directive , reports may also be sent by the web . all reports of adverse reactions will merge into the european database eudravigilance , but with different timing depending on the severity of the reaction ( within 15 days for serious reports and within 90 days for non - serious ones ) . reports from pharmaceutical companies will also be transmitted to the eudravigilance database . regarding some medicinal products authorized through the centralized procedure , it is already possible to consult the european database of adverse reactions at the following website http://www.adrreports.eu/. in summary , directive 2010/84/eu and regulation ( eu ) 1235/2010 aim to maintain , reinforce and further develop the eudravigilance database as the single or at least the main- point of receipt , storage and exchange of information concerning the safety of medicinal products for human use authorized in the eu . the data collected in the eudravigilance database will be monitored by the ema in cooperation with the member states , while monitoring of the data originated at the national level will be carried out by the member state involved ; these activities are aimed at the identification of changes in risk or new risks through the signals analysis . the methodology for the identification and management process of the signal were defined in the implementing regulation ( eu ) n. 520/2012 of 19 june 2012 , concerning the performance of pharmacovigilance activities provided for in regulation ( ec ) n.726/2004 of the european parliament and of the council and directive 2001/83/ec of the european parliament and of the council . as afore mentioned , the new legislation aims to guarantee greater transparency and improve communication . in fact , on its web portal , aifa makes available to the public at least the following information : public assessment reports , together with a summary there of;summaries of product characteristics and package leaflets;summaries of risk management plans;list of medicinal products that are subject to additional monitoring;information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . public assessment reports , together with a summary there of ; summaries of product characteristics and package leaflets ; summaries of risk management plans ; list of medicinal products that are subject to additional monitoring ; information about the various procedures for the reporting of suspected adrs to the competent authorities by the healthcare professionals and patients , including forms with web data entry mask . medicines subject to additional monitoring will be the products containing new active substances not included in the authorized medicines in the eu before the january 1 2011 ; in particular , biologics and biosimilars , but the list may also include products whose license is subject to particular conditions or products authorized in exceptional circumstances ; e.g. , products subject to safety studies after the granting of the marketing authorization ( post - authorisation safety studies ) . these medicinal products subject to additional monitoring will be identifiable from the package leaflet that will bear the statement this medicinal product is subject to additional monitoring . this statement will be preceded by a black symbol and followed by an explanatory sentence explaining the concept of additional monitoring . the additional monitoring list will be regularly kept up to date by the ema in collaboration with member states . the new legislation has empowered the relevant authorities to impose on the marketing authorization holders the obligation to perform post - authorization safety and/or efficacy studies . these studies would be required at the time of the granting of the marketing authorization or later . according to another key point of the 2010 reform , each member state and each marketing authorization holder is obliged to adopt a pharmacovigilance system with the aim of analyzing suspected adrs and implementing provisions in order to prevent them . at last , the new eu pharmacovigilance legislation has established within the ema a new scientific committee , the pharmacovigilance risk assessment committee ( prac ) in which all member states are represented . the role of the prac in protecting public health requires close interaction with the agency 's committee for medicinal products for human use ( chmp ) and the coordination group for mutual recognition and decentralized procedures - human ( cmdh ) . its members include experts from regulatory authorities in eu member states and independent scientific experts who provide additional expertise in particular scientific areas . members from the patient organizations and healthcare professionals will be included in the near future further to appointment from the european commission . in its role in monitoring the safety of human medicines , the prac assesses all aspects of risk management , including the detection , assessment , minimization and communication of the risk of adrs , giving due consideration to the benefits of the medicine . the prac is also involved in designing and evaluating post - authorization safety studies and in conducting pharmacovigilance audits . when the prac has concluded an evaluation related to the safety of medicines , it prepares a recommendation that is sent either to the chmp ( if it concerns at least one centrally authorized medicine ) or to the cmdh ( if it only concerns medicines that are not centrally authorized in the eu ) . recently , the financial law no . 296/2006 has provided to the aifa an ad hoc fund to be allocated for activities related to pharmacovigilance . the regional authorities are responsible for the development and promotion of active pharmacovigilance programs , which agreed with aifa and subsequently , allow the regions to access to funds . in this context , aifa has promoted several projects and studies in italy , with the aim of increasing knowledge on drugs and the culture of pharmacovigilance the funding of pharmacovigilance projects by aifa has contributed to the improvement of spontaneous reporting . in italy , the situation of spontaneous adrs reports in 2010 showed an increase of 39% in comparison to 2009 . this result is in line with the average annual increase of 30% observed in the last five years , and it also demonstrates the crucial role of active pharmacovigilance programs , funded by aifa , in increasing knowledge on drugs , to better define their safety profile and to improve their safe use in clinical practice . in conclusion , the italian pharmacovigilance system is based on local activities coordinated at national level by aifa under the control of the ministry of health . this national system represents a link among local experiences and its counterparts at the european level . therefore , aifa has the double opportunity to transfer and receive knowledge from other eu countries ; furthermore , it is responsible in italy of the entire campaign to improve pharmacovigilance knowledge and increase the number of adrs reports up to the gold standard level established by the who . this system represents a good structure , however , several points need to be improved . this is particularly relevant for italy , where too many differences exist between regional and local activities . the financial support received by aifa and the future projects should help to achieve a more comprehensive pharmacovigilance activity .

heparin - binding protein ( hbp ) , also known as azurocidin , has multiple functions in the inflammatory process , especially during severe infections . beside its antimicrobial properties , hbp may induce vascular leakage leading to extravascular efflux , which is an important pathophysiologic event in the development of septic shock . not surprisingly , high hbp plasma levels are found in severe sepsis patients and in septic shock patients as well as in serious infections associated with endothelial damage . in the present issue of critical care , linder and colleagues demonstrate new aspects of hbp daily monitoring in icu patients . the authors observed that high hbp plasma levels are associated with an increased mortality rate in both septic and nonseptic critically ill patients , indicating that hbp may be a reliable prognostic biomarker . however , there are some limitations hindering rapid translation of these interesting findings into the daily routine . first , the group of nonseptic critically ill patients ( n = 28 ) enrolled in the study was rather small as compared with the septic group ( n = 151 ) . moreover , 50% of nonseptic patients developed infection while hospitalized in the icu , and to classify them as truly nonseptic patients is problematic . second , there is a lack of a routine diagnostic method for hbp analysis . nevertheless , if the results of the present study are validated in large clinical trials in different icu populations and cost - effectiveness data become available , the serial hbp measurements will have a promising future . introduction : spontaneous reporting of adverse drug reactions ( adrs ) is the basis of pharmacovigilance . in fact , adrs are associated with a high degree of morbidity and mortality . however , underreporting by all healthcare professionals remains the major problem in italy and in the rest of the world . the dissemination of pharmacovigilance knowledge among italian healthcare professionals , and the new pharmacovigilance regulations may promote the early detection and reporting of adrs . this review examines the legislative framework concerning the pharmacovigilance in italy.materials and methods : the information was collected from scientific articles and the websites of the italian ministry of health and the italian medicines agency ( agenzia italiana del farmaco , aifa).results : the pharmacovigilance system , both in italy and europe , has undergone profound changes . european legislation on pharmacovigilance has been changed in 2010 according to the eu regulation 1235/2010 and directive 2010/84/eu . basically , the changes tend to increase the efficiency , speed and transparency of pharmacovigilance activities . the new regulation ( 1235/2010 ) and the directive ( 2010/84/eu ) aim to strengthen the system of pharmacovigilance , establish more precisely who is obliged to do what , and allow faster and easier circulation and retrieval of information about adrs.conclusion:a greater knowledge on what is the italian pharmacovigilance legislation will be useful to improve the status of adrs reporting and spread the culture of spontaneous reporting .

periapical pathoses of pulpal origin develop in response to microbial irritants in the root canal systems . bacterial cell wall components react with monocytes , macrophages , other cells of the immune system , as well as with fibroblasts , leading to the production of proinflammatory cytokines , such as il-1 , il-1 , tnf- , il-6 , and il-8 . irritants may be mechanical or chemical , but are most often bacterial . the interaction between these irritants and host defensive cells results in release of numerous mediators that , through the root canal system , are capable of initiating immunologic reactions in periapical tissues resulting in the formation on inflammatory periapical lesions . these reactions include immune response mediated by cells , through the actions of t lymphocytes and cytokines , and hummoral immune response mediated by antibodies , activated by b - lymphocyte products . tnf- is a monocyte - derived protein that has a wide range of proinflammatory and immunomodulatory effects on a number of different cell populations . tnf- is a cytokine which stimulates bone resorption , prostaglandin synthesis , and protease production by many cell types , including fibroblasts and osteoblasts . the local production of pge2 , il1- , and tnf- has been demonstrated in periapical lesions [ 4 , 5 ] . tnf- is a cytokine initially identified as a causative of hemorrhagic necrosis in certain tumors and was later shown to be the same molecule as cachectin , a serum product earlier known as a mediator of wasting in chronic disease . in addition to its diverse bioactivities , tnf- is the only molecule other than il-1 that is presently known to have osteoclast - activating function . although extensive research has been performed in the area of periapical inflammatory mediators , only few studies focused on the role and levels of tnf- in periapical exudate [ 6 , 7 ] . the aim of this study was to determine the levels of tnf- in periapical exudates and to evaluate possible relationships between this cytokine and radiological findings in the involved teeth . the patients with noncontributory medical histories and diagnosed with apical periodontitis were selected for the study as follows . group 1 consisted of 20 subjects whose single - rooted teeth had the diagnosis of chronic apical periodontitis , with dull or absent pain , and size of the lesion < 1 cm . group 2 consisted of 20 subjects whose single - rooted teeth were diagnosed with chronic apical periodontitis , with dull or absent pain , but with the lesion size of > 1 cm.group 3 was control and comprised of 20 subjects whose investigated single - rooted teeth showed symptoms of acute periapical condition accompanied by excruciating pain , necrotic pulp , and radiological thickening of the apical periodontal ligament . primary access to the pulp chamber was gained using slow - speed round dental burs , with placement of rubber dam isolation . after determination of the working length , the necrotic pulp was removed and the root canal was enlarged to iso size 40 . a sterile paper point was inserted into the root canal up to the instrumentation length and held in place for 1 minute to absorb the exudate for the sample [ 6 , 7 ] . paper points were immersed into sterile ependorf vials containing phosphate buffered saline ( pbs ) and stored at 70c until further analysis [ 8 , 9 ] . the volume of the fluid was calculated from a standard curve and expressed as l . size of the radiolucent area was determined by two independent examiners at the time exudate samples were collected . periapical lesions were classified as small , when the longer diameter was shorter than 1 cm , while the remaining lesions were classified as large . in the control group , the radiological thickening of the periodontal ligament was present . group 1 consisted of 20 subjects whose single - rooted teeth had the diagnosis of chronic apical periodontitis , with dull or absent pain , and size of the lesion < 1 cm . group 2 consisted of 20 subjects whose single - rooted teeth were diagnosed with chronic apical periodontitis , with dull or absent pain , but with the lesion size of > 1 cm . group 3 was control and comprised of 20 subjects whose investigated single - rooted teeth showed symptoms of acute periapical condition accompanied by excruciating pain , necrotic pulp , and radiological thickening of the apical periodontal ligament . determination of the tnf- levels in supernatant of periapical exudate is based on utilization of monoclonal antibodies specific for tnf- which bind competitively to tnf-. investigation is based on sandwich all subjects were informed of the aims and procedures of research . within the research , they are guaranteed in respect of their basic ethical and bioethical principles : personal integrity ( independence , righteousness , well - being , and safety ) as regulated by nrnberg codex and the most recent version of helsinki declaration . only those subjects who gave a written permission in the form of informed consent the present study demonstrated the presence of tnf- in all periapical exudate samples . differences in concentrations of tnf- between control group ( 40 , 57 28 , 15 pg / ml ) , the group with large radiolucent areas ( 2365 , 79 582 , 95 pg / ml ) , as well as between control and the canals associated with small radiolucent areas ( 507 , 66 278 , 97 ) were statistically significant ( p < .05 ) . there was also statistically significant difference between the samples with large radiolucent areas and small radiolucent areas . eia data for tnf- were categorized according to radiological diagnosis , using the nonparametric kruskal - wallis test ( see figure 1 ) . soluble mediators produced and secreted by various inflammatory , immunologically - active , and structural cells , commonly referred to as cytokines , play the leading role in pathogenesis of infectious disease , acting side - by - side with other inflammatory mediators : kinines , vasoactive amines , components of the complement system , and metabolites of arachidonic acid . secretion of the cytokines is initiated with the purpose of activating immunological response to irritants and increasing local concentrations of inflammatory cells in order to prevent further colonization of bacteria within the tissues . enhanced reaction of the host to various antigens results in bone resorption and formation of granulomatous tissue , which are the typical features of periapical lesion . in this investigation , levels of the proinflammatory cytokine tnf- in periapical exudate were analyzed due to its leading role in pathogenesis of periapical lesions . these exudates present the inflammatory exudate from the periapical tissues and theoretically contain locally produced and secreted factors . they resulted from the inflammatory response and contain host mediators related in response to the infection . highest concentrations of tnf- were detected in periapical lesions with large radiolucent areas , while these levels were significantly lower in the periapical lesions with smaller radiolucent areas . in the control group diagnosed as acute periodontitis , with radiological changes consistent with periodontal ligament thickening , which differs in clinical symptoms from the other two experimental groups , and is characterized by radioloical changes consistent with periodontal ligament thickening , the levels of the investigated cytokine demonstrated the lowest levels . these results show different values from the results obtained by ataoglu et al . ; however , these authors compared the levels of two kinds of cytokines : il-1 and tnf-. in the work of ataoglu et al . , il-1 was detected in all samples of periapical exudates and its levels were significantly higher than tnf- levels ; however , the authors focused their attention only on the tnf- levels in periapical exudates demonstrated in all tissue specimens with significant differences regarding certain experimental groups . a study by kjeldsen et al . using elisa technique also showed significantly higher concentrations of tnf- in crevicular fluid in patients with chronic adult periodontitis . it is likely that chronic periodontal infection may evoke an immune response that may result in the production of slightly higher levels of tnf-. it was proved that tnf- leads to bone resorption through osteoclast activation and stimulation of the secretion of proteolytic enzymes , plasminogen activator ( pa ) , and matrix metalloproteinases ( mmp ) , which are in charge of destroying extracellular matrix of the bone tissue . . showed that there is a small fraction of tnf--positive cells within the granuloma with a macrophage - like morphology . ultrastructural analyses showed that there are some macrophages which have adjusted the extracellular secretion ; therefore , these macrophages might be the main source of cytokines in the tissue . wang et al . showed that both tnf- and il-1 are secreted in the infected rat pulps and periapical lesions . the cells appeared as soon as 2 days following the pulp chamber opening and their numbers increased steadily until the day 30 . these findings demonstrate the presence of il-1- and tnf--secreting cells in the pulp and periapical tissues immediately following the pulp exposure , which supports the assumption that the abovementioned cytokines play a role in pathogenesis of pulp and periapical pathoses . in the analysis of tnf- levels in healthy , symptomatic , and asymptomatic human pulps , the highest concentrations of tnf- , with regard to clinical classification , were found in symptomatic reversible pulpitis , and the difference was statistically significant in comparison with irreversible asymptomatic pulpitis and healthy pulp . the highest concentrations of tnf- , with regard to histological classification , were found in histologically confirmed moderate inflammation , followed by severe inflammation , mild inflammation , and finally healthy pulp . conclusion can be drawn that cytokine tnf- could be an objective marker in radiologically confirmed periapical lesions . the lowest levels were demonstrated in control group , which was characterized by radiological thickening of the periodontal ligament , while somewhat greater concentrations were found in teeth with periapical radiolucencies of < 1 cm diameter ; finally , the greatest concentrations were measured in teeth with radiolucent lesions > 1 cm , which confirms the relationship between tnf- and the emergence and development of periapical lesions . objective analysis of tnf- levels enables establishment of a relationship between different concentrations of this cytokine and different radiological changes . the traditional management of gallstone pancreatitis ( gp ) has been to perform cholecystectomy during the same hospital admission after resolution . however , when gp develops in the immediate postoperative period from a major colorectal operation , cholecystectomy may be fraught with difficulty due to the inflammatory response that occurs . thus , delaying cholecystectomy until the inflammatory response subsides may be worthwhile , and it maximizes the chances of completing the cholecystectomy laparoscopically . the best management strategy for gp under these circumstances , we have described our management of 2 patients with gp occurring after colorectal operations requiring proximal diverting ileostomy . a 31-year - old male with adenomatous polyposis coli gene mutation and familial adenomatous polyposis presented prophylactic surgical treatment . he underwent an uncomplicated laparoscopic total proctocolectomy with ileal j - pouch anal anastomosis and diverting loop ileostomy . on postoperative day ( pod ) 8 , he developed acute epigastric and right upper quadrant ( ruq ) pain . laboratory studies revealed a white blood cell ( wbc ) count of 14,000 , serum lipase and amylase of 1300 , and normal bilirubin . he was placed on bowel rest with iv hydration until pancreatitis improved clinically and biochemically . cholecystectomy was deferred until planned ileostomy reversal . to decrease the risk of recurrent episodes of gp , the patient then underwent ileostomy reversal and cholecystectomy as described below 6 weeks later . a 44-year - old male with a history of sigmoid colectomy for endoscopically unresectable adenoma presented to us with rectal bleeding . laboratory studies revealed a wbc count of 13,000 , lipase and amylase above 400 , and normal bilirubin . gp was diagnosed and then managed non - operatively , similar to our first case . he refused ercp / s . after discharging , the patient returned in 6 weeks for ileostomy reversal and cholecystectomy as described below . in both cases , anastomosis was found to be healed on pouchogram and pouchoscopy at 6 weeks postoperatively . after establishing general anesthesia and administration of pre - incisional antibiotics , the proximal and distal portions were marked with different colored sutures and placed within the abdomen . a 12-mm balloon - tipped port was placed through the residual defect for the laparoscope . three 5 mm ports were placed in the ruq away from any intra - abdominal adhesions [ figure 1 ] . the two limbs of ileum were eviscerated in proper anatomic position using the colored sutures . a 31-year - old male with adenomatous polyposis coli gene mutation and familial adenomatous polyposis presented prophylactic surgical treatment . he underwent an uncomplicated laparoscopic total proctocolectomy with ileal j - pouch anal anastomosis and diverting loop ileostomy . on postoperative day ( pod ) 8 , he developed acute epigastric and right upper quadrant ( ruq ) pain . laboratory studies revealed a white blood cell ( wbc ) count of 14,000 , serum lipase and amylase of 1300 , and normal bilirubin . he was placed on bowel rest with iv hydration until pancreatitis improved clinically and biochemically . cholecystectomy was deferred until planned ileostomy reversal . to decrease the risk of recurrent episodes of gp , endoscopic retrograde cholangiopancreatography with sphincterotomy ( ercp / s ) was performed . a 44-year - old male with a history of sigmoid colectomy for endoscopically unresectable adenoma presented to us with rectal bleeding . laboratory studies revealed a wbc count of 13,000 , lipase and amylase above 400 , and normal bilirubin . gp was diagnosed and then managed non - operatively , similar to our first case . he refused ercp / s . after discharging , the patient returned in 6 weeks for ileostomy reversal and cholecystectomy as described below . in both cases , anastomosis was found to be healed on pouchogram and pouchoscopy at 6 weeks postoperatively . after establishing general anesthesia and administration of pre - incisional antibiotics , the proximal and distal portions were marked with different colored sutures and placed within the abdomen . a 12-mm balloon - tipped port was placed through the residual defect for the laparoscope . three 5 mm ports were placed in the ruq away from any intra - abdominal adhesions [ figure 1 ] . the two limbs of ileum were eviscerated in proper anatomic position using the colored sutures . about 20% of patients harboring asymptomatic gs develop symptoms requiring cholecystectomy 3 - 5 years following colorectal surgery . the mechanism is unclear , but studies have implicated changes in the enterohepatic circulation and cholesterol metabolism . the potential for gs related symptoms in these patients caused some to recommend cholecystectomy as an incidental procedure during the time of the index colorectal operation . proponents feel that this practice spares patients future complications and the morbidity of another operation . previous studies do not focus on the immediate postoperative period or comment on gp specifically . therefore , the true incidence of this particular postoperative complication is unknown . in the absence of good data , most patients presenting with gp undergo cholecystectomy during the same hospital admission to prevent the potential morbidity of future bouts . in our cases , ercp / s can be used as a temporizing procedure to prevent recurrent episodes of gp while awaiting cholecystectomy . in addition , both of our patients had stomas that were planned to be reversed 6 - 8 weeks after the initial operation and , thus , provided the opportunity for a combined operation . ports for laparoscopic procedures are routinely placed at sites of planned stomas in order to minimize the number of incisions . using the stoma site for the camera avoids an extra incision and is away from the midline , where adhesions are expected from previous operations . we feel that delaying the completion of the anastomosis until after cholecystectomy and marking the proximal and distal limbs are important to avoid trauma to the anastomosis and to prevent un - intended twisting of the bowel . complications attributable to gs in the immediate postoperative period following colorectal surgery are difficult to manage . when the complication is gp , the episode is mild and resolves with non - operative measures , it may be preferable to delay cholecystectomy until the postoperative inflammatory response subsides . when the index operation is the first stage of a planned 2-stage operation , delaying the cholecystectomy seems even more attractive . as seen in these two cases , the fascial aperture of the ileostomy site can be easily used for the laparoscope , and combining the two operations is straightforward .

aim . the aim of this study was to determine tumor necrosis factor - alpha ( tnf- ) levels in periapical exudates and to evaluate their relationship with radiological findings . methodology . periapical exudates were collected from root canals of 60 single - rooted teeth using absorbent paper points . tnf- levels were determined by enzyme - linked immunosorbent assays . the samples were divided into three groups according to the periapical radiolucent area . results . nonparametric kruskal - wallis test revealed significant differences between tnf- concentrations in control group ( 40 , 5728 , 15 pg / ml ) and group with larger radiolucent areas ( 2365 , 79582 , 95 pg / ml ) , as well as between control and canals with small radiolucent areas ( 507 , 66278 , 97 ) ( p<.05 ) . conclusions . the levels of tnf- increase significantly in teeth with periapical pathosis , from smaller to bigger lesions . this research and its results have shown that objective analysis of the tnf- levels enables establishment of a relationship between different concentrations of tnf- and different radiological changes . traditional management of gallstone pancreatitis ( gp ) has been to perform cholecystectomy during the same hospital admission after resolution . however , when gp develops in the immediate postoperative period from a major colorectal operation , cholecystectomy may be fraught with difficulty due to the inflammatory response that occurs . thus , delaying cholecystectomy until the inflammatory response subsides may be worthwhile , and it maximizes the chances of completing the cholecystectomy laparoscopically . we have described our management of 2 patients with gp occurring after colorectal operations , which required proximal diverting ileostomy . in both cases , we deferred management of gp with either endoscopic retrograde cholangiopancreatography ( ercp ) or medical conservative measures during the acute attack and performed laparoscopic cholecystectomy during ostomy reversal surgery utilizing the existing ostomy takedown site for port placement . both patients tolerated this management well .

high - grade gliomas , who grade iii or iv [ 1 , 2 ] , are the most common primary brain tumors in adults [ 1 , 3 ] . who grade iv tumors are almost exclusively ( 8090% ) glioblastoma multiforme ( gbm ) , which are the most common high - grade glioma ( 4045% ) [ 1 , 3 ] , the most common form of brain tumor overall ( 1215% ) [ 1 , 3 ] , and the most aggressive malignant primary brain tumor . despite limited significant advances in standard therapy , notably temozolomide , median overall survival ( os ) remains low : 15 months for newly - diagnosed gbm [ 5 , 6 ] and 57 months for recurrent / relapsed gbm [ 710 ] . in a clinical trial setting , the current standard of care ( radiotherapy plus temozolomide followed by 6 cycles of adjuvant temozolomide ) provided 2- and 5-year survival rates of 27 and 10% for patients with newly diagnosed gbm . given the poor prognosis of gbm , the primary objectives of therapy are to reduce morbidity , restore or preserve neurologic functions and the capacity to perform daily activities as long as possible . the aim of this review is to examine the impact of gbm therapy on qol , neurocognitive function , and their correlates . it is well recognized that impairment of neurocognitive functioning , resulting in behavioral , emotional , and intellectual difficulties , occurs in nearly all patients with brain tumors and eventually compromises their independence . this impairment is related to a combination of various factors , including the tumor itself , tumor - related epilepsy , treatment , and patient - related factors ( e.g. , age , psychological distress ) [ 1316 ] . however , there have been relatively few well - powered longitudinal studies of neurocognitive function in patients with high - grade glioma . not surprisingly , neurocognitive function assessments have been incorporated as major components of patient assessments , along with common and widely used questionnaires to assess health - related qol ( hr - qol ) , e.g. , european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 ) , and functional assessment of cancer therapy ( fact ) cancer - specific scales . indeed , neurocognitive function has been shown to be a valid predictor of long - term qol [ 2224 ] . as life expectancy in high - grade glioma , and particularly in recurrent gbm , is so short , issues relating to qol are immensely important to patients and their caregivers . this is especially important in relation to new treatments in recurrent gbm that do not yet have evidence supporting their contribution to extended survival but may significantly delay the expected steep qol deterioration occurring after progression following standard therapies . unfortunately , qol data are difficult to collect in cancer patients because they may be unwilling to complete the questionnaire when they are feeling unwell . furthermore , repeated application of lengthy , formal hr - qol questionnaires can represent a major and impractical burden for patients . also , the analysis of qol data is challenging due to the high rates of non - random missing qol values that may be linked to patients qol status , and if ignored may introduce bias in the interpretation of results . interpretation of the impact of standard and new therapies on qol in gbm patients is consequently problematic , even when attempting to classify their effect into the three broad categories of negative , positive , or neutral . to overcome these challenges , changes in neurocognitive functioning assessment of neurocognitive function can therefore represent a practical surrogate for formal qol assessment of patients with recurrent gbm . in addition , besides grade and age , performance status in patients with newly diagnosed glioma is an independent prognostic marker . thus , it is plausible to assume that neurocognitive function , irrespective of clinical stage , may also have prognostic implications even after initiation of therapy and during the course of treatment . hr - qol in patients with high - grade glioma has recently been reviewed in detail , which noted problems associated with interpretation of different studies and the paucity of robust hr - qol information derived from well - powered randomized controlled trials . among the seven randomized controlled trials of new treatments published from 2002 to 2007 they identified for high - grade glioma , there was no or little difference between treatment groups at baseline or follow - up evaluation . these authors suggested that standard multidimensional hr - qol questionnaires might therefore contain too many items and consequently lack sensitivity to detect qol changes in patients with high - grade glioma . simpler , practical , and more sensitive instruments ( such as cognitive function ) are therefore needed to study qol changes in relation to therapy in high - grade glioma , and , thus , the confounding factor of missing substantial follow - up data ( primarily related to dropouts ) needs to be addressed . neurosurgery and/or radiotherapy are still fundamental elements of standard therapy for patients with high - grade glioma . it is well recognized that surgery may initially , at least temporarily , improve qol dramatically in a significant proportion of patients with severe symptoms related to increased intracranial pressure . conversely , radiotherapy may decrease qol in some patients from adverse effects such as hair loss , fatigue , somnolence , or cognitive problems . it is clear that tumor recurrence and short - term survival are confounding variables , but it is generally agreed that short - term memory and progression of dementia are observed in many patients treated with brain irradiation . the impact of adjunctive therapy with corticosteroids and antiepileptic medication has also been extensively studied . while the presence and severity of epileptic seizures and/or the use of antiepileptic medication have been significantly associated with cognitive deficits in patients with low - grade gliomas , more so than the effects of radiotherapy [ 2830 ] , the effects of antiepileptic medication on neurocognitive functioning and qol have been less extensively studied in patients with high - grade glioma , although some studies have reported a negative impact . the negative effects of corticosteroid use on neurocognitive function and/or qol are well documented in healthy subjects [ 32 , 33 ] and in various disease states , such as leukemia . while it appears accepted that the use of high - dose corticosteroids has a clinically significant negative impact on neurocognitive function in glioma , there would appear to be no published information in this indication . it is documented that corticosteroid use in primary brain tumors and/or metastases caused adverse effects [ 35 , 36 ] which would be expected to decrease qol . assessment of qol is important with cytotoxic chemotherapy , particularly when survival benefit may be marginal and has to be balanced against any negative contribution of significant toxicity . among newly diagnosed gbm patients randomized to radiotherapy alone or radiotherapy plus temozolomide , the addition of temozolomide had no significant negative effect on qol measures , except on social functioning ( p = 0.0052 ) . similarly , among first - relapse gbm patients , temozolomide had no significant negative effect on qol , although responders to temozolomide had improvement in most qol domain scores , e.g. , global , motor dysfunction , emotional function , drowsiness , future uncertainty , and communication deficit , until eventual disease progression [ 3840 ] . these overall findings with temozolomide in gbm are concordant with the recent cochrane review that evaluated randomized controlled trial data assessing temozolomide in patients with high - grade glioma . when used as a comparator for temozolomide among the aforementioned trials , procarbazine was reported to have a negative impact on hr - qol domain scores , e.g. , drowsiness , communications deficit , motor dysfunction , role functioning , social functioning , and physical functioning , regardless of disease progression status [ 38 , 39 ] . the impact of combined procarbazine , ccnu ( lomustine ) , and vincristine ( pcv ) chemotherapy after radiotherapy compared with radiotherapy alone on hr - qol measures was determined in the eortc 26951 trial of patients with anaplastic oligodendroglioma ( n = 368 ) : a major negative impact on hr - qol ( nausea / vomiting , appetite loss , and drowsiness ) was found during and shortly after pcv treatment . however , when hr - qol measures were used to assess the impact of pcv chemotherapy after radiotherapy compared with radiotherapy alone in the radiation therapy oncology group ( rtog ) trial 94 - 02 of patients with mixed anaplastic oligodendroglioma , scores were similar longitudinally and between treatments for survivors . hr - qol ( spitzer quality of life index ) score showed continual deterioration when measured longitudinally in the rtob 98 - 03 study of escalating doses of conformal three - dimensional radiation and carmustine in gbm patients . supplementation of surgery and radiotherapy with ccnu chemotherapy provided no benefit in terms of quality of life or change in clinical performance . finally , there are very limited qol data from randomized controlled trials with implanted carmustine - impregnated wafers in primary or recurrent high - grade glioma according to a recent cochrane review . bevacizumab ( avastin ) , a humanized monoclonal antibody that binds to and inhibits the activity of vegf , is the first approved antiangiogenic cancer treatment . bevacizumab acts synergistically with cytotoxic chemotherapy or biological agents in the treatment of various tumors , e.g. , colorectal , lung , renal , and breast cancer [ 4750 ] . recently , studies in patients with recurrent high - grade glioma or gbm have indicated promising results with durable response using the combination of bevacizumab and irinotecan [ 5167 ] , and additionally in combination with etoposide , nitrosourea , fotemustine , or erlotinib . positive results have also been reported with single - agent bevacizumab in recurrent high - grade glioma [ 57 , 61 , 7274 ] , and in combination with adjuvant temozolomide in newly diagnosed gbm [ 7579 ] . during these studies of bevacizumab - based therapy in high - grade glioma , it was reported that one of the consequences of bevacizumab therapy is a steroid - sparing effect in a proportion of patients , which would be expected to positively impact qol . various studies have indicated a reduction or elimination of corticosteroid use with bevacizumab - based therapy in patients with recurrent high - grade glioma after prior treatment [ 53 , 5661 , 63 , 64 , 73 ] . for example , in the randomized phase ii clinical trial comparing bevacizumab alone or in combination with irinotecan in 167 patients with recurrent , treatment - refractory gbm , patients who were taking corticosteroids at baseline showed a trend to take stable or decreasing doses over time , e.g. , median corticosteroid dose was reduced by about 75% after 6 months ( fig . 1 ) . the steroid - sparing effect associated with bevacizumab - based therapy appeared associated with clinical response in high - grade glioma [ 58 , 64 ] and clinical benefit such as improved neurological symptoms in high - grade glioma or recurrent gbm .fig . 1change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) a potentially positive impact of bevacizumab - based therapy on neurocognitive function , performance status and/or qol has also started to emerge from reports of clinical studies among gbm patients [ 52 , 59 , 63 , 64 ] . for example , in a retrospective analysis of recurrent gbm patients treated with bevacizumab ( n = 44 ) or without bevacizumab ( n = 79 ) at a single us institution , it was reported that bevacizumab - treated patients took longer to increase dexamethasone dose ( median 149 vs. 130 days , p = 0.04 ) and also maintained their karnofsky performance status ( kps ) for longer ( median 252 vs. 120 days , p = 0.006 ) ; subgroup analysis indicated that the difference in these effects were more pronounced in patients aged > 55 years . in another study of 22 consecutive patients with recurrent gbm treated with bevacizumab plus irinotecan , cognitive function was assessed by the blessed orientation - memory - concentration test ( bomc ) and functional status was assessed by kps , barthel index ( bi ) , and instrumental activities of daily living ( iadl ) prior to each cycle of treatment . improvement in bomc score was seen in 15 patients ( 62% ) , with median improvement of 7 points . improvement in functional status was seen in 18 patients ( 85.7% ) , with median improvement in kps by 10 points , bi by 8 points , and iadl by 2 points . the overall clinical response rate with bevacizumab plus irinotecan was 95% and was associated with significant improvements in cognitive functional and functional status . larger scale controlled clinical trials of bevacizumab - based therapy in gbm patients were consequently undertaken or are planned / on - going . among these is the recently reported brain study ( avf3708 g ) , an open - label , multicenter , randomized , phase ii trial of two concurrent arms treated with single - agent bevacizumab ( n = 85 ) or bevacizumab plus irinotecan ( n = 82 ) in patients with first- or second - relapse gbm who had been previously treated with temozolomide initially or at relapse . primary endpoint results have been reported : estimated 6-month pfs rates were 43 and 50% in single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively , and objective response rates were 28 and 38% , respectively . these results supported the activity of bevacizumab in recurrent gbm patients given that the 6-month pfs rate was considerably higher than the 15% rate expected for salvage chemotherapy and/or chemotherapy alone . the authors noted a trend for patients who were taking corticosteroids at baseline to take stable or decreasing doses over time , but they made no formal comparison . more compellingly data on corticosteroid use during this study have recently been reported . at baseline , 51 and 53% of patients received systemic corticosteroids in the single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively . more than 75 and 65% of patients in the single - agent and combination arms , respectively , who did not receive corticosteroids at baseline continued to receive no corticosteroids after baseline . sustained reduction in corticosteroid use was defined in this study as a 50% dose reduction for 50% of time on study treatment up to 52 weeks . among patients with complete or partial response in the single - agent bevacizumab and bevacizumab plus irinotecan arms , 57 and 65% , respectively , had a sustained reduction in corticosteroid use compared with 17 and 38% , respectively , among those with stable or progressive disease . neurocognitive function of patients treated with single - agent bevacizumab in the brain study ( n = 85 ) has been analyzed . patients were assessed with the hopkins verbal learning test - revised ( hvlt - r ) , trail making test parts a ( tmta ) and b ( tmtb ) , and the controlled oral word association ( cowa ) test . assessments were conducted at baseline and then every 6 weeks while patients remained on study treatment up to 52 weeks . change in neurocognitive function from baseline to week 6 was categorized as improved , stable , or declined , using the reliable change index . between 93 and 98% of patients completed each test at baseline and 7378% completed each test at both baseline and week 6 . the majority of patients demonstrated stable performance on each test at week 6 , relative to baseline . with the exception of the cowa test , 1628% of patients demonstrated improved performance on one or more tests at week 6 ( table 1 ) . preliminary results suggest that the majority of patients with recurrent gbm who were treated with bevacizumab alone in the brain study demonstrated stable or improved neurocognitive function during the first 6 weeks of treatment ( 81100% across all tests ) . changes across tasks and associations with measures of clinical efficacy were also explored ( table 2).table 1neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studyassessmentchange in performance at week 6 relative to baseline ( per reliable change index)improved ( % ) declined ( % ) stable ( % ) hvlt - r total recall ( n = 74)161866hvlt - r delayed recall ( n = 70)141670hvlt - r delayed recognition ( n = 69)221959tmta ( n = 73)231166tmtb ( n = 65)281162cowa ( n = 70)3097hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association testtable 2stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studystable / improved neurocognitive function on all tests n ( % ) deterioration in neurocognitive function in at least one test n ( % ) responders at time of irf response ( n = 24)18 ( 75%)6 ( 25%)pfs > 6 months at week 24 ( n = 27)19 ( 70%)8 ( 30%)patients at time of investigator pd ( n = 49)15 ( 31%)34 ( 69%)irf independent radiology facility , pfs progression - free survival , pd progressive diseasetwo patients had missing neurocognitive data and were dropped from the analysiseight patients had missing neurocognitive data and were dropped from the analysis neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association test stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study irf independent radiology facility , pfs progression - free survival , pd progressive disease two patients had missing neurocognitive data and were dropped from the analysis eight patients had missing neurocognitive data and were dropped from the analysis in a recent retrospective analysis , 12 patients with gbm and poor neurologic function ( kps < 60% ) due to bulky disease were treated in an up - front setting with bevacizumab following induction temozolomide in an attempt to improve their ability to tolerate chemoradiation . median kps improved from 50 to 70% and their median dexamethasone dose was reduced from 12 to 4 mg / day . five of 11 evaluable patients ( 45% ) had a partial response , 4 ( 35% ) a minor response , and 1 ( 10% ) stable disease . the tumor response , rapidly improved neurologic function , and reduced steroid requirement allowed the majority of patients ( 75% ) to complete definitive radiotherapy . little significant information appears to have been published on the effects of other investigational biological agents ( e.g. , cilengitide , cediranib , sorafenib , sunitinib ) on corticosteroid use , neurocognitive function , or qol in patients with high - grade glioma or more specifically those with gbm . in a recent phase ii study of cediranib , an oral pan - vegfr tyrosine kinase inhibitor , was administered as monotherapy ( 45 mg / day ) in 31 patients with recurrent gbm and resulted in encouraging proportions of radiographic partial responses of 57 and 27% on 3- and 2-dimensional mri , respectively ) and 6-month pfs ( 26% ) . furthermore , among 15 patients receiving corticosteroids on study entry , the dose was reduced ( n = 10 ) or discontinued ( n = 5 ) . neurosurgery and/or radiotherapy are still fundamental elements of standard therapy for patients with high - grade glioma . it is well recognized that surgery may initially , at least temporarily , improve qol dramatically in a significant proportion of patients with severe symptoms related to increased intracranial pressure . conversely , radiotherapy may decrease qol in some patients from adverse effects such as hair loss , fatigue , somnolence , or cognitive problems . it is clear that tumor recurrence and short - term survival are confounding variables , but it is generally agreed that short - term memory and progression of dementia are observed in many patients treated with brain irradiation . the impact of adjunctive therapy with corticosteroids and antiepileptic medication has also been extensively studied . while the presence and severity of epileptic seizures and/or the use of antiepileptic medication have been significantly associated with cognitive deficits in patients with low - grade gliomas , more so than the effects of radiotherapy [ 2830 ] , the effects of antiepileptic medication on neurocognitive functioning and qol have been less extensively studied in patients with high - grade glioma , although some studies have reported a negative impact . the negative effects of corticosteroid use on neurocognitive function and/or qol are well documented in healthy subjects [ 32 , 33 ] and in various disease states , such as leukemia . while it appears accepted that the use of high - dose corticosteroids has a clinically significant negative impact on neurocognitive function in glioma , there would appear to be no published information in this indication . however , it is documented that corticosteroid use in primary brain tumors and/or metastases caused adverse effects [ 35 , 36 ] which would be expected to decrease qol . assessment of qol is important with cytotoxic chemotherapy , particularly when survival benefit may be marginal and has to be balanced against any negative contribution of significant toxicity . among newly diagnosed gbm patients randomized to radiotherapy alone or radiotherapy plus temozolomide , the addition of temozolomide had no significant negative effect on qol measures , except on social functioning ( p = 0.0052 ) . similarly , among first - relapse gbm patients , temozolomide had no significant negative effect on qol , although responders to temozolomide had improvement in most qol domain scores , e.g. , global , motor dysfunction , emotional function , drowsiness , future uncertainty , and communication deficit , until eventual disease progression [ 3840 ] . these overall findings with temozolomide in gbm are concordant with the recent cochrane review that evaluated randomized controlled trial data assessing temozolomide in patients with high - grade glioma . when used as a comparator for temozolomide among the aforementioned trials , procarbazine was reported to have a negative impact on hr - qol domain scores , e.g. , drowsiness , communications deficit , motor dysfunction , role functioning , social functioning , and physical functioning , regardless of disease progression status [ 38 , 39 ] . the impact of combined procarbazine , ccnu ( lomustine ) , and vincristine ( pcv ) chemotherapy after radiotherapy compared with radiotherapy alone on hr - qol measures was determined in the eortc 26951 trial of patients with anaplastic oligodendroglioma ( n = 368 ) : a major negative impact on hr - qol ( nausea / vomiting , appetite loss , and drowsiness ) was found during and shortly after pcv treatment . however , when hr - qol measures were used to assess the impact of pcv chemotherapy after radiotherapy compared with radiotherapy alone in the radiation therapy oncology group ( rtog ) trial 94 - 02 of patients with mixed anaplastic oligodendroglioma , scores were similar longitudinally and between treatments for survivors . hr - qol ( spitzer quality of life index ) score showed continual deterioration when measured longitudinally in the rtob 98 - 03 study of escalating doses of conformal three - dimensional radiation and carmustine in gbm patients . supplementation of surgery and radiotherapy with ccnu chemotherapy provided no benefit in terms of quality of life or change in clinical performance . finally , there are very limited qol data from randomized controlled trials with implanted carmustine - impregnated wafers in primary or recurrent high - grade glioma according to a recent cochrane review . bevacizumab ( avastin ) , a humanized monoclonal antibody that binds to and inhibits the activity of vegf , is the first approved antiangiogenic cancer treatment . bevacizumab acts synergistically with cytotoxic chemotherapy or biological agents in the treatment of various tumors , e.g. , colorectal , lung , renal , and breast cancer [ 4750 ] . recently , studies in patients with recurrent high - grade glioma or gbm have indicated promising results with durable response using the combination of bevacizumab and irinotecan [ 5167 ] , and additionally in combination with etoposide , nitrosourea , fotemustine , or erlotinib . positive results have also been reported with single - agent bevacizumab in recurrent high - grade glioma [ 57 , 61 , 7274 ] , and in combination with adjuvant temozolomide in newly diagnosed gbm [ 7579 ] . during these studies of bevacizumab - based therapy in high - grade glioma , it was reported that one of the consequences of bevacizumab therapy is a steroid - sparing effect in a proportion of patients , which would be expected to positively impact qol . various studies have indicated a reduction or elimination of corticosteroid use with bevacizumab - based therapy in patients with recurrent high - grade glioma after prior treatment [ 53 , 5661 , 63 , 64 , 73 ] . for example , in the randomized phase ii clinical trial comparing bevacizumab alone or in combination with irinotecan in 167 patients with recurrent , treatment - refractory gbm , patients who were taking corticosteroids at baseline showed a trend to take stable or decreasing doses over time , e.g. , median corticosteroid dose was reduced by about 75% after 6 months ( fig the steroid - sparing effect associated with bevacizumab - based therapy appeared associated with clinical response in high - grade glioma [ 58 , 64 ] and clinical benefit such as improved neurological symptoms in high - grade glioma or recurrent gbm .fig . 1change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) change in median corticosteroid use following treatment with bevacizumab with or without irinotecan in patients with recurrent glioblastoma multiforme ( adapted with permission from friedman et al . ) a potentially positive impact of bevacizumab - based therapy on neurocognitive function , performance status and/or qol has also started to emerge from reports of clinical studies among gbm patients [ 52 , 59 , 63 , 64 ] . for example , in a retrospective analysis of recurrent gbm patients treated with bevacizumab ( n = 44 ) or without bevacizumab ( n = 79 ) at a single us institution , it was reported that bevacizumab - treated patients took longer to increase dexamethasone dose ( median 149 vs. 130 days , p = 0.04 ) and also maintained their karnofsky performance status ( kps ) for longer ( median 252 vs. 120 days , p = 0.006 ) ; subgroup analysis indicated that the difference in these effects were more pronounced in patients aged > 55 years . in another study of 22 consecutive patients with recurrent gbm treated with bevacizumab plus irinotecan , cognitive function was assessed by the blessed orientation - memory - concentration test ( bomc ) and functional status was assessed by kps , barthel index ( bi ) , and instrumental activities of daily living ( iadl ) prior to each cycle of treatment . improvement in bomc score was seen in 15 patients ( 62% ) , with median improvement of 7 points . improvement in functional status was seen in 18 patients ( 85.7% ) , with median improvement in kps by 10 points , bi by 8 points , and iadl by 2 points . the overall clinical response rate with bevacizumab plus irinotecan was 95% and was associated with significant improvements in cognitive functional and functional status . larger scale controlled clinical trials of bevacizumab - based therapy in gbm patients were consequently undertaken or are planned / on - going . among these is the recently reported brain study ( avf3708 g ) , an open - label , multicenter , randomized , phase ii trial of two concurrent arms treated with single - agent bevacizumab ( n = 85 ) or bevacizumab plus irinotecan ( n = 82 ) in patients with first- or second - relapse gbm who had been previously treated with temozolomide initially or at relapse . primary endpoint results have been reported : estimated 6-month pfs rates were 43 and 50% in single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively , and objective response rates were 28 and 38% , respectively . these results supported the activity of bevacizumab in recurrent gbm patients given that the 6-month pfs rate was considerably higher than the 15% rate expected for salvage chemotherapy and/or chemotherapy alone . the authors noted a trend for patients who were taking corticosteroids at baseline to take stable or decreasing doses over time , but they made no formal comparison . more compellingly data on corticosteroid use during this study have recently been reported . at baseline , 51 and 53% of patients received systemic corticosteroids in the single - agent bevacizumab and bevacizumab plus irinotecan arms , respectively . more than 75 and 65% of patients in the single - agent and combination arms , respectively , who did not receive corticosteroids at baseline continued to receive no corticosteroids after baseline . sustained reduction in corticosteroid use was defined in this study as a 50% dose reduction for 50% of time on study treatment up to 52 weeks . among patients with complete or partial response in the single - agent bevacizumab and bevacizumab plus irinotecan arms , 57 and 65% , respectively , had a sustained reduction in corticosteroid use compared with 17 and 38% , respectively , among those with stable or progressive disease . neurocognitive function of patients treated with single - agent bevacizumab in the brain study ( n = 85 ) has been analyzed . patients were assessed with the hopkins verbal learning test - revised ( hvlt - r ) , trail making test parts a ( tmta ) and b ( tmtb ) , and the controlled oral word association ( cowa ) test . assessments were conducted at baseline and then every 6 weeks while patients remained on study treatment up to 52 weeks . change in neurocognitive function from baseline to week 6 was categorized as improved , stable , or declined , using the reliable change index . between 93 and 98% of patients completed each test at baseline and 7378% completed each test at both baseline and week 6 . the majority of patients demonstrated stable performance on each test at week 6 , relative to baseline . with the exception of the cowa test , 1628% of patients demonstrated improved performance on one or more tests at week 6 ( table 1 ) . preliminary results suggest that the majority of patients with recurrent gbm who were treated with bevacizumab alone in the brain study demonstrated stable or improved neurocognitive function during the first 6 weeks of treatment ( 81100% across all tests ) . changes across tasks and associations with measures of clinical efficacy were also explored ( table 2).table 1neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studyassessmentchange in performance at week 6 relative to baseline ( per reliable change index)improved ( % ) declined ( % ) stable ( % ) hvlt - r total recall ( n = 74)161866hvlt - r delayed recall ( n = 70)141670hvlt - r delayed recognition ( n = 69)221959tmta ( n = 73)231166tmtb ( n = 65)281162cowa ( n = 70)3097hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association testtable 2stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain studystable / improved neurocognitive function on all tests n ( % ) deterioration in neurocognitive function in at least one test n ( % ) responders at time of irf response ( n = 24)18 ( 75%)6 ( 25%)pfs > 6 months at week 24 ( n = 27)19 ( 70%)8 ( 30%)patients at time of investigator pd ( n = 49)15 ( 31%)34 ( 69%)irf independent radiology facility , pfs progression - free survival , pd progressive diseasetwo patients had missing neurocognitive data and were dropped from the analysiseight patients had missing neurocognitive data and were dropped from the analysis neurocognitive changes in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study hvlt - r hopkins verbal learning test - revised , tmta trail making tests part a , tmtb trail making test part b , cowa controlled oral word association test stabilization or improvement in neurocognitive function in patients with recurrent glioblastoma receiving single - agent bevacizumab in the brain study irf independent radiology facility , pfs progression - free survival , pd progressive disease two patients had missing neurocognitive data and were dropped from the analysis eight patients had missing neurocognitive data and were dropped from the analysis in a recent retrospective analysis , 12 patients with gbm and poor neurologic function ( kps < 60% ) due to bulky disease were treated in an up - front setting with bevacizumab following induction temozolomide in an attempt to improve their ability to tolerate chemoradiation . median kps improved from 50 to 70% and their median dexamethasone dose was reduced from 12 to 4 mg / day . five of 11 evaluable patients ( 45% ) had a partial response , 4 ( 35% ) a minor response , and 1 ( 10% ) stable disease . the tumor response , rapidly improved neurologic function , and reduced steroid requirement allowed the majority of patients ( 75% ) to complete definitive radiotherapy . little significant information appears to have been published on the effects of other investigational biological agents ( e.g. , cilengitide , cediranib , sorafenib , sunitinib ) on corticosteroid use , neurocognitive function , or qol in patients with high - grade glioma or more specifically those with gbm . in a recent phase ii study of cediranib , an oral pan - vegfr tyrosine kinase inhibitor , was administered as monotherapy ( 45 mg / day ) in 31 patients with recurrent gbm and resulted in encouraging proportions of radiographic partial responses of 57 and 27% on 3- and 2-dimensional mri , respectively ) and 6-month pfs ( 26% ) . furthermore , among 15 patients receiving corticosteroids on study entry , the dose was reduced ( n = 10 ) or discontinued ( n = 5 ) . maintenance of qol in patients with high - grade glioma is an important endpoint during treatment , and more so for gbm because of the particularly poor prognosis with short life expectancy at this stage of the disease . however , reliable serial measurement of qol in patients with high - grade glioma is notoriously difficult , relating to many factors but particularly dropout bias or inability to repeatedly complete complex forms . it would appear that there is a progressive decrease in qol during the course of high - grade glioma that substantially accelerates once the disease relapses . this is also expressed as deterioration peaks driven by the administered therapies ( e.g. , radiotherapy ) or by the exacerbation of accompanying syndromes ( e.g. , brain edema , neurological symptoms , psychiatric disturbances ) . it has proven difficult to identify new therapies that extend os and pfs in patients with recurrent gbm after failure of previous therapy . most alternative cytotoxic chemotherapeutic agents do not seem to significantly impact response or survival , yet may produce adverse effects that have a likely negative impact on qol . however , among the new biological agents , bevacizumab has been shown to induce a clinically meaningful proportion of durable responses among patients with an acceptable safety profile . furthermore , data are emerging that bevacizumab induces improvement or preservation of neurocognitive function , suggestive of qol improvement , in the majority of poor - prognosis patients who would otherwise be expected to show a sudden , rapid deterioration in qol . further studies are underway to confirm these findings and better understand the natural history of the qol of these patients . adrenal myelolipoma is a tumor - like lesion composed of variable amounts of mature adipose tissue and bone marrow elements . in spite of wbc and rbc precursors such as megakaryocytes giercke in 1905 first described and 24 years later , oberling coined the term myelolipoma. only two cases under the age of 16 are reported and to the best of our knowledge , this patient is the youngest pediatric case of adrenal myelolipoma reported in the english literature . an 11-year - old girl presented with recurrent pain abdomen for 1 year , which was gradually increasing in frequency and severity for last 5 months . ultrasonography ( usg ) of abdomen showed a well - encapsulated and hypo echoic lesion in right suprarenal region measuring 27.1 24.3 mm . contrast enhanced computed tomography ( cect ) scan of abdomen confirmed the lesion to be arising from right adrenal gland with fat density measuring 1.8 2.3 cm [ figure 1 ] . gross examination of the specimen showed reddish yellow colored friable mass , soft in consistency ; and on microscopy , islands of hematopoietic tissue were found scattered among the fat cells [ figure 2 ] . cect reveal small fat containing lesion in right adrenal suggestive of myelolipoma histopathology showing fat lobules ( f ) and megakaryocyte ( m ) adrenal myelolipoma is a tumor - like lesion composed of variable amounts of mature adipose tissue and bone marrow elements . in spite of wbc and rbc precursors such as megakaryocytes myelolipoma may occur in retroperitoneum around kidney and pre - sacral space or in liver ; however , adrenal gland is the most common site . most of the cases of myelolipomas are asymptomatic . usually diagnosed incidentally at imaging for other purposes and the reported incidence are reaching up to 7% of the adrenal masses . majority of incidentally discovered myelolipomas are small and asymptomatic though large symptomatic lesions are also reported but in adult population . symptoms may be due to large tumor causing the pressure effect , tumor necrosis and intralesional hemorrhage . malignant potential of adrenal myelolipoma has not been reported . in our study , the child presented with pain abdomen and , on investigation diagnosis was confirmed . the lesion is itself hormonally inactive but may be associated with metabolically active adrenal lesion like cushing syndrome , conn 's syndrome , congenital adrenal hyperplasia etc . myelolipoma can be regarded as an exception to the mandatory metabolic work - up of a newly discovered adrenal mass . large myelolipoma usually associated with hematological disorders like hereditary spherocytosis , thalassemia intermedia , thalassemia major , sickle cell anemia and usually bilateral if we get a case of large or bilateral adrenal myelolipoma we have to investigate for chronic hematological disorders . before removal the of tumor , it may be necessary for bone marrow transplantation to avoid aggravation of hemolytic anemia . most accepted theory regarding etiologies are that it arises in response to infection , stress or necrosis causing metaplasia of reticuloendothelial cells of the capillaries of adrenal gland . focal fat density , peripheral calcification and hemorrhage within the tumor can be better identified with cect scan . myelolipoma contains adipocytes with hematopoietic elements , consisting of myeloid and erthyroid precursors , as well as , megakaryocytes . these hematopoietic elements are scattered in the adrenal tissue and separated by sheets and large clusters of mature adipocytes admixed with hemorrhagic foci . though open surgery is the classical method , laparoscopic adrenalectomy has now become the standard of care for the treatment of functioning and non - functioning adrenal tumors , introduced by gagner in 1992 . small asymptomatic myelolipomas can be treated conservatively but the symptomatic one should be treated with surgical excision and/or adrenalectomy .

the maintenance of quality of life ( qol ) in patients with high - grade glioma is an important endpoint during treatment , particularly in those with glioblastoma multiforme ( gbm ) given its dismal prognosis despite limited advances in standard therapy . it has proven difficult to identify new therapies that extend survival in patients with recurrent gbm , so one of the primary aims of new therapies is to reduce morbidity , restore or preserve neurologic functions , and the capacity to perform daily activities . apart from temozolomide , cytotoxic chemotherapeutic agents do not appear to significantly impact response or survival , but produce toxicity that is likely to negatively impact qol . new biological agents , such as bevacizumab , can induce a clinically meaningful proportion of durable responses among patients with recurrent gbm with an acceptable safety profile . emerging evidence suggests that bevacizumab produces an improvement or preservation of neurocognitive function in gbm patients , suggestive of qol improvement , in most poor - prognosis patients who would otherwise be expected to show a sudden and rapid deterioration in qol . myelolipoma is a rare benign tumor of adrenal gland and rarer in children . myelolipoma contains adipose tissue and myeloid precursor producing white blood cells ( wbc ) , red blood cells ( rbc ) and megakaryocytes . asymptomatic tumor does not require treatment whereas symptomatic tumor needs operation . we are reporting a rare adrenal myelolipoma in a child with review of literature .

nutcracker syndrome is generally referred to as the compression of the left renal vein between the superior mesenteric artery and aorta ( 1 ) . this compression leads to lrv hypertension and results in direct communication of the renal vein with the calyceal cavity and the varicosities of the collateral pathway of the lrv ( 2 ) . renal varicosities or abnormal branching of the superior mesenteric artery from the aorta are the most common factors considered responsible for this condition ( 1 , 2 ) . the other causes , such as compression of the retroaortic lrv between the aorta and spine ( which is referred to as ' posterior ncs ' in contrast to the more common ' anterior ncs ' ) or aberrant right renal artery constricting the lrv , are uncommon ( 3 , 4 ) . however , three recently reported cases of nutcracker phenomenon associated with the left inferior vena cava ( livc ) suggest that some anomalies of the inferior vena cava ( ivc ) can also impair lrv blood outflow and result in ncs ( 4 - 6 ) . here we reported a case of complicated malformation of livc with interrupted ivc and azygos continuation and retroaortic right renal vein causing posterior ncs , which has been diagnosed by both color doppler ultrasound and computed tomographic angiography ( cta ) . to the best of our knowledge , this is the first report of an association between a complicated anomaly of the livc and ncs . a 42-year - old female patient was referred to our hospital for renal ultrasonographic scanning . she had a past medical history of intermittent microscopic hematuria or clots for nearly 10 years ' duration . the morphology of urinary red cells by phase - contrast microscopy revealed 53% dysmorphic cells . however , oblique transverse scanning of the upper abdomen showed two tubular structures on both sides of the abdominal aorta above the level of the renal vein . we traced the vessels in an effort to scrutinize every segment of these two tubular structures and found that the right common iliac vein crossed the vertebra and formed the left - sided infrarenal ivc ( l - ivc ) along with the left common iliac vein . the right renal vein ( rrv ) traveled retroaortically and joined the l - ivc ( fig . the post - anterior diameter of the hilar portion and that of the retroaortic stenotic portion of the rrv were 8.8 mm and 2.0 mm , respectively , revealed by grey - scale ultrasound , which , with peak velocities , was demonstrated by a spectral doppler of 26 cm / s and 87 cm / s , respectively . the diameter ratio and the peak velocity ratio between the dilated portion and the stenotic portion were 4.4 and 3.4 , respectively . after collecting blood from the right and left common iliac veins and the renal veins , the l - ivc crossed the midline retroaortically and flowed into the right - sided suprarenal ivc ( r - ivc ) with turbulent , high - velocity flow ( fig . the diameters of the l - ivc at the level of the lrv and the dilated portion of the lrv near the hilus and the diameter of the ivc at the stenotic portion were 18.6 mm , 14.3 mm and 2.3 mm , respectively , with the peak velocities of 30 cm / s , 27 cm / s and 149 cm / s , respectively . correspondingly , the diameter ratio between the dilated portion of lrv and the stenotic portion of l - ivc was 6.2 , accompanied by the peak velocity ratio of 5.5 . 1d ) . below the level of the rrv , no tubular structure was found at the right lateral portion of the aorta . the results demonstrated that the rrv traveled retroaortically to the l - ivc . after collecting blood from the right and left common iliac veins and the renal veins , the l - ivc traversed retroaortically to join the r - ivc above the level of the celiac artery . the r - ivc ascended with the azygos instead of joining to the hepatic vein . in addition , paravertebral collateral veins in the right lumbar region traveled upward tortuously and drained into the r - ivc ( fig . 1e - g ) . the findings of both color doppler ultrasound and cta revealed that the anomaly of the ivc in our patient was livc with continuation of the azygos , existence of a retroaortic rrv , and absence of the posthepatic segment of the ivc ( fig . cystoscopy was performed to evaluate otherwise asymptomatic episodes of microscopic hematuria , which demonstrated no positive findings . analysis the data obtained by image findings , together with the patient 's typical history and other laboratory findings , she was diagnosed as the posterior ncs associated with a complicated malformation of the livc . the normal ivc is composed of four segments : hepatic , suprarenal , renal , and infrarenal . its evolutionary process involves the development , regression , and anastomosis of three pairs of fetal veins : the posterior cardinal veins , subcardinal veins , and supracardinal veins , in the embryonic life during the sixth to eighth week of gestation ( 7 ) . the livc is the result of the persistence of the left supracardinal vein with regression of the right supracardinal vein . moreover , this case was more complicated because of the continuation with the azygos , the existence of the retroaortic rrv , and the absence of the posthepatic segment of the ivc . consequently , the pathology may be accompanied by the atrophy of the right subcardinal vein , the persistence of the dorsal limb as well as the regression of the ventral limb of the right renal collar , and the failure to form the right subcardinal - hepatic anastomosis ( 7 ) . furthermore , interruption of the ivc with azygos continuation is often associated with congenital heart disease , polysplenia , and less commonly , asplenia . in our patient no congenital heart diseases or other anomalies were found , so we have reason to believe that this condition is extremely rare . diagnosis of the anomalies of ivc may have significant clinical implications , especially during retroperitoneal surgery or in the treatment of thromboembolic diseases ( 5 - 7 ) . the majority of abnormal ivcs are clinically asymptomatic and only recognized incidentally during radiologic investigations performed for other reasons or during surgeries on the aorta or retroperitoneal structures or during postmortem examinations . in our literature review , we found only three cases of the anomalies of the ivc presenting as ncs . ( 5 ) first described data on an 80-year - old woman , who had a liver metastatic adenocarcinoma , and was incidentally found to have the anomaly of the livc . the authors believed that the compression of the livc between the aorta and superior mesenteric artery was responsible for her hematuria , a symptom of ncs . regardless of the various causes of ncs , an outlet obstruction of the lrv is the outcome of ncs . its typical symptoms , such as hematuria , left flank and abdominal pain , and varicoceles , are closely related to a backward venous renal hypertension due to the outflow impairment of the lrv and the development of compensating collateral circulation . in our present case , the aberrant vessels of the ivc resulted in two potential entrapments of the renal vein : 1 ) the pathway where the l - ivc drains into the r - ivc between the aorta and vertebra indirectly causing the hypertension of the lrv and 2 ) the location where the rrv runs to the l - ivc retroaortically . the data obtained by sonography revealed that the ratios of the diameter and of the peak velocity in rrv between the dilated portion and the stenotic portion were 4.4 and 3.4 , respectively , whereas those between the dilated segment of lrv and the narrowest segment of l - ivc were 6.2 and 5.5 , respectively . ct angiography also demonstrated the existence of obvious dilatation of lrv , which we believed to be the main factor of the occurrence of the hematuria . in addition , the distension of the lrv and tortuously paravertebral collateral veins in the right lumbar region could explain the patient 's feeling of a continuous dull lumbago for a long time . clinically , the diagnosis of ncs is based on the assessment of the pathologic anatomy and physiology ( 8) . the newer imaging modalities such as cta and magnetic resonance angiography have powerful abilities in providing accurate , three - dimensional reconstructive images in any desired plane and allowing better identification of these anomalies . spectral doppler ultrasound can give more details of the hemodynamics of the lrv at the site of compression . the case we presented here was first diagnosed by color doppler ultrasound and then identified by cta scanning with a high - quality delineated picture of the anomaly of the ivc . the dilated lrv as well as the l - ivc draining into the r - ivc in our patient were clearly visualized by ct and color doppler ultrasound . kim 's study showed that the cutoff values for ncs as the ratios of both antero - posterior diameter and peak velocity between the distended and narrowed segments of the lrv are greater than 5 , with sensitivity of 69% , 80% and specificity of 89% , 94% , respectively . but when combined with the cutoff value of more than 5 of the ratio of the antero - posterior diameter and the ratio of peak velocities divided by 2 , the sensitivity and the specificity can be elevated to 90% and 100% , respectively ( 9 ) . in our case , both the diameter ratio and the peak velocity ratio between the dilated segment of lrv and the narrowest segment of l - ivc were greater than 5 with a combined value of 5.9 . moreover , the morphology of urinary red cells is less than 75% , which is thought to be non - specific in nature ( 10 ) . in review of our patient 's typical history and clinical and radiologic findings , the diagnosis was most likely posterior ncs associated with complicated malformation of the livc . in conclusion , a congenital anomaly of the ivc , although rare , may be one of the contributing factors for the development of ncs . with the aid of ultrasonography and the newer cross - sectional imaging modalities , variant venous anatomy should be identified and its clinical significance , which had not been mentioned in previous reports , should be evaluated thoroughly . since the international subarachnoid trial , coil embolization of aneurysms is now accepted as a primary optional treatment19 ) . however , endovascular treatment ( evt ) of middle cerebral artery ( mca ) aneurysms has been less often applied in many institutions because of the feasibility of surgical clipping afforded by the superficial anatomy of mca aneurysms and favorable outcomes8,24 ) . a large series of evt of mca aneurysms was recently reported with favorable results and a low recurrence rate4,11,27 ) , but evt of wide - necked and fusiform mca aneurysms remains still challenging . after self - expandable stents became available , many of complex aneurysms have been treated by coil embolization1,3,17,18,21,23 ) . to our knowledge , there are a few reports regarding stent - assisted evt of mca aneurysms , but they treated complex mca aneurysms with one self expandable intracranial stent6,26,29 ) . the aim of this study was to evaluate the feasibility and clinical and angiographic outcomes of stent - assisted coiling for complex mca aneurysms using more than one stent . the institutional review board of our hospital approved this retrospective study , and the requirement for written informed consent was waived . we treated 101 mca aneurysms with coil embolization in 98 patients between april 2006 and august 2011 . twenty - four aneurysms in 23 patients were treated with stent - assisted coil embolization . our study involved 13 males and 10 females , and the mean age of the patients was 56.9 years ( range , 42 - 70 years ) . nineteen of 24 were incidental aneurysms that had not been treated , and two were recanalized aneurysms previously coiled , and two were recurred after surgical clipping . the number of unruptured aneurysms were 23 and ruptured aneurysm was only one which was ruptured after surgical clipping . the mean size of the aneurysms was 7 mm ( range , 3 - 13.4 mm ) , and mean size of the aneurysm neck was 6.2 mm ( range , 2.4 - 12.7 mm ) . the mean sac - to - neck ratio ( snr ) was 1.02 ( range , 0.53 - 1.45 ) and the mean height - to - neck ratio was 0.75 ( range , 0.48 - 1.3 ) . all aneurysms had wide neck and/or shallow depths , and six aneurysms were " ultra - wide necked " circumferential aneurysm which is involving more than 180 degree of parent artery lumen with no visible neck . in circumferential five aneurysms had incorporated parent vessel branch . there were 17 aneurysms in mca bifurcation , 3 proximal inferior divisional branch , 2 distal m1 , and one proximal superior divisional branch . patients were premedicated with 75 mg of clopidogrel ( plavix ; bristol - myers squibb , new york , ny , usa ) and 100 mg of aspirin for 5 - 7 days . if antiplatelet premedication was not done , on a day before treatment , patients were premedicated with a loading dose of clopidogrel ( 300 mg ) and aspirin ( 100 mg ) . after coiling , patients were then kept under dual antiplatelet therapy daily for 6 months ( clopidogrel , 75 mg , and aspirin , 100 mg ) followed by aspirin daily alone for 12 months . in ruptured aneurysm , all procedures were performed using a monoplane and biplane angiographic system ( integris v-5000 or allura xper , philips healthcare , best , the netherlands ) with the patient under local anesthesia . anticoagulation was initiated by injection of a bolus of 3000 to 5000 iu heparin intravenously at the beginning of the procedure , followed by continuous infusion of heparin at the rate of 1000 iu / h . activated coagulation time was maintained between two and three times the baseline value during and for the next 24 to 48 hours after the procedure . in one ruptured aneurysm , a bolus of 3000 iu heparin was injected intravenously after two coils were inserted in the aneurysm , and followed by same heparinization . endovascular coiling was performed via a single femoral route in all cases with an envoy 6 f ( cordis endovascular systems , miami lakes , fl , usa ) or a shuttle 6 f guiding catheter ( cook , inc . , after completion of coiling , a closing device ( angioseal ; st jude medical , st paul , mn , usa , or perclose ; abott , diegem , belgium ) was used to seal the access site in all cases . we initially tried to treat all complex aneurysms using single or double microcatheter or balloon - assisted techniques without stent - assistance . if parent artery or branching vessel is compromised by frame coils or coil frame is unstable , we performed stent - assisted coiling during same procedure . fifteen aneurysms were treated using one self - expandable intracranial stent ( neuroform stent , boston scientific , natick , ma , usa ; or enterprise self - expanding stent , cordis , miami lakes , fl , usa ) . a stent placed into the parent vessel , bridging the aneurysm neck . stent placement was performed either before coiling ( taking advantage of the open cell design of neuroform stents ) or with the jailing technique ( using enterprise stents ) when the size of the artery allowed safe navigation of two simultaneous microcatheters . in selected cases , the stent was deployed after coiling at the end of the procedure with the aim of securing the parent artery . six aneurysms were treated with a stent - within - a - stent ( overlapping two stents ) when aneurysm treatment was incomplete with one stent due to very wide neck or fusiform aneurysm for more insertion of coils and effective flow diversion . the stent - within - a - stent technique involves stent - assisted coiling followed by overlapping stent(s ) . a 0.021-inch microcatheter is navigated across the aneurysm neck portion to a distal branch of the parent artery . a second microcatheter is inserted into the aneurysm sac , and the first enterprise stent is deployed , bridging the aneurysm neck . after the first stent is deployed , the microcatheter is re - advanced to the initial position over the stent - loading wire , which is left in situ within the deployed stent . when the first deployed stent was neuroform stent , a micorwire was re - advanced through the deployed stent into the m2 branch and the microcatheter was repositioned in the distal portion of the first deployed stent . then coil embolization is performed as compactly as possible , including the aneurysm neck portion , which encompasses the parent artery more than 180 degrees . finally , a second enterprise stent is introduced and is deployed in an overlapping manner . two mca bifurcation aneurysms were treated with y - stent if one stent was insufficient for covering the aneurysm neck which was involving both branches . a microcatheter was navigated into the mca bifurcation aneurysm with or without frame coiling and another microcatheter was placed in the distal superior or inferior divisional branch at the most acute angle from the mca bifurcation . a 021 prowler select plus microcatheter was placed at the distal mca through a long exchangeable wire , and the first stent was deployed from the m2 and the m1 of mca . the second stent was navigated through the interstices of the initial stent with its distal end in the another bifurcation branch of mca and the proximal portion overlapping the first stent in the m1 of mca . coiling was performed using axium coils ( ev3 , plymouth , ma , usa ) , guglielmi detachable coils and target coils ( boston scientific , natick , ma , usa ) , hydrocoils ( microvention , aliso viejo , ca , usa ) , or a combination of coils . in the event of a periprocedural thromboembolic complication , tirofiban ( aggrastat , medicure pharma , somerset , nj , usa ) was slowly injected intra - arterially as a loading dose ( 500 mcg/10 ml ) and then administered intravenously , at an initial rate of 0.4 mcg / kg / min for 30 minutes and then continued at 0.1 mcg / kg / min . the clinical outcomes of patients were evaluated during hospitalization after procedure and then re - evaluated at the first time of angiographic control , which was generally performed at 6 - 12 months by dsa . additional controls by mra were then scheduled at 2 years , 3 years , and 5 years . in our institution , the mra sequences ( time - of - flight ) were analyzed by using source images ; maximum - intensity - projection and contrast enhanced mra images were irrelevant due to major artifacts caused by the stents . in cases of unreliable mr imaging findings , dsa was performed . morbidity - mortality rates and angiographic results were retrospectively analyzed at initial and midterm follow - ups . we used the raymond classification scale22 ) to compare the initial and midterm angiographic results . we defined the midterm results as follows : 1 ) a stable result showed no change from the initial results ; 2 ) the improved state demonstrated further occlusion of the remnant neck or residual aneurysm ; 3 ) a minor recurrence revealed no need for retreatment ( aneurysms changed from the class 1 to class 2 ) ; and 4 ) a major recurrence required requiring retreatment ( aneurysms changed from class 1 to class 3 or from class 2 to class 3 ) . the mean size of the aneurysms was 7 mm ( range , 3 - 13.4 mm ) , and mean size of the aneurysm neck was 6.2 mm ( range , 2.4 - 12.7 mm ) . the mean sac - to - neck ratio ( snr ) was 1.02 ( range , 0.53 - 1.45 ) and the mean height - to - neck ratio was 0.75 ( range , 0.48 - 1.3 ) . all aneurysms had wide neck and/or shallow depths , and six aneurysms were " ultra - wide necked " circumferential aneurysm which is involving more than 180 degree of parent artery lumen with no visible neck . in circumferential aneurysm , five aneurysms had incorporated parent vessel branch . there were 17 aneurysms in mca bifurcation , 3 proximal inferior divisional branch , 2 distal m1 , and one proximal superior divisional branch . patients were premedicated with 75 mg of clopidogrel ( plavix ; bristol - myers squibb , new york , ny , usa ) and 100 mg of aspirin for 5 - 7 days . if antiplatelet premedication was not done , on a day before treatment , patients were premedicated with a loading dose of clopidogrel ( 300 mg ) and aspirin ( 100 mg ) . after coiling , patients were then kept under dual antiplatelet therapy daily for 6 months ( clopidogrel , 75 mg , and aspirin , 100 mg ) followed by aspirin daily alone for 12 months . in ruptured aneurysm , all procedures were performed using a monoplane and biplane angiographic system ( integris v-5000 or allura xper , philips healthcare , best , the netherlands ) with the patient under local anesthesia . anticoagulation was initiated by injection of a bolus of 3000 to 5000 iu heparin intravenously at the beginning of the procedure , followed by continuous infusion of heparin at the rate of 1000 iu / h . activated coagulation time was maintained between two and three times the baseline value during and for the next 24 to 48 hours after the procedure . in one ruptured aneurysm , a bolus of 3000 iu heparin was injected intravenously after two coils were inserted in the aneurysm , and followed by same heparinization . endovascular coiling was performed via a single femoral route in all cases with an envoy 6 f ( cordis endovascular systems , miami lakes , fl , usa ) or a shuttle 6 f guiding catheter ( cook , inc . , after completion of coiling , a closing device ( angioseal ; st jude medical , st paul , mn , usa , or perclose ; abott , diegem , belgium ) was used to seal the access site in all cases . we initially tried to treat all complex aneurysms using single or double microcatheter or balloon - assisted techniques without stent - assistance . if parent artery or branching vessel is compromised by frame coils or coil frame is unstable , we performed stent - assisted coiling during same procedure . fifteen aneurysms were treated using one self - expandable intracranial stent ( neuroform stent , boston scientific , natick , ma , usa ; or enterprise self - expanding stent , cordis , miami lakes , fl , usa ) . stent placement was performed either before coiling ( taking advantage of the open cell design of neuroform stents ) or with the jailing technique ( using enterprise stents ) when the size of the artery allowed safe navigation of two simultaneous microcatheters . in selected cases , the stent was deployed after coiling at the end of the procedure with the aim of securing the parent artery . six aneurysms were treated with a stent - within - a - stent ( overlapping two stents ) when aneurysm treatment was incomplete with one stent due to very wide neck or fusiform aneurysm for more insertion of coils and effective flow diversion . the stent - within - a - stent technique involves stent - assisted coiling followed by overlapping stent(s ) . a 0.021-inch microcatheter is navigated across the aneurysm neck portion to a distal branch of the parent artery . a second microcatheter is inserted into the aneurysm sac , and the first enterprise stent is deployed , bridging the aneurysm neck . after the first stent is deployed , the microcatheter is re - advanced to the initial position over the stent - loading wire , which is left in situ within the deployed stent . when the first deployed stent was neuroform stent , a micorwire was re - advanced through the deployed stent into the m2 branch and the microcatheter was repositioned in the distal portion of the first deployed stent . then coil embolization is performed as compactly as possible , including the aneurysm neck portion , which encompasses the parent artery more than 180 degrees . finally , a second enterprise stent is introduced and is deployed in an overlapping manner . two mca bifurcation aneurysms were treated with y - stent if one stent was insufficient for covering the aneurysm neck which was involving both branches . a microcatheter was navigated into the mca bifurcation aneurysm with or without frame coiling and another microcatheter was placed in the distal superior or inferior divisional branch at the most acute angle from the mca bifurcation . a 021 prowler select plus microcatheter was placed at the distal mca through a long exchangeable wire , and the first stent was deployed from the m2 and the m1 of mca . the second stent was navigated through the interstices of the initial stent with its distal end in the another bifurcation branch of mca and the proximal portion overlapping the first stent in the m1 of mca . coiling was performed using axium coils ( ev3 , plymouth , ma , usa ) , guglielmi detachable coils and target coils ( boston scientific , natick , ma , usa ) , hydrocoils ( microvention , aliso viejo , ca , usa ) , or a combination of coils . in the event of a periprocedural thromboembolic complication , tirofiban ( aggrastat , medicure pharma , somerset , nj , usa ) was slowly injected intra - arterially as a loading dose ( 500 mcg/10 ml ) and then administered intravenously , at an initial rate of 0.4 mcg / kg / min for 30 minutes and then continued at 0.1 mcg / kg / min . the clinical outcomes of patients were evaluated during hospitalization after procedure and then re - evaluated at the first time of angiographic control , which was generally performed at 6 - 12 months by dsa . additional controls by mra were then scheduled at 2 years , 3 years , and 5 years . in our institution , the mra sequences ( time - of - flight ) were analyzed by using source images ; maximum - intensity - projection and contrast enhanced mra images were irrelevant due to major artifacts caused by the stents . in cases of unreliable mr imaging findings , dsa was performed . morbidity - mortality rates and angiographic results were retrospectively analyzed at initial and midterm follow - ups . we used the raymond classification scale22 ) to compare the initial and midterm angiographic results . we defined the midterm results as follows : 1 ) a stable result showed no change from the initial results ; 2 ) the improved state demonstrated further occlusion of the remnant neck or residual aneurysm ; 3 ) a minor recurrence revealed no need for retreatment ( aneurysms changed from the class 1 to class 2 ) ; and 4 ) a major recurrence required requiring retreatment ( aneurysms changed from class 1 to class 3 or from class 2 to class 3 ) . a total of 23 patients having 24 mca aneurysms were eligible for evt with the stent assisted coiling technique . treatment techniques , initial and follow - up angiographic outcomes , and treatment related complication after stent assisted coiling of mca aneurysms are shown in table 1 . fifteen aneurysms were treated by the use of a single stent , six aneurysms by a stent - within - a - stent procedure , and two using the y - stent technique . twenty - three aneurysms were successfully treated with a stent - assisted technique ( 95.9% ) and one aneurysm embolization was failed due to an inability to catheterize the inferior divisional branch due to acute angle and complex geometry . in - stent thrombosis was noted in five patients ( 20.8% ) with symptomatic thromboembolism in two patients , and they showed complete or nearly complete resolution of the thrombi after intra - arterial and intravenous administration of glycoprotein iib / iiia inhibitor ( tirofiban , 12.5 to 25 mg ) . two symptomatic patients with dysarthria and left side weakness showed complete recovery without permanent deficit . one aneurysm was treated with single stent technique and achieved complete embolization of the mca bifurcation aneurysm , but the superior divisional branch was occluded due to in - stent thrombosis . another stent was deployed within the stent after 20 minutes following partial recanalization of the occlusion by tirofiban injection and immediate control angiogram showed complete recanalization of the superior divisional branch ( fig . immediate thromboembolic complication was seen in 5 patients ( 20.8% ) during or after procedure showing motor weakness ( grade ii - iv ) , dysarthria , and facial weakness . two symptomatic thromboembolisms were resolved by tirofiban injection and one left side weakness was disappeared after conservative treatment . permanent morbidity was noted in 2 patients ( 8.3% ) with partial aphasia and grade iii motor weakness [ ( modified rankin scale ( mrs ) 1 and 2 ] . periprocedural thromboembolic complication was noted in 3 single stent - assisted coiling , 1 a stent - within - a - stent technique , and 1 y - stent technique . intraprocedural aneurysm rupture occurred in one patient who was treated with a single - stent assisted technique . the rupture was immediately controlled by the insertion of further coils and left no sequelae except for transient post - procedural headaches . postembolization control angiograms revealed complete occlusion in 15 ( 65.2% ) , residual neck in 5 ( 21.7% ) , and a residual aneurysm in 3 ( 13.1% ) patients . clinical follow - up was available in all patients ( mean , 28 months ; range , 5 - 60 months ) . there were no cases of sah and no deaths by the recurrence of the coiled aneurysms . none of the surviving 23 patients had any additional deterioration of functional neurological outcomes ( mrs , 0 - 2 ) . follow - up angiography was available in 17 of the 23 treated aneurysms ( 73.9% ) by using either mra ( n=5 , 29.4% ) or dsa ( n=12 , 70.6% ) at a mean period of 13.2 months ( 6 - 31 months ) . an artifact of the stent disturbed mra interpretation in two cases for which dsa was performed . one major recurrent aneurysm treated by the stent - within - a - stent technique was retreated using a stent - assisted technique without any complications ( fig . fifteen aneurysms were treated by the use of a single stent , six aneurysms by a stent - within - a - stent procedure , and two using the y - stent technique . twenty - three aneurysms were successfully treated with a stent - assisted technique ( 95.9% ) and one aneurysm embolization was failed due to an inability to catheterize the inferior divisional branch due to acute angle and complex geometry . in - stent thrombosis was noted in five patients ( 20.8% ) with symptomatic thromboembolism in two patients , and they showed complete or nearly complete resolution of the thrombi after intra - arterial and intravenous administration of glycoprotein iib / iiia inhibitor ( tirofiban , 12.5 to 25 mg ) . two symptomatic patients with dysarthria and left side weakness showed complete recovery without permanent deficit . one aneurysm was treated with single stent technique and achieved complete embolization of the mca bifurcation aneurysm , but the superior divisional branch was occluded due to in - stent thrombosis . another stent was deployed within the stent after 20 minutes following partial recanalization of the occlusion by tirofiban injection and immediate control angiogram showed complete recanalization of the superior divisional branch ( fig . immediate thromboembolic complication was seen in 5 patients ( 20.8% ) during or after procedure showing motor weakness ( grade ii - iv ) , dysarthria , and facial weakness . two symptomatic thromboembolisms were resolved by tirofiban injection and one left side weakness was disappeared after conservative treatment . permanent morbidity was noted in 2 patients ( 8.3% ) with partial aphasia and grade iii motor weakness [ ( modified rankin scale ( mrs ) 1 and 2 ] . periprocedural thromboembolic complication was noted in 3 single stent - assisted coiling , 1 a stent - within - a - stent technique , and 1 y - stent technique . intraprocedural aneurysm rupture occurred in one patient who was treated with a single - stent assisted technique . the rupture was immediately controlled by the insertion of further coils and left no sequelae except for transient post - procedural headaches . postembolization control angiograms revealed complete occlusion in 15 ( 65.2% ) , residual neck in 5 ( 21.7% ) , and a residual aneurysm in 3 ( 13.1% ) patients . clinical follow - up was available in all patients ( mean , 28 months ; range , 5 - 60 months ) . there were no cases of sah and no deaths by the recurrence of the coiled aneurysms . none of the surviving 23 patients had any additional deterioration of functional neurological outcomes ( mrs , 0 - 2 ) . follow - up angiography was available in 17 of the 23 treated aneurysms ( 73.9% ) by using either mra ( n=5 , 29.4% ) or dsa ( n=12 , 70.6% ) at a mean period of 13.2 months ( 6 - 31 months ) . an artifact of the stent disturbed mra interpretation in two cases for which dsa was performed . one major recurrent aneurysm treated by the stent - within - a - stent technique was retreated using a stent - assisted technique without any complications ( fig . since the advent of the detachable coil , endovascular coil embolization has been an important treatment method for both ruptured and unruptured aneurysms . however , coil embolization of aneurysms with wide neck and complex geometry remains a challenging procedure despite the use of several techniques such as balloon - assisted20,30 ) , double2 ) or multiple microcatheter12 ) , microcatheter protective15 ) , coil protective , and stent - assisted techniques1,3,6,7,17,18,21,23,26,29 ) . ever since the advent of self - expandable intracranial stents for coil embolization of aneurysms , especially closed cell design stent ( enterprise and leo stent , balt co. , montmorency , france ) , stent - assisted embolization has been widely used due to ease of deployment of the stent and high success rates10 ) . recently , many of complex aneurysms is treated by stent - assisted technique rather than balloon - assisted and multiple microcatheter techniques . stent - assisted embolization of the complex mca aneurysms was feasible , safe and durable , and could be considered as an alternative treatment26 ) . however , surgical clipping of mca aneurysms is preferred in many institutions due to relative ease and effectiveness of aneurysm clipping . the initial complete or near - complete embolization rate in our series ( 65.2% ) was similar to other studies ( 67 - 71%)6,26,29 ) . we performed the jailing technique in 17 aneurysms and repositioning of the microcatheter through the strut of the stent in the remnant neck or aneurysm during procedure if the jailed microcatheter was kickback in the parent artery . a stent - within - a - stent technique in six aneurysms allowed more compact packing and efficient flow diversion than single stent - assisted embolization . flow diverter might be used , especially in the m1 aneurysm without branching parent vessels , but we could not use them because those stents were not available in our country . in the present study , in - stent thrombosis ( 20.8% ) and the symptomatic thromboembolic complication rate ( 20.8% ) were high despite the use of dual antiplatelet therapy . we did n't clopidogrel and aspirin resistance test and high thromboembolic complication rate may be antiplatelet resistance . we also think that our initial high complication rate may arise from more complex procedures such as a stent - within - a - stent or y - stent technique than single stent - assisted technique . the permanent morbidity and mortality rates were decreased as result of complete thrombolysis by immediate tirofiban injection and leaved two mild disabilities ( mrs 1 and 2 ) . however , this morbidity - mortality rate ( 8.3% ) was higher than those of other series ( 0 - 12% ) ( table 2 ) and may not be acceptable for treatment method . during recent 3 years , 10 aneurysms was treated and showed no permanent morbidity and one immediate symptomatic complication . in this result , experience of neurointerventionist and careful case selection are important factors for reducing complication rate of stent - assisted coil embolization of the mca aneurysms . the recurrence rate for stent - assisted coil embolization of the aneurysms was relatively high in other studies ( non - stenting ) , ranging from 14.6% to 29%4,11,27 ) , and our results were either comparable ( 11.8% ) with previous stent - assisted coil embolization studies ( table 2 ) . coil embolization with a stent - within - a - stent technique is a feasible and reconstructive treatment option for ruptured dissecting aneurysms25 ) and effectively prevents rebleeding and regrowth of ruptured blood blister - like aneurysms without requiring sacrifice of the internal carotid artery14 ) . therefore , we expected high packing density and lower recurrence rates for large and wide - necked aneurysms while preserving the parent artery , especially ultra - wide necked circumferential mca aneurysms . in addition to the neck covering role of the stent - assisted technique during embolization , advantages of a stent - within - a - stent technique included prevention of possible protrusion of the coils into the parent artery after first stenting , coil insertion of a very wide - necked circumferential aneurysms due to poor discrimination of the parent artery and neck of the aneurysm due to complex anatomic geometry . although a down - the - barrel view may help to differentiate the parent artery lumen from coils8 ) , it is not always available as a working projection . another advantage is prevention of the recurrence of incomplete embolization of the aneurysm due to more effective flow diversion than the single stent , resultant aneurysmal thrombosis , and eventual endothelialization around the neck5,16,28 ) . finally , blood flow may be compromised due to in - stent thrombosis or coil protrusion within the parent vessel during coiling , so a second stent can easily restore blood flow through the stented parent artery if tirofiban injection could not recanalized sufficiently ( fig . lawson et al.13 ) reported that stent - assisted coiling causes the progression of occlusion and complete thrombosis of incompletely coiled aneurysms , possibly by a flow remodeling effect , and is therefore associated with lower rates of aneurysm recurrence . there was also a larger series ( 216 aneurysms ) that show a statistically significant lower recurrence rate of stenting ( 14.9% ) compare to coiling ( 33.5%)21 ) . hwang et al.9 ) insisted their study did not show that additional hemodynamic and biologic effects of stents designed for neck remodeling were sufficient to enhance progressive occlusion and to prevent the recanalization of unruptured aneurysms . their findings also suggested that stent placement provides no better long - term angiographic outcomes for unruptured aneurysms with an unfavorable configuration for coiling as compared with nonstent - assisted ( multiple microcatheter technique or balloon - assisted technique ) coil embolization groups . we believe that selection bias between the stenting and non - stenting groups in each study that were non - randomized and retrospective study , caused the discrepancies in results . we also believe that the most important factor for lower recanalization rate was complete embolization and compact packing of the aneurysms irrespective of the techniques used . another limitation was that we included no comparable non - stenting group or surgical clipping group with same geometry of the aneurysms because unruptured mcas are not treated by surgical clipping at our hospital . our limited number of angiographic follow up cases and limited follow up times were another major limitation . mca aneurysms with wide neck or fusiform could be treated by stent - assisted coiling with more than one stent , and showed good initial angiographic outcomes and lower recanalization rate during mid - term follow - up by effective flow diversion . our results warrant further study with a longer follow - up period in a larger sample .

various anatomic anomalies have been considered the causes of nutcracker syndrome ( ncs ) . posterior ncs refers to the condition , in which vascular narrowing was secondary to the compression of the retroaortic left renal vein while it is crossing between the aorta and the vertebral column . here , we report an unusual case of posterior ncs associated with a complicated malformation of the interrupted left inferior vena cava with azygos continuation and retroaortic right renal vein , diagnosed by both color doppler ultrasonography and ct angiography . objectiveto evaluate the feasibility and clinical and angiographic outcomes of stent - assisted embolization for complex middle cerebral artery ( mca ) aneurysms.methodsthe records of 23 consecutive patients with 24 mca aneurysms , who underwent stent - assisted embolization of the aneurysm , were retrospectively evaluated.resultsfifteen aneurysms were treated with one stent and 8 were treated using more than two stents ( 5 a stent - within - a - stent , 1 triple stents , and two y - stent ) . angiographically , complete or near complete occlusion was achieved in 15 aneurysms ( 65.2% ) , residual neck in five ( 21.7% ) , and residual aneurysm in three ( 13.1% ) . five aneurysms demonstrated thrombosis within the stent during the procedure and hospitalization , and were resolved by intraarterial and intravenous tirofiban injection . symptomatic thromboembolic complications were developed in five patients and permanent deficits demonstrated in two patients with modified rankin scale 1 and 2 , respectively . treatment - related permanent morbidity and mortality rates were 8.3% and 0% with relatively high complication rate . angiographic follow - up was available in 17 aneurysms at 6 - 31 months ( mean , 13.2 months ) and showed stable or improved in 15 ( 88.2% ) and major and minor recurrence in one , respectively.conclusioncomplex mca aneurysms could be treated by stent - assisted coiling and showed lower recanalization rate during mid - term follow - up by effective flow diversion due to various stent - assisted techniques . our results warrant further study with a longer follow - up period in a larger sample .

co2mnsi samples were prepared and investigated completely in situ in an ultrahigh vacuum cluster consisting of sputtering chambers , an molecular beam epitaxy ( mbe ) chamber , and a srups chamber equipped with a he gas discharge lamp ( h=21.2 ev ) and a hemispherical energy analyzer with multi - channel spin filter27 ( energy resolution 400 mev , sherman function s=0.420.05 ( ref . first , an epitaxial buffer layer of the heusler compound co2mnga ( 30 nm ) was grown on the mgo(100 ) substrate by radio frequency ( rf)-sputtering at room temperature . by an optimized additional annealing process at 550 c l21 order is obtained as shown by high energy electron diffraction ( rheed ) and x - ray diffraction ( xrd ) . . induced by the buffer layer the co2mnsi thin films show already some degree of l21 surface order as deposited . by additional annealing the order is improved as demonstrated for the film surface by rheed ( fig . a low annealing temperature of ta=300c results in a significantly increased intensity of the characteristic rheed l21 superstructure peaks . however , by xrd no ( 111 ) peak , which is indicative for l21 order , is observed for ta<400 c . this suggests that l21 order is present at the film surface , but not in the bulk of the thin film . for ta400 c the ( 111 ) peak appears in xrd . for ta500 c some ga from the buffer layer is observed by core - level haxpes to have diffused to the co2mnsi surface . the magnetic moments of all samples amount to 5 b per formula unit at 4 k and is reduced by 3% at room temperature , in agreement with theoretical predictions and experimental values measured on bulk samples29 . figure 2 shows in situ ups spectra of co2mnsi thin films annealed at different temperatures ta without spin analysis . the large acceptance angle of the spectrometer ( 10 ) and applied sample bias voltage of 10 v result in k|| values which cover the complete brillouin zone . the spectra of all samples are almost identical , only the broad hump at eef=2,900 mev vanishes and the peak at eef=1,150 mev is slightly broadened for the deposited and the ta=550 c sample . however , by spin analysis clear differences between the samples are revealed . figure 3 shows the spin polarization of mgo / co2mnga(30 nm)/co2mnsi(70 nm ) thin films annealed at different temperatures ta as measured by srups . a huge room temperature spin polarization of 9093% at the fermi energy at room temperature was obtained for samples annealed between 300 c and 450 c . in combination with the ups calculations discussed below , these exceptionally high values are the first direct observation of half - metallicity in the surface region of any heusler compound , which provide strong evidence for 100% spin polarization in the bulk of the thin films . with lower annealing temperatures the spin polarization is reduced at ef and slightly increased at higher binding energies , which can be explained by an energy broadening of the electronic states owing to reduced structural order . with higher annealing temperatures ups is a surface sensitive method and thus the results can not be directly associated with electronic bulk band structure properties . however , as will be shown below , band structure based calculations of photoemission spectra provide this link . as additional experimental input for such calculations , a comparison of spin - integrated ex situ haxpes with a photon energy of 6 kev of alox capped ( oxidation protection ) co2mnsi thin films and spin - integrated in situ ups ( uncapped films ) was carried out . owing to the increased information depth of haxpes , true surface states are typically not observed by this method . as shown in fig . 4 , the in situ spin - integrated ups and the haxpes results fundamentally agree although the information depth of both experiments varies from 2 nm to 20 nm . this provides evidence that true surface states like shockley or tamm states , which are mainly located at the first atomic layer30 , do not contribute to the ups data . we calculated the spin resolved bulk dos of co2mnsi using the spin polarized relativistic korringa kohn rostoker ( spr - kkr ) green function method implemented in the munich spr - kkr band structure programme package employing the perdew ernzerhof functional32 shifts the upper edge to higher energies , but leaves the lower edge almost unchanged . for a comparison of our experimental data with ups- and haxpes calculations this electronic structure provides the basis for a one - step model of photoemission , which includes all matrix - element effects , multiple scattering in the initial and final states33 , and all surface - related effects in the excitation process . we used a recently developed relativistic generalization for excitation energies ranging from about 10 ev to more than 10 kev ( ref . 34 ) realized in the full spin - density matrix formulation for the photocurrent35 . in fig . 4 the calculations and the experimental spin - integrated ups and haxpes results are compared . nearly quantitative agreement for both , uv and hard x - ray photon energies , is obtained . particularly with regard to the small dos just below the fermi energy the agreement of the calculations with the high ups and haxpes intensities in this energy range is remarkable and is traced back to a bulk - like surface resonance as will be discussed below . the obtained agreement between the spin - integrated ups / haxpes experiments and calculations based on a half metallic bulk band structure represents already evidence for half - metallicity . additional strong evidence is provided by the analysis of the srups data . for the surface region we can estimate the position of the lower band edge of the minority gap directly from the experimental data by taking the maximum of the derivative of the minority spin intensity with respect to the energy , which is found at eef 500 mev . from previous surface sensitive x - ray magnetic circular dichroism experiments we estimated the position of the upper band edge to be at eef+400 mev ( ref . 5 the highest experimentally obtained spin polarization is shown together with the spin polarization derived directly from the calculated dos , the calculated photoemission asymmetry including all broadening effects considering bulk contributions only , and the calculated photoemission asymmetry including surface - related effects . the correspondence between the dos and calculated pure bulk - like ups spectrum becomes clear , if the influence of intrinsic life time broadening owing to electronic correlations and included experimental energy resolution ( e=400 mev ) is considered . it is obvious that these broadening effects within the bulk calculations reduce the expected ups spin polarization although the dos is half - metallic . however true surface states contribute to the layer - resolved photocurrent with an intensity distribution that is nonzero for the first atomic layer only . consequently , their contribution to the total spectral weight decreases with increasing number of layers generating the photocurrent . thus in general with increasing photon energies the combined effect of energy - dependent cross - sections and larger inelastic mean free path results in a reduced weight of surface state photoemission . however , the situation is very different for co2mnsi , where we identified in our calculations a resonance on the ( 001)-surface , which is embedded in the bulk continuum with a strong coupling to the majority bulk states . in our case this surface resonance extends over the first six atomic layers , which is similar to the case of w(110 ) , where we found a surface resonance revealing a considerable bulk contribution35 as well . the spectral weight of this surface resonance is much larger than that of a true surface state resulting in a significant contribution to the total intensity even at hard x - ray energies . co2mnsi samples were prepared and investigated completely in situ in an ultrahigh vacuum cluster consisting of sputtering chambers , an molecular beam epitaxy ( mbe ) chamber , and a srups chamber equipped with a he gas discharge lamp ( h=21.2 ev ) and a hemispherical energy analyzer with multi - channel spin filter27 ( energy resolution 400 mev , sherman function s=0.420.05 ( ref . first , an epitaxial buffer layer of the heusler compound co2mnga ( 30 nm ) was grown on the mgo(100 ) substrate by radio frequency ( rf)-sputtering at room temperature . by an optimized additional annealing process at 550 c l21 order is obtained as shown by high energy electron diffraction ( rheed ) and x - ray diffraction ( xrd ) . . induced by the buffer layer the co2mnsi thin films show already some degree of l21 surface order as deposited . by additional annealing the order is improved as demonstrated for the film surface by rheed ( fig . a low annealing temperature of ta=300c results in a significantly increased intensity of the characteristic rheed l21 superstructure peaks . however , by xrd no ( 111 ) peak , which is indicative for l21 order , is observed for ta<400 c . this suggests that l21 order is present at the film surface , but not in the bulk of the thin film . for ta400 c the ( 111 ) peak appears in xrd . for ta500 c some ga from the buffer layer is observed by core - level haxpes to have diffused to the co2mnsi surface . the magnetic moments of all samples amount to 5 b per formula unit at 4 k and is reduced by 3% at room temperature , in agreement with theoretical predictions and experimental values measured on bulk samples29 . figure 2 shows in situ ups spectra of co2mnsi thin films annealed at different temperatures ta without spin analysis . the large acceptance angle of the spectrometer ( 10 ) and applied sample bias voltage of 10 v result in k|| values which cover the complete brillouin zone . the spectra of all samples are almost identical , only the broad hump at eef=2,900 mev vanishes and the peak at eef=1,150 mev is slightly broadened for the deposited and the ta=550 c sample . however , by spin analysis clear differences between the samples are revealed . figure 3 shows the spin polarization of mgo / co2mnga(30 nm)/co2mnsi(70 nm ) thin films annealed at different temperatures ta as measured by srups . a huge room temperature spin polarization of 9093% at the fermi energy at room temperature was obtained for samples annealed between 300 c and 450 c . in combination with the ups calculations discussed below , these exceptionally high values are the first direct observation of half - metallicity in the surface region of any heusler compound , which provide strong evidence for 100% spin polarization in the bulk of the thin films . with lower annealing temperatures the spin polarization is reduced at ef and slightly increased at higher binding energies , which can be explained by an energy broadening of the electronic states owing to reduced structural order . with higher annealing temperatures ups is a surface sensitive method and thus the results can not be directly associated with electronic bulk band structure properties . however , as will be shown below , band structure based calculations of photoemission spectra provide this link . as additional experimental input for such calculations , a comparison of spin - integrated ex situ haxpes with a photon energy of 6 kev of alox capped ( oxidation protection ) co2mnsi thin films and spin - integrated in situ ups ( uncapped films ) was carried out . owing to the increased information depth of haxpes , true surface states are typically not observed by this method . as shown in fig . 4 , the in situ spin - integrated ups and the haxpes results fundamentally agree although the information depth of both experiments varies from 2 nm to 20 nm . this provides evidence that true surface states like shockley or tamm states , which are mainly located at the first atomic layer30 , do not contribute to the ups data . we calculated the spin resolved bulk dos of co2mnsi using the spin polarized relativistic korringa kohn rostoker ( spr - kkr ) green function method implemented in the munich spr - kkr band structure programme package employing the perdew ernzerhof functional32 shifts the upper edge to higher energies , but leaves the lower edge almost unchanged . for a comparison of our experimental data with ups- and haxpes calculations this electronic structure provides the basis for a one - step model of photoemission , which includes all matrix - element effects , multiple scattering in the initial and final states33 , and all surface - related effects in the excitation process . we used a recently developed relativistic generalization for excitation energies ranging from about 10 ev to more than 10 kev ( ref . 34 ) realized in the full spin - density matrix formulation for the photocurrent35 . in fig . 4 the calculations and the experimental spin - integrated ups and haxpes results are compared . nearly quantitative agreement for both , uv and hard x - ray photon energies , is obtained . particularly with regard to the small dos just below the fermi energy the agreement of the calculations with the high ups and haxpes intensities in this energy range is remarkable and is traced back to a bulk - like surface resonance as will be discussed below . the obtained agreement between the spin - integrated ups / haxpes experiments and calculations based on a half metallic bulk band structure represents already evidence for half - metallicity . additional strong evidence is provided by the analysis of the srups data . for the surface region we can estimate the position of the lower band edge of the minority gap directly from the experimental data by taking the maximum of the derivative of the minority spin intensity with respect to the energy , which is found at eef 500 mev . from previous surface sensitive x - ray magnetic circular dichroism experiments we estimated the position of the upper band edge to be at eef+400 mev ( ref . 5 the highest experimentally obtained spin polarization is shown together with the spin polarization derived directly from the calculated dos , the calculated photoemission asymmetry including all broadening effects considering bulk contributions only , and the calculated photoemission asymmetry including surface - related effects . the correspondence between the dos and calculated pure bulk - like ups spectrum becomes clear , if the influence of intrinsic life time broadening owing to electronic correlations and included experimental energy resolution ( e=400 mev ) is considered . it is obvious that these broadening effects within the bulk calculations reduce the expected ups spin polarization although the dos is half - metallic . true surface states contribute to the layer - resolved photocurrent with an intensity distribution that is nonzero for the first atomic layer only . consequently , their contribution to the total spectral weight decreases with increasing number of layers generating the photocurrent . thus in general with increasing photon energies the combined effect of energy - dependent cross - sections and larger inelastic mean free path results in a reduced weight of surface state photoemission . however , the situation is very different for co2mnsi , where we identified in our calculations a resonance on the ( 001)-surface , which is embedded in the bulk continuum with a strong coupling to the majority bulk states . in our case this surface resonance extends over the first six atomic layers , which is similar to the case of w(110 ) , where we found a surface resonance revealing a considerable bulk contribution35 as well . the spectral weight of this surface resonance is much larger than that of a true surface state resulting in a significant contribution to the total intensity even at hard x - ray energies . as shown in fig . 5 , the inclusion of the complete surface - related photoexcitation in the ups calculation results in perfect agreement with the experiment . if the surface resonance were not present , half - metallic behaviour would persist but the finite experimental resolution in photoemission would hinder the observation of a high spin polarization . because the surface resonance is strongly coupled to the band structure of the bulk , this provides evidence for the validity of our calculated half metallic bulk band structure of co2mnsi . and , from the spintronics applications point of view it is the room temperature spin polarization in the thin film surface region , which is relevant . in conclusion , investigating optimized thin films of the compound co2mnsi by in situ srups , we were able to demonstrate for the first time half - metallicity in combination with directly measured ( ) % spin polarization at room temperature in the surface region of a heusler thin film . novel band structure and photoemission calculations including all surface - related effects show that the observation of a high spin polarization in a wide energy range below the fermi energy is related to a stable surface resonance in the majority band of co2mnsi extending deep into the bulk of the material . our results show that careful thin film preparation can indeed result in a high spin polarization with a sufficient degree of stability in a surface region of several atomic layers . in particular it shows that the observed tunnelling magnetoresistance values are not limited by the intrinsic spin polarization of the heusler alloy and that potentially much larger values can be obtained by carefully optimized growth . fundamentally our observation paves the way for most powerful future spintronic devices on the basis of heusler materials . m.j . initiated and coordinated the project and wrote the paper . a.k . and m.j mucocutaneous lymph node syndrome , which had been affecting infants and young children in japan . diagnosis requires fever and 4 out of 5 principal clinical features ( conjunctivitis ; erythematous lips , oral cavity ; palms and soles ; polymorphous exanthema of the body trunk ; swelling of the cervical lymph nodes ) or if fewer than 4 , patients should also have documented coronary artery disease diagnosed by echocardiography or coronary angiography . cardiac , particularly coronary artery complications are well - known fact in this syndrome . here , we are reporting 5 cases [ table 1 ] with past history of kawasaki disease presenting with coronary artery abnormalities . 7-year - old boy with documented history of kawasaki disease at the age of 1 year , treated conservatively without using intra - venous immunoglobulin ( ivig ) , presented to us with shortness of breath ( nyha class 3 ) . echocardiography revealed feature suggestive of dilated cardiomyopathy with left ventricular ejection fraction ( lvef ) of 30% . angiogram [ figure 1 ] revealed normal right coronary artery ( rca ) and left main coronary artery ( lmca ) cut - off at mid part with collateral filling from right coronary . further evaluation by cardiac stress testing revealed presence of provocable ischemia . after stabilization of heart failure , he was referred to cardiothoracic surgery for coronary artery bypass grafting ( cabg ) . non selective coronary angiogram showing normal rca and lmca cut - off from mid part with collateral filling from right system 11-year - old boy with history of kawasaki disease at 3 years of age treated without any ivig presented with non - specific chest discomfort . coronary angiography revealed aneurysm of proximal rca with internal diameter of 9.2 mm [ figure 2a ] . he was started with aspirin 5 mg / kg body weight and warfarin keeping inr between 2 to 2.5 . on more than 1 year follow - up ( a ) coronary angiogram showing aneurism of osteo - proximal rca without any feature of stenosis of lumen . ( b ) coronary angiogram showing osteo - proximal lmca aneurism involving upto bifurcation , without any flow limiting obstruction of lumens . ( c ) coronary angiogram showing aneurismal dilatation of lcx prior to 1st obtuse marginal ( om 1 ) branch followed by severe stenosis of lcx and om 1 15-year - old boy presented with exertional chest discomfort and occasional palpitation . he had a history of kawasaki disease at the age of 1.5 years . coronary angiogram revealed giant aneurysm of lmca with maximum diameter of 12 mm [ figure 2b ] . he was similarly put on oral aspirin and anti - coagulation therapy and keeping well on 6 months follow - up . 19-year - old male presented to us with exertional chest pain and dyspnea for last 6 months . he had a history of fever for 3 weeks - duration at the age of 4 years associated with conjunctival redness and skin rash over trunk . coronary angiography showed aneurysm of left circumflex ( lcx ) artery with maximum internal diameter measuring 8.8 mm , followed by severe stenosis of lcx and 1 obtuse marginal branch [ figure 2c ] . though no documents were available , his childhood exanthem was postulated to be kawasaki disease . on dobutamine stress echo he was kept on oral anti - coagulation , aspirin , and enalapril along with other anti - anginal medications and later referred for surgical correction in the form of aneurysm closure and bypass grafting to coronaries distal to stenosis . 6-year - old boy with history of kawasaki disease at the age of 3 years was complaining of occasional chest tightness . he had dilatation of lmca in the initial echocardiography , which persisted on serial evaluations . a 64 slice ct coronary angiography done this time revealed fusiform dilatation ( 5 mm diameter ) of lmca at its trifurcation up to ostio - proximal segment of left anterior descending ( lad ) artery without any plaque or stenosis [ figure 3a and b ] . he was continued with oral aspirin 5 mg/ kg body weight and was asymptomatic thereafter . ( a ) and ( b ) 64 slice ct coronary angiography showing fusiform dilatation ( 5 mm ) of lmca at its trifurcation upto osteo - proximal lad without any significant stenosis . 7-year - old boy with documented history of kawasaki disease at the age of 1 year , treated conservatively without using intra - venous immunoglobulin ( ivig ) , presented to us with shortness of breath ( nyha class 3 ) . echocardiography revealed feature suggestive of dilated cardiomyopathy with left ventricular ejection fraction ( lvef ) of 30% . angiogram [ figure 1 ] revealed normal right coronary artery ( rca ) and left main coronary artery ( lmca ) cut - off at mid part with collateral filling from right coronary . further evaluation by cardiac stress testing revealed presence of provocable ischemia . after stabilization of heart failure , he was referred to cardiothoracic surgery for coronary artery bypass grafting ( cabg ) . non selective coronary angiogram showing normal rca and lmca cut - off from mid part with collateral filling from right system 11-year - old boy with history of kawasaki disease at 3 years of age treated without any ivig presented with non - specific chest discomfort . coronary angiography revealed aneurysm of proximal rca with internal diameter of 9.2 mm [ figure 2a ] . he was started with aspirin 5 mg / kg body weight and warfarin keeping inr between 2 to 2.5 . on more than 1 year follow - up , he is doing fine with these medicines . ( a ) coronary angiogram showing aneurism of osteo - proximal rca without any feature of stenosis of lumen . ( b ) coronary angiogram showing osteo - proximal lmca aneurism involving upto bifurcation , without any flow limiting obstruction of lumens . ( c ) coronary angiogram showing aneurismal dilatation of lcx prior to 1st obtuse marginal ( om 1 ) branch followed by severe stenosis of lcx and om 1 coronary angiogram revealed giant aneurysm of lmca with maximum diameter of 12 mm [ figure 2b ] . he was similarly put on oral aspirin and anti - coagulation therapy and keeping well on 6 months follow - up . 19-year - old male presented to us with exertional chest pain and dyspnea for last 6 months . he had a history of fever for 3 weeks - duration at the age of 4 years associated with conjunctival redness and skin rash over trunk . coronary angiography showed aneurysm of left circumflex ( lcx ) artery with maximum internal diameter measuring 8.8 mm , followed by severe stenosis of lcx and 1 obtuse marginal branch [ figure 2c ] . though no documents were available , his childhood exanthem was postulated to be kawasaki disease . on dobutamine stress echo he was kept on oral anti - coagulation , aspirin , and enalapril along with other anti - anginal medications and later referred for surgical correction in the form of aneurysm closure and bypass grafting to coronaries distal to stenosis . 6-year - old boy with history of kawasaki disease at the age of 3 years was complaining of occasional chest tightness . he had dilatation of lmca in the initial echocardiography , which persisted on serial evaluations . a 64 slice ct coronary angiography done this time revealed fusiform dilatation ( 5 mm diameter ) of lmca at its trifurcation up to ostio - proximal segment of left anterior descending ( lad ) artery without any plaque or stenosis [ figure 3a and b ] . he was continued with oral aspirin 5 mg/ kg body weight and was asymptomatic thereafter . ( a ) and ( b ) 64 slice ct coronary angiography showing fusiform dilatation ( 5 mm ) of lmca at its trifurcation upto osteo - proximal lad without any significant stenosis . although most commonly found in asian population , particularly in japan , kawasaki disease occurs worldwide . in many areas , it is more common than rheumatic fever and viral myocarditis . there is suggestion that both genetic susceptibility and environmental factors may play a role in kawasaki disease . its rarity in the first few months of life and in adults suggests an agent to which the latter are immune and from which very young infants are protected by passive maternal antibodies . kawasaki disease has 2 phases : an acute phase lasting 1 to 2 weeks , followed by a chronic ( convalescent ) phase . kawasaki disease is a generalized systemic vasculitis involving medium and small size blood vessels throughout the body , virtually always involving the coronaries . progressive stenosis in the disease results from active remodeling with an intimal proliferation and neoangiogenesis ; the intima is markedly thickened and consists of linearly - arranged microvessels , a layer that is rich in smooth muscle cells , and fibrous layers . several growth factors are prominently expressed at the inlet and outlet of aneurysms where they are activated by high shear stress . they display normal findings on electrocardiograms and stress tests and are at low risk of subsequent myocardial infarction or sudden death . sometimes , aneurysms persist and become occlusive , thereby increasing the risk of myocardial infarction or sudden death . the risk factors to predict the presence of coronary aneurysms in kawasaki disease include boys less than 1 year of age , fever lasting longer than 2 weeks , elevated erythrocyte sedimentation rate persisting for more than 4 weeks , and palpable axillary artery aneurysms . the size less than 4 mm in diameter regresses spontaneously within a short time , whereas those larger than 8 mm in diameter are often associated with a stenotic lesion . factors associated with regression of aneurysm in kawasaki disease include age less than 1 year , saccular as opposed to fusiform morphology , and distal location . myocardial infarction can occur early or late after the acute phase ; therefore , patients should be counseled to avoid atherosclerotic risk factors , and the progress of these patients should be followed into adulthood . the longer the aneurysms or stenotic lesions persist , the less likely they are to resolve . massive thrombosis of the aneurysm is seen predominantly in the right coronary artery , usually within 1 year after disease onset . progressive localized stenosis at the aneurysm inlet or outlet is seen predominantly in the left coronary artery , again usually within 1 year . our patients presented with coronary complications 3 to 14 years after the initial attack of kawasaki disease . many a time , the initial diagnosis is either missed or not documentable as is the case in our 4 patient . the laboratory findings of incomplete cases appear to be similar to those of classic cases . therefore , although laboratory findings in kawasaki disease are non - diagnostic , they may prove useful in heightening or reducing the suspicion of incomplete kawasaki disease . the goal in treating kawasaki disease is to control acute inflammation and to prevent serious cardiovascular complications , such as coronary artery disease . during the acute phase of illness , aspirin is administered at 80 to 100 mg / kg per day in 4 divided doses together with ivig 2 g/ kg in a single infusion . high dose aspirin continued until day 14 of illness or 48 to 72 hours after fever cessation . when high - dose aspirin is discontinued , clinicians begin low - dose aspirin ( 3 to 5 mg / kg per day ) and maintain it until the patient shows no evidence of coronary changes by 6 to 8 weeks after the onset of illness . for children who develop coronary abnormalities other forms of treatment including glucocorticoids , pentoxyfylline , plasma exchange , abciximab , infliximab , cyclophosphamide etc . the most common anti - thrombotic regimen for patients with giant aneurysms is low - dose aspirin together with warfarin , maintaining an international normalized ratio ( inr ) of 2.0 to 2.5 . the treatment of acute coronary occlusion in patients with kawasaki disease should target multiple steps in the coagulation cascade . because no randomized controlled trials have been performed in children , the treatment of infants and children with coronary thrombosis should be done similar to adults . attempts at excision or plication of the coronary artery aneurysm have not been successful and have caused deaths . surgical management in kawasaki disease coronary revascularization is recommended for patients with giant or multiple coronary artery aneurysms or significant stenosis . overall mortality is low for such operations , and graft target sites are easily accessible since most coronary artery lesions occur in proximal segments with little distal involvement . in situ arterial grafts the indications for coronary bypass graft procedures in children have not been established in clinical trials , but such surgery should be considered when reversible ischemia is present on stress - imaging test results , the myocardium to be perfused through the graft is still viable , and no appreciable lesions are present in the artery distal to the planned graft site . according to one older panel of experts , surgical revascularization may be considered under the following conditions : severe occlusion of the main trunk of the lmca , severe occlusion of > 1 major coronary arteries , severe occlusion in the proximal segment of the lad , collateral coronary arteries in jeopardy , or all of the above . along with the technical advancement , more and more cases of kawasaki diseases are going to be diagnosed . therefore , we shall need to follow up more such cases and upgrade our knowledge regarding this condition . as coronary artery sequelae of kawasaki disease can be a cause of ischemic heart disease even in adults , heightened awareness of this possibility is required for adults with coronary lesions but without coronary risk factors .

ferromagnetic thin films of heusler compounds are highly relevant for spintronic applications owing to their predicted half - metallicity , that is , 100% spin polarization at the fermi energy . however , experimental evidence for this property is scarce . here we investigate epitaxial thin films of the compound co2mnsi in situ by ultraviolet - photoemission spectroscopy , taking advantage of a novel multi - channel spin filter . by this surface sensitive method , an exceptionally large spin polarization of ( ) % at room temperature is observed directly . as a more bulk sensitive method , additional ex situ spin - integrated high energy x - ray photoemission spectroscopy experiments are performed . all experimental results are compared with advanced band structure and photoemission calculations which include surface effects . excellent agreement is obtained with calculations , which show a highly spin polarized bulk - like surface resonance ingrained in a half metallic bulk band structure . kawasaki disease is a generalized systemic vasculitis of unknown etiology involving medium and small size blood vessels throughout the body , virtually always involving the coronaries . in many part of this world , it is more common than rheumatic fever and viral myocarditis . here , we are reporting 5 cases with history suggestive of kawasaki disease in the early life , presenting with different coronary artery abnormalities . one of the patients had left main coronary artery cut - off , and the remaining 4 had some form of coronary artery aneurysms . 3 patients were given medical management , 1 patient had coronary bypass grafting , and the remaining 1 was planned for surgical correction . all the patients were stable on subsequent follow - ups .

expression of genes is regulated at many different levels , transcription of dna being one of the most critical stages . specific configurations of transcription factors ( tfs ) that interact with gene promoter regions are recruited to activate or modulate the production of a given transcript . many of these tfs possess the ability to recognize a small set of genomic sequence footprints called tf - binding sites ( tfbss ) . these motifs are typically 615 bp long and in some cases , they show a high degree of variability . in addition because of these flexible binding rules , computational methods for the identification of regulatory elements in a promoter sequence tend to produce an overwhelming amount of false positives . however , the identification of conserved regulatory elements present in orthologous gene promoters ( also called phylogenetic footprinting ) has proved to be more effective to characterize such sequences ( 13 ) . in fact , the ever - growing availability of more genomes and the constant improvement of bioinformatics algorithms hold great promise for unveiling the overall network of gene interactions of each organism ( 4 ) . typically , computational methods to detect regulatory elements use their own training set of experimental annotated tfbss . these annotations are usually collected from bibliography or from general repositories of gene regulation information , such as jaspar ( 5 ) and transfac ( 6 ) . however , each program establishes different criteria and formats to retrieve and display the data that forms the final training set , which makes the comparison between different methods very difficult . the construction of a good benchmark to evaluate the accuracy of several pattern discovery methods is therefore not a trivial procedure ( 7 ) . although important efforts are being carried out to standardize the construction of collections of promoter regions ( 8) or the presentation of experimental data ( 9 ) , there is a clear necessity to provide stable and common datasets for future algorithmic developments . in this direction , we present here the release 1.0 of the abs database constructed from literature annotations that have been experimentally verified in human , mouse , rat or chicken . we have gathered from the literature a collection of experimentally validated binding sites that are conserved in at least two orthologous vertebrate promoters . the compiled data are suitable for training both classical pattern discovery programs and new emerging comparative methods . flat files accomplishing the gff standard format were used to store and query the information . the genbank accession number of the sequences in each bibliographical reference was utilized to retrieve the promoters . such sequences were mapped on to the corresponding refseq annotations to ensure we were retrieving the actual promoter . the dbtss database ( 10 ) was finally used to refine the annotation of the tsss . since it is the region in which most experimental studies have been focused on , we considered the sequence 500 bp immediately upstream the annotated tss , as the promoter region in this first release . for each annotated promoter , we only included experimentally tested sites in this proximal region whose motifs were correctly identified in at least two species , i.e. orthologous sites . every known binding site was mapped on to the corresponding promoter sequence by blastn ( 11 ) . those matches that exhibited < 80% of identity between the sequence of the original site and the mapped motif in the promoter region were rejected . we computed blastn ( 11 ) , clustalw ( 12 ) , avid ( 13 ) and lagan ( 14 ) alignments of the orthologous promoters from each gene . moreover , we produced a dotplot of word matches with emboss ( 15 ) to visualize unusually conserved regions . for comparative assessments , computational predictions using the jaspar ( 5 ) , transfac ( 6 ) and promo ( 16 ) collections of position weight matrices were calculated . a very restrictive threshold of 0.85 was used to remove those predicted tfbss whose score was below this value , creating a first group of more reliable predictions . a second group of predictions was produced using a more flexible threshold of 0.70 ( see the abs website for further information about the scoring method ) . release 1.0 of abs database contains 100 annotated orthologous genes , each entry corresponding to two or more species . there is a clear predominance of human and mouse annotations : 73 entries contain at least annotations for human mouse orthologs . a total of 650 experimental binding sites from 68 different tfs are associated with abs entries , covering 8624 nt . in average , three tfbss per sequence have been mapped on to the promoters with an average length of 13.3 nt per site . the tfs that appear more frequently are tbp ( 14.6% of sites ) , sp1 ( 13.6% ) and cebp ( 5.6% ) . those tfs are known to be part of the core of many eukaryotic promoters ( see the abs documentation for further details about the contents of the database ) . the abs database can be accessed through a simple cgi / perl - based web interface at . the following functionalities are implemented in the current release ( see figure 1 ) : for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation.retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters.retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs.the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation . retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters . retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs . the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . the abs database aims to become a platform to evaluate new algorithms for the discovery of novel regulatory elements in a set of related gene promoters ( e.g. orthologous promoters or co - regulated genes from microarray experiments ) . in addition to the data retrieval functions , two on - line applications are available to perform the benchmarking of such algorithms ( see figure 2 ) : constructor is a web server to produce synthetic datasets based on the abs annotations . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18).evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18 ) . evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the abs database can be accessed through a simple cgi / perl - based web interface at . the following functionalities are implemented in the current release ( see figure 1 ) : for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation.retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters.retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs.the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . for each gene in the collection , show the orthologous promoters and a list of experimentally verified tfbss annotated on the corresponding sequence . promoter sequence alignments , computational predictions , dotplots and cross - references to other well - known databases , such as genbank , entrez gene and pubmed , are also provided for each annotation . retrieve all of the binding motifs associated with a given tf , filtering by species . moreover , a global alignment of the motifs is provided and the corresponding sequence logo representation is displayed by using weblogo ( 17 ) . this information could be used to produce new profiles for subsequent detection of this tf in other promoters . retrieve all of the promoter sequences in which at least one binding site for a given tf was annotated . these sequences and the associated motifs could be used to generate datasets based on known sites to train motif - finding programs . the gene catalogue , the promoter sequences , the collection of annotations , the sequence alignments and the computational predictions are also individually distributed in several flat files . the abs database aims to become a platform to evaluate new algorithms for the discovery of novel regulatory elements in a set of related gene promoters ( e.g. orthologous promoters or co - regulated genes from microarray experiments ) . in addition to the data retrieval functions , two on - line applications are available to perform the benchmarking of such algorithms ( see figure 2 ) : constructor is a web server to produce synthetic datasets based on the abs annotations . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18).evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the design of the benchmark is highly flexible allowing to customize the number of sequences , their length , the background nucleotide distribution , the number of motifs to plant on them , the probability to plant a motif , the species and the tfs for which the associated motifs will be randomly selected from the known sites collection . the output consists of the artificial sequences with the embedded motifs , the list of motifs and a graphical representation of the occurrences in the sequences produced with the program gff2ps ( 18 ) . evaluator is a web server to determine the accuracy of a set of predicted motifs in several sequences using a list of known binding sites as a reference set . a complete accuracy assessment at both nucleotide and site levels is computed using the standard measures in the field ( 7,19 ) . the abs database has been developed to fill the existing gap in the availability of consistent datasets to train and compare different pattern discovery programs . the lack of standard collections of tfbss is specially serious in the case of phylogenetic footprinting data . the collection described here contains 650 experimental tfbss identified in human , mouse , rat and chicken genes . in addition , two web applications ( constructor and evaluator ) are included in this first release to facilitate the development of new motif - finding programs using the abs annotations . in the next release , we plan to increase the number of annotations adding known sites in regulatory regions different from the proximal promoter and eventually incorporate binding motifs from other species . examples of the abs data retrieval system showing the annotation of a gene , the set of binding motifs from a given tf in human and mouse and the extraction of the promoter sequences containing such annotations . protocol to evaluate the accuracy of an external motif - finding program on a synthetic dataset generated by planting motifs from abs in randomly generated sequences . it is well known that job stress is responsible for poor work performance , high absenteeism , less work productivity , and several diseases.15 recently , results from thirteen independent cohort studies in europe indicated that job strain is responsible for coronary heart disease , as are lifestyle and orthodox risk factors.6 the population s attributable risk for job strain was 3.4% , which is lower than that for smoking habits ( 36% ) , abdominal obesity ( 20% ) , and physical inactivity ( 12% ) . however , the interheart study7 showed that work stress doubled the risk of coronary heart disease . in finland , the increase in job strain was associated with an increase in the risk of requiring a disability pension . moreover , as regarding men , a positive association between cardiovascular diseases and an increased risk of disability pension was found.8 mental health caused disabling conditions in 9% of the population in the uk , and estimations for the 20012002 prevalence of self - reported illness caused or made worse by work classified stress , depression , or anxiety as the second most commonly reported illness.9 within this context , in 2010 , in italy , a governmental commission published several indications for the evaluation of stress . the commission identified a preliminary phase and a second phase for in - depth examination . instruments for self - evaluation such as psychometric tests are recommended during the second phase . in this context of changes in the cultural and legislative background , the authors have taken the opportunity to contribute to the development of models for the identification and assessment of stress , responding to the demand for specific measurement instruments . data from the literature as well as normative requirements suggest the need for methods for the early identification of stress in workers . some instruments have already been developed , such as the job content questionnaire,1012 the work organization assessment questionnaire,13 and the effort reward imbalance questionnaire.14,15 these instruments are considered complementary sources since they are based on different concepts of the work - related stress . they are mainly focused on measuring the job climate , the facets of job organization , or the balance between effort and reward , but an in - depth examination of individual resources and capabilities is lacking . newman and beehr16 underlined that job stress is a situation wherein job - related factors interact with the worker to change his / her psychological and/or physiological condition , forcing a person to deviate from normal functioning . from this viewpoint , the focus is not the stressful situation or the subjective perception , but the interaction between these two components . in agreement with this rationale , we developed a new multidimensional instrument called the maugeri stress index ( masi ) designed to investigate individual resources for coping with stressful situations on the job . the instrument was developed for an in - depth examination , but it is also usable in the preliminary phase and for monitoring homogeneous groups of workers when individual resources play a critical role in coping with the demands arising from organizational changes . this group of workers represents a category particularly at risk for the development of job stress , and for this reason they can be considered a representative sample of the target population . the first version was tested on a restricted sample ( n=329 ) and has already been published with some clinical qualities and some limitations.17 the aims of this study were to develop a shorter form of the questionnaire using an item reduction procedure and to assess the psychometric properties of the resulting instrument using the rasch measurement model . the masi ( figure s1 ) requires participants to express their level of agreement with 51 items on a five - point likert scale ( never , little , enough , much , very much ) . the questionnaire is composed of four main scales including 47 items : wellness ( eleven items ) , resilience ( 20 items ) , perception of social support ( nine items ) , and negative coping styles ( seven items ) . further , a lie scale ( four items ) is included . a high score on the index shows perceived eustress . previously , giorgi et al17 studied the dimensionality of the instrument by using exploratory factor analysis . in the study here presented , we analyzed the internal consistency of the masi and its factorial structure by using structural equation models ( sems).18 therefore , starting with these results , we developed a reduced form of the instrument , the maugeri stress index reduced form ( masi - r ) , which is described in this paper . the psychometric properties of the masi - r are analyzed using the rasch measurement model.19 the scientific and technical committee ( irb ) of the care and research institute of tradate ( varese , italy ) , salvatore maugeri foundation irccs , discussed and approved the study ( 5/5/2015 cod . the sample comprised 1,182 participants ( females : 57.6% ; males : 42.4% ) with a mean age of 45.7 years ( standard deviation = 9.0 ) . other subjects engaged in the study were factory workers ( 15.1% ) , teachers ( 4.6% ) , and managers ( 4.2% ) . of the sample , 93.9% had a permanent work contract and 38.0% were shift workers . all the participants filled in the questionnaire voluntarily after a short motivational presentation made by a psychologist . each participant provided written informed consent for the use of data in anonymous form . participants who omitted at least 25% of responses ( 13 items ) were not considered in the analysis . therefore , seven subjects were removed , and 1,175 units were used for analysis . regarding sem analysis , the remaining missing data were replaced by the mean of the responses for each item . by contrast , for the rasch analysis , only the complete cases were considered , leading to a total sample of 904 subjects . the confirmatory factor analysis was used to test and improve the fit of the data for the hypothesized four - factor model ( see missing data section ) . the confirmatory factor analysis is part of the general methodology of the sem and allows us to test causal relations between latent and observed variables . the analysis was started by building an independence model , assuming uncorrelated latent factors . as a second step , we tested the presence of relations between the latent factors , performing a model comparison . step by step , we imposed covariance relations among factors , looking for an improvement in the fit . a similar methodology was applied to select the items in order to reduce their number and to simplify the instrument . to make model comparisons , we used the akaike information criterion ( aic)20 and the bayesian information criterion ( bic)21 to identify which model was better.22,23 since the best model has the lowest indices , we compared the information criteria of a complex model with those of the simpler one , obtaining two differences : aic and bic . a negative difference indicates that the complex model is better than the simpler one ; a difference of bic 2 is generally accepted as positive evidence for the difference between two models.24 in contrast to the aic , the bic penalizes greatly complex models : a very complex model would have to improve the fit substantially in order for bic to choose it as the best . the item properties were analyzed using the partial credit model,25 a polytomous formulation of the rasch model . the rasch model is a well - known one - parameter logistic model , commonly applied for the evaluation of the properties of a unidimensional psychometric test with an ordinal response scale . it assumes that the probability of a person producing a positive outcome to an item depends on the distance between the person s ability and the item s difficulty . in contrast to the classical test theory and the sem approach , the rasch model is centered on the study of items and their properties . the model tests the calibration of the instrument on a population , putting persons abilities and item difficulties into the same scale . it can distinguish between the easiest and hardest items and evaluate if the instrument leads to a reliable measure of the latent trait . the scales of the masi were studied separately , fitting one partial credit model for each scale . the reliability was evaluated using the separation index g for persons ( gp ) and items ( gi ) and the person separation reliability r , which corresponds to cronbach s .26 a high variability in persons abilities and in item difficulties is necessary so that the measurement is reliable . gp quantifies the number of strata of abilities that the instrument can separate and identify as different ; a low value ( gp < 2 ) is a sign that the instrument may not be sensitive enough to distinguish between individuals with high and low performance abilities.27 by contrast , a low gi value ( gi <3 ) indicates that either the variance of item difficulties is too small or the sample is not large enough . item properties have been evaluated using two popular indices:27 infit ( information - weighted fit ) and outfit ( outlier - sensitive fit ) . both indices are calculated based on the mean square of standardized residuals for items , but outfit is more sensitive to outliers than infit . acceptable values range between 0.6 and 1.4;28 lower values indicate underfit ( ie , the presence of unexplained variance ) and higher values indicate overfit ( ie , redundancy in the set of items ) . the unidimensionality of each scale was assessed by evaluating the strength of correlations between residuals and the results of the principal component analysis performed on the residuals.29 since the rasch analysis extracts the first , and theoretically , the only latent component within each scale , residuals should be constituted only by random noise . a simple rule of thumb27 is that if the residuals are constituted by pure random noise , the eigenvalue of the first extracted component must be less than 2 ( ie , a strength of less than two items ) . furthermore , we used the martin - lf test,30 which splits the scale into two clusters of items on the basis of the median difficulty and performs a likelihood ratio test . analyses were performed in the r environment,31 using the packages lavaan 0.51132 for the confirmatory factor analysis and erm 0.15033 for the rasch analysis . the sample comprised 1,182 participants ( females : 57.6% ; males : 42.4% ) with a mean age of 45.7 years ( standard deviation = 9.0 ) . other subjects engaged in the study were factory workers ( 15.1% ) , teachers ( 4.6% ) , and managers ( 4.2% ) . of the sample , all the participants filled in the questionnaire voluntarily after a short motivational presentation made by a psychologist . each participant provided written informed consent for the use of data in anonymous form . participants who omitted at least 25% of responses ( 13 items ) were not considered in the analysis . therefore , seven subjects were removed , and 1,175 units were used for analysis . regarding sem analysis , the remaining missing data were replaced by the mean of the responses for each item . by contrast , for the rasch analysis , only the complete cases were considered , leading to a total sample of 904 subjects . analyses were conducted to study the reliability of the masi and its factorial structure . the confirmatory factor analysis was used to test and improve the fit of the data for the hypothesized four - factor model ( see missing data section ) . the confirmatory factor analysis is part of the general methodology of the sem and allows us to test causal relations between latent and observed variables . the analysis was started by building an independence model , assuming uncorrelated latent factors . as a second step , we tested the presence of relations between the latent factors , performing a model comparison . step by step , we imposed covariance relations among factors , looking for an improvement in the fit . a similar methodology was applied to select the items in order to reduce their number and to simplify the instrument . to make model comparisons , we used the akaike information criterion ( aic)20 and the bayesian information criterion ( bic)21 to identify which model was better.22,23 since the best model has the lowest indices , we compared the information criteria of a complex model with those of the simpler one , obtaining two differences : aic and bic . a negative difference indicates that the complex model is better than the simpler one ; a difference of bic 2 is generally accepted as positive evidence for the difference between two models.24 in contrast to the aic , the bic penalizes greatly complex models : a very complex model would have to improve the fit substantially in order for bic to choose it as the best . the item properties were analyzed using the partial credit model,25 a polytomous formulation of the rasch model . the rasch model is a well - known one - parameter logistic model , commonly applied for the evaluation of the properties of a unidimensional psychometric test with an ordinal response scale . it assumes that the probability of a person producing a positive outcome to an item depends on the distance between the person s ability and the item s difficulty . in contrast to the classical test theory and the sem approach , the rasch model is centered on the study of items and their properties . the model tests the calibration of the instrument on a population , putting persons abilities and item difficulties into the same scale . it can distinguish between the easiest and hardest items and evaluate if the instrument leads to a reliable measure of the latent trait . the scales of the masi were studied separately , fitting one partial credit model for each scale . the reliability was evaluated using the separation index g for persons ( gp ) and items ( gi ) and the person separation reliability r , which corresponds to cronbach s .26 a high variability in persons abilities and in item difficulties is necessary so that the measurement is reliable . gp quantifies the number of strata of abilities that the instrument can separate and identify as different ; a low value ( gp < 2 ) is a sign that the instrument may not be sensitive enough to distinguish between individuals with high and low performance abilities.27 by contrast , a low gi value ( gi <3 ) indicates that either the variance of item difficulties is too small or the sample is not large enough . item properties have been evaluated using two popular indices:27 infit ( information - weighted fit ) and outfit ( outlier - sensitive fit ) . both indices are calculated based on the mean square of standardized residuals for items , but outfit is more sensitive to outliers than infit . acceptable values range between 0.6 and 1.4;28 lower values indicate underfit ( ie , the presence of unexplained variance ) and higher values indicate overfit ( ie , redundancy in the set of items ) . the unidimensionality of each scale was assessed by evaluating the strength of correlations between residuals and the results of the principal component analysis performed on the residuals.29 since the rasch analysis extracts the first , and theoretically , the only latent component within each scale , residuals should be constituted only by random noise . a simple rule of thumb27 is that if the residuals are constituted by pure random noise , the eigenvalue of the first extracted component must be less than 2 ( ie , a strength of less than two items ) . furthermore , we used the martin - lf test,30 which splits the scale into two clusters of items on the basis of the median difficulty and performs a likelihood ratio test . analyses were performed in the r environment,31 using the packages lavaan 0.51132 for the confirmatory factor analysis and erm 0.15033 for the rasch analysis . the internal consistency , measured by the cronbach s , appeared good for the scales wellness ( =0.94 ) and resilience ( =0.89 ) . in contrast , for the scales perception of social support ( =0.64 ) and negative coping styles ( =0.65 ) , the values were not so good , although they were acceptable . we considered the correlation between each item and the total score of the respective scale ( calculated excluding the target item ) . within the scale of perception of social support these items were : 4 , 13 , 17 , and 28 ( the content of items are reported in figure s1 ) . whereas the item - total correlation for items 4 and 28 were near 0.3 , the indices for items 13 and 17 were lower than 0.2 . the most problematic item was 13 , because the cronbach s of the respective scale increased if this item was deleted . we observed a medium - sized pearson s correlation between the scales resilience and wellness ( r=0.53 , p<0.001 ) and between resilience and perception of social support ( r=0.45 , p<0.001 ) . the confirmatory factor analysis started with the estimation of the independence model , assuming uncorrelated latent dimensions . all parameters were estimated to be significantly different from zero for p<0.001 . for the wellness scale , the standardized parameters ranged between 0.61 and 0.87 , while the values for the resilience scale were between 0.33 and 0.71 . in the perception of social support scale , the coefficients ranged between 0.11 and 0.66 ( item 13 was the only one that was significant for p<0.01 ) . finally , in the negative coping styles scale , the values ranged between 0.35 and 0.55 . the goodness - of - fit indices highlighted a poor fit ( comparative fit index [ cfi ] = 0.78 , nonnormed fit index [ nnfi ] = 0.77 , root mean squares error of approximation [ rmsea ] = 0.06 ) . therefore , we proceeded to free the covariance relations between the latent dimensions one by one , looking for an improvement . we released , in sequence , the covariance parameters for wellness , resilience , and perception of social support . in the last step , we freed the covariance relations with the negative coping styles scale at each step , the new model was compared with the previous one using the difference in aic and bic . the progressive deletion of constraints in covariance parameters improved the model ( cfi = 0.82 , nnfi = 0.81 , rmsea = 0.06 ) . in order to develop a short form of the masi as a first step , starting from the identified model in the confirmatory factor analysis of the masi section ( correlated factors ) , we deleted selected items one by one , looking for a better internal consistency . the candidate items for deletion were those with an item - total correlation of less than 0.3 and those that , if deleted , caused an increase in the cronbach . we deleted three items from the scale perception of social support ( 13 , 17 , and 4 ) , three items from the scale resilience ( 36 , 8 , and 22 ) , and two items from the negative coping styles scale ( 38 and 15 ) . the resulting model showed a better fit than the starting model ( cfi = 0.86 , nnfi = 0.85 , rmsea = 0.06 ) . as the second step , using sem , we tested the presence of covariance between errors . inspecting modification indices , we identified several candidate parameters to free , concentrating our attention on items with semantic similarities in content . one at a time , aic and bic showed an improvement of the fit for each step ; further , the final model showed better fit indices ( cfi = 0.89 , nnfi = 0.88 , rmsea = 0.05 ) than the starting one . since the semantic content of several items was very similar , we kept only one item from each pair , considering the relevance of the item in a clinical perspective as driving principle for the choice . competing items are reported in table 2 , with bold items being included in the final model . in one case ( 6s and 11s ) , we deleted both items , and in two cases , ( items 33 and 25 and items 29 and 39 ) we kept both items . the final model considered item 37 , marked in bold in figure s1 , which constitutes the short version of the tool , called masi - r . figure 1 is a graphical description of the model with estimated parameters ; fit indices indicate an acceptable fit with the data ( cfi = 0.88 , nnfi = 0.87 ) and a good error of approximation of the model ( rmsea = 0.05 ) . preliminarily , we evaluated the possible presence of a differential item functioning on the scales . using the regression approach , we tested the invariance of item scores across several clusters of subjects , taking into account the sex of participants , their age , the presence / absence of job shifts , and the length of service . for each scale , f - tests sometimes showed significant results at the 5% level , but the effect sizes were always very low ( < 0.05 ) . since the effect of these variables on the total scores was not noticeable , they were not considered significant . regarding the issue of reliability , the results of the analyses on the masi - r were similar to the results of the analyses on the masi . the person reliability appeared good for the scales wellness ( r=0.89 ) and resilience ( r=0.89 ) and were acceptable for the scales perception of social support ( r=0.70 ) and negative coping styles ( r=0.50 ) . in the two latter scales , the persons separation indices seemed critical : gp = 1.52 for perception of social support and gp = 0.99 for negative coping styles . in contrast , for the wellnesss ( gp = 2.87 ) and resilience ( gp = 2.79 ) scales , the persons separation indices were more adequate . both infit and outfit were always good ( table 3 ) and neither underfit nor overfit affected the items . figure 2 depicts the calibration of the instrument , comparing the distribution of the persons abilities and item difficulties along the logit scale . for each panel , the scores on the left side represent persons with high stress ( ie , low ability ) , while the right side represents persons with low stress ( ie , high ability ) . the matched distributions of persons abilities and item difficulties show that difficulties cover the abilities quite well . this is confirmed by the separation indices gi , for which the values are always good ( wellness : 15.10 , resilience : 6.80 , perception of social support : 11.01 , negative coping styles : 4.55 ) , showing that the variances of item difficulties are adequate for measuring latent dimensions because they cover the entire spectrum of each trait . finally , we tested the actual unidimensionality of the masi - r scales . for each one , all of the correlations between residuals were lower than 0.4 ( rarely did they exceed 0.3 ) . further , the eigenvalues estimated by the principal component analyses were always lower than 2 . moreover , the martin - lf test confirmed that no other components appeared to be present within the four scales : values were never significant . the internal consistency , measured by the cronbach s , appeared good for the scales wellness ( =0.94 ) and resilience ( =0.89 ) . in contrast , for the scales perception of social support ( =0.64 ) and negative coping styles ( =0.65 ) , the values were not so good , although they were acceptable . we considered the correlation between each item and the total score of the respective scale ( calculated excluding the target item ) . within the scale of perception of social support these items were : 4 , 13 , 17 , and 28 ( the content of items are reported in figure s1 ) . whereas the item - total correlation for items 4 and 28 were near 0.3 , the indices for items 13 and 17 were lower than 0.2 . the most problematic item was 13 , because the cronbach s of the respective scale increased if this item was deleted . we observed a medium - sized pearson s correlation between the scales resilience and wellness ( r=0.53 , p<0.001 ) and between resilience and perception of social support ( r=0.45 , p<0.001 ) . the confirmatory factor analysis started with the estimation of the independence model , assuming uncorrelated latent dimensions . for the wellness scale , the standardized parameters ranged between 0.61 and 0.87 , while the values for the resilience scale were between 0.33 and 0.71 . in the perception of social support scale , the coefficients ranged between 0.11 and 0.66 ( item 13 was the only one that was significant for p<0.01 ) . finally , in the negative coping styles scale , the values ranged between 0.35 and 0.55 . the goodness - of - fit indices highlighted a poor fit ( comparative fit index [ cfi ] = 0.78 , nonnormed fit index [ nnfi ] = 0.77 , root mean squares error of approximation [ rmsea ] = 0.06 ) . therefore , we proceeded to free the covariance relations between the latent dimensions one by one , looking for an improvement . we released , in sequence , the covariance parameters for wellness , resilience , and perception of social support . in the last step , we freed the covariance relations with the negative coping styles scale at each step , the new model was compared with the previous one using the difference in aic and bic . the progressive deletion of constraints in covariance parameters improved the model ( cfi = 0.82 , nnfi = 0.81 , rmsea = 0.06 ) . in order to develop a short form of the masi , we implemented an item selection procedure to reduce the length of the questionnaire . as a first step , starting from the identified model in the confirmatory factor analysis of the masi section ( correlated factors ) , we deleted selected items one by one , looking for a better internal consistency . the candidate items for deletion were those with an item - total correlation of less than 0.3 and those that , if deleted , caused an increase in the cronbach . we deleted three items from the scale perception of social support ( 13 , 17 , and 4 ) , three items from the scale resilience ( 36 , 8 , and 22 ) , and two items from the negative coping styles scale ( 38 and 15 ) . the resulting model showed a better fit than the starting model ( cfi = 0.86 , nnfi = 0.85 , rmsea = 0.06 ) . as the second step , using sem , we tested the presence of covariance between errors . inspecting modification indices , we identified several candidate parameters to free , concentrating our attention on items with semantic similarities in content . one at a time , aic and bic showed an improvement of the fit for each step ; further , the final model showed better fit indices ( cfi = 0.89 , nnfi = 0.88 , rmsea = 0.05 ) than the starting one . since the semantic content of several items was very similar , we kept only one item from each pair , considering the relevance of the item in a clinical perspective as driving principle for the choice . competing items are reported in table 2 , with bold items being included in the final model . in one case ( 6s and 11s ) , we deleted both items , and in two cases , ( items 33 and 25 and items 29 and 39 ) we kept both items . the final model considered item 37 , marked in bold in figure s1 , which constitutes the short version of the tool , called masi - r . figure 1 is a graphical description of the model with estimated parameters ; fit indices indicate an acceptable fit with the data ( cfi = 0.88 , nnfi = 0.87 ) and a good error of approximation of the model ( rmsea = 0.05 ) . preliminarily , we evaluated the possible presence of a differential item functioning on the scales . using the regression approach , we tested the invariance of item scores across several clusters of subjects , taking into account the sex of participants , their age , the presence / absence of job shifts , and the length of service . for each scale , f - tests sometimes showed significant results at the 5% level , but the effect sizes were always very low ( < 0.05 ) . since the effect of these variables on the total scores was not noticeable , they were not considered significant . regarding the issue of reliability , the results of the analyses on the masi - r were similar to the results of the analyses on the masi . the person reliability appeared good for the scales wellness ( r=0.89 ) and resilience ( r=0.89 ) and were acceptable for the scales perception of social support ( r=0.70 ) and negative coping styles ( r=0.50 ) . in the two latter scales , the persons separation indices seemed critical : gp = 1.52 for perception of social support and gp = 0.99 for negative coping styles . in contrast , for the wellnesss ( gp = 2.87 ) and resilience ( gp = 2.79 ) scales , the persons separation indices were more adequate . both infit and outfit were always good ( table 3 ) and neither underfit nor overfit affected the items . figure 2 depicts the calibration of the instrument , comparing the distribution of the persons abilities and item difficulties along the logit scale . for each panel , the scores on the left side represent persons with high stress ( ie , low ability ) , while the right side represents persons with low stress ( ie , high ability ) . the matched distributions of persons abilities and item difficulties show that difficulties cover the abilities quite well . this is confirmed by the separation indices gi , for which the values are always good ( wellness : 15.10 , resilience : 6.80 , perception of social support : 11.01 , negative coping styles : 4.55 ) , showing that the variances of item difficulties are adequate for measuring latent dimensions because they cover the entire spectrum of each trait . finally , we tested the actual unidimensionality of the masi - r scales . for each one , all of the correlations between residuals were lower than 0.4 ( rarely did they exceed 0.3 ) . further , the eigenvalues estimated by the principal component analyses were always lower than 2 . moreover , the martin - lf test confirmed that no other components appeared to be present within the four scales : values were never significant . this paper presents the masi - r , a new psychometric instrument that aims to screen for job stress . the instrument is structured as a self - report questionnaire that is relatively short but psychometrically comprehensive . the masi - r allows us to obtain a reliable measure of exposure to workplace stress and its impact on workers . using four correlated subscales ( wellness , resilience , perception of social support , negative copying styles ) and a lie index , the questionnaire measures several dimensions that determine the total index . the total index can be obtained as the sum of responses ( coded from 1 to 5 ) . when the response pattern presents missing items , these values can be estimated by the mean of the provided responses . the masi - r originates from a reduction of the original longer masi , whose scales include an overall pool of 47 items . using an item selection procedure , we selected and removed those items that did not adequately contribute to the measurement . the structure of the instrument was further improved using an sem . the rasch model indicated good item properties with neither underfit , overfit , nor differential item functioning between population strata . furthermore , item difficulties covered the persons abilities quite well . the first one refers to a general condition of wellness , serenity , and satisfaction ; it is defined by the absence of negative symptoms and by the presence of optimism regarding the work experience . the resilience scale evaluates skills such as tenacity , flexibility , and the ability to deal with problems on the job . the other two scales have a restricted number of items . perception of social support explores a component that plays a fundamental role in stress and burnout , consisting of the perception of adequate support and assistance from colleagues and superiors.34 by contrast , the negative coping styles scale investigates the presence of social isolation , behavioral disengagement , anxiety , or irritability in reaction to stressor situations . for both scales this was probably due to the small number of items included in the scales , but it might also indicate a restricted ability range within the selected sample.27 the masi - r assumes stress as a general and unidimensional latent factor that shows itself in a broad spectrum of facets . these facets are clustered in the four identified components . according to this point of view , the correlation between the four latent traits suggests that the number of items could be further reduced and the total score could be used as a uniform one - factorial measure . recently , a study regarding the discriminant validity of the masi - r was published35 and the results show that workers exposed to workplace mobbing showed higher work stress levels compared to the control group in all aspects measured . in summary , using only 37 items , the masi - r can measure the perception of wellness and support , the resilience capability , and the behavioral reactions of individuals coping with stressful situations in the workplace . further , unlike other instruments , the masi - r provides a lie index capable of identifying cheating responders , a phenomenon that can occur in workplace contexts . it is focused on the relation between the worker and stressful situations on the job , and it could be used to drive interventions centered on the interaction between the work environment and individual perceptions and resources . list of the items included into the masi , the full version of the questionnaire . notes : only the bold items are included in the masi - r ( 33 items ) . items from the lie control scale ( 4 items ) are marked with an asterisk .

information about the genomic coordinates and the sequence of experimentally identified transcription factor binding sites is found scattered under a variety of diverse formats . the availability of standard collections of such high - quality data is important to design , evaluate and improve novel computational approaches to identify binding motifs on promoter sequences from related genes . abs ( ) is a public database of known binding sites identified in promoters of orthologous vertebrate genes that have been manually curated from bibliography . we have annotated 650 experimental binding sites from 68 transcription factors and 100 orthologous target genes in human , mouse , rat or chicken genome sequences . computational predictions and promoter alignment information are also provided for each entry . a simple and easy - to - use web interface facilitates data retrieval allowing different views of the information . in addition , the release 1.0 of abs includes a customizable generator of artificial datasets based on the known sites contained in the collection and an evaluation tool to aid during the training and the assessment of motif - finding programs . introduction and objectivesa multidimensional self - report questionnaire to evaluate job - related stress factors is presented . the questionnaire , called maugeri stress index reduced form ( masi - r ) , aims to assess the impact of job strain on a team or on a single worker by considering four domains : wellness , resilience , perception of social support , and reactions to stressful situations.material and methodsthe reliability of a first longer version ( 47 items ) of the questionnaire was evaluated by an internal consistency analysis and a confirmatory factor analysis . an item reduction procedure was implemented to obtain a short form of the instrument , and the psychometric properties of the resulting instrument were evaluated using the rasch measurement model.resultsa total of 14 items from the initial pool were deleted because they were not productive for measurement . the analysis of internal consistency led to the exclusion of eight items , while the analysis performed using structural equation models led to the exclusion of another six items . according to the rasch model , item properties and the reliability of the instruments appear good , especially for the scales for wellness and resilience . in contrast , the scales for perception of social support and negative coping styles show a lower internal consistency.conclusionsthe maugeri stress index reduced form provides a reliable and valid measure , useful for early identification of stress levels in workers or in a team along the eustress vadistress continuum .

techniques that allow the detection of single base changes in genomic dna have had a major impact on our understanding of human genetic diseases , both by identifying specific mutations that result in disease[1 , 2 ] and by identifying dna polymorphisms that are used as genetic markers in linkage studies[35 ] . detection of beta globin gene lesions in thalassemic patients and carriers is based on screening for the mutations most often found in the relevant ethnic group . but in circumstances in which an unknown mutation is encountered , another difficulty arises when at - risk couples present at a late stage of pregnancy with an unusual beta globin mutation asking for prenatal diagnosis . since the nature of the parental beta thalassemia alleles must be determined before fetal sampling , it is fundamental to find either parental specific mutations or dna polymorphisms in beta globin gene cluster for linkage studies . in some instances base changes have been identified as restriction fragment length polymorphisms ( rflps ) near or within beta globin gene[6 , 7 ] . rflps are detected by determining the presence or absence of a restriction enzyme cleavage site within a dna fragment . this concerns rs713040 or codon2 ( c - t ) recognized by hgiai , rs10768683 or ivs2 - 16 ( c - g ) recognized by avaii and rs1609812 or ivs2 - 666 recognized by sspi[8 , 9].oas there are hot spots for recombination upstream of beta globin gene[10 , 11 ] and few rflps within beta globin gene , it is wiser to study either polymorphism in both sides of beta globin gene or look for polymorphisms within beta globin gene for prenatal diagnosis . moreover in many instances rflps are not informative due to the low degree of polymorphism and each restriction site 's study necessitates a separate pcr reaction . the combined pcr and denaturing gradient gel electrophoresis ( dgge ) can be used to detect the presence of uncommon mutations or natural polymorphisms within beta globin genes[12 , 13 ] . this method permits the separation of strands of small dna fragments ( 200 - 700bp ) according to the nature of their sequence . it involves the electrophoresis of double stranded dna fragments through an acrylamide gel containing a linear gradient of dna denaturants , such as formamide and urea . using this method , 4 different beta globin gene frameworks called fw1 , fw2 , fw3 and fw3a representing combinations of 5 natural sequence changes at exon one ( one site ) and intron two ( 4 sites ) of beta globin can be distinguished upon a single pcr reaction . these five neutral sequence changes occur in the human beta globin gene in codon 2 , ivs 2 ( nt 16 , 74 , 81 and 666 ) . the five neutral sequence changes at codon 2 c / t , ivs 2 nt 16 c / g , nt 74 g / t , nt 81 c / t , nt 666 t / c occurring in the human beta globin gene define four frameworks called : 1 , 2 , 3 and 3a ( table 1 ) that differ from each other by at least one nucleotide . to detect these polymorphisms , we analyzed the dgge pattern of the fragment corresponding to nucleotides + 188 to + 621 of beta globin and containing ivs2 ( nt 16 , 74 , 81 ) and report the frequency of these frameworks among 92 beta globin alleles belonging to 46 thalassemia major patients for the first time in northern iran . blood samples were collected from 46 unrelated regularly transfused thalassemic children of mazandaran province , iran . for each sample , dna was obtained from 0.5 ml blood after alkaline lysis of white blood cells . all patients were compound heterozygote with one or both mutations being unknown or present at a frequency lower than 2% in the population studied . dna amplification : pcr was performed using a techne progene thermal cycler ( uk ) . the primer sequences and pcr conditions are as described in ghanem et al , for fragment g amplification . a 474 bp dna fragment corresponding to nucleotides + 188 to + 621 of beta globin gene was amplified using forward 5'ctgggcatgtggagacagag3 primer containing an additional 5'gc rich oligomer which creates a high temperature melting domain : 5'gcgggcggggcggggcgcggcgggcggggc called gc clamp and a reverse 5'cactgatgcaatcattcgtc3 primer ( bioneer company , south korea ) . beta globin gene representation : a portion of beta globin gene spanning from nucleotide + 188 to + 621 and covering exon1 and 2 was amplified using pcr . the position of the 5 single nucleotide polymorphisms ( one in exon 1 and four in intron 2 ) is also presented . three polymorphic nucleotides ivs2(nt 16,74,81 ) are present in the amplicon , and their association defines the beta globin frameworks . dgge was accomplished according to ghanem et al : 10 l of pcr product was subjected to electrophoresis at 160 v for 5 hours in a 6.5% polyacrylamide gel containing a linearly increasing denaturant gradient of 10% - 60% in a water bath at 60c . after migration the gel was stained by ethidium bromide and the position of different bands visualized under 312 nm uv wavelength . as dgge is based on the principle that two double - stranded dna fragments of the same size , but differing in sequence , melt at different points in a denaturing gradient and can be distinguished by differential migration , a uniform temperature with a linear denaturant gradient formed by urea and formamide creates the denaturing environment in dgge . dgge analysis of amplicons belonging to 46 thalassemia major patients revealed that framework 2 is the most abundant among mutant alleles analyzed and represents 45.7% of the cases . frameworks 1 and 3a represent each 23.9% of the cases and framework 3 is the less frequent , namely 6.5% . table 2 shows the frequency of each framework in the population studied and fig 2 shows dgge patterns for individuals with different combinations of these frameworks . as expected , in individuals homozygous for a particular framework , heteroduplex can not form upon renaturation of the amplicon 's strand and only homoduplexes corresponding to complete matching between the two complementary strands of amplicons are possible and consequently a single band is visible upon electrophoresis in a gradient gel and under denaturing conditions ( fig . 4 bands are detectable under similar conditions : 2 bands migrating faster and representing homoduplexes corresponding to each framework and 2 other bands which migrate slowly comparing to homoduplexes due to mismatches between complementary dna strands belonging to different frameworks . each framework combination has its particular pattern of heteroduplexes and homoduplexes migration as nuclotidic sequences and consequently dna migration pattern under denaturing gradient gel electrophoresis varies in each framework . the higher the number of g and c in the polymorphic sites of the amplican , the faster is the migration of homoduplexes . thus , amplicons corresponding to framework 1 which contains ivs2 nucleotide polymorphisms c , g and c respectively at positions 16 , 74 and 81 migrate faster than amplicons corresponding to framework 3a with nucleotides g , t and c and framework 2 with nucleotides c , t and c at those polymorphic positions . amplicons corresponding to framework 3 with nucleotides g , t and t at the above mentioned positions are the slowest ( fig . 2 ) . a : schematic representation of relative migration pattern expected for different frameworks homoduplex after denaturing gradient gel electrophoresis . lanes 1 , 3 : 2/3a lane 2 : 1/2 lane 4 : 1/3a lane 5 : 1/3 lane 6 : 2/2 . in each lane , lower bands migrate faster and thus correspond to homoduplexes while upper bands correspond to heteroduplexes . although extensive studies were performed for molecular characterization of beta globin mutations in northern iran[1517 ] , but as in any prenatal diagnosis program , new or undefined mutations may be encountered . different restriction site polymorphisms have been described near beta globin gene[6 , 7 ] , but few of them are intragenic markers[8 , 9 ] . here we report for the first time polymorphisms for five intragenic markers in north iran using dgge analysis . , we have determined the frequency of these 4 different frameworks among mutant beta globin alleles present in northern iran . their frequency vary between 45.7% for the most abundant ( framework 2 ) to 6.5% for the less abundant ( framework 3 ) . dgge analysis of beta globin gene was also performed in other populations . in india where framework 3a is the most prevalent with a frequency of 66.5% followed by framework 1 with a frequency of 25.2% , frameworks 2 and 3 represent 5.8% and 2.5% respectively of the 206 mutant alleles studied . another study accomplished in north thailand showed that the frequency of fw1 and fw3a was 73.3% and 26.7% respectively in 30 mutant alleles studied . similar frequencies were also reported in a limited number of mutant alleles analyzed in korean population . the difference in framework distribution and their frequencies among population studied reflect the differences in both mutation spectrum and the degree of heterogeneity within a population . indeed framework 2 was the most prevalent in north iran with a frequency of 45.7% while it was absent in east asians and rather rare in indian population studied . as ivsii-1(g - a ) mutation is the most prevalent mutation in north iran with a frequency of around 60%[15 , 16 ] and compound heterozygotes presenting ivsii-1(g - a ) alleles included in this study were also possessing the framework 2 ( data not presented ) , ivsii-1(g - a ) may be associated to framework 2 . this could explain the difference in framework distribution and frequencies observed in iranian and indian or east asian populations where ivsii-1(g - a ) was not reported . although direct mutation detection is routinely performed either by using arms pcr or reverse dot blot analysis or sequencing analysis in molecular diagnosis of thalassemia , this indirect method which necessitates segregation analysis may be used either in cases where unknown mutations are encountered or as a complementary method to confirm the genotype . many diagnostic laboratories apply traditional haplotype analysis by co - segregation studies within a family either in order to detect an unknown mutation for carrier screening or prenatal diagnosis in families with a previous history of disease or as a complementary analysis to direct mutation methods in prenatal diagnosis to confirm the genotype of the fetus . however , most polymorphic sites studied routinely using restriction enzymes are either located outside the gene which may be subject to errors in genotype determination at beta globin gene locus in rare cases of recombination due to the presence of a hotspot for recombination located upstream of beta globin gene[10 , 11 ] or may be located within the beta globin gene with a low degree of polymorphism and therefore non - informative in many families . the frameworks characterized in the present study represent a combination of 5 single nucleotide polymorphisms all located within the beta globin gene which abolishes errors due to recombination . moreover a single pcr reaction allows the simultaneous evaluation of the genotype in all 5 intragenic polymorphic sites determining each specific framework , while in segregation analysis using rflp method , genotyping of each polymorphic site necessitates a separate pcr reaction followed by enzymatic digestion . regarding their abundance these polymorphisms could be studied either as frameworks or as single polymorphic sites using restriction endonuclease analysis : alw44 for codon 2 , avaii for ivs2 - 16 , maeiii for ivs2 - 81 and sspi for ivs2 - 666 as intragenic markers for detection of beta thalassemia by linkage analysis . polymorphism at ivs2 nt666 was studied separately in north iran as the t nucleotide at this position is recognized by sspi . our previous findings show that ivs2 nt 666(t ) is present in about 85.7% of mutant alleles . this nucleotide is present in frameworks 1 and 2 which together represent about 70% of studied mutant alleles . as table 1 indicates the c allele at codon 2 is in the same linkage phase than ivs2 nt 16(c ) and ivs2 nt 666(t ) . the two former polymorphisms can also be distinguished by restriction analysis using alw44 and avaii respectively . thus as alw44 is commercially less expensive than avaii , we recommend the use of the former enzyme in segregation analysis using rflp . all 4 defined frameworks were present in beta thalassemia chromosomes at a frequency higher than 5% and consequently may be useful for tracking mutant alleles in prenatal diagnosis programs . tendinopathy is an overuse tendon injury that occurs in loaded tendons and results in pain and functional impairment , a major problem in sports and occupational medicine . characteristic changes in tendon structure include loss of type i collagen , increase in proteoglycan content , and neovascularization . this has been termed failed healing response and results in the tendon being less capable of sustaining repeated tensile load . recently , a new pathology model has been accepted to explain the clinical course of tendinopathy . the cook and purdam model states there is a continuum of tendon pathology that , for easier clinical use , has been divided into 2 clear groups : reactive tendinopathy / early tendon disrepair and degenerative tendinopathy / late tendon disrepair . because the same intervention to all presentations of tendinopathy is unlikely to show efficacy , applying this model might be useful to rationally place specific treatments through the evolution of the pathology . although many treatments are described for painful tendons , the scientific evidence for most of the conservative and surgical treatments is not always conclusive . eccentric exercise has evidential basis obtained from randomized controlled trials and is currently included in most treatment algorithms.4 , 5 it involves active lengthening of the muscle tendon unit and short - term improvements in tendon pain6 , 7 , 8 and normalization of tendon structure have been demonstrated . nevertheless , why eccentric exercise reduces pain in tendinopathy and what is the correct intensity , speed , load , and frequency remains unclear.2 , 10 other therapeutic options that have shown some positive outcomes for tendinopathies treatment include shockwaves , sclerosing polidocanol injections , topical nitroglycerin therapy , and cooling therapies . costicosteoid injections have also shown very good outcomes in the short term but should be avoided due to the cortisone s negative influence on the tendon structure . a composition containing mucopolysaccharides , type i collagen , and vitamin c ( mcvc ) demonstrated efficacy both in in vitro models and human studies.13 , 14 , 15 mcvc induces changes in tenocytes metabolism in vitro , thereby helping to preserve the structure of the extracellular matrix . results indicate that this formulation promotes the endogenous synthesis of collagen type i , avoiding the accumulation of collagen type iii and aggrecan , and therefore may interfere with the degeneration of tendon tissue . these findings suggest that mcvc may improve tendinopathy symptomatology , reducing discomfort and improving tendon function . indeed , in 3 studies in humans , 2 observational and 1 controlled , the consumption of the supplement might be associated with clinical and structural improvements in the recovery of tendinopathies.13 , 14 , 15 the goal of this trial was to determine whether daily consumption of a supplement containing mcvc , added to an eccentric exercise program , provided some additional benefit in improving symptoms of chronic noninsertional tendinopathy in patients with a gradually evolving painful condition in the midportion of the achilles tendon for at least 3 months . in parallel , the specific effect of mcvc when combined with 2 different physical therapies was compared : eccentric training ( ec ) or passive stretching ( ps ) . finally , whether there is a different response pattern to the aforementioned treatments depending on the pathology stage of the patient ( reactive tendinopathy vs degenerative tendinopathy ) was assessed . this was a nutrition - based , randomized , controlled , multicenter trial to assess the efficacy and safety of a supplement containing mcvc combined with either an ec or ps program , and compared with ec alone . patients were randomly assigned to 1 of 3 treatment groups : ec , combination of ec and mcvc , and combination of ps and mcvc . patients were assigned a randomization number taken from a computer - generated randomization list following the chronological order by which they were included in the study , independent of the stage of their tendinopathy . it was conducted in men and nonpregnant women , aged 18 to 70 years , with painful noninsertional achilles tendinopathy of the midportion for at least 3 months . diagnosis was based on clinical examination showing a painful thickening of the achilles tendon located 2 to 6 cm above the tendon insertion , and confirmed by ultrasonography ; specifically , local thickening of the tendon , irregular tendon structure with hypoechoic areas , and irregular fiber orientation . clinical suspicions of insertional tendinopathy , tendon rupture ( thomson test and palpable gap ) , neural disorder , systemic disease , like gout , spondylarthropaty , rheumatoid arthritis and sarcoidosis , or pregnancy were considered exclusion criteria . patients which already received a previous treatment with eccentric training or prp for the studied injury were also excluded . the stage of the tendinopathy was classified into 2 subgroups according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . based on ultrasonographic assessment , reactive tendinopathy is characterized by swollen tendon and diffuse hypoechogenicity occurring between intact collagen structures ( figure 1a ) , whereas degenerative tendinopathy shows increased matrix disorganization represented by hypoechoic regions with few reflections from collagen fascicles and clear neovascularization usually visible on doppler ultrasound ( figure 1b ) . stage of tendinopathy was assessed using imaging criteria . to avoid variations between centers , a single specialist blinded to the study group of the participants evaluated the imaging across all study sites . fifty - nine potentially eligible patients were recruited between september 2012 and june 2013 in 5 spanish sports medicine centers . in each clinical center , a team composed of an orthopedist and a physiotherapist was in charge of the study . a consolidated standards of reporting trials diagram of participant flow through the trial is shown in figure 2 . a total of 58 participants fulfilled the inclusion criteria and consented to take part in the study . the intention to treat ( itt ) population was defined by all the participants who took at least 1 unit of the trial product . the subjects did not receive any financial incentive to participate in the study but received the assigned treatment free of charge . the study was approved by the official clinical research ethical committees of consell catal de lesport , barcelona , spain . patients allocated in the ec training group were instructed to perform a training protocol according to alfredson et al . to guarantee the consistency of the procedures among different research centers , the protocol was agreed upon by all the researchers in prestudy meetings , and its correct execution was confirmed by a physiotherapist every week . the gastrocnemius muscle was eccentrically loaded both with the knee straight and , to maximize the activation of the soleus muscle , also with the knee bent . this protocol was repeated twice per day , 7 days a week , for 12 weeks . patients in the ec + mcvc group consumed 3 capsules per day containing 435 mg mucopolysaccharides , 75 mg collagen type i , and 60 mg vitamin c ( tendoactive ; bioiberica sa , palafolls , spain ) and followed the ec protocol . patients in the ps + mcvc group consumed the dietary supplement and were instructed to perform a personalized protocol of passive stretching that was supervised weekly by a physiotherapist . ps routines were included in the program to guarantee the same weekly monitoring in all the study groups . a standard protocol was applied , including 2 exercises to stretch the gastrocnemius muscle and 2 for the soleus muscle . patients were evaluated at baseline , 6 weeks , and 12 weeks after the beginning of the treatment protocol with the original english version of the validated victorian institute of sports assessment - achilles ( visa - a ) questionnaire . the visa - a questionnaire scores range from 0 to 100 , with higher scores corresponding with less pain and increased activity . every week , pain was evaluated at rest and during activity using a visual analog scale ( vas ) . the evolution of the affected tendons was monitored ultrasonographically on every visit and bilateral thickness was recorded . bilateral thickness was measured in the transversal section of the more thickened cross point at a level of 2 to 6 cm above the tendon insertion . the statistical analysis followed the statistical plan according to the principles specified in the guidelines of the international council for harmonization of statistical principles for clinical trials and of the committee for proprietary medicinal products with the efficacy working party points to consider on multiplicity issues in clinical trials . no interim analyses of efficacy were planned for this study and , therefore , no multiplicity adjustments were needed . the primary efficacy analysis was conducted to evaluate the absolute change from baseline to last visit of the visa - a score , using an ancova model with the baseline value as a covariate and a baseline observation carried forward approach on itt . all secondary efficacy variables were assessed similarly as primary variables using an ancova model with the baseline value as a covariate . treatment effect was also evaluated in 2 subgroups designed according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . this was a nutrition - based , randomized , controlled , multicenter trial to assess the efficacy and safety of a supplement containing mcvc combined with either an ec or ps program , and compared with ec alone . patients were randomly assigned to 1 of 3 treatment groups : ec , combination of ec and mcvc , and combination of ps and mcvc . patients were assigned a randomization number taken from a computer - generated randomization list following the chronological order by which they were included in the study , independent of the stage of their tendinopathy . it was conducted in men and nonpregnant women , aged 18 to 70 years , with painful noninsertional achilles tendinopathy of the midportion for at least 3 months . diagnosis was based on clinical examination showing a painful thickening of the achilles tendon located 2 to 6 cm above the tendon insertion , and confirmed by ultrasonography ; specifically , local thickening of the tendon , irregular tendon structure with hypoechoic areas , and irregular fiber orientation . clinical suspicions of insertional tendinopathy , tendon rupture ( thomson test and palpable gap ) , neural disorder , systemic disease , like gout , spondylarthropaty , rheumatoid arthritis and sarcoidosis , or pregnancy were considered exclusion criteria . patients which already received a previous treatment with eccentric training or prp for the studied injury were also excluded . the stage of the tendinopathy was classified into 2 subgroups according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . based on ultrasonographic assessment , reactive tendinopathy is characterized by swollen tendon and diffuse hypoechogenicity occurring between intact collagen structures ( figure 1a ) , whereas degenerative tendinopathy shows increased matrix disorganization represented by hypoechoic regions with few reflections from collagen fascicles and clear neovascularization usually visible on doppler ultrasound ( figure 1b ) . stage of tendinopathy was assessed using imaging criteria . to avoid variations between centers , a single specialist blinded to the study group of the participants evaluated the imaging across all study sites . fifty - nine potentially eligible patients were recruited between september 2012 and june 2013 in 5 spanish sports medicine centers . in each clinical center , a team composed of an orthopedist and a physiotherapist was in charge of the study . a consolidated standards of reporting trials diagram of participant flow through the trial is shown in figure 2 . a total of 58 participants fulfilled the inclusion criteria and consented to take part in the study . the intention to treat ( itt ) population was defined by all the participants who took at least 1 unit of the trial product . the subjects did not receive any financial incentive to participate in the study but received the assigned treatment free of charge . the study was approved by the official clinical research ethical committees of consell catal de lesport , barcelona , spain . patients allocated in the ec training group were instructed to perform a training protocol according to alfredson et al . to guarantee the consistency of the procedures among different research centers , the protocol was agreed upon by all the researchers in prestudy meetings , and its correct execution was confirmed by a physiotherapist every week . the gastrocnemius muscle was eccentrically loaded both with the knee straight and , to maximize the activation of the soleus muscle , also with the knee bent . this protocol was repeated twice per day , 7 days a week , for 12 weeks . patients in the ec + mcvc group consumed 3 capsules per day containing 435 mg mucopolysaccharides , 75 mg collagen type i , and 60 mg vitamin c ( tendoactive ; bioiberica sa , palafolls , spain ) and followed the ec protocol . patients in the ps + mcvc group consumed the dietary supplement and were instructed to perform a personalized protocol of passive stretching that was supervised weekly by a physiotherapist . ps routines were included in the program to guarantee the same weekly monitoring in all the study groups . a standard protocol was applied , including 2 exercises to stretch the gastrocnemius muscle and 2 for the soleus muscle . patients were evaluated at baseline , 6 weeks , and 12 weeks after the beginning of the treatment protocol with the original english version of the validated victorian institute of sports assessment - achilles ( visa - a ) questionnaire . the visa - a questionnaire scores range from 0 to 100 , with higher scores corresponding with less pain and increased activity . every week , pain was evaluated at rest and during activity using a visual analog scale ( vas ) . the evolution of the affected tendons was monitored ultrasonographically on every visit and bilateral thickness was recorded . bilateral thickness was measured in the transversal section of the more thickened cross point at a level of 2 to 6 cm above the tendon insertion . patients were evaluated at baseline , 6 weeks , and 12 weeks after the beginning of the treatment protocol with the original english version of the validated victorian institute of sports assessment - achilles ( visa - a ) questionnaire . the visa - a questionnaire scores range from 0 to 100 , with higher scores corresponding with less pain and increased activity . every week , pain was evaluated at rest and during activity using a visual analog scale ( vas ) . the evolution of the affected tendons was monitored ultrasonographically on every visit and bilateral thickness was recorded . bilateral thickness was measured in the transversal section of the more thickened cross point at a level of 2 to 6 cm above the tendon insertion . the statistical analysis followed the statistical plan according to the principles specified in the guidelines of the international council for harmonization of statistical principles for clinical trials and of the committee for proprietary medicinal products with the efficacy working party points to consider on multiplicity issues in clinical trials . no interim analyses of efficacy were planned for this study and , therefore , no multiplicity adjustments were needed . the primary efficacy analysis was conducted to evaluate the absolute change from baseline to last visit of the visa - a score , using an ancova model with the baseline value as a covariate and a baseline observation carried forward approach on itt . all secondary efficacy variables were assessed similarly as primary variables using an ancova model with the baseline value as a covariate . treatment effect was also evaluated in 2 subgroups designed according to the cook and purdam pathology model : patients with reactive tendinopathy and patients with degenerative tendinopathy . three participants were excluded from the randomized population for not having attended the inclusion visit ( 1 in the ps + mcvc group and 2 in the ec + mcvc group ) . fifty - five participants ( 94.83% of those randomized ) entered the itt population ; 23 had reactive tendinopathy and 32 degenerative tendinopathy . a consort diagram of participant flow through the trial is shown in figure 2 . the average ( sd ) age was 38.9 ( 6.6 ) years for the ec group , 43.5 ( 14.5 ) years for the ec + mcvc group , and 40.2 ( 10.6 ) years for the ps + mcvc group . the participants were mostly men : 78% , 82% , and 80% in the ec , ec + mcvc , and ps + mcvc groups , respectively . there were 5 , 9 , and 9 patients with reactive tendinopathy and 15 , 8 , and 9 patients with degenerative tendinopathy in the ec , ec + mcvc , and ps + mcvc groups , respectively . there were no significant differences at baseline in any of the clinical outputs of the study . when analyzing the obtained data regardless of the patient s stage of disease , a significant improvement in visa - a score was detected in all 3 treatment groups at 6 and 12 weeks follow - up , when compared with baseline . there was no between - group effect for the primary outcome of visa - a questionnaire between baseline and the end of the study ( p > 0.1 ) . in all the studied groups , vas - rated pain was reduced both at rest and during activity after the 12-week treatment . differences in pain reduction among treatments were found between the ps + mcvc and the ec groups , being significant at rest : for ps + mcv ( 3.7 [ 0.8 ] cm ) and ec ( 2.7 [ 1.3 ] cm ) ( p < 0.05 ) , and tending toward significance during activity : ps + mcv ( 4.4 [ 1.1 ] cm ) and ec ( 3.5 [ 1.4 ] cm ) ( p = 0.074 ) . although no significant variation was found in the tendon bilateral thickness among treatments ( p > 0.1 ) , reduction from baseline was only detected in the ps + mcvc treatment group ( 0.63 [ 0.73 ] mm ) ( p < 0.05 ) . in patients with reactive tendinopathy , the improvement of the visa - a score at 12 weeks follow - up was 56% for the ps + mcv group , 44% for the ec + mcv group , and 30% for the ec group ( table i ) ( p = 0.154 ) . although no statistically significant results were found , a tendency toward significance was found in the ec and ec + mcvc groups ( p = 0.069 ) . in patients with degenerative tendinopathy , improvement in visa - a score had the same magnitude among treatments ( table ii ) . similarly , pain at rest improved in all 3 groups of treatment in all randomized patients . in patients with reactive tendinopathy , the pain reduction at 12 weeks follow - up was greater for the group ps + mcvc ( 3.815 [ 0.8 ] cm ) compared with the ec group ( 2.80 [ 1.0 ] cm ) ( p < 0.05 ) . for the ec + mcvc group the pain reduction can not be compared in absolute values because the mean pain value at rest was not balanced at baseline ( table i ) . in patients with degenerative tendinopathy , the magnitude of the pain reduction was similar among the study groups ( table ii ) . the evaluation of pain during activity followed the same pattern of response as pain at rest , although differences among treatment groups failed to reach statistical significance , either in reactive or in degenerative tendinopathy . the bilateral thickness of the affected tendon remained constant during the study period in patients in the ec and ec + mcvc groups from the itt population . in contrast , a significant reduction ( 0.63 [ 0.3 ] mm ; p < 0.05 ) was detected in patients in the ps + mcvc group . in patients with degenerative tendinopathy , a significant reduction was registered in the ps + mcvc group ( p < 0.05 ) , whereas no differences were detected in patients with reactive tendinopathy ( table iii ) . in this study , 3 different interventions have been evaluated : ec alone , the combination of ec with a dietary supplement containing mcvc , and an alternative physical therapy such as ps combined with the dietary supplement in patients with achilles tendinopathy . the main goal was to determine the additional benefit of mcvc to a physical therapy program . after 90 days , all 3 treatment groups experienced significant improvements from baseline in visa - a , vas - rated pain at rest , and vas - rated pain during activity . these improvements were not only statistically , but also clinically , significant because the minimum clinically important difference is 9 mm for vas - rated pain and 6.5 points in the visa - a scale.17 , 18 the clinical improvements registered as a result of the ec training agree with those previously described16 , 19 and were expected because ec is currently the gold standard for achilles tendinopathy treatment . the addition of mcvc to ec significantly improved pain , but not function - related outcomes . when patients were divided according to severity of tendinopathy , a statistically significant clinical benefit from mcvc in patients with reactive but not degenerative tendinopathy , as determined by vas - rated pain , was clearly observed . although functionality , assessed with the visa - a test , did not reach a statistically significant improvement it showed a tendency toward significance , although statistical significance was not reached due to low patient number . however , it reached a clinically significant improvement because it increased more than 6.5 points in both mcvc groups compared with controls . according to the model described by cook and purdam , in reactive tendinopathy a homogeneous , noninflammatory cell response to load that leads to metaplastic change in cells and cell proliferation is found . theoretically , this cellular deregulation could be counterbalanced by the effects of mcvc on tenocyte metabolism , as described by shakibaei et al . in degenerative tendinopathy , areas of cell death due to apoptosis , trauma , or tenocyte exhaustion in tendons have been reported . in this stage of the pathology the efficacy of mcvc could be limited by the reduced viability of the tenocytes . in this study , a protocol of ps supervised by a physiotherapist and adjusted personally and supplemented with mcvc was shown to improve pain reduction and function recovery as good as a standard ec in both reactive and degenerative tendinopathy . although the efficacy of the eccentric gastrocnemius muscle training has been widely described , less data about the efficacy of a protocol of ps are available . it has been recently published that isometric exercises could induce pain reduction in patellar tendinopathy . because ps implies a certain degree of isometric contractions , the potential analgesic effect of this intervention could partially explain the clinical findings for this group . to elucidate the contribution of the ps protocol and the mcvc supplement to the clinical improvement , an additional group of patients following the ps program but without the mcvc supplement would have been required . from our study the placebo effect can not be assessed because the groups not receiving the mcvc supplement did not receive an inert capsule . another weakness from the study is the lack of blinding of the therapists treating the patients . although the lack of blinding could potentially interfere with the results , we think that this should not have had a major impact on the patients evaluation and reported outcomes as greater improvements were expected from the ec+mcvc treatment compared to the ps+mcvc group and the obtained data did not match the expectations . regarding the structural changes during the study period , it has been previously described that a program of eccentric exercise is valid in the symptomatic nonoperative management of tendinopathy , probably by promoting collagen fiber cross - linkage formation within the tendon and , thereby , facilitating tendon remodeling . previous studies report increased type i collagen synthesis and reduction of fluid within the tendon after eccentric training offering a possible explanation for the mechanism of tendon healing.24 , 25 nevertheless , actual histologic adaptations after a program of ec or ps remain unclear . in this trial , the stretching program improved tendon structure when compared with baseline , as evidenced by a reduction in tendon thickness in patients with degenerative tendinopathy . during the study period this might be caused by the harshness of ec , which applies some degree of aggression to the tendon structure , and could maintain longer a temporary reactive disorganization of the tendon tissue . eccentric training is not the only kind of exercises that have been used for treating tendinopathies , other exercises like concentric ones have been tested for tendinopathy management . some studies have compared the effectiveness of eccentric vs. concentric training but conflicting results have been obtained ; some studies found that eccentric training was statistically superior to concentric training and others did not find any statistical difference between the treatments.8 , 26 , 27 some protocols combine eccentric and concentric training in the same sessions , like the silbernagel - combined program or the stanish and curwin programs . besides concentric exercises , one study found that isometric muscle contractions reduce pain in patellar tendinopathy . further research on this field is required as the effectiveness on the achilles tendon can not be assessed . due to the existing uncertainty in the programs equivalence , , a dietary supplement containing mcvc seems to present a high safety profile and to be useful for managing tendinopathies , providing some additional benefit to ec , especially in early stages of the disease . in the reactive phase , tendons present a short - term adaptation to overload with thickening and increased stiffness , but without severe matrix and vascular changes . a reactive tendon has the potential to revert to normal if the overload is sufficiently reduced or if there is enough time between loading sessions . in this type of patient , conversely , in degenerative pathology the tendon presents areas of cell death , vessel - filled matrix , matrix breakdown products , and little collagen , and therefore the reversibility of pathologic changes is limited . although a nutrition - based approach is less likely to offer a clinical benefit at this stage , certain clinical improvements were observed in the ps - mcvc group during our study . however , further studies with larger sample sizes are required to confirm the efficacy of mcvc for the dietary management of tendinopathy . the data obtained from this study confirm the therapeutic potential of ec for the management of tendinopathy , both at reactive and degenerative stages . the novelty of this study is that it shows the effects of a new therapeutic approach consisting of personalized stretching routines and dietary supplementation with mcvc . in patients with reactive tendinopathy , supplementation with mcvc further decreased pain levels compared to ec alone , so it could be included in the therapeutic arsenal for tendinopathy . in patients with degenerative tendinopathy , despite the lack of blinding and placebo treatment , the obtained results suggested that for patients with difficulties in performing ec due to advanced age , illness , or extreme tendon injury , a ps program supplemented with mcvc could be an alternative treatment to be taken into account . financial support for this work was provided by bioiberica , sa , palafolls , spain . bioiberica sa is the trademark owner , manufacturer , and distributor of tendoactive , the supplement used in this study . the authors have indicated that they have no other conflicts of interest regarding the content of this article .

objectivefour combinations of five neutral sequence changes at rs713040 , rs10768683 , rs7480526 , rs7946748 , and rs1609812 occurring in the human beta globin gene defined as frameworks have been reported in beta globin gene . here we report for the frequency of these frameworks in thalassemia major patients of north iran.methodsbeta globin gene frameworks of 46 thalassemia major patients of north iran were determined using denaturing gradient gel electrophoresis.findingsall these frameworks called framework 1 , 2 , 3 , 3a were present at the frequency of 23.9% , 45.7% , 6.5% and 23.9% respectively.conclusionthese frameworks may be used for tracking mutant alleles in prenatal diagnosis programs . backgroundtendinopathy is an overuse tendon injury that occurs in loaded tendons and results in pain and functional impairment . although many treatments for painful tendons are described , the scientific evidence for most of the conservative and surgical treatments is not always conclusive.objectivesthis study was designed to evaluate the efficacy of 3 different interventions in patients with achilles tendinopathy . the interventions include the combination of 2 physical therapy programs ( eccentric training [ ec ] or passive stretching [ ps ] ) with a supplement containing mucopolisaccharides . the efficacy of the interventions was evaluated depending on the stage of the disease.methodsfifty-nine patients were randomly assigned to 1 of 3 treatment groups , and classified according to the disease stage : reactive versus degenerative tendinopathy . treatment groups were ec ; ec + a dietary supplement containing mucopolisaccharides , type i collagen , and vitamin c ( mcvc ) ; and a passive stretching program + mcvc . patients were evaluated at baseline , 6 weeks , and 12 weeks with the victorian institute of sports assessment - achilles questionnaire for function , a visual analog scale for pain , and ultrasound characterization for the evolution of tendon structure.resultsa significant improvement in victorian institute of sports assessment - achilles questionnaire score , pain at rest , and pain during activity were detected in all 3 treatment groups at 6 and 12 weeks follow - up when compared with baseline . in patients with reactive tendinopathy , the reduction in pain at rest was greater in the groups who took the supplemental mcvc than in the ec alone group ( p < 0.05).conclusionsmcvc seems to be therapeutically useful for management of tendinopathies , providing some additional benefit to physical therapy . this is especially evident in early stages of the disease , when the tendon does not present severe matrix and vascular changes.clinicaltrials.gov identifiernct01691716 .

livestock animals are subjected to a huge sort of stress factors , such as environmental conditions , animal management or diets that may disrupt the intestinal ecosystem and increase the risk of pathogenic infections . in the swine industry , transition from the liquid to a solid diet during weaning and post - weaning are critic processes due to an important decrease of feed intake , impairing animal performance , immune function , microbiota and therefore , gut homeostasis . concerning the immune system , these performance stages lead to the end of transference of passive maternal immunity from the sows to the piglets which still have an immature or developing immune system . this means that piglets become highly susceptible to intestinal pathogens such as enterotoxigenic e.coli ( etec ) or salmonella enterica ser . antibiotic growth promoters ( agps ) have been extensively used as dietary additives to minimize the risk of infections , especially during the critical periods of animal production , like weaning . however , after european ban of agps ( ec 1831/2003 ) several studies proposed the use of probiotics and prebiotics as natural alternatives to agps . in that sense , the health benefits of probiotics and prebiotics have been widely accepted as gut microbiota stabilizers because they are believed to avoid pathogenic colonization and therefore improving animal welfare . our group recently published in vitro results that demonstrated the potential of probiotic saccharomyces cerevisiae var boulardii and prebiotic -galactomannan ( gm ) to prevent intestinal infections by e.coli and salmonella in pigs . here , we aimed to extend the knowledge about the prebiotic role of gm against salmonella challenge according to its polysaccharide structure . the porcine small intestine epithelial cell line ipi-2i ( ecacc 93100622 ) was established from the ileum of an adult boar ( slad / d haplotype ) . ipi-2i cells were maintained in dmem glutamax ( invitrogen , spain ) supplemented with 10% fcs ( invitrogen ) , insulin 10 g / ml ( sigma aldrich , saint - quentin , france ) . in all experiments , cells were cultured in 6-well plates ( nunc , labclinics , spain ) to confluence . before the addition of pre / probiotics and/or infection , cells were washed three times and the cell culture was replaced with dmem media supplemented with insulin 10 g / ml ( sigma aldrich ) . cells were used between passages 30 and 70 and periodically tested to avoid mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza ) . to assess the functionality of mannans according to their structure we compared the prebiotic gm ( salmosan , patent wo2009/144070 a2 , licensed to industrial tcnica pecuaria , itpsa , barcelona , spain ) to another mannanoligosaccharide purified from saccharomyces cerevisiae ( mannan sc , sigma ) and to d - mannose monosaccharide ( sigma - aldrich ) in relation to a negative control ( medium ) . prebiotic gm , mannan sc and d - mannose were diluted in dmem glutamax ( 1 mg / ml ) , vortexed and incubated 30 min at 37c . immediately before the salmonella infection , gm , mannan sc and d - mannose were respectively added to each well at 10g / ml . pathogenic salmonella enterica serovar typhimurium ( salmonella ) with antigenic formulae ( 4,12:i:1,2 ) , resistant to ampicillin , chloramphenicol , streptomycin , sulfonamide and tetracycline was provided by dr . ignacio badiola from centre recerca en sanitat animal ( cresa , irta - uab , bellaterra , spain ) . aliquots of salmonella were provided in bacterial cryo - preservers ( technical service consultants , ferrer international , spain ) and stored at -80c until use . before infection iec cells , a single salmonella cryo - preserver was added to 20 ml of luria - bertani media ( lb ) and cultured for 34h at 37c with 180 rpm rotational agitation ( multitron ht , infors ) . for infection , salmonella was used during the exponential growth phase as determined by absorbance at 600 nm . after 3 h of host cell - pathogen assay performed as described before , 75g / ml of gentamycin ( sigma - aldrich ) were added to each well to avoid bacterial overgrowth . cytokine protein determination in the culture supernatant was performed by enzyme - linked immunosorbent assays ( elisas ) . swine il-6 and cxcl8 duoset elisa kits ( r&d systems , vitro sp ) were used according to manufacturer s recommendations . all statistical analyses were performed using glm procedure ( proc glm ) by sas software version 9.1.3 ( sas institute ) . means for protein secretion were considered in a 2 * 3 factorial design ( two infection levels * 3 experimental treatments ) with duncan s post - test for group analysis . superscript letters are used to designate statistical significance : bars with different superscript letters are significantly different ( p < 0.05 ) and bars with same superscript letters indicate no statistical difference ( n.s ) . the porcine small intestine epithelial cell line ipi-2i ( ecacc 93100622 ) was established from the ileum of an adult boar ( slad / d haplotype ) . ipi-2i cells were maintained in dmem glutamax ( invitrogen , spain ) supplemented with 10% fcs ( invitrogen ) , insulin 10 g / ml ( sigma aldrich , saint - quentin , france ) . in all experiments , cells were cultured in 6-well plates ( nunc , labclinics , spain ) to confluence . before the addition of pre / probiotics and/or infection , cells were washed three times and the cell culture was replaced with dmem media supplemented with insulin 10 g / ml ( sigma aldrich ) . cells were used between passages 30 and 70 and periodically tested to avoid mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza ) . to assess the functionality of mannans according to their structure we compared the prebiotic gm ( salmosan , patent wo2009/144070 a2 , licensed to industrial tcnica pecuaria , itpsa , barcelona , spain ) to another mannanoligosaccharide purified from saccharomyces cerevisiae ( mannan sc , sigma ) and to d - mannose monosaccharide ( sigma - aldrich ) in relation to a negative control ( medium ) . prebiotic gm , mannan sc and d - mannose were diluted in dmem glutamax ( 1 mg / ml ) , vortexed and incubated 30 min at 37c . immediately before the salmonella infection , gm , mannan sc and d - mannose were respectively added to each well at 10g / ml . pathogenic salmonella enterica serovar typhimurium ( salmonella ) with antigenic formulae ( 4,12:i:1,2 ) , resistant to ampicillin , chloramphenicol , streptomycin , sulfonamide and tetracycline was provided by dr . ignacio badiola from centre recerca en sanitat animal ( cresa , irta - uab , bellaterra , spain ) . aliquots of salmonella were provided in bacterial cryo - preservers ( technical service consultants , ferrer international , spain ) and stored at -80c until use . before infection iec cells , a single salmonella cryo - preserver was added to 20 ml of luria - bertani media ( lb ) and cultured for 34h at 37c with 180 rpm rotational agitation ( multitron ht , infors ) . for infection , salmonella was used during the exponential growth phase as determined by absorbance at 600 nm . after 3 h of host cell - pathogen assay performed as described before , 75g / ml of gentamycin ( sigma - aldrich ) were added to each well to avoid bacterial overgrowth . cytokine protein determination in the culture supernatant was performed by enzyme - linked immunosorbent assays ( elisas ) . swine il-6 and cxcl8 duoset elisa kits ( r&d systems , vitro sp ) were used according to manufacturer s recommendations . all statistical analyses were performed using glm procedure ( proc glm ) by sas software version 9.1.3 ( sas institute ) . means for protein secretion were considered in a 2 * 3 factorial design ( two infection levels * 3 experimental treatments ) with duncan s post - test for group analysis . the p value 0.05 was considered to be significant . superscript letters are used to designate statistical significance : bars with different superscript letters are significantly different ( p < 0.05 ) and bars with same superscript letters indicate no statistical difference ( n.s ) . many intestinal pathogenic strains of salmonella and e.coli share type i fimbriae , although from different evolutionary origin , which have high affinity for mannose residues of glycoproteins present on cell surface of the host . the recognition of these residues leads to bacterial adherence to enterocyte surface and intestinal colonization by the pathogen . our recent articles showed how probiotic strain of saccaromyces cerevisiae var boulardii ( scb ) and a recently developed prebiotic -galactomannan ( gm ) , derived from fruit seed of the ceratonia silliqua tree , inhibit in vitro adhesion of salmonella and etec to pig intestinal cell line ipi-2i . indeed , both products decreased the overall proinflammatory profile induced by etec or salmonella , with the least expenditure of resources for innate immune response to deal with each pathogen . mannan derived products , such as scb and gm , are widely accepted to prevent salmonella colonization by binding to the adhesin fim h of the type i fimbriae . the interaction of probiotic scb and prebiotic gm was already confirmed in our previous work . however , enterotoxigenic e.coli k88 , which is one on the most important causes of neonatal and post - weaning diarrhea ( pwd ) in piglets , bears type iv fimbriae or pilli . the latter has been described as mannose resistant to erythrocyte agglutination test in the presence of d - mannose ( 5% ) . nevertheless , the k88 adhesins ( ab , ac and ad ) bind to -galactose residues of receptors of syalic glycoprotein and to neutral glycosphingolipids expressed on the surface of the intestinal mucosa . the presence of -galactose in scb and gm may mimic host receptor for k88 adhesin and therefore inhibit etec attachement to cell surface . in addition to the residue composition , prebiotic functionality is determined by the size and molecular structure of the saccharides . here , we evaluated functionality of gm according to their polysaccharide structure . we compared gm to monosaccharide d - mannose and to another mannan oligosaccharide purified from saccharomyces cerevisiae yeast ( mannan sc ) at a final concentration of 10 g / ml . we observed that in vitro challenge with salmonella induces the secretion of proinflammatory cytokine il6 ( 4-fold increase : 224 vs. 897 pg / ml ; figure 1a ) and chemokine cxcl8 ( 2-fold increase : 2645 vs. 5192 pg / ml ; figure 1b ) compared with control without infection . both , gm and mannan sc showed a 40% reduction of salmonella - induced secretion of il6 ( 897 vs. 563 and 585 pg / ml , figure 1a ) . concerning cxcl8 , gm reduce chemokine secretion by 30% ( 5192 vs 3697 pg / ml ; figure 1b ) . however , mannan sc shows a trend to reduce cxcl8 secretion induced by salmonella ( fig . interestingly , the secretion of proinflammatory il6 and cxcl8 in iecs treated with monosaccharide d - mannose was similar to the control of infection ( fig . present data confirms early results shown by oyofo et al . and vil et al . who showed higher efficiency of oligomeric mannoses compared with monomeric d - mannose to inhibit in vivo salmonella infection in other productive species like chickens . in pigs , the addition of a commercial source of mannanoligosaccharides in the diet increased weight gain during the starter period , improved feed efficiency in vivo together with a reduction of enterobacteria counts in the jejunum ( see ref . indeed , mannanoligosaccharide additives have been shown to modify the composition of the gastrointestinal microbiota , resulting in increased populations of bacteroidetes spp and lactobacillus spp after salmonella infection in vivo , leading to a greater compensatory weight gain of pigs ( see ref . cytokine il6 ( a ) and chemokine cxcl8 ( b ) concentration in supernatants from ipi-2i cells ( 1 10 cells / well ) co - cultured for 24 h with salmonella ( moi = 4 ) is decreased by gm and mannan sc ( 10 g / ml ) . regard , our previous in vitro works showed that scb has a higher anti - inflammatory role in etec infections . in contrast , gm is a potent prophylactic agent against salmonella infections . therefore , the preference of the use of each probiotic or prebiotic additive as a dietary alternative to agps , should be determined , if possible , according to the specific conditions of each farm . this means the prevalence of etec or salmonella of each productive facility or region . in that sense , considering that etec infections often occur during weaning , while salmonella usually colonizes post - weaning and finishing growing pigs , to enrich diets with scb at early stages to prevent etec infections and with gm to reduce salmonella prevalence at final growing stages should be considered . in addition , we hypothesize that the prebiotic gm may enhance probiotic scb survival and colonization of the intestinal tract improving host health . future studies should determine the suitability to combine gm and scb to become a symbiotic additive to prevent intestinal infections . nowadays in eu , products such as mannan sc and gm are not authorized as zootechnical feed additives under the commission regulation ( ec ) 1831/2003 . in the future , both products could be authorized following concepts described in the opinion of efsa ( see ref . 23 ) concerning potential new categories of zootechnical feed additives . in this case it would be additives which improve product quality as microbial contamination controllers . however , today it is also possible to introduce them in the european feed legislation according to the regulation ( ec ) 767/2009 where there is a description of how ingredients can be used for particular purposes . in conclusion , we demonstrated that oligosaccharide structure is crucial for mannans to develop their prebiotic role to inhibit pathogenic adhesion and therefore to reduce proinflamatory immune response induced by the pathogen . transposable elements ( tes ) dispersed in the mammalian genome are on the rise , as completion of the whole human genome sequence . contrary to our expectations , tes , thought to be junk dna , were estimated to account for approximately 45% of the human genome ( international human genome sequencing consortium , 2001 ) . tes can be categorized by various features such as the capacity to mobilize themselves , dna / rna intermediation , and the existence of a long terminal repeat ( ltr ) . endogenous retroviruses ( ervs ) and long interspersed elements ( lines ) have gene - encoding enzymatic machinery that could mobilize to other genomic regions as autonomous elements . however , nonautonomous elements , especially alu elements , which are classified as short interspersed elements ( sine ) and sine - variable number tandem repeat - alu - like sequences ( sva ) , mobilize using the reverse transcriptase of the line . all of the tes described above are integrated into the host genome by their rnas , but the integration of dna transposons is mediated by dna . ervs and ltr retrotransposons have ltrs , but sines and lines are non - ltr elements [ 13 ] . one group of retrotransposon - like elements , mammalian apparent ltr retrotransposons ( malrs ) , accounts for approximately 3.65% and 4.82% , including solitary ltrs , of the human and mouse genome , respectively . . typical ervs consist of an internal region , gag , pol , and env with ltrs on both sides . ltrs are very important for integration and transcription because they function as a promoter element , transcription start site , and polyadenylation signal and site . compared to ervs , malrs have short internal sequences and ltrs at the 5 and 3 ends , but they do not have a transcription initiation site , reverse transcriptase , and primer binding site . recent studies have shown that tes contributed to primate genome evolution through gene structure variation and regulation by integrating into an intron , gene - flanking region , and an exon [ 7 , 8 ] . additionally , tes that integrate into introns could make alternative exons or cassette exons in a process termed exonization within human and mouse protein - coding genes ; in contrast , tes can make new introns in a process called intronization . these events could help with the understanding of transcriptome and proteome complexities [ 911 ] . the exonization of the primate - specific alu element is well established in the divergence of human and chimpanzee genomes as a major mechanism of exon creation [ 12 , 13 ] . ( 2003 ) reported that the alu consensus sequence could provide a splice site , leading to exonization . in the case of ltrs , 2007 ) reported that 256 of 1057 ltr retrotransposons related to human genes were observed in protein - coding regions , and 50 protein - coding exons of 45 genes were derived wholly from ltr retrotransposons . furthermore , they analyzed alternative exons of the interleukin 22 receptor , the alpha 2 gene ( il22ra2 ) , which was derived from malrs that provided splice sites , indicating the important roles of tes in human evolution ; this result coincided with those of previous studies [ 1 , 5 ] . importantly , the supt16h gene , also known as factp140 , is a component of the facilitates chromatin transcription ( fact ) complex . in eukaryotic cells , the transcription of genes requires rna polymerase ii and transcription factors with altered chromatin structure . in order to initiate transcription , dna needs to be in an open structure that is moderated by the alteration of chromatin with accessory factors , facilitating access to the dna . as described above , the supt16h gene has an original transcript consisting of 26 exons . however , the integration of an malr created a new exon that was expressed transcriptionally ( bx333035 ) . furthermore , we found a new alternative transcript , including the malr - derived exon . we assumed the time of integration of the malr into the genome by pcr amplification . all of the primates assayed had the malr in the supt16h gene , which was a natural result of its conservation in the mammalian genome . the expression patterns of the 2 transcripts in several tissues of humans , crab - eating monkey , marmoset , and squirrel monkey were ubiquitous with no significant tissue specificity or species specificity . structurally , the 2 transcripts included the exon - derived malr , but they adopted different splice sites , leading to exons of different sizes . taken together , we showed that the malr could regulate gene expression at the transcriptional level , that is , tes could provide transcriptome diversity , resulting in the proteome 's capacity for diversification , which potentially occupies an important role in the host genome . to identify the sequences related to the malr , we scanned the human reference genome sequence , human ests , and human refseq mrnas . using the malr consensus sequence as a query , we screened nonredundant databases through blast version 2.2.26 + to identify the novel hybrid transcripts of the malr and supt16h . then , we aligned the transcripts of the supt16h gene to identify the precise splicing patterns . tes included in the supt16h transcripts and their genomic loci were identified using the repeatmasker program ( http://www.repeatmasker.org/ ) , referencing a library containing the consensus sequences from repbase update . using the est and refseq mrnas , we accurately reconstructed the supt16h gene structure . to analyze the sequences of the various supt16h transcripts , we aligned them using the clustalw program . using the trizol reagent ( invitrogen ) , we extracted the total rna from the cerebrum , cerebellum , pituitary gland , heart , lung , spleen , liver , pancreas , kidney , and urinary bladder of the crab - eating monkey ; the heart , lung , brain , stomach , liver , kidney , pancreas , colon , spleen , and small intestine of the marmoset ; and the heart , esophagus , diencephalon , small intestine , lung , pancreas , cerebrum , colon , and stomach of the squirrel monkey . the total rna from human tissues , including the adrenal gland , cerebellum , whole brain , heart , kidney , liver , lung , testis , trachea , bone marrow , fetal brain , fetal liver , placenta , prostate , salivary gland , skeletal muscle , spinal cord , thymus , thyroid , and uterus , were purchased from clontech . we isolated the mrna from the total rna by using polyatract mrna isolation systems ( promega ) . we used the genomic dna of the following species : ( 1 ) hominoids : common chimpanzee ( pan troglodytes ) , gorilla ( gorilla gorilla ) , orangutan ( pongo pygmaeus ) , and gibbon ( hylobates agilis ) ; ( 2 ) old world monkeys : japanese monkey ( macaca fuscata ) , rhesus macaque ( m. mulatta ) , bonnet macaque ( m. radiata ) , mandrill ( mandrillus sphinx ) , mangabey ( cercocebus agilis ) , african green monkey ( chlorocebus aethiops ) , colobus ( colobus guereza ) , and langur ( trachypithecus cristatus ) ; ( 3 ) new world monkeys : night monkey ( aotus trivirgatus ) , squirrel monkey ( saimiri sciureus ) , and common marmoset ( callithrix jacchus ) ; and ( 4 ) prosimian : ring - tailed lemur ( lemur catta ) and a galago ( otolemur crassicaudatus ) . these genomic dna were isolated from the heparinized blood samples according to a standard protocol . after anticoagulant , and then , proteinase k and phenol equilibrated with tris - cl were treated for extraction . the viscous aqueous was transferred and ammonium acetate and ethanol were added to form a precipitate . lastly , the dna precipitate was washed , dried , and dissolved in te ( tris and edta ) . takenaka ( primate research institute , kyoto university ) kindly provided genomic dna of primates after conducting this procedure . using the genomic dna of humans and 17 primates , the malr in the supt16h gene m - mlv reverse transcriptase ( promega ) with an annealing temperature of 42c and an rnase inhibitor ( promega ) were used for the reverse transcriptions of the transcripts . the supt16h transcripts were amplified with a primer pair from genbank ( accession number nm001033560 ) consisting of s1 ( 5-aattggcggaaaggagaag-3 ) and as1 ( 5-gaggcagtcattccagctct-3 ) . the supt16h transcripts related to the malr were amplified with a primer pair from genbank ( bx_333035 ) consisting of s2 ( 5-tccagttctctgttctgcca-3 ) and as2 ( 5-tgtgtggagcttcaagcaag-3 ) . the pcr samples were subjected to an initial denaturation step of 4 min at 94c , followed by 30 cycles of pcr at 40 s of denaturation at 95c , 40 s of annealing at 55c , and 90 s of extension at 72c , followed by a final extension step of 7 min at 72c . as a standard control , gapdh was amplified with a primer pair consisting of gph - s ( 5-gagccccagccttctccatg-3 ) and gph - as ( 5-gaaatcccatcaccatcttccagg-3 ) from human gapdh ( genbank accession number nm002046 ) . the supt16h gene located on chromosome 14q11.2 encodes accessory factors that may facilitate access to dna by unpackaging the chromatin structure . it contains 26 exons with several transposable elements such as malrs ( ltr ) , alus ( sine ) , and l1s ( line ) distributed in the intronic regions . the supt16h transcript related to the malr ( malr - derived transcript ) has 3 exons , and its last exon was created by the integration of the malr ltr element in the opposite direction of the host gene ( figure 1 ) . malrs are considered to function in the mammalian genome by providing alternative splice sites , promoters , and other cis - regulatory elements , similar to other tes that have been conserved during primate evolution . for example , rnf19 has an alternative transcript resulting from the exonization of malr and alujo . specifically , the malr in the first exon has promoter activity in the sense orientation in the african green monkey fibroblast cell line , cos7 , and the human colorectal carcinoma cell line , hct116 . as described above , malrs are known to be present in genomes of rodents to primates and humans , indicating that they were distributed before the divergence of eutherian mammals . in order to compare the integration time of the malr in the supt16h gene with that from previous reports , we conducted pcr amplification using the genomic dna of human and other primates ( figure 2 ) . we identified an amplicon that was 481 bp long in all of the primates that we assessed , indicating that the malr in the supt16h gene integrated into the genome of a common ancestor before the divergence of the new world monkey and prosimians . in addition , from the sequencing alignment , we also identified 40 bp and 4 bp of specific sequences in japanese macaque ( figure 2(b ) ) . these sequences were not detected in any other primate , including the rhesus macaque , which belongs to the old world monkey family . tes distributed in the mammalian genome have played important roles in gene regulation and evolution . one of the typical characteristics of tes which is that they affect the transcription of cellular genes by exonization , which creates an alternatively spliced exon by integration into exonic or intronic regions , has been reported in many studies [ 24 , 25 ] . actually , these alternative transcripts are transient and flexible according to the cellular environment and stimuli . however , alternatively spliced variants can be stabilized and increased without selection , and exonization can lead to not only transcriptome diversity but also proteome diversity [ 2 , 10 ] . furthermore , these events occur differently or specifically according to the tissue , developmental stage , and disease ; thus , alternative transcripts are used to mine disease diagnostic markers [ 9 , 26 ] . to confirm whether the malr sequences are expressed and could regulate transcription , we conducted rt - pcr analysis of the refseq mrna ( original transcript ) and malr - derived transcript ( bx333035 ) from several human tissues . we analyzed its expression in tissues of other primates , including the crab - eating monkey , common marmoset , and squirrel monkey ( figure 3 ) . like the expression in humans , it was ubiquitously expressed in the tissues of these primates . because we identified japanese monkey - specific sequences , we conducted rt - pcr analysis in rhesus macaque samples to compare the expression in old world monkeys . however , we could not detect a difference in the supt16h expression between the japanese monkey and rhesus macaque ( data not shown ) . gene expression is a process that produces rna or proteins through transcription , splicing , and translation . the supt16h gene is a subunit of the fact complex that acts as a chromatin - specific transcription elongation factor and interacts with histone h2a / h2b to disassemble nucleosomes and promote transcription elongation . namely , the broad expression of supt16h in human and other primate tissues indicated its essential function for gene expression . as shown in figure 4 , a transcript related to the malr was also expressed broadly in human tissues . this product was also detected in tissues of crab - eating monkey , common marmoset , and squirrel monkey . there was hardly any difference in the expression of 2 transcripts which indicated malr did not affect the expression of the supt16h gene . its product was longer than that of the original transcript because alternative splicing retained an intronic region . the malr in the intron was not spliced and exonized ( exon 2 ) wholly between exons 2 and 3 ( figure 3 ) . comparing the new alternative transcript and the malr - derived transcript ( bx_333035 ) , we identified specific features of alternative splicing . entire sequences of the new exon of the alternative transcript identified in rt - pcr were derived from the malr . as shown in figure 5 , the malr provided both typical alternative splice sites , the acceptor site ( ag ) and a nucleotide ( g ) of the donor site ( gt ) . although these splicing sites were typical sequences recognized by the spliceosome , the splicing pattern could be changed . in case of the transcript related to the malr ( bx_333035 ) , the malr in the intronic region was also exonized to form a new alternative transcript . . only 1 site , the acceptor site , was recognized by the spliceosome , and the donor site was skipped . additionally , the elongated exon included malr - derived sequences and sequences of intron and exon 3 of the original and new alternative transcript . in spite of the different splicing pattern , the new alternative transcript was expressed broadly in tissues of humans and other primates , similar to the original and malr - related transcripts ( figures 3 and 4 ) . alternative splicing is a common mechanism by which most genes are processed [ 27 , 28 ] . in a previous report alternative splicing can be controlled by genetic mutation and epigenetic alteration [ 30 , 31 ] . however , the spliceosome can recognize splicing sites created by mutation and cryptic splicing sites , leading to alternative splicing . in addition to point mutations , the integration of transposable elements can induce alternative splicing as genetic mutations [ 2 , 25 ] . if the 2 transcripts showed different and tissue - specific expression patterns , we could assume that tissue - specific factors contributed to alternative splicing ; however , both transcripts were expressed broadly in tissues of humans and primates . various mechanisms can be considered to understand the differences between the 2 transcripts . compared to original transcript , however , the new exon includes intronic sequences by the typical splice site , ag - gt ( 35 ) , in the new alternative transcript , and ag ( 3 ) in the malr - derived transcript existing in the intron . if so , we could guess that the acceptor site and donor site of the new exon could be marked as a weak splice site by epigenetic regulation , such as histone modifications and dna methylationan , although it was the typical splice site . in a previous study , histone modification was reported as important factor for alternative splicing . among histone modifications such as h3k36me3 , h3k79me1 , h2bk5me1 , h3k27me1 , h3k27me2 , and h3k27me3 enriched in exon , the donor site of the new exon could be recognized weakly by the spliceosome with h3k36me3 which is found in weakly expressed exon [ 35 , 36 ] . additionally , the dna methylation and the cg content in the new exonic region could affect the expression of the 2 transcripts . zhou et al . ( 2012 ) reported that exclusive exons have lower levels of cg and methylated cg , whereas retained intron has higher levels . we suggest that the malr integrated into an intron of the supt16h gene before the divergence of new world monkeys and prosimians , and it played a biological role in the evolution of the supt16h gene during primate evolution by providing an alternative splice site . although further analyses are required to conclusively elucidate the functions of the transcriptional variants of the supt16h gene in primate genomes , the results of this study will help us understand the characteristics of the transposable element insertion driving the transcript diversification and its effect on the evolution of the host gene .

prebiotics and probiotics are considered natural alternatives to dietary antibiotics in animal production . plant extracts and yeast cell walls are mannose rich products that can be used as substrate for adhesion of gram - negative bacteria . we assessed whether the structure of these saccharides is relevant to develop their role as prebiotics and therefore , their suitability to be used as alternatives to antibiotics to prevent intestinal infections in pigs . the prebiotic functionality of -galactomannan ( gm ) , mannanoligosaccharide from yeast saccharomyces cerevisiae ( mannan sc ) and monosaccharide d - mannose were studied in porcine intestinal epithelial cells ( ipi-2i ) challenged with salmonella enterica ser . typhimurium . results showed that in vitro challenge with salmonella induces the secretion of proinflammatory cytokine il6 and chemokine cxcl8 compared with control without infection . both gm and mannan sc , attenuate salmonella - induced secretion of il6 and cxcl8 . interestingly , cells treated with d - mannose showed similar levels of proinflammatory il6 and cxcl8 compared with the control of infection . these data suggest that prebiotic role of gm is related to its oligosaccharide structure . the supt16h gene known as factp140 is required for the transcription of other genes . for transcription , genes need to be complexed with accessory factors , including transcription factors and rna polymerase ii . one such factor , fact , interacts with histones h2a / h2b for nucleosome disassembly and transcription elongation . the supt16h gene has a transcript and many expressed sequence tags ( ests ) . we were especially interested in an malr - derived transcript ( est , bx333035 ) that included a new exon introduced by a transposable element , a mammalian apparent ltr retrotransposon ( malr ) . the malr was detected ranging from humans to galagos , indicating the malr in the supt16h gene is integrated into the primate ancestor genome . a new exon was created by alternative donor site provided by the malr . the original transcript and the malr - derived transcript were expressed in various human , rhesus monkey , and other primate tissues . additionally , we identified a new alternative transcript that included the malr , but there was no significant difference in the expression of the original transcript and the malr - derived transcript . interestingly , the new alternative transcript and the malr - derived transcript had the malr sequence in the new exon , but they had different structures by adopting different 3 splice sites . from this study , we verified transposable elements that contributed to transcriptome diversity .

sarcomas are a group of very uncommon malignant tumors that compromise 1% of all adult malignancies.1 approximately 11,590 new diagnoses and 3,560 deaths from soft tissue sarcomas ( sts ) and bone sarcomas occur annually in the united states.1 isolated pulmonary metastases from sts occur in 20 - 50% of these patients , and 70% of these patients will have disease limited only to the lungs.2 once systemic metastatic disease develops , the prognosis is typically poor.3 patients with extremity sarcomas are more likely to develop distant metastatic disease as the initial site of recurrence , whereas those with retroperitoneal and visceral sarcomas tend to present local recurrence.4 although considerable progress has been achieved in both chemotherapy and surgical treatment of sts , pulmonary metastases remain a major cause of mortality . given that a diagnosis of pulmonary metastases significantly affects outcome , treatment and prognosis , the detection of such pulmonary metastases is of vital importance.5 surgical resection is well accepted as a standard approach for the treatment of metastases from sts isolated to the lungs , and many studies investigating this technique have reported an overall 5-year survival ranging from 30 - 40% 6,7 . chemotherapy has not been shown to increase survival following the complete resection of pulmonary metastases.6 pulmonary metastasectomy is currently indicated for patients with the following criteria : primary tumor controlled , possibility of complete resection verified by computed tomography ( ct ) of the chest , pulmonary function and performance status compatible with the proposed lung resection , and lack of another available treatment that would be more effective than the surgical procedure.8 although the results obtained for pulmonary metastasectomy are encouraging , there is a need to better select and identify the patients who would benefit most from surgical removal of pulmonary metastases to avoid unnecessary surgical procedures . many authors have reviewed their experience in an attempt to clearly identify the variables associated with better survival . important predictors of survival include disease - free interval , histology of the tumor , the number of metastases and complete resection.5 - 7 among these , the most consistent predictor of survival is complete resection.6,7,9 the number of metastatic nodules and their impact on survival was initially considered to be an independent predictor of survival.10 however , if complete resection can be achieved , the number of pulmonary metastatic nodules does not seem to influence survival.6,7,11 since the early 1990s , our institution has employed a prospective protocol - oriented approach for the treatment of patients with resectable lung metastases to define factors related to outcome following lung metastasectomy . the aim of this study was to report the outcomes of patients with pulmonary metastases from sts who were submitted to surgical resection and to identify prognostic factors that affect overall survival in a group of patients from a single institution . the patients were referred to the thoracic surgery department of ac camargo hospital between 1990 and 2006 . the following characteristics of the primary tumors were recorded : primary tumor site ( extremity vs. others ) , completeness of surgical resection , histological type and grade , and neoadjuvant and adjuvant treatment . all patients were evaluated for the presence of lung nodules before treatment of the primary sts , followed by evaluations at routine follow - up appointments . the disease - free interval was defined as the time period between treatment of the primary tumor and the diagnosis of pulmonary metastases . patients with the following characteristics were considered eligible for pulmonary resection : 1 ) primary tumor controlled , 2 ) nodules confined to the lung parenchyma , 3 ) nodules that were amenable to complete surgical resection , 4 ) pulmonary function and clinical condition compatible with the scheduled operation , and 5 ) the unavailability of more effective treatment options for the patient at that time.in addition to the evaluations , we collected variables related to the pulmonary metastases , including the number of pulmonary nodules on the ct scan , the number of malignant pulmonary nodules resected , the size of the largest nodule resected and the laterality of the pulmonary nodules . all patients underwent a chest ct scan prior to surgery to evaluate the possibility of complete resection . radiological characteristics were analyzed in each patient as follows : the number of pulmonary nodules , the size of the largest nodule and the laterality of the nodules . the number of nodules reported in this study was the final number confirmed by the surgeon after complete resection . this procedure was typically initiated on the side where complete resection was deemed more difficult and included the number and size of the nodules identified by chest ct scan , the anatomical position of one or more nodules relative to the pulmonary hilum , and the potential need for a more complex and extensive lung resection . patients underwent surgery with general anesthesia and single lung ventilation . in all cases , we attempted a complete resection while preserving the lung parenchyma with a margin of 10 mm . , mediastinal lymph node dissection is not routinely performed in patients with lung metastases . in cases of a single pulmonary nodule at surgery , frozen sections were collected . if the histological evidence suggested the possibility of primary lung cancer , the patient was submitted to lobectomy with radical mediastinal lymph node dissection whenever possible . the date of surgery , type of resection ( complete or incomplete ) , number of malignant or benign nodules resected , size of the largest nodule , and type of lung resection ( wedge , segmentectomy , lobectomy or pneumonectomy ) were documented . some patients received chemotherapy based on the discretion of the medical oncologist , before or after surgical resection of the lung metastases . the type of radiological response ( partial , stable , complete , or progression ) as defined by recist12 criteria was documented . after surgery , the patients were discharged from the hospital following chest tube removal and in good clinical condition . after hospital discharge , the patients were followed - up with clinical and radiological examinations ( chest x - ray and ct scan ) every three months during the first two years and every six months subsequently until the fifth year . an annual radiological ( chest x - ray ) the observed postoperative complications were fever > 38c , bleeding , air leakage for more than seven days , intensive care unit stay for more than two days , cardiac complications ( arrhythmia , ischemia , and infarction ) , pulmonary atelectasis , embolism or respiratory failure , stroke and infection . whenever necessary , a histological confirmation of the new tumor was performed . if the recurrence was confined to the lungs and if the nodules were considered resectable by the thoracic surgeon , the patient was offered another attempt at complete disease resection . survival was calculated from the time of resection of the primary tumor and from the time of first metastasectomy to death or until the last follow - up . the log - rank and breslow tests were used to compare survival differences for each variable . multivariate analyses to determine the independent prognostic factors for overall survival were performed using the cox proportional hazard model14 as identified by the univariate analyses . survival time was determined whenever more than one pulmonary metastasectomy was performed , with time zero denoting the first thoracotomy . survival was calculated from the time of resection of the primary tumor and from the time of first metastasectomy to death or until the last follow - up . the log - rank and breslow tests were used to compare survival differences for each variable . multivariate analyses to determine the independent prognostic factors for overall survival were performed using the cox proportional hazard model14 as identified by the univariate analyses . survival time was determined whenever more than one pulmonary metastasectomy was performed , with time zero denoting the first thoracotomy . the patients included in this retrospective study ( n = 77 ) underwent a total of 122 thoracotomies ( mean of 1.6 thoracotomies / patient ) ; 273 nodules were resected , with a mean of 3.5 nodules per thoracotomy . five of the nodules were carcinomas and were treated as primary lung tumors , and more than four nodules were resected in each of 18 ( 18.1% ) patients . according to the primary sts , 66 ( 85.7% ) patients had extremity tumors , and 11 patients ( 14.3% ) had visceral or retroperitoneal tumors . in 64 patients ( 83.1% ) , the primary sts histology ( table 1 ) and grade ( table 2 ) were registered as well . in the 13 remaining patients , we could not find sufficient detailed information regarding the histological type and grade of the primary tumors in the medical records because the primary tumors of these patients had been resected at other institutions . based on the final confirmation of sarcomas from the pathology of the pulmonary metastases , these tumors were classified as undetermined and excluded from statistical analyses in which histology was considered . the median follow - up time was 36.7 months ( range : 10 - 138 months ) . twenty - nine patients were alive at the time of the final analyses , and 13 ( 16.9% ) showed no evidence of disease . thirty patients ( 39% ) died due to metastatic sts , and 18 ( 23.4% ) patients were considered lost to follow - up after missing two medical evaluations . more than one complication occurred in some patients , including fever , atelectasis , prolonged air leakage ( > 7 days ) , infection and a prolonged hospital stay ( > 30 days ) . the median overall survival for all patients was 36.7 months , with an estimated 5-year survival rate of 34.7% ( figure 1 ) . the median survival for patients with one , two , three or more pulmonary metastasis resections was 15.1 , 44.8 and 48.1 months , respectively . no significant difference in survival was detected between the groups with respect to the number of thoracotomies ( p = 0.07 ) . all other factors evaluated in the univariate analyses , including the response to adjuvant chemotherapy , type of resection , number of resected nodules , laterality of the nodules on the ct scan , number of thoracotomies and size of the largest nodule resected , were not statistically significant ( p>0.05 ) . multivariate analyses identified the number of pulmonary nodules resected ( p = 0.003 ) , the disease - free interval ( p<0.001 ) and complete resection ( p<0.001 ) as independent prognostic factors for overall survival ( table 4 ) . the resection of pulmonary sarcomatous metastasis remains the cornerstone for treatment of isolated pulmonary metastases in patients with sts.14 in the absence of other effective treatments and of contraindications , patients with lung metastases from sts benefit from surgical therapy.16,17 approximately 20 - 30% of patients undergoing pulmonary resection can experience long - term survival.5,6,18 improved survival following the resection of lung metastases has increased the surgical indications for such lesions , thus confirming that surgery itself affects the outcomes of patients who are candidates for a complete resection of nodules confined to the lungs . therefore , resectability should be evaluated preoperatively according to specific criteria . the recent introduction of positron emission tomography using 18-fluorodeoxyglicose ( fdg pet - ct ) has proven to be of clinical value for the proper staging of cancer patients to better select the best candidates for resection . according to pastorino et al . , this method can eliminate 21% of patients who are otherwise considered as surgery candidates.19 unfortunately , pet - ct was not available at our institution for most of the patients in this study . 5 - 7,17 the factors evaluated included the disease - free interval number of metastatic lesions resected , tumor doubling time , laterality , number of thoracotomies and resectability of the lesions.20 - 23 stss are an extremely heterogeneous group of tumors with a variable biological behavior . therefore , the histological type and grade of malignancy are the most important prognostic factors in the treatment of primary tumors.24 moreover , other studies have found histology to be predictive of pulmonary metastasis , resectability , and overall survival in extremity sts.6,25 the 77 patients included in this study underwent 122 thoracotomies for the treatment of lung metastases . the estimated 5-year overall survival of our patients was 34.7% at an average follow - up of 36.7 months . this outcome is similar to previous reports .5,7,9 in a review of the national cancer institute , the overall 5-year survival rate was 28% , with a 35% 3-year survival rate for patients who underwent complete resection . in that series , patients who experienced incomplete resection demonstrated a median survival of 9 months.26 with a median follow - up period of 27 months , the international registry of pulmonary metastases reported a 5- and 10-year survival rate in 936 patients with sts lung metastases of 30% and 22% , respectively .7 however , this result does not compare to the 14 - 21% overall survival reported by some studies with longer follow - up periods.6 several reports have demonstrated the prognostic importance of the number of nodules detected in the preoperative workup . the sensitivity of detecting pulmonary nodules varies depending on the technique used.23 in a prospective study including 182 patients with cancer and pulmonary metastases by younes et al . , ct scan detected the exact number of nodules resected in 47% of the patients . however , ct scan underestimated the actual number of lung nodules found during surgery in 28% of the cases and overestimated that number in 24%.27 in the present study , we discovered a poorer outcome in patients in which 4 nodules had been resected , as shown in the multivariate analysis . for some authors , the number of pulmonary metastases does not preclude resection because the ability to achieve complete resection is of greater prognostic value than the number of lesions removed.28 in a review of the european organization for research and treatment of cancer ( eortc ) database , the majority of patients undergoing resection ( 53% ) had only one lesion , although 15% of the patients had more than four lesions at the time of pulmonary metastasectomy.29 previous studies have examined the number of nodules resected and the presence of unilateral and bilateral metastatic disease as prognostic factors . casson et al . demonstrated that patients with three or fewer nodules survived significantly longer than did patients with four or more nodules resected9 . similar findings have been noted by putnam et al.5 who found that patients with four or fewer nodules survived longer than did patients with more than four nodules resected . the same group demonstrated that unilateral versus bilateral disease is not a significant indicator for prognosis . in most studies published to date , no differences have been observed in the survival of patients with metastases confined to one lung compared to bilateral lesions , despite the histology of the primary tumor 7 . the current study did not identify laterality as an independent indicator of prognosis . in the present study , the number of nodules resected had a significant effect on long - term survival in both univariate and multivariate analyses ( p = 0.003 ) . although the maximum number of pulmonary metastases often predicts survival,5,10 subgroup analyses have found that complete resection is a stronger predictor of survival , regardless of the number or size of the nodules.31 based on multivariate analysis , patients with synchronous metastases ( dfi<16 months ) presented with a significantly lower survival rate ( p<0.001 ) than did patients with metachronous disease , as reported in other studies.6,7,21,22,32 the correlation between dfi and survival is not consistent in the published data . depending on the study , dfi ranging from 8 months to as long as 5 years have been a significant prognostic factor ; however , the results are vague and inconclusive according to some authors.29,31 previous reports have been unable to consistently demonstrate a correlation between the primary site of sts and survival.29,33 in the current series , univariate analyses demonstrated better survival for patients with non - extremity primary tumors . in the multivariate analyses , however , the site of the resected primary tumor did not affect the long - term survival of patients submitted to pulmonary metastasectomy.6 similarly , no significant effect on outcome was observed between extremity tumors and those present in other primary sites . preoperative and postoperative systemic therapy had no significant effect on the long - term survival rate in this patient population , which is consistent with previously published data.34,35 although the patients ( n = 26 ) submitted to neoadjuvant treatment with chemotherapy prior to primary tumor resection demonstrated a numeric difference in the univariate analyses ( p = 0.018 ) , this variable did not have a significant effect in the multivariate analyses . this variable was a strong prognostic indicator in the multivariate analyses ( p<0.001 ) , which is consistent with previous reports.5 - 7,10,11,21,22 the median survival for patients who underwent complete resection was 44.8 months , as compared to 9.5 months in the group who underwent incomplete resection ( n = 15 ) . in the present study , 15 ( 19.5% ) patients were resubmitted to surgery for recurrent metastases , which ranged from one to four thoracotomies for complete resection . curiously , these patients demonstrated better survival ( median survival : 48.1 months ) than did those who underwent one thoracotomy for metastasectomy ( median survival : 15.1 months ) , which is consistent with previously published data.21,22,31,36 weiser et al . investigated a series of 86 patients who had undergone repeat resections . a poorer survival after repeat resection was associated with the following factors : more than three nodules , lesions larger than 2 cm , and high - grade histopathology . the presence of 0 - 1 adverse factors was associated with a disease - specific survival of 65 months . in comparison , the presence of 2 - 3 factors was associated with a disease - specific survival of 10 months.37 the type of surgical resection and the total number of thoracotomies failed to significantly influence patient outcome . the majority of our patient population ( 81.8% ) underwent a sublobar resection , and ten ( 15.6% ) patients required a lobectomy for complete resection . as reported previously , limited resection with lung parenchyma preservation is considered adequate to achieve clear margins and thus to provide local control of the disease . recently , cerfolio et al . showed that among 37% of patients who were candidates for video - assisted thoracic surgery ( vats ) , intraoperative palpation during a thoracotomy detected more nodules than had been identified using preoperative imaging by ct scan.38 there may be a role for minimally invasive surgery in the pulmonary metastasectomy setting ; however , additional prospective studies are required . although randomized controlled studies comparing observations to surgery are impossible , the current data suggest that the patients most likely to benefit from metastasectomy have a longer disease - free interval , single metastases , and complete resection of the metastases . the administration of neoadjuvant or adjuvant therapy may allow for further prolonged survival in some instances , despite the finding that surgery alone fails to treat lung metastases in the majority of patients . it should be emphasized that surgery does not change the biology of the tumor or the metastatic process , and a definitive cure for most patients represents the combination of host histology , tumor spread , response to systemic therapy , and surgical resection , which together render the patient free of disease . all patients with resectable nodules evaluated by ct scan and selected by the inclusion criteria should be considered as candidates for surgical resection because they might reveal an interesting overall survival , as shown by the present study . obsessive compulsive disorder ( ocd ) is a common psychiatric disorder ( 2% - 3% of the general population ) which is diagnosed easily most of the time by the psychiatrist or even the physician . it is sometimes very difficult to properly diagnose a person for ocd based only on an interview . the presentation of ocd may be so atypical or unusual that the patients may be referred to different specialties before the actual diagnosis can be made . neuropsychological studies have consistently found cognitive impairment in the domains of memory and attention in patients of ocd . anxiety , lack of confidence , indecisiveness , associated clinical symptoms of ocd , along with impaired memory and attention , may further complicate or interfere in the delivery of information . a 36-year - old man used to request multiple repetitions of spoken words in order to understand what was said . he was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptoms . even after multiple consultations with the ent , neurology and medicine specialists , no organic pathology to explain his symptoms could be detected . the patient lost considerable time and money because of delay in diagnosis caused by the atypical presentation to specialties other than psychiatry . at last , the patient was referred to the department of psychiatry for further evaluation . the patient 's chief complaints were difficulty in hearing , grasping or comprehending the components of conversations , interactions or clinical interviews at once . he had this problem for the last 6 months . during the interview , almost every question or the instruction had to be repeated slowly 2 to 3 times or more to make him understand . he was anxious and expressed regret repeatedly for the inconvenience caused to the doctor because of his disability . initially the patient was put on mirtazapine 15 mg / day and clonazepam 0.75 mg / day , keeping in mind the possibility of anxiety disorder nos ( not otherwise specified ) . mental state examination of the patient revealed impaired attention and concentration with depressive and anxiety symptoms . the possibility of ocd , predominantly obsessive type , was kept on the basis of answers of leading questions , and he was put on fluoxetine 60 mg / day ( titrated from 40 to 60 mg ) and clonazepam 0.5 mg / day . there was drastic improvement in all the presenting symptoms , including his ability to grasp and comprehend conversation , along with improvement in anxiety symptoms and clarity in thinking over a period of 6 weeks . the patient was then able to report his pre - treatment problem and the level of improvement comprehensively . he accepted that he was unable to understand or express his own problem before treatment . according to him , there were thoughts keeping his mind busy all the time , to the extent that he was unable to think or communicate his problem effectively . according to him , initially he used to make frequent unsuccessful attempts to get rid of these excessive and unnecessary thoughts . according to the wife of the patient , after treatment the patient was able to hear clearly . the diagnosis could be confirmed as ocd on the basis of patient 's spontaneous elaboration of symptoms and the level of improvement . in this case , the presentation of ocd is atypical , as the chief complaint was problem in hearing . he was able to hear on normal volume of sound , but the sentences needed to be repeated multiple times . moreover , no ent - related or neurological cause of hearing impairment was found in the patient . though hearing difficulty was not his chief subjective problem , he consulted the ent doctor only after repeated and forced advice by his wife and relatives . the hearing problem as perceived by the patient 's friends and relatives was basically a misinterpretation of the patient 's problem secondary to his psychiatric illness . also the patient was unable to express his actual problem at the time of presentation . the patient used to ask for repetition of the spoken sentences multiple times in order to grasp the components of interaction because of his significantly impaired attention . however , there could be the other possibility that this behavior could be a compulsive act of the patient to confirm his doubts . the patient could not express his problem elaborately , which was one of the reasons for not being able to make an early diagnosis . there were multiple factors that had complicated and hindered the narration of history by the patient . lack of clarity of thoughts and associated clinical symptoms of ocd ( especially predominantly obsessive type ) , indecisiveness , anxiety , lack of confidence , along with impaired memory and attention , were the possible factors in this case , as also reported in different studies . the patient could express details of his illness comprehensively once his above - mentioned signs and symptoms improved significantly . his wife and relatives also reported improvement in his hearing , which was actually the improvement in his attention and ocd symptoms . such an atypical presentation may lead to misdiagnosis , delay in diagnosis , loss of time and money as the patient may present to other specialties instead of psychiatry . high suspicion for diagnosis of ocd and response to treatment may help in managing such cases . obsessive compulsive disorder is usually easily recognized , but sometimes its presentation is so atypical or bizarre that the problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties . but if proper evaluation is done , such cases can easily be recognized and treated effectively .

introduction : isolated pulmonary metastases from soft tissue sarcomas occur in 20 - 50% of these ( the issue is about metastases , not lung cancer ) patients , and 70% of these patients will present disease limited only to the lungs . surgical resection is well accepted as a standard approach to treat metastases from soft tissue sarcomas isolated in the lungs , and many studies investigating this technique have reported an overall 5-year survival ranging from 30 - 40% . the most consistent predictor of survival in these patients is complete resection . the aim of the present study was to determine the demographics and clinical treatment - related variables associated with long - term ( 90-month ) overall survival in patients with lung metastases undergoing pulmonary metastasectomy from soft tissue sarcomas.methods:we performed a retrospective review of patients admitted in the thoracic surgery department with lung metastases who underwent thoracotomy for resection following treatment of the primary tumor . data regarding primary tumor features , demographics , treatment , and outcome were collected.results:one hundred twenty - two thoracotomies and 273 nodules were resected from 77 patients with previously treated soft tissue sarcomas . the median follow - up time of all patients was 36.7 months ( range : 10 - 138 months ) . the postoperative complication rate was 9.1% , and the 30-day mortality rate was 0% . the 90-month overall survival rate for all patients was 34.7% . multivariate analysis identified the following independent prognostic factors for overall survival : the number of metastases resected , the disease - free interval , and the number of complete resections.conclusion:these results confirm that lung metastasectomy is a safe and potentially curative procedure for patients with treated primary tumors . a select group of patients can achieve long - term survival after lung resection . obsessive compulsive disorder ( ocd ) is a common psychiatric disorder which is easily recognized . however , sometimes patients of ocd present in such an atypical or bizarre way that their problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties . we are reporting a case of a man who had first sought opinion in the department of ear , nose and throat ( ent ) for hearing impairment . he was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptom . as no pathology related to ent or neurology could be detected , he was referred to the department of psychiatry . the patient 's chief complaints were difficulty in hearing and inability to understand at once . he could be diagnosed as a case of ocd after meticulous evaluation and studying his response to treatment . there was significant improvement in all the presenting symptoms over a period of 6 weeks on 60 mg of fluoxetine .

the new drg - based prospective payment system ( pps ) for medicare is a revolutionary concept in health care finance . it introduces a new approach to reimbursement , which is based upon a defined clinical product . in contrast , under retrospective cost - based reimbursement , each component of the process of care ( each visit , x - ray , procedure , and day of care ) was paid for separately . that open - ended payment system has proved too inflationary ; federal outlays for medicare have increased from $ 1.1 billion in 1966 to $ 51 billion in 1982 , a rate of inflation which has outpaced the gross national product , the consumer price index , the median family income , and other benchmarks of the economy 's growth . under the prospective payment system the shifts in the medicare system should stimulate major efficiencies , as providers of care begin to consider which components should be introduced into the clinical production process , and which should be rejected . it is expected that there will be a major break in the upward - spiraling overutilization of services which produce no improvement in clinical care . it is hoped that the drg system will create incentives for physicians and hospitals to shorten hospital lengths of stay and incentives to decrease resource consumption per episode of illness . however , considerable debate has arisen over whether the current drg formulations in the prospective payment system represent the best approach for achieving these objectives . with tens of billions of dollars at stake each year , the health care financing administration ( hcfa ) will face innumerable challenges to the design and administration of the prospective payment system . these challenges are likely to encompass a broad spectrum : on one hand , there may arise overt challenges to the equity of the drg 's , particularly with respect to their ability to adjust fully for differences in severity of illness . on the other hand , there is the possibility of covert challenges , in particular , those which involve clinical opportunism . for example , under the pps , providers may seek to : 1 ) admit larger numbers of patients , especially patients with easily treated illnesses and short anticipated lengths of stay ; 2 ) split illnesses into two parts , in order to spread a patient 's care over two hospital admissions ; 3 ) unbundle diagnostic procedures , shifting some to the ambulatory setting ( outside the pps ) ; 4 ) upgrade primary and secondary diagnostic codes , in order to obtain a higher - paying drg assignment ; 5 ) perform more complex surgical procedures to inflate the drg ( procedure inflation ) ; and 6 ) prolong the hospital stay of patients with lingering illnesses , as the outlier trim point is approached . when a patient has more than one clinical problem , there arises the possibility of gaming the data about the correct principal diagnosis ; there also arises the possibility of outright fraudulent representations . hcfa can respond to these challenges by improvements in design of the basic drg formulation and administrative rules and procedures . it is likely that changes in drg structure and design can best deal with the equity and overt challenges to the system ; administrative controls will likely have their greatest strength in dealing with the covert challenges . a cornerstone of the new prospective payment system for medicare is the use of a diagnosis - related group case classification system . the drg system employs existing discharge abstract data on the patient 's principal diagnosis , secondary diagnoses , surgical procedures , age , sex , and discharge status , in order to classify patients into different groups that are clinically coherent and homogenous with respect to resource use the current drg 's have been criticized for failing to account for severity of illness . controversies about severity are based upon the assumption that , within each drg , there are sicker patients who will cost more . hospital administrators and physicians have expressed concern that they will be unfairly penalized if their patients are sicker and consume greater resources . medicare administrators have concerns of their own : to the extent that providers can measure or anticipate patient severity , they may reject patients with severe illnesses in order to maximize their profit . among policy analysts and economists , severity has become the buzzword to describe the sources of otherwise unexplained variations in resource use within the drg 's . it is the authors ' opinion that the objectives of a system for defining severity of illness under drg 's ( with respect to medicare reimbursement ) should be threefold : 1 ) to place patients into categories which are homogeneous with respect to resource need ; 2 ) to categorize patients in such a way that manipulations of data are minimized ; and 3 ) to avoid producing distortions of provider behavior that could adversely affect the outcome of patient care . ( for example , with respect to the third objective , hcfa has refrained from using death as a classification factor in most drg 's . unless this is a common outcome , in heart attacks hcfa does not want to reward hospitals for letting patients die . ) one of the major assumptions by many critics of the drg system , particularly those who point to large variations in resource use within drg 's , is that the major source of this variation is the absence of equitable severity of illness measures . . these may be responsible for a substantial portion ( or even the majority ) of the variation in costs and length - of - stays observed to date in drg 's and may be even more important as covert challenges to restraining medicare costs . for example : 1 ) situations exist where there are no clear rules for determining an individual 's correct principal diagnosis or the validity of the presence of a complication ; 2 ) existing practice patterns frequently involve substantial proportions of medically inappropriate use , and 3 ) clinical practice patterns exist where definition of an appropriate episode of medical care can be split to produce two or more reimbursable episodes . stated another way , variation in resource use within drg 's may be due not only to inadequacies of measurements and adjustments , but also to the above types of problems . in this article , these three basic sources of confounding will be reviewed , and the measurement of severity of illness will be discussed , focusing on an applied research paradigm for testing new methods of incorporating such measurement approaches into the medicare prospective payment system . the major factor which determines the drg assignment , and thus determines the payment of the provider , is the physician 's assessment of the patient 's diagnosis . the accuracy and completeness of the diagnostic and procedure data are fundamental to the drg system . regulations require that physicians record all diagnostic and procedures data on the face sheet in an honest and responsible manner . peer review organizations are charged with monitoring the validity of diagnostic information provided by the hospital for the purposes of payment . however , hcfa has accumulated evidence suggesting serious errors in the description and coding of the principal diagnoses and principal procedures . according to studies by the institute of medicine and the department of management and research of the university hospitals of cleveland , errors and discrepancies in the listing of the principal diagnoses range from 20 - 40 percent ( demlo et al . , 1978 , much of this error was probably due to sloppi - ness and to use of unqualified record abstractors . these errors represent a threat to the validity of all analytic studies employing historical hospital diagnostic data , and almost certainly contribute to the variance found in some previous drg analyses . the extent of the sloppiness problem should , however , rapidly diminish under pressure from concerned administrators and from hcfa oversight through professional review organizations ( pro 's ) and fiscal intermediaries . creation of each drg category starts with consideration of the patient 's primary diagnosis and the performance or nonperformance of an operating room surgical procedure . a substantial number of the drg categories are then further divided , depending upon the presence or absence of significant complications or comorbid conditions . a complication is a condition other than the principal diagnosis which occurs after the onset of the principal diagnosis ( and usually after the patient is admitted to the hospital ) . many ( if not most ) medicare patients admitted to the hospital have one or more comorbid or complicating conditions in addition to their primary diagnosis . in the planning of the drg system , it was reasoned that the length of stay and the consumption of resources ( and , of course , reasonable reimbursement ) are likely to be higher if a substantial complication or comorbidity is present . therefore , specific lists of such conditions have been developed , based upon the judgment of the physician teams who assisted the drg design team at yale . a substantial comorbidity or complication is defined as any secondary diagnosis likely to prolong the hospital stay of at least 75 percent of patients by one day or more ( yale university , 1982 ) . one example of a substantial comorbid - complicating condition is international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) code 202.98 ( hodgkin 's disease ) . if one or more of these is present and is listed on the discharge data abstract sheet as a secondary diagnosis , the patient 's care needs are considered more resource - intensive . therefore , he or she is usually placed in a drg category for which the hospital is awarded a higher reimbursement for the episode of illness . the concept of a complicating condition or a comorbid condition requiring additional resources has validity and should contribute to a reduction in variance among drg categories . at the same time , operationalization of the concept in a prospective payment system raises some serious medical and definitional problems . one obvious problem is that the source of the information used to classify each patient into a drg is the face sheet of the medical record . for example , the presence or absence of a substantial complication or comorbid condition can only be determined from the list of all secondary diagnoses on the face sheet . however , as noted earlier , there is considerable error in hospital discharge data . another serious problem is whether rewarding hospitals for more complications is a fundamentally sound approach . if a comorbid condition is present upon admission , its inclusion as a factor discriminating between types of patients may be valid . for complications which arise after admission , however , their inclusion may reward less careful practitioners , while penalizing those who make investments to prevent such untoward events . might a hospital which reduces its infection control procedures benefit if its infection rates subsequently rose ( thus sending additional patients into higher reimbursement drg categories ) ? the current drg classification system , however , does not distinguish a comorbid problem present at the time of admission from a complicating condition which occurs subsequently . this dichotomy , although conceptually attractive , would be extremely difficult to operationalize ; identification of many comorbid conditions at admission might require a series of investigational procedures , which are not normally done and which might involve additional cost and risk for patients . an even more difficult problem is how to define and validate the actual presence of a substantial comorbidity or complication . for example , diabetes mellitus with renal manifestations is one of the drg substantial comorbid / complicating conditions . does the presence of an abnormal glucose tolerance test and the finding of trace proteinuria constitute icd-9-cm 250.4 for the purposes of reimbursement ? similarly , icd-9-cm 780.0 , the diagnosis of coma ( including stupor , drowsiness and somnolence ) is a substantial comorbid / complicating condition . how is drowsiness validated by the pro ? how is respiratory failure ( icd-9-cm 799.1 ) defined ? would the presence of a positive culture from the wound site for any organism be sufficient ? would it depend upon the colony count of infecting organisms or the types of organisms found ? would it require that the infection be significant to the point where fever occurs or antibiotic therapy is instituted ? if the lowest level of this hierarchy of definitions is chosen , every patient will have their wound site cultured , and many with clinically insignificant bacterial organisms ( many of which are normally present on the skin ) will suddenly start to become included in the complicated condition category . if sufficient evidence is antiobiotic treatment , we are likely to see many elderly patients ( who are already at higher risk of adverse drug reactions ) having such agents administered more frequently , because of the bias introduced by the drg reimbursement system . as long as the definitions of comorbid and complicating conditions remain vague , clinicians and data abstracters have an incentive to include larger numbers of such conditions on the data abstract sheet , whether or not such conditions are clinically relevant . indeed , most of the current drg scheme makes no distinction between complicating / comorbid conditions related and not related to principal diagnosis . related comorbidities or complications may contribute more ( or less ) to resource consumption than unrelated conditions ( louis and heineccius , 1983 ) . it should be noted that these definitional issues plague not only the assessment of complicating or comorbid conditions , but also affect the determination of the principal diagnosis . for example , when does a patient with peptic ulcer disease fit into drg 175 ( gastrointestinal hemorrhage , age less than seventy without c / c ) , rather than into one of the other peptic ulcer categories ( drg 176 or drg 178 ) ? is hemorrhage evidenced by a positive stool guaiac test for blood ? is it , instead , any aspiration of hematest - positive material through a nasogastric tube ( and if so , does it count any fresh bleeding which may have been induced by insertion of the nasogastric tube ) ? or is adjacent drg categories are frequently separated only by such terms as hemorrhage or complication . different parties are likely to define such terms differently , depending upon economic and other pressures . obviously , many of the adjacent drg 's require more precise separation and definition , for they rank among the high cost diagnoses for medicare patients . the boundaries separating many such drg 's are indistinct and , potentially , malleable . in maryland , for example , the mean charge for drg 140 ( angina ) was $ 2,409 in 1981 versus a mean charge for drg 143 ( chest pain ) of $ 1,925 . there are no well - accepted rules delineating the correct assignment of a patient to one or the other of these categories . thus , the incentives to label a case as angina , with a 25 percent greater reimbursement , will be substantial . there will still remain , however , a more serious basic issue how to relate a patient 's clinical reasons for hospitalization to the icd-9-cm coding system used in drg groupings . the international classification of diseases , now in its ninth revision , is still based on an anatomic approach to disease developed in the late nineteenth century by the great french forensic pathologist , dr . jacques bertillon . this system , despite its decennial updating and an american clinically modified version , was never designed to deal with clinical severity of illness . it is still not well - suited to this task . nor was the icd-9-cm coding structure even intended to accommodate a reimbursement system or withstand the pressures for precision that this requires . icd-9-cm 's central flaw is that there are no clear rules for determining when a patient has a particular problem which fits an icd-9-cm code . while a pro policing effort may prevent massive definitional abuse , the pressing need is for a research and development program to develop at least consensus guidelines and criteria for use of icd-9-cm terms . where there are objectively verifiable criteria , these should be disseminated by hcfa throughout the hospital and pro community nationwide . where there are no such criteria , definitional guidelines can be generated through consensus conferences ( such as the national institutes of health utilizes ) , opinion surveys of specialists and generalists , and other such means . certainly , the medical community will continue to be plagued with serious definitionally - induced variance unless such steps are initiated . in the current health system patients are often admitted to the hospital for problems such as low back pain , which can be treated at home , and cancer chemotherapy , which may be safely administered on an outpatient basis . if these current patterns of unnecessary admissions those not severe enough to warrant hospital admission are allowed to continue , medicare will continue to spend billions of dollars unnecessarily . many of the most common drg 's , particularly those not associated with surgery , involve conditions with a wide spectrum of clinical manifestations and needs for acute care . the decision whether or not patients in these categories require hospitalization instead of outpatient treatment involves a set of physiologic and clinical assessments which are often not captured by the diagnostic , procedure , and demographic factors used to designate drg 's . for example , the category heart failure ( drg 127 ) might include a patient with such excess fluid load that he or she was having severe difficulty breathing and was in need of aggressive therapy in a hospital environment . however , this drg category would also apply to a patient with long - standing heart disease who had gained some extra fluid weight as a result of dietary indiscretion and was noticing a mild increase in shortness of breath when walking a short distance . the latter case would not require hospitalizaion in most circumstances , but with the drg system there might be an incentive to admit such a patient for several days of therapy for the patient 's convenience and for the financial benefit of the provider . this situation involves no manipulation of the diagnosis as suggested by the term drg creep , but rather involves playing on a central weak point of the drg system a lack of severity of illness standards which distinguish inappropriate hospital admissions . other high frequency categories such as drg 182 ( gastroenteritis and miscellaneous digestive diseases ) and drg 132 ( artherosclerosis in patients over 70 ) involve the same potential problem of classification of disease by diagnosis , by procedure , and by age which could run the gamut from life - threatening acute situations to chronic , stable manifestations which can be effectively treated on an ambulatory basis . while it may be fraudulent to deliberately change the diagnosis of a patient so as to gain higher drg reimbursements , there are no accepted legal or procedural barriers to physicians lowering their severity standards for admitting patients within these medical drg categories . a study of over 8,000 medicare and non - medicare patients hospitalized in 41 massachusetts hospitals in 1973 and 1978 found that almost two out of every five days for medical patients with a length of stay ( los ) under ten days were inappropriate a rate higher than that of medical patients staying longer than ten days ( gertman et al . , 1982 ) . studies done by the boston university school of medicine indicated that in 1980 , among selected professional standards review organizations , levels of inappropriate hospital admissions among medicare beneficiaries ranged from a low of 12 percent to over 31 percent ( restuccia et al . , 1984 ) . both of these studies employed the appropriateness evaluation protocol methodology developed for hcfa ( gertman and restuccia , 1981 ) . part of the mysterious variation in days of care per 1,000 medicare beneficiaries across the country may represent variation in inappropriate admissions . assessments by systemetrics incorporated , using their standardized medreview instrument ( smi ) found that a smaller but still substantial amount of medicare admissions were inappropriate and that levels varied geographically ( systemetrics , inc . , january 1983 ) . given that neither of these instruments challenged the necessity of elective surgery ( i.e. , was the hysterectomy indicated ) , these must be conservative estimates . the joint massachusetts blue cross - massachusetts hospital association 's ancillary services review program has documented average levels of inappropriate laboratory tests , electrocardiograms and respiratory therapy in the 20 percent to 30 percent range ( hughes et al . , 1984 ) . after adjustments for certain severity of illness factors and for pricing differences , the dollar amount of inappropriate use of an ancillary service for a specific diagnosis varied over tenfold from the highest to the lowest hospital . these data suggest that a major source of variation among institutions for any given diagnostic grouping may not be due to inadequacies in the grouping method , but rather , largely due to inappropriate admissions , inappropriate days of care and inappropriate ancillary utilization . this possibility can be addressed by a major program of research on the correlation of unexplained drg variation with measures of inappropriate use . if this , along with the other basic factors , is the principal source of variation , then the current drg system may , in the near term , need only modest refinement rather than a dramatic overhaul . the drg case - mix classification system defines the relevant unit of service for reimbursement as the illness episode , which lasts from the time of admission to the hospital until discharge . the greater the number of admissions a hospital has for a fixed number of bed days and available staff , the greater its revenue will be . this could create incentives for shortening hospital lengths of stay and decreasing consumption of resource per episode . for some conditions a medical episode of illness has a clear beginning and end point which correspond fairly closely with initial admission to the hospital and subsequent discharge . for many conditions , however , the potential exists for deliberate abuse of the admission payment concept , by artificially dividing an episode of illness into two hospitalizations . in other words , patients may be admitted for several days or weeks , then discharged , and later readmitted for a procedure or for continued medical treatment . the medicare regulations recognize the potential for such deliberate abuses , and the admission pattern monitoring program is specifically charged with looking for such attempts . unfortunately , for some conditions , the proper end points of hospital care are less clearly defined . the single admission - to - discharge concept remains most problematic in the following two areas . there are a substantial number of common conditions where medical opinion may differ and individual physician judgment is used to determine whether a patient 's course of treatment should be completed during one or more hospitalizations . some physicians believe that patients should first be admitted for medical treatment , which includes fluids , antibiotics and nasogastric suction . other physicians and surgeons believe it is safe and proper to operate on the patient who is improving during a single hospitalization . at the present time , no medical review panel could fairly penalize competent physicians for choosing one course over the other . similarly , a patient hospitalized with symptoms of coronary insufficiency may undergo cardiac catheterization during his hospitalization and be discharged for a period of recovery and risk - factor modification ( e.g. , stopping smoking ) prior to coronary bypass surgery ; alternatively , these procedures might occur in the same admission . another example is the procedure - linked diagnosis of benign prostatic hypertrophy ; patients may be admitted with symptoms of urinary obstruction and then scheduled for prostatectomy either during the same hospitalization or at a subsequent admission in the near future . to illustrate this issue , the authors reviewed the top 100 drg 's and identified 32 examples of medically acceptable situations where , in their opinion , multiple procedure - linked admissions might occur ; these are listed in table 1 . in each case , for some physicians , it might seem preferable to complete the medical treatment , the diagnostic evaluation and the operative therapy in a single hospitalization . however , depending upon the habits and style of practice of the physicians in charge and depending upon the economic incentives which are allowed to come to the fore , a single process of diagnosis and treatment might be split into two care episodes . there are usually no well established guidelines to determine whether split admissions are appropriate for a given clinical problem . the older the patient and the more complicated his medical care , the more often physicians may decide to partition care into two or more hospitalizations . the second problematic area related to illness episode involves chronic diseases requiring episodic hospital care . in managing many illnesses , such episodes of illness are very difficult to monitor or regulate under the drg system . particular areas of difficulty are psychiatric illnesses such as alcoholism . if , after seven days , an alcoholic patient signs out of the hospital against medical advice , how hard should the hospital or physician fight to convince him to stay ? similarly , if a psychiatric patient is almost well , a potentially inappropriate incentive exists to allow him to return home too early . in such cases , there is a high probability the patient will return in the days or weeks to come . indeed , there are no clearly defined lengths of stay for inpatient treatment of alcoholism and psychiatric illnesses . such patients are usually treated to the point where they are able to function outside of the hospital environment . the more vigorously the acute episode of heart failure or chronic lung disease is treated , the less likely that the patient will be readmitted in the near future . a study conducted at university hospital and boston city hospital demonstrated that readmissions for heart failure were inversely related to the vigor with which excess fluid was removed during the hospital stay ( gertman and stanton , 1975 ) . patients who were discharged early with persisting but modest signs of fluid accumulation had a higher rate of readmission in the ensuing year . yet , examining only an individual case after the fact , a medical panel could not have successfully proven undertreatment of any single patient . relatively little research has been done , even at a descriptive level , on patterns of care for older individuals over time ; this deficiency is particularly great for those with multiple active chronic health problems . prior use , as discussed later , may in part explain subsequent hospital admission resource use . how patterns of care affect resource use and outcomes is a topic which requires extensive research . at the least , such research is necessary to minimize confounding efforts to properly adjust for severity of illness ; beyond this , any study of drg impacts on quality of care must develop such information . there is considerable concern among providers about whether the current drg system optimally achieves the stated objectives of the medicare prospective payment system . specifically , many fear that the pps may not equitably deal with systematic differences in severity of patient illnesses across all hospitals . as much as one might wish that 467 drg 's controlled for interhospital differences in patient severity , the large amount of unexplained variance in some drg 's is disturbing . of course , much of it may be simply random variation , with zero net effect across all admissions . on the other hand , there may be systematic variations by one or more hospital characteristics ( e.g. , teaching status , ownership , location , etc . ) , resulting in windfall gains or unfair losses . inevitably , however , an inaccurately defined output leads to inequities and , what is worse , gaming of the system . while all parties to this debate over the equity of drg 's generally understand the term severity and would agree that in any group of patients there are those with more severe and less severe illnesses , there is no agreement on how to : quantify differences in severity on a continuous scale of measurement ; translate such severity measures into uniform resource need measures ; and fairly monetize the resource measures in medicare reimbursement procedures . in fact , the authors would argue that there is no fully valid way to accomplish any one of these three measurement tasks . the best one can do is make approximations of the concept which reduce the extent of disagreement . recognizing that , even if one could remove all confounding factors , no perfect severity measure exists , hcfa and health care providers must move forward to make the best approximations possible subject to a host of practical constraints . the old drg system devised by the yale university group was subjected to extensive methodological criticism for not adequately dealing with major differences in severity among patients . several research organizations , including blue cross of western pennsylvania , systemetrics , inc . , susan horn 's team at the johns hopkins school of public health and others have demonstrated that they can achieve superior reductions in length of stay variance by alternative methods compared to the drg 's used in the old system ( young , 1979 ; garg et al . , 1978 ; horn , 1983 ) . horn has compared four existing classifications systems ( icd-9-cm drg 's , the old new jersey drg 's , disease staging , and generalized patient management paths ) with her severity of illness index for their respective variance reduction characteristics on a disease - specific basis across different hospitals . generally , horn 's chart abstract method achieves far greater subgroup homogeneity ( total charges and length of stay ) than do any of the other computerized , discharge abstract - based classification systems . horn and her co - workers have also tested whether their approach is superior to the new medicare drg system which has attempted to address some of the prior criticisms on severity adjustment . in a study of ten new drg 's at four hospitals , they have reportedly been able to achieve better than 40 percent additional reduction in variance by their severity of illness index ( horn et al . , 1983 ) . the issue for hcfa is not whether to simply stand pat with the drg system as currently defined versus attempting to identify possible improvements . rather , the issue is : what is the most cost effective way to proceed in enhancing the drg 's . while basic research must proceed on the sources of confounding , such as definitional imprecision , inappropriate use and variance in practice patterns , efforts to explore alternative severity of illness adjustments in the medicare pps must move forward as well . to assist in illustrating how a drg / severity of illness policy research program could be organized , figure 1 shows a decision tree model for questions which might be explored in the pps severity of illness revisions . the authors believe that the central issue structuring a practical severity of illness research paradigm is the amount and type of information available . the most important practical question to address is whether to limit enhancements of the case - mix method to those measures available only in the uniform hospital discharge data set ( uhdds ) . while some might argue that this is only a procedural constraint and should not have such an important place in the research agenda paradigm , the availability of information and the logistics of acquiring that information is in fact the most critical decision that medicare must address now if it wants to have an operationally revised system in the near future . within the current information set available to medicare , the next question which must be addressed is whether the focus of revisions should be to fine - tune the existing drg system or to alter it ? the former course leads to research option area a ; here the principal research work would be to evaluate better trim points , test use of dollar ( cost ) dependent variables more extensively than was possible in the initial drg design , structure definitional requirements more tightly to prevent abuse , develop a system of potential correction factors for inappropriate admissions , etc . alternatively , under research option area b , hcfa could decide to modify the drg 's by creating additional drg subcategories . applying the primary staged conditions and staging levels to selectively create subcategories where there is a clear need for reduction in currently large variances in costs per admission ( systemetrics , inc . , revisions might also be designed that anticipate issues of major quantitative importance in the near future . a pioneering institution in the field , brigham and women 's hospital in boston , has now started to extensively perform multiple major joint replacements in a single hospital admission episode rather than having two or more admissions . in their own internal , drg - oriented management information system , they have now subdivided drg 209 into two groups based on whether there is a single major joint replacement or multiple major joint replacements . this is an important , common surgical procedure where cost - effective and quality - of - care advances in surgical practice , involving multiple joint replacements per admission , are likely to disseminate fairly rapidly over , the next several years . research option area c , using the current uhdds dataset , would set aside the current yale drg framework and evaluate completely different ways of defining severity of illness differentials between institutions for purposes of reimbursement . these research options might involve use of multivariate quantitative models as opposed to a step - wise classification methodology to determine severity - adjusted reimbursement formulas . carol fernow at health care systems international , for example , has taken commission on professional and hospital activity ( cpha ) data and developed log - linear models which apply specific quantitative coefficients to factors such as specific secondary diagnoses ( e.g. , diabetes as a secondary diagnosis in cholelithiasis ) , types of secondary operation procedures , etc . , to compute case - mix intensity and expected lengths of stay ( fernow , 1983 ) . the potential flexibility of a multivariate model based on medicare uhdds data sets is shown in tables 2 , 3 , and 4 which were part of a teaching example developed at the boston university school of medicine in the spring of 1983 . in this hypothetical case situation , 27 patients who were discharged alive were admitted for heart attacks to three different hospitals in the same community . the current drg algorithm categorizes such live discharges sequentially by whether or not they have had a permanent pacemaker implant , and whether or not they have had cardiovascular complications . under the existing drg framework for classifying heart attacks , the only surgical data used is whether or not there is a permanent pacemaker implant and the complications factors are related solely to the cardiovascular system . as shown in table 2 , after adjusting for the highly significant class variable ( p = .0004 ) of the current drg category , no differences could be demonstrated among the hospitals ( p = .27 ) . in contrast , a simple multivariate model incorporated an age factor , a factor for whether or not the patient had any nonoperating room procedures ( such as insertion of a swan - ganz catheter , an endoscopic procedure , etc . ) and whether or not the patient had a prior history of a heart attack ( which could be determined fairly readily in a longitudinal data file ) . in this model , while the pacemaker variable is still the most important single factor explaining variation in charges , all of the other factors contributed and produce an r value 50 percent greater than a drg hospital comparison . additionally , the conclusion of the analysis using the multivariate model is that there are significant differences in severity among the three different hospitals ; in fact , it reverses the unadjusted results . what appears to be the most expensive hospital in terms of the unadjusted charges is actually the least expensive hospital after adjustment through the multivariate model . while this is obviously a simplified example , it does illustrate the potential to use more clinical diagnostic and procedure data than is employed in the current drg framework and to potentially obtain superior reductions in variance through these techniques . revisions of severity adjusters potentially unfettered by the serious clinical constraints of current drg branched groupings might provide substantial additional explanatory power and could , if useful , be quickly implemented because the data is readily at hand . another example of a factor which could be incorporated is whether there is a history of any socio - medical problem such as alcohol abuse , even though that may not be an active clinical issue during the admission . data collected by the health data institute , inc . on auto workers indicated that for major nonsubstance abuse admissions , individuals with a history of alcoholism have more expense per admission than persons admitted without such a prior history , controlling for all other relevant case - severity factors ( mcguire and fair bank , 1984 ) . if the research on revision and enhancement of drg 's is not constrained to working with just the current uhdds data items , a series of other research options become available . again , data issues are key . the next practical question is whether any expansion of the items needs to be limited to a small number of additional fields ( e.g. , five to ten characters ) or whether the process can be more open - ended in the types of data elements that are used . a further subdivision is whether common elements are to be used as adjusters for all diagnoses or procedures ; if so , then the focus would be on generic , nondiagnostic clinical measures such as vital signs ( research option area d ) . factors like the admission temperature , the highest peak temperature , the lowest systolic blood pressure during the hospitalization , etc . , would frequently indicate the severity of a condition , the presence of comorbid conditions or the development of complications . table 5 shows how one of these clinical factors , peak temperature , can considerably enhance the explanation of variance within a single drg , uncomplicated heart attacks . in a project for alcoa , cpha data sets were obtained for six southern community hospitals which were the sole source of health care in the community , as part of an effort to understand why the alcoa corporation 's length of stay experience at its main aluminum plant in marysville , tennessee was so high ( lind et al . , 1979 ) . as part of that effort , an analysis was done for a set of common conditions to pinpoint differences in resource utilization which employed standard diagnostic and procedure information . the analysis also attempted to adjust for severity of illness by use of the medical audit component of the cpha hospital discharge abstract form . peak temperature was by far the most significant factor in explaining variations in length of stay among hospitals for this drg and also turned out to be the most significant factor in explaining variations in charges among physicians at alcoa 's principal community hospital . table 4 shows that it is possible to obtain additional reductions in variance by use of other treatment and clinical factors within a defined drg category . clinical variables not currently in the uhdds data set may be conceptually more attractive as severity adjusters because : 1 ) they are more directly tied to a patient 's actual clinical status than is a diagnosis , and 2 ) they can be selected to more likely be exogenous rather than endogenous variables . thus , an admission blood pressure level can not readily be directed by the provider , while performing surgery can be done with considerable discretion . if variable severity elements are allowed for different conditions ( research option area e ) , type and extent - of - disease spread variables could be incorporated for malignancies , medically derived cardiovascular classes could be identified ( e.g. , american heart association functional rating for general cardiac patients , killip classifications for heart attacks , plum - pozner codes for strokes ) , trauma severity index for injuries and other limited data elements shown in clinical trials to be important prognostic severity of illness measures . if variable special elements could be incorporated , the availability of four , six or ten additional characters of data might dramatically improve the severity of illness adjusters and thus the clinical homogeneity of the groups created for reimbursement purposes . in fact , hcfa need not be limited to adding data fields to the uhdds data set , but rather might replace fields that are currently reserved for other purposes . for example , by dropping the five digits reserved for the fifth diagnosis an item with potentially extremely small marginal utility one could encode in two character fields the peak temperature ( e.g. , 99.6 , 103.5 , etc . ) and the lowest systolic blood pressure ( which would occupy only three character fields ) . if extensive additional data elements could feasibly be added to the uhdds dataset for medicare , then much more clinically - oriented physiologic parameters could be used in the severity adjustment methodology ( research option area f ) . one example of this is the acute physiology and chronic health evaluation ( apache ) system used at george washington university . in apache , more than thirty additional clinical measurements , including blood gas determinations , are incorporated into the classification weighting scheme for intensive care patients ( knaus et al . , 1981 ) . finally , if one is not limited to the uhdds dataset , severity of illness classification based on judgmental criteria such as the severity of illness index of susan horn and her associates and the relative intensity measures ( rims ) nursing care weighted system , etc . , could be tested ( research option area g ) . a critical policy decision on the approach to incorporating severity measures into the drg revision process is whether an attempt should be made to develop a comprehensive set of revisions which would be introduced en masse , or whether revisions should be promulgated through a piecemeal or phased - in approach . ( by the latter , we do not mean a phase - in of a comprehensive set of revisions because , if evidence was present of a superior method of classification , it would probably be politically unacceptable to the losers under the old approach for hcfa to not introduce all of the revisions at once . ) the arguments for and against either of these approaches are basically political rather than technical . yet , the decision on which is the preferable course to the government and the health care industry is important to provide guidance for an efficient research strategy and would prioritize among the areas . some approaches , such as the severity of illness index technique , are essentially comprehensive approaches and do not lend themselves to partial introduction for the drg list of categories . on the other hand , approaches which would further subdivide or minimally reorganize terminal drg 's within a major diagnostic category , such as the systemetrics approach , using adjacent diagnosis - related groups or additional age splits , are more reasonable on a selective introduction basis . there is a final issue which will plague the drg reimbursement system even if all data confounding and severity problems could be resolved perfectly the issue of effectiveness of treatment . there has been an assumption in many quarters that if any group of patients had exactly the same clinical needs , their physicians or any outside physicians would agree on the product to be delivered . the actual situation today in american medicine is that there are sometimes vast , well - reasoned and sincere differences among outstanding physicians about the best course of diagnosis and treatment . if these differences are associated with substantial and systematic differences in resource use within a drg or other adjustment schemes , then american medicine will continue to resist the system , labelling it as inequitable and as potentially containing adverse quality of care consequences . obstructive sleep apnea ( osa ) is a highly prevalent and underdiagnosed disease.1 a recent large clinical trial established osa as a novel risk factor for sudden cardiac death ( scd).2 after 20 years of followup , the busselton health study cohort showed that moderate to severe sleep apnea was independently associated with an increased risk of allcause mortality.3 in a longitudinal study of 10 701 adults , osa was associated with scd , and the magnitude of risk varied with osa severity.2 qt prolongation is an independent risk factor for scd,4 , 5 , 6 and osa is associated with longer qt intervals.7 , 8 this electrocardiographic effect is thought to be caused by systemic hypoxia , hypercarbia , or acidosis . the most effective treatment for osa is continuous positive airway pressure ( cpap ) therapy , which improves oxygenation and decreases sleep fragmentation . cpap improves prolonged cardiac repolarization9 and decreases the rate of death.10 cpap withdrawal is associated with prolongation of the corrected qt interval.11 the genetic link of scd and qt interval to potassium channels has been well established in inherited long qt syndromes.12 lossoffunction gene mutations of cardiac potassium channels contribute to different types of long qt syndromes , such as kcnq1 ( long qt syndrome type 1 [ lqt1 ] ) , kcnh2 ( lqt2 ) , kcne1 ( lqt5 ) , kcne2 ( lqt6 ) , and kcnj2 ( lqt7).13 , 14 other potassium channels expressed in the heart include kcnd3 , encoding the potassium channel that underlies the transient outward current ito15 ; kcna5 , encoding the potassium channel that underlies ikur 16 ; and kcnj11 , encoding kir6.2 , which underlies the inward rectifying channel.17 in summary , osa is associated with qt prolongation and scd . cpap improves qt prolongation.9 , 11 qt interval is influenced by potassium channels . in our previously published study , the levels of circulating cardiac sodium channel fulllength mrna and splicing variants in white blood cells were representative of levels in the myocardium.18 based on these observations , we reasoned that circulating white blood cells may experience conditions similar to those of cardiomyocytes and may reveal insights into the mechanism of qt changes with osa and cpap . we hypothesized that prolonged qt in osa represented altered potassium channel regulation and that this regulation would be reflected in circulating white blood cells . sodium channel splicing in obstructive sleep apnea ( socsosa ) and was conducted at the lifespan health system ( rhode island hospital and the miriam hospital ) in providence , rhode island , and at the university of illinois at chicago ( uic ) in chicago , illinois . participants were adults ( aged 18 years ) with osa newly diagnosed by polysomnogram who agreed to cpap treatment ( osa group ) or adults without osa ( control group ) . all trial participants were screened with the stopbang questionnaire19 before proceeding to the sleep study . based on the severity of apnea hypopnea index ( ahi ) , osa patients were assigned to 3 groups : mild ( ahi 515 ) , moderate ( ahi 1530 ) , or severe ( ahi > 30).20 the ahi was defined as the number of apneas and hypopneas per hour and calculated by adding the total number of apnea and hypopnea events and dividing by the total number of minutes of actual sleep time multiplied by 60 . the overnight sleep study consisted of continuous polygraphic recordings from 10 pm to 7 am , and the baseline parameters were measured . arterial oxygen saturation was recorded by digital pulse oximeter , and the nadir arterial oxygen saturation levels ( nadir o2 ) were obtained . the oxygen desaturation index is the number of times per hour of sleep that the blood 's oxygen level drops by 3% or 4% ( ie , odi4 ) from baseline . to quantify overall nocturnal desaturation , we used the cumulative percentages of sleep time spent at saturations < 90% or < 85% . respiratory disturbance index was defined as the average number of episodes of apnea , hypopnea , and respiratory event related arousals per hour of sleep documented in the polysomnography . after 4 weeks of cpap therapy , an ahi score was measured and documented as residual ahi , which was used to assess the efficacy of cpap therapy . the osa patients were evaluated with the epworth sleepiness scale ( ess)21 and the functional outcome of sleep questionnaire 10 ( fosq10)22 at the time of initial diagnosis . improvement of the symptoms were reevaluated by the ess and fosq10 after 1 month of cpap treatment . the exclusion criteria included chronic use of hypnotics ; current drug or alcohol addiction ; a rhythm other than sinus at enrollment ; mandatory and biventricular pacing ; a history of heart transplant or left ventricular assist device ; active use of intravenous vasodilators , vasopressors , or inotropes ; hemodialysis or peritoneal dialysis ; active infection ; acute coronary syndrome ; major trauma or surgery ; malignant neoplastic disease on active treatment including chemotherapy and radiation therapy ; life expectancy < 1 year ; collagen vascular disease on active treatment including steroids and other immunomodulating drugs ; systemic steroid use ; or concomitant use of an investigational drug . blood samples were collected in paxgene blood rna tubes ( qiagen ) and were stored in a freezer at 80c before processing . total rna was isolated with the use of the paxgene blood rna isolation kit and was then converted to cdna with the use of the superscript iii cdna reverse transcription kit ( life technologies ) . only samples with an optical density of 260/280>2.0 and 260/230>1.7 quantitative reverse transcriptase polymerase chain reaction was performed to detect the abundance of potassium channels using iqsybr green supermix ( biorad laboratories ) and the 7500 fast realtime pcr system ( life technologies ) . the primer sequences used were hkcnq1f ( 5ttgggaagccctcactgttc3 ) , hkcnq1r ( 5ctggtg aagcatgtcggtga3 ) , hkcnh2f ( 5tcaccgccctgtacttcatct3 ) , hkcnh2r ( 5caggccttgcataca ggttca3 ) , hkcne1f ( 5gaaccccaccactggctaaa3 ) , hkcne1r ( 5taatcca cccctcacccctt3 ) , hkcne2f ( 5atttcatcctgcccacacact3 ) , hkcne2r ( 5 ccagcgtctgtgtgaaattgg3 ) , hkcna5f ( 5agaggagtccagcggaaggt3 ) , hkcna5r ( 5actgtctgcattctccagggt3 ) , hkcnj11f ( 5tccagggtgttacaa ggcac3 ) , hkcnj11r ( 5gaattgggttgggaggagca3 ) , hkcnj2f ( 5tgtcacgg atgaatgcccaa3 ) , hkcnj2r ( 5ctgcgccaatgatgaaagca3 ) , hkcnd3f ( 5t gtacgaacctccaccatcaa3 ) , and hkcnd3r ( 5atgggtagttctgcattgaact ct3 ) . quantitative reverse transcriptase polymerase chain reaction thermal cycling conditions were initial incubation at 50c for 60 minutes and then termination of the reactions at 85c for 5 minutes . polymerase chain reaction was performed with an initial denaturation step at 95c for 20 seconds , followed by 40 cycles of denaturation at 95c for 3 seconds and annealing and nucleotide prolongation at 60c for 30 seconds . each sample was measured for the target genes kcnq1 , kcnh2 , kcne1 , kcne2 , kcna5 , kcnj11 , kcnj2 , kcnd3 , and actin . continuous variables were described in terms of descriptive statistics ( mean and standard deviation ) . independent samples t tests and pearson chisquare tests were used for bivariate analysis comparisons of continuous and categorical variables , respectively . the pearson correlation coefficient was used to assess the correlation between the parameters measured in the sleep study , the severity of osa , and the mrna levels of potassium channels . differences between gene expression at baseline and at the followup visit were examined by means of paired wilcoxon tests . sodium channel splicing in obstructive sleep apnea ( socsosa ) and was conducted at the lifespan health system ( rhode island hospital and the miriam hospital ) in providence , rhode island , and at the university of illinois at chicago ( uic ) in chicago , illinois . participants were adults ( aged 18 years ) with osa newly diagnosed by polysomnogram who agreed to cpap treatment ( osa group ) or adults without osa ( control group ) . all trial participants were screened with the stopbang questionnaire19 before proceeding to the sleep study . based on the severity of apnea hypopnea index ( ahi ) , osa patients were assigned to 3 groups : mild ( ahi 515 ) , moderate ( ahi 1530 ) , or severe ( ahi > 30).20 the ahi was defined as the number of apneas and hypopneas per hour and calculated by adding the total number of apnea and hypopnea events and dividing by the total number of minutes of actual sleep time multiplied by 60 . the overnight sleep study consisted of continuous polygraphic recordings from 10 pm to 7 am , and the baseline parameters were measured . arterial oxygen saturation was recorded by digital pulse oximeter , and the nadir arterial oxygen saturation levels ( nadir o2 ) were obtained . the oxygen desaturation index is the number of times per hour of sleep that the blood 's oxygen level drops by 3% or 4% ( ie , odi4 ) from baseline . to quantify overall nocturnal desaturation , we used the cumulative percentages of sleep time spent at saturations < 90% or < 85% . respiratory disturbance index was defined as the average number of episodes of apnea , hypopnea , and respiratory event related arousals per hour of sleep documented in the polysomnography . after 4 weeks of cpap therapy , an ahi score was measured and documented as residual ahi , which was used to assess the efficacy of cpap therapy . the osa patients were evaluated with the epworth sleepiness scale ( ess)21 and the functional outcome of sleep questionnaire 10 ( fosq10)22 at the time of initial diagnosis . improvement of the symptoms were reevaluated by the ess and fosq10 after 1 month of cpap treatment . the exclusion criteria included chronic use of hypnotics ; current drug or alcohol addiction ; a rhythm other than sinus at enrollment ; mandatory and biventricular pacing ; a history of heart transplant or left ventricular assist device ; active use of intravenous vasodilators , vasopressors , or inotropes ; hemodialysis or peritoneal dialysis ; active infection ; acute coronary syndrome ; major trauma or surgery ; malignant neoplastic disease on active treatment including chemotherapy and radiation therapy ; life expectancy < 1 year ; collagen vascular disease on active treatment including steroids and other immunomodulating drugs ; systemic steroid use ; or concomitant use of an investigational drug . blood samples were collected in paxgene blood rna tubes ( qiagen ) and were stored in a freezer at 80c before processing . total rna was isolated with the use of the paxgene blood rna isolation kit and was then converted to cdna with the use of the superscript iii cdna reverse transcription kit ( life technologies ) . only samples with an optical density of 260/280>2.0 and 260/230>1.7 were used . quantitative reverse transcriptase polymerase chain reaction was performed to detect the abundance of potassium channels using iqsybr green supermix ( biorad laboratories ) and the 7500 fast realtime pcr system ( life technologies ) . the primer sequences used were hkcnq1f ( 5ttgggaagccctcactgttc3 ) , hkcnq1r ( 5ctggtg aagcatgtcggtga3 ) , hkcnh2f ( 5tcaccgccctgtacttcatct3 ) , hkcnh2r ( 5caggccttgcataca ggttca3 ) , hkcne1f ( 5gaaccccaccactggctaaa3 ) , hkcne1r ( 5taatcca cccctcacccctt3 ) , hkcne2f ( 5atttcatcctgcccacacact3 ) , hkcne2r ( 5 ccagcgtctgtgtgaaattgg3 ) , hkcna5f ( 5agaggagtccagcggaaggt3 ) , hkcna5r ( 5actgtctgcattctccagggt3 ) , hkcnj11f ( 5tccagggtgttacaa ggcac3 ) , hkcnj11r ( 5gaattgggttgggaggagca3 ) , hkcnj2f ( 5tgtcacgg atgaatgcccaa3 ) , hkcnj2r ( 5ctgcgccaatgatgaaagca3 ) , hkcnd3f ( 5t gtacgaacctccaccatcaa3 ) , and hkcnd3r ( 5atgggtagttctgcattgaact ct3 ) . quantitative reverse transcriptase polymerase chain reaction thermal cycling conditions were initial incubation at 50c for 60 minutes and then termination of the reactions at 85c for 5 minutes . polymerase chain reaction was performed with an initial denaturation step at 95c for 20 seconds , followed by 40 cycles of denaturation at 95c for 3 seconds and annealing and nucleotide prolongation at 60c for 30 seconds . each sample was measured for the target genes kcnq1 , kcnh2 , kcne1 , kcne2 , kcna5 , kcnj11 , kcnj2 , kcnd3 , and actin . continuous variables were described in terms of descriptive statistics ( mean and standard deviation ) . independent samples t tests and pearson chisquare tests were used for bivariate analysis comparisons of continuous and categorical variables , respectively . the pearson correlation coefficient was used to assess the correlation between the parameters measured in the sleep study , the severity of osa , and the mrna levels of potassium channels . differences between gene expression at baseline and at the followup visit were examined by means of paired wilcoxon tests . overall , 28 osa patients and 6 participants without osa met eligibility criteria and completed the baseline and followup visits . the mean age ( sd ) was 50.710.1 years in osa patients and 43.616.3 years in participants without osa . the baseline body mass index was 36.48.1 in osa patients and 39.510.7 in participants without osa . at baseline , there was no significant difference in demographic characteristics between groups ( table 1 ) . clinical characteristics of the socsosa study ahi indicates apnea hypopnea index ; bmi , body mass index ; cpap , continuous positive airway pressure ; ct85 , the cumulative percentages of sleep time spent at saturations < 85% ; ct90 , the cumulative percentages of sleep time spent at saturations < 90% ; odi3 , the number of times per hour of sleep that the blood 's oxygen level drops by 3% from baseline ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline ; osa , obstructive sleep apnea ; rdi , respiratory disturbance index ; residual ahi , the apnea hypopnea index after 4 weeks of continuous positive airway pressure therapy . p value from t tests for continuous variables and chisquare tests for categorical variables . the sleep characteristics and cpap treatment among participants with osa are shown in table 1 . as expected , cpap therapy was associated with significant reductions in ahi . nevertheless , compared with patients with mild or moderate osa , patients with severe osa had significantly higher residual ahi ( mild 2.01.8 , moderate 1.00.9 , severe 5.85.6 ; p=0.015 ) . the stopbang questionnaire was used to screen for osa . compared with patients with mild osa , patients with moderate osa had markedly higher scores ( mild 4.11.4 , moderate 6.01.3 , severe 5.41.1 ; p=0.022 ) . there was no statistically significant difference between moderate and severe osa , and that might be explained by use of the stopbang questionnaire , which is highly sensitive at low scores and highly specific at high scores for osa.23 the osa patients were evaluated with the ess questionnaire to measure daytime sleepiness before and after cpap therapy . the ess score significantly decreased after 4 weeks of cpap in mild and moderate osa patients ( mild 12.65.2 versus 9.26.1 , p=0.028 ; moderate 11.74.5 versus 7.35.7 , p=0.034 ; severe 12.36.6 versus 10.75.3 , p=0.497 ) . we observed a significant inverse correlation between the severity of osa stratified by ahi and the mrna expression of the main potassium channels assessed : kcnq1 ( r=0.486 , p=0.007 ) , kcnh2 ( r=0.437 , p=0.016 ) , kcne1 ( r=0.567 , p=0.001 ) , kcnj2 ( r=0.442 , p=0.015 ) , and kcna5 ( r=0.468 , p=0.009 ) ( figure 1 and table 2 ) . in addition , kcnq1 ( r=0.404 , p=0.027 ) , kcnh2 ( r=0.416 , p=0.022 ) , and kcne1 ( r=0.465 , p=0.010 ) inversely correlated with odi4 , indicating the oxygen desaturation status ( figure 2 and table 2 ) . a , correlation between mrna level of kcnq1 ( ordinate ) and ahi ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and ahi ( abscissa ) . correlation of the expression of baseline potassium channels with hypoxia ahi indicates apnea hypopnea index ; nadir o2 , nadir arterial oxygen saturation levels ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . a , correlation between mrna level of kcnq1 ( ordinate ) and odi4 ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and odi4 ( abscissa ) . odi4 indicates the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . for patients with newly diagnosed osa initiating cpap for the first time , circulating kcnq1 and kcnj2 were increased 1.40.4fold ( p=0.040 ) and 2.11.4fold ( p=0.046 ) , respectively , after 4 weeks of cpap therapy in the moderate group ( figure 3 ) . although they did not reach statistical significance , cpap improved most potassium channel gene expression in moderate osa : kcne1 1.5fold , kcne2 2.9fold , kcna5 1.9fold , kcnd3 1.3fold , and kcnj11 1.6fold . p<0.05 comparing the postcpap fold change of kcnq1 or kcnj2 to the baseline in the moderate osa group . overall , 28 osa patients and 6 participants without osa met eligibility criteria and completed the baseline and followup visits . the mean age ( sd ) was 50.710.1 years in osa patients and 43.616.3 years in participants without osa . the baseline body mass index was 36.48.1 in osa patients and 39.510.7 in participants without osa . at baseline , there was no significant difference in demographic characteristics between groups ( table 1 ) . clinical characteristics of the socsosa study ahi indicates apnea hypopnea index ; bmi , body mass index ; cpap , continuous positive airway pressure ; ct85 , the cumulative percentages of sleep time spent at saturations < 85% ; ct90 , the cumulative percentages of sleep time spent at saturations < 90% ; odi3 , the number of times per hour of sleep that the blood 's oxygen level drops by 3% from baseline ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline ; osa , obstructive sleep apnea ; rdi , respiratory disturbance index ; residual ahi , the apnea hypopnea index after 4 weeks of continuous positive airway pressure therapy . p value from t tests for continuous variables and chisquare tests for categorical variables . the sleep characteristics and cpap treatment among participants with osa are shown in table 1 . as expected , cpap therapy was associated with significant reductions in ahi . nevertheless , compared with patients with mild or moderate osa , patients with severe osa had significantly higher residual ahi ( mild 2.01.8 , moderate 1.00.9 , severe 5.85.6 ; p=0.015 ) . the stopbang questionnaire was used to screen for osa . compared with patients with mild osa , patients with moderate osa had markedly higher scores ( mild 4.11.4 , moderate 6.01.3 , severe 5.41.1 ; p=0.022 ) . there was no statistically significant difference between moderate and severe osa , and that might be explained by use of the stopbang questionnaire , which is highly sensitive at low scores and highly specific at high scores for osa.23 the osa patients were evaluated with the ess questionnaire to measure daytime sleepiness before and after cpap therapy . the ess score significantly decreased after 4 weeks of cpap in mild and moderate osa patients ( mild 12.65.2 versus 9.26.1 , p=0.028 ; moderate 11.74.5 versus 7.35.7 , p=0.034 ; severe 12.36.6 versus 10.75.3 , p=0.497 ) . we observed a significant inverse correlation between the severity of osa stratified by ahi and the mrna expression of the main potassium channels assessed : kcnq1 ( r=0.486 , p=0.007 ) , kcnh2 ( r=0.437 , p=0.016 ) , kcne1 ( r=0.567 , p=0.001 ) , kcnj2 ( r=0.442 , p=0.015 ) , and kcna5 ( r=0.468 , p=0.009 ) ( figure 1 and table 2 ) . in addition , kcnq1 ( r=0.404 , p=0.027 ) , kcnh2 ( r=0.416 , p=0.022 ) , and kcne1 ( r=0.465 , p=0.010 ) inversely correlated with odi4 , indicating the oxygen desaturation status ( figure 2 and table 2 ) . a , correlation between mrna level of kcnq1 ( ordinate ) and ahi ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and ahi ( abscissa ) . correlation of the expression of baseline potassium channels with hypoxia ahi indicates apnea hypopnea index ; nadir o2 , nadir arterial oxygen saturation levels ; odi4 , the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . a , correlation between mrna level of kcnq1 ( ordinate ) and odi4 ( abscissa ) . b , correlation between mrna level of kcnj2 ( ordinate ) and odi4 ( abscissa ) . odi4 indicates the number of times per hour of sleep that the blood 's oxygen level drops by 4% from baseline . for patients with newly diagnosed osa initiating cpap for the first time , circulating kcnq1 and kcnj2 were increased 1.40.4fold ( p=0.040 ) and 2.11.4fold ( p=0.046 ) , respectively , after 4 weeks of cpap therapy in the moderate group ( figure 3 ) . although they did not reach statistical significance , cpap improved most potassium channel gene expression in moderate osa : kcne1 1.5fold , kcne2 2.9fold , kcna5 1.9fold , kcnd3 1.3fold , and kcnj11 1.6fold . p<0.05 comparing the postcpap fold change of kcnq1 or kcnj2 to the baseline in the moderate osa group . the main findings of this prospective study are that the mrna expression of at least 5 of 8 cardiac potassium channels correlated with the severity of osa and that cpap treatment improved circulating kcnq1 and kcnj2 in patients with moderate osa . there were trends toward increases in 7 of 8 potassium channels in patients with moderate osa . qt prolongation is an independent risk factor for scd . in the oregon sudden unexpected death study , idiopathic abnormal prolongation of the corrected qt interval was associated with 5fold increased odds of scd.5 recent genetic studies have established a clear inverse relationship between qt interval and expression and function of potassium channels . gene defects resulting in loss of function of voltagegated potassium channels , for example , are associated with long qt syndromes : lqt1 ( kcnq1 ) and lqt2 ( kcnh2 ) , encoding subunits of the potassium channels iks and ikr ; lqt5 ( kcne1 ) and lqt6 ( kcne2 ) , encoding subunits of the potassium channels iks and ikr ; and lqt7 ( kcnj2 ) , encoding the inward rectifier potassium channel kir2.1.12 , 13 , 14 , 15 , 16 , 17 osa is associated with scd and long qt . osa predicted incident scd in a longitudinal study of 10 701 adults.3 in osa patients who had no evidence of underlying cardiac , pulmonary , or central nervous system disease , the qt interval was prolonged at the onset of apnea.7 because osa is associated with long qt , and long qt can be caused by potassium channel loss of function , we tested whether osa was associated with circulating potassium channel changes . a total of 8 potassium channel genes were studied , and we found that the mrna levels of at least 5 of 8 potassium channel genes assessed correlated inversely with hypoxemia and osa severity . of these channel genes , 2 were statistically significantly increased and 5 more showed trends toward an increase with cpap therapy . this could be explained if correction of hypoxia were the major driver for improvements in channel levels . consistent with this idea , in a population of 10 701 adults , the magnitude of scd risk was predicted by ahi and nocturnal oxygen desaturation.3 in our study , even with cpap , severe osa patients had higher residual ahi with statistical difference , and there was no change in potassium channel levels with cpap . this supports the idea that the degree of hypoxia , rather than some other aspect of cpap , was mediating the changes in mrna abundance . in our patients with mild osa , hypoxia was not severe and changed little with cpap . the transcription factor hypoxiainducible factor 1 ( hif1 ) plays a key role in cellular response to systemic oxygen levels in humans . hif1 is a subunit of a heterodimeric hif1.24 , 25 there are 3 putative hif1 transcription factor binding sites located in the upstream sequence of the human kcnq1 gene and 4 hif1 binding sites located in the upstream sequence of the human kcnj2 gene ( data not shown ) . the systemic oxygen level affects hif1 through multiple mechanisms , such as hif1 conformational changes and stability , subcellular localization , and transcriptional activity . hif1 regulates the expression of > 60 genes involved in angiogenesis , cell proliferation , cell survival , and glucose and iron metabolism.26 the presence of hif1 binding sites in the promoters of potassium channels may provide the mechanism by which systemic oxygen level affects the gene expression of potassium channels . although the paired design controlled for interparticipant variability , the small sample size precluded comprehensive multivariable adjustment . in addition , because of the short study duration , it is not clear if the changes in potassium channel expression with cpap will be durable or if they correlate with improved outcomes . furthermore , we did not correlate potassium channel mrna levels in white blood cells with levels or currents in the myocardium . the mrna expression of most potassium channels inversely correlated with the severity of osa and hypoxemia . if downregulations in circulating potassium channel genes with osa are mirrored in the heart , as may happen if systemic hypoxemia is a causal factor , then downregulation of potassium channel genes in the heart may explain the long qt and increased risk of scd in osa . moreover , it suggests that potassium channel gene levels may represent a new circulating marker for arrhythmic risk in osa . the present work was supported in part by national institutes of health grants r01 hl104025 and hl106592 .

the new medicare prospective payment system has been challenged with regard to its fairness in reimbursing hospitals adequately , given the true resource needs in caring for patients . most of these criticisms are now labelled as issues about adjustments for severity of illness . critics point to the large amount of unexplained variation in charges and length of stay within the existing drg 's as indirect support for their contentions about inadequate adjustments.a paradigm is presented which argues that the key questions on the types of severity of illness measures to be utilized in future refinements of drg 's revolve around the extent and type of data which can feasibly be included in any workable reimbursement approach . a paradigm is presented on how these questions about information define a series of research options in the severity of illness arena . backgroundcardiac arrhythmias and sudden cardiac death are more frequent in patients with obstructive sleep apnea ( osa ) . osa is associated with qt prolongation , and qt prolongation is an independent risk factor for sudden cardiac death . because qt prolongation can be mediated by potassium channel loss of function , we tested whether osa or continuous positive airway pressure therapy altered mrna expression of circulating white blood cell potassium channels.methods and resultsin total , 28 patients with osa newly diagnosed by polysomnogram and 6 participants without osa were enrolled . potassium channel levels in white blood cells at baseline and at a 4week followup visit were compared . there was a significant inverse correlation between the severity of the osa stratified by apnea hypopnea index and mrna expression of the main potassium channels assessed : kcnq1 ( r=0.486 , p=0.007 ) , kcnh2 ( r=0.437 , p=0.016 ) , kcne1 ( r=0.567 , p=0.001 ) , kcnj2 ( r=0.442 , p=0.015 ) , and kcna5 ( r=0.468 , p=0.009 ) . in addition , kcnq1 , kcnh2 , and kcne1 inversely correlated with the oxygen desaturation index 4 . after 4 weeks of continuous positive airway pressure therapy , circulating kcnq1 and kcnj2 were increased 1.40.4fold ( p=0.040 ) and 2.11.4fold ( p=0.046 ) in the moderate osa group . compared with patients with mild or moderate osa , patients with severe osa had a persistently higher apnea hypopnea index ( mild 2.01.8 , moderate 1.00.9 , severe 5.85.6 ; p=0.015 ) , perhaps explaining why the potassium channel changes were not seen in the severe osa group.conclusionsthe mrna expression of most potassium channels inversely correlates with the severity of osa and hypoxemia . continuous positive airway pressure therapy improves circulating kcnq1 and kcnj2 in patients with moderate osa .

parkinson 's disease ( pd ) is a debilitating neurologic disorder characterized clinically by bradykinesia , resting tremor , rigidity , and postural instability with a therapeutic response to levodopa . pathophysiologically , pd is characterized by the loss of dopamine neurons in the substantia nigra ( sn ) pars compacta and by the presence of intracellular inclusions known as lewy bodies , which are composed primarily of alpha - synuclein protein aggregates . although the disease etiology remains largely unclear , generation of reactive oxygen species during oxidation of dopamine ( da ) could be one of the factors leading to the selective loss of nigral dopaminergic neurons in pd ( spina and cohen , 1989 ) . the vesicular monoamine transporter type 2 ( vmat2 , slc18a2 ) uptakes cytosolic monoamines , including dopamine , into intracellular secretory vesicles , preventing their toxicity in the cytosol and discharging them into the extracellular space by exocytosis . altered function of vmat2 proteins may therefore cause cytoplasmic accumulation of free da , leading to dopaminergic neuron death , therefore being a risk factor for pd . vmat2 is expressed in central , peripheral , and enteric neurons as well as in platelets ( peter et al . , 1995 ) . vmat2-deficient mouse exhibits increased oxidative stress , progressive loss of da terminals and cell bodies in the sn pars compacta , alpha - synuclein accumulation ( caudle et al . , 2007 ) , and increased sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) toxicity ( mooslehner et al . , 2001 ) ; interestingly , this model displays also motor and nonmotor symptoms of pd ( taylor et al . , 2011 ) . in humans , the involvement of vmat2 in pd pathogenesis is supported by positron emission tomography studies showing significantly lower vmat2 densities in the putamen , caudate , and sn of pd patients ( bohnen et al . , 2006 ; , 2008 ; okamura et al . , 2010 ) ; interestingly , reduced vmat2 messenger rna ( mrna ) levels were observed in platelets from patients ( sala et al . , 2010 ) . polymorphisms in the vmat2 gene or in its regulatory regions that affect its quantitative expression or protein function might therefore represent genetic risk factors for pd . some common gain - of - function haplotypes in vmat2 promoter have been reported to confer a protective effect against pd in women ( glatt et al . , 2006 ) , whereas no studies have so far addressed specifically the association of intragenic vmat2 variability with pd in caucasian populations . in light of these premises , we carried out an association study to evaluate whether genetic variants in vmat2 gene and in its regulatory regions might influence either susceptibility to pd or age of onset of the disease in a series of italian sporadic pd patients and unrelated healthy controls . the study included 704 pd patients recruited consecutively from the outpatient clinic for movement disorders at the department of neurology of san gerardo hospital in monza and at the parkinson institute in milan . pd patients were diagnosed according to the standard criteria ( gelb et al . , 1999 ) . among patients , 45 ( 7.5% ) reported a family history of pd in at least a first - degree or second - degree relative . pd cases with an age at onset ( aao ) of < 40 years were screened to exclude that they were carriers of recognized pd - causing mutations ( parkin , pink1 , and lrrk2 genes ) ; the remaining familial cases were screened for the highly penetrant g0219s mutation in the lrrk2 gene that was absent in all the examined subjects . all the 45 cases with a family history of pd were included in the analysis . control population consisted of 678 individuals , comprising healthy spouses and former blood donors . both patients and controls were caucasians , and the controls none of the controls had clinical evidence of neurologic disease ( as assessed by neurologic examination ) or familial history of neurodegenerative diseases . none of the patients and of the controls had a current or former history of alcohol use disorders . the present analysis comprised a total of 8 single - nucleotide polymorphisms ( snps ) . snps were selected according to the intermarker distance , their minor allele frequency ( maf > 5% ) , the illumina design scores ( > 0.7 ) , and the previous publications showing associations with other dopamine - related traits . in detail , the 2 snps in the promoter region ( rs363371 and rs363324 ) previously identified by resequencing and included in haplotypes have been shown to be associated with alcoholism ( lin et al . , 2005 ) , rs363333 and rs363387 have been previously shown to be associated with substance dependence ( schwab et al . , 2005 ) , and rs363399 and rs4752045 with depression ( christiansen et al . , 2007 ) . lastly , considering a possible role in vmat2 mrna stability , an additional candidate snp in the 3-untranslated region ( rs363236 ) was also selected . table 1 highlights the genomic positions and the type of the genotyped markers . genomic dna was extracted from peripheral venous blood using a qiagen blood kit ( qiagen , milan , italy ) . snps were typed using the veracode goldengate genotyping assay on illumina beadxpress reader platform according to the manufacturer 's protocol ( illumina , san diego , ca , usa ) . goodness of fit of the genotype frequencies to hardy weinberg ( hw ) expected proportions in control subjects was examined . chi - square tests were used to compare sex distribution and genotype frequencies between patients and controls and to test for deviation from hw equilibrium . we used logistic regression analysis to test for the association between the vmat2 gene selected variants and pd risk . the estimated odds ratios ( ors ) and relative 95% confidence intervals ( cis ) were adjusted for sex , age at enrollment , and smoking ( ever / never ) . we performed analyses assuming autosomal dominant , log - additive , and recessive models of inheritance . in the test , all p values for individual snps , or , and 95% ci are presented as noncorrected for multiple testing . analyses were performed for subjects overall and stratified by family history of pd , age at study ( divided in quartiles ) , and sex . the association of each genetic variant with aao of pd was assessed using cox proportional hazard models , adjusted for sex . for each genetic variant , we calculated a hazard ratio , a 95% ci , and a 2-tailed p value . power calculations were carried out using the quanto software version 1.2 , under a dominant model of inheritance . the pairwise linkage disequilibrium ( ld ) between markers the ensembl genome browser ( http://www.ensembl.org/index.html ) was used to identify and to annotate nearby snps in ld ( proxies ) based on hapmap ceu . the overall difference in haplotype frequencies between cases and controls was assessed using a global score test . haplotype - specific tests were performed only including haplotypes with frequencies > 1% , using the additive model with the most frequent haplotype as the referent group in the regression analysis . correction for multiple testing was performed by applying the simulate = true parameter in haplo.score that gives simulated p values based on 10,000 permutations . the demographic data for the 704 pd patients were as follows : mean age at study was 65.14 9.3 years ( range 3290 ) and mean aao of symptoms was 57.28 10 years ( range 2787 ) . the 678 control subjects consisted of 375 men and 303 women ; their mean age at examination was 64.2 7.7 years ( range 4490 ) . there was no statistical difference in the age at study and sex between patients and controls ( p > 0.05 ) . the control genotype distributions were in agreement with the hw equilibrium for all markers ( hw p value > 0.05 ) . table 3 lists the estimated ors and associated 95% cis from the association analysis between each snp and pd , assuming autosomal dominant and trend ( additive ) models of inheritance . two out of the 8 investigated snps , both located in the putative promoter region at the 5 untranslated region , were inversely associated to pd when assuming dominant ( rs363371 : or 0.72 , ci 0.620.97 , p = 0.004 ; rs363324 : or 0.76 , ci 0.610.95 , p = 0.01 ) and trend model of inheritance ( rs363371 : or 0.78 , ci 0.650.94 , p = 0.008 ; rs363324 : or 0.85 , ci 0.720.92 , p = 0.04 ) . after bonferroni correction for multiple comparisons , only rs363371 remained significantly associated with pd ( corrected p value = 0.03 , dominant model ) . the remaining intragenic snps were not significantly associated with pd overall or in strata defined by age at study , sex , and family history ( data not shown ) . furthermore , we tested the possible association among all the different combinations of alleles at the 8 polymorphic loci with pd . four common haplotypes ( gatttagt , frequency in cases / controls = 0.30/0.29 ; gatttact , frequency = 0.23/0.21 ; gatttacc , frequency = 0.07/0.07 ; and agtctagt , frequency = 0.05/0.06 ) and several rare ones ( with frequency < 5% ) were inferred . none of the common haplotypes was associated with the overall risk for pd ( global haplotype association p value = 0.56 ) . given the observed ld pattern between the studied snps ( with poor linkage between the 5 half and the 3 half markers ) , we then partitioned the whole haplotype into smaller segments ( composed of 24 adjacent markers ) and tested them for association with pd . consistent with the individual snp analysis , a significant association with pd was observed for the haplotype including the 2 promoter variants , rs363371 and rs363324 ( global p value = 0.04 ) . in particular , the all - minor allele haplotype ag ( the second most frequent haplotype in both case and control populations ) was significantly underrepresented in cases ( frequency 19% ) as compared with controls ( frequency 23% ) , hence being associated with a reduced pd risk as compared with the most frequent haplotype ga ( or 0.78 , 95% ci 0.650.94 , permutation - based p value = 0.03 , additive model ) . finally , in analyses restricted to pd cases , none of the studied snps displayed a significant association with age at disease onset under additive or dominant models , either in the overall sample or after stratification by sex ( data not shown ) . in our study , we found that 2 polymorphisms located in the putative promoter region of the vmat2 gene were inversely associated to pd , whereas the other snps within the gene were not related to the risk of developing pd or to disease aao . the human vmat2 gene consists of 16 exons and 15 introns and has been localized to chromosome 10q25 . the recently launched website pdgene ( http://www.pdgene.org/ ) , which systematically reviews genetic association studies of pd including genome - wide association studies ( gwas ) , confirms that information regarding the association of vmat2 gene with pd is still deficient . a single case control study comprising 190 japanese patients and 190 controls failed to identify any significant association among 3 intronic snps in the gene ( rs2072362 , rs3523 , and rs363334 ) and pd ( mizuta et al . , 2006 ) . although none of the snps analyzed in the present study was specifically tested in the publicly accessible gwas ( as assessed using the national center for biotechnology information database of genotypes and phenotypes , http://view.ncbi.nlm.nih.gov/dbgap ) , the national institute of neurological disorders and stroke ( ninds ) genome - wide genotyping in parkinson 's disease ( dbgap study accession number : phs000089.v3.p2 ) and the center of inherited disease research ( cidr ) : gwas in familial parkinson disease ( phs000126.v1.p1 ) contained respectively 2 ( rs363327 and rs1860404 ) and 1 ( rs1860404 ) snps in high ld with our variants rs363343 and rs363333 , which similarly were not associated with pd . hence the present study , extending the analysis to more snps inside the gene ( both in coding and noncoding regions ) , adds to previous evidence showing no association between intragenic vmat2 variability and pd . a few more studies focused on the association of variants in the vmat2 promoter region with pd . in stark contrast to the very low degree of variability of the coding sequence , a high degree of genetic variability in the vmat2 promoter region has been identified ( glatt et al . , 2001 , 2006 ) . ( 2005 ) sequenced the 17.4-kb vmat2 promoter region in 23 caucasian individuals and identified 47 polymorphisms conferring 13 haplotypes associated to different promoter activities , as suggested by in vitro luciferase - transport assays . of note , both the snps in the promoter region ( rs363371 and rs363324 ) genotyped in our study were among the 4 haplotype - defining snps identified by lin et al . the g allele ( wild type ) of rs363371 is embedded in a consensus sequence for transcriptional repressor gc - binding factor ( gcf ) that is expressed in many tissues , whereas the minor a allele is not ( kageyama and pastan , 1989 ) . on the other hand , rs363324 is in considerable ld with rs363371 ( d = 1 , r = 0.61 ) and with another variant at the 5 untranslated region , 2504t > c ( rs2619096 ) , which is probably a transcription factor binding site , because the switching from t ( wild - type allele ) to c ( variant allele ) could double the promoter activity in luciferase assays in shsy cells ( lin et al . , 2005 ) . interestingly , regulatory haplotypes including these variants have been associated also with alcohol dependence , a different dopamine - related phenotype , supporting the hypothesis that vmat2 gene expression has in vivo physiological relevance to dopamine homeostasis ( lin et al . , 2005 ) . another study screened the vmat2 gene promoter region for common haplotypes and tested their functional effects in reporter gene assays , showing that only the gain - of - function haplotypes ( which displayed significantly increased transcriptional activity in vitro ) were associated to a reduced risk of developing pd ( glatt et al . , 2006 ) in women . in a previous work , we analyzed the relationship between the 2 variants in the promoter region ( rs363371 and rs363324 ) and vmat2 mrna levels in platelets from a subgroup of the subjects genotyped for the present study , but we failed to detect any association ( sala et al . , 2010 ) . nevertheless , because this was not the primary aim of that study , we were probably underpowered to detect subtle differences in mrna platelet levels according to distinct genotypes / haplotypes ; furthermore , the in vitro data may not necessarily reflect the snp activity in vivo because of other unknown activities and known mechanisms such as dna methylation . although the quantitative effect of the variants in vmat2 promoter region on transcriptional activity is expected to be small , and it is unclear how our findings might relate to in vivo rna and protein levels , the reported inverse association might still be biologically meaningful : even slight variations in vmat2 expression might affect pathogenic mechanisms involved in nigrostriatal degeneration ( chen et al . , 2008 ) or in the defense against pd - related environmental toxins ( lee et al . , 2000 ) . in contrast to glatt 's and our findings , 2 polymorphic sequences upstream the vmat2 promoter region were not associated to pd in the japanese population ( kariya et al . , 2005 ) ; however , the different type ( polymorphic microsatellites ) and location of the studied polymorphisms as well as the relevant ethnic differences previously observed in the genotype and allele frequencies of the polymorphisms in / around this gene ( crowley et al . , 2008 ; glatt et al . , the aforementioned gwas ninds and cidr examined only 2 variants ( rs10886051 and rs2532798 ) in the vmat2 putative promoter region , which were in high ld with rs363371 and rs363324 , respectively . whereas in the ninds study both variants were not significantly associated with pd ( association p values = 0.41 and 0.47 , respectively ) , in the cidr study only the snp in ld with rs363371 showed a tendency toward inverse association with pd ( or = 0.8 , p = 0.08 ) , which is in favor of a possible involvement of rs363371 rather than rs363324 in disease risk . although our reported associations of the promoter variants with pd may be spurious , it is also recognized that efforts to minimize the false - positive rates in gwas may lead to missed associations . replication of our findings in independent populations as well as marker saturation of this still understudied vmat2 regulatory region will be required to determine if and how variability in vmat2 promoter affects susceptibility to pd . we adjusted our analyses for possible confounders ( sex , age at study , and smoking ) . the sample size employed and the maf for the selected snps provided sufficient statistical power for the main effect analyses . assuming the population susceptibility allele frequency to be the values observed in controls and a population prevalence of 0.02 , our study had 80% power ( alpha = 0.05 , uncorrected for multiple testing ) to detect ors as small as 1.43 ( or 0.71 or smaller ) for the snp with the highest maf and as small as 1.5 ( or 0.63 or smaller ) for the snp with the lowest maf under a dominant model of inheritance . we also considered multiple genetic models and stratified our sample for multiple variables to explore possible effect modifications , in particular by sex . although we planned our study to include most haplotype - defining snps based on the previous data from the literature , our coverage of vmat2 variability is inevitably incomplete , given the low ld across and around the gene , as inferred from hapmap ceu data ; furthermore , nonsynonymous variants in coding regions of the gene ( most likely to alter its function ) , as well as rare polymorphisms ( maf < 5% ) and copy number variations , have been excluded from our study . hence , the possibility remains that intragenic vmat2 variability contributes to pd susceptibility or interacts epistatically with other genes . another limitation of our study might be that , because we did not conduct correction for multiple testing , false - positive findings remain possible ; hence , our results must be considered as preliminary until replication in additional series is obtained . with use of the bonferroni criteria to adjust for multiple comparisons , the association with pd of only 1 of the 2 snps in the promoter ( rs363371 ) remained significant ( corrected p value = 0.03 , dominant model ) . however , the bonferroni correction for multiple testing takes no account of ld between adjacent variants , thus leading to overcorrection and possible rejection of true - positive findings ( attia et al . , 2009 ) . in conclusion , the current study suggests that variability within the vmat2 promoter region may confer a reduced risk of developing pd . if confirmed and supported by further functional studies , genetic association of these variants with pd susceptibility might also support molecular targeting of the vmat2 gene or gene products as a therapeutic strategy for the disease . therapeutic transrectal or transcolonic drainages with endosonography ( ( endoscopic ultrasound ) eus ) are now considered as a mini - invasive option for the treatment of pelvic abscesses . the aim of our case series is to confirm that eus transrectal drainage is effective , safe , and induces a short duration of hospitalization . we did a single - center retrospective analysis of seven patients with pelvic abscess treated by eus transrectal drainage between january 2010 and august 2014 . there were four men and three women , with a median age of 50 years ( range 22 - 68 ) . a concomitant intravenous antibiotic treatment was always administered be for eus and for a minimum of 7 days after drainage . the method of drainage was done with a linear interventional echoendoscope ( pentax ) ( eg 3870utk pentax - hitachi , hambourg , germany ) . eus - doppler evaluation was first performed to exclude the presence of intercalated blood vessels before puncture with a 19-gauge access needle ( cook ) ( 19 g , echotip access needle , cook ireland ltd . , limerick , ireland ) . the puncture tract was then enlarged with a 10 french diathermic cystostomy over a tetrafluoroethylene ( tfe)-coated 0.035-inch guidewire ( cook endoscopy , winston - salem , nc , usa ) . a second guidewire was then put into the cavity , and two 7 french double pigtail plastic stents were positioned ( 4 or 7 cm length ) . eus drainages were done for a majority of abscesses post surgery ( n = 5/7 ) . the treatment was feasible and effective in 100% of cases [ figures 17 ] , without supplementary radiological or surgical intervention [ table 1 ] . the median time of hospitalization was 10 days ( range 4 - 25 days ) . the main difficulty related to this technique is the positioning of the double pigtail plastic stents , induced by pus outflow that can reduce the endoscopic visibility . two of our patients were embarrassed by the length of the double pigtail plastic stents which protruded in the anal canal . pelvic abscess before drainages pelvic abscess after drainage sagittal view of pigtail stent in pelvic abscess fluoroscopic view of first pigtail insertion endoscopic ultrasound ( eus ) view of douglas abscess eus view of abscess puncture endoscopic view with first guidewire insertion etiology and outcomes since 2003 , eus pelvic abscesses drainage is performed ; given the proximity between the rectal lumen and the abscess , with a mini - invasive technique compare to radiological or surgical drainage . moreover , percutaneous drainage may be difficult to realize and uncomfortable , especially if the collection is in deep location which needs a posterior drainage . our technique is safe , without adverse events observed ; and is actually well standardized , in analogy with 10 years of practice for eus drainage of pancreatic pseudocysts . the use of the fluoroscopy and cystostomy facilitates the procedure and can be easily used by experienced operators . the length of double pigtail plastic stents should be as short as possible , especially if drainage is done in the lower rectum , because it may cause discomfort if it touches the anal canal . we can also conceive that the length of hospital stay could be further reduced if the antibiotic treatment is continued orally in selected patients . eus transrectal drainage is a safe and an efficient method for therapeutical treatment of pelvic abscess . in our experience , we recommend eus transrectal drainage for pelvic abscess as the first - line technique for experienced hands .

generation of reactive oxygen species during dopamine ( da ) oxidation could be one of the factors leading to the selective loss of nigral dopaminergic neurons in parkinson s disease ( pd ) . vesicular monoamine transporter type 2 ( vmat2 ) proteins in nerve terminals uptake dopamine into synaptic vesicles , preventing its cytoplasmic accumulation and toxic damage to nigral neurons . polymorphisms in vmat2 gene and in its regulatory regions might therefore serve as genetic risk factors for pd . in the present study , we have analyzed 8 single - nucleotide polymorphisms ( snps ) located within / around the vmat2 gene for association with pd in an italian cohort composed of 704 pd patients and 678 healthy controls . among the 8 snps studied , only the 2 located within the promoter region ( rs363371 and rs363324 ) were significantly associated with pd . in the dominant model , odds ratios were 0.72 ( 95% confidence interval [ ci ] : 0.60.9 , p < 0.005 ) for rs363371 and 0.76 ( 95% ci : 0.60.9 , p = 0.01 ) for rs363324 ; in the additive model , odds ratios were 0.78 ( 95% ci : 0.650.94 , p = 0.008 ) for rs363371 and 0.85 ( 95% ci : 0.720.92 , p = 0.04 ) for rs363324 . there were no significant relationships between the remaining snps ( rs363333 , rs363399 , rs363387 , rs363343 , rs4752045 , and rs363236 ) and the risk of sporadic pd in any genetic model . this study adds to the previous evidence suggesting that variability in vmat2 promoter region may confer a reduced risk of developing pd , presumably via mechanisms of gene overexpression . background and objective : pelvic abscesses are a well - known complication of intestinal diseases or abdominal surgery . we report our case series concerning transrectal drainage by endoscopic ultrasound ( eus).methods : between january 2010 and august 2014 , seven patients received transrectal drainage by endoscopic ultrasound ( eus ) were selected and analyzed.results:two pigtails was positioned under fluoroscopic and eus control . the success rate was 100% and complication rate was 0% . the median time of hospitalization was 10 days [ range 4 - 25].conclusions : the technique appears to be safe and feasible in all etiologies . in our experience , we can considerate transrectal drainage by eus like a first - line technique in experienced hands .

the reovirus 2 protein catalyzes mrna capping , that is , addition of a guanosine to the 5 ' end of each transcript in a 5'-to-5 ' orientation , as well as transfer of a methyl group from s - adenosyl - l - methionine ( adomet ) to the n7 atom of the added guanosyl moiety and subsequently to the ribose 2'-o atom of the first template - encoded nucleotide . the structure of the human reovirus core has been solved at 3.6 resolution , revealing a series of domains that include a putative guanylyltransferase domain and two putative methyltransferase ( mtase ) domains . it has been suggested that the order of domains in the 2 protein corresponds to the order of reactions in the pathway and that the mg ( cap 0 ) and the 2'-o - ribose ( cap 1 ) mtase activities may be exerted by the mtase 1 and the mtase 2 domains , respectively . we show that the reovirus mtase 1 domain shares a putative active site with the structurally characterized 2'-o - ribose mtases , including vaccinia virus cap 1 mtase , whereas the mtase 2 domain is structurally similar to glycine n - mtase . on the basis of our analysis of the structural details we propose that the previously suggested functional assignments of the mtase 1 and mtase 2 domains should be swapped . methylated 5'-terminal cap structures have been described in most eukaryotic and many viral mrnas . in all cap structures , including the ' minimal ' cap 0 ( mg(5')ppp(5')n ) , an n7-methylguanosine ( mg ) is attached through a 5'-5 ' triphosphate bridge to the penultimate nucleoside . cap 0 is usually synthesized in the nucleus by the sequential action of three enzymatic activities : mrna triphosphatase , guanylyltransferase ( gtase ) , and mg - methyltransferase ( mtase ) ; this pathway was defined originally during studies of vaccinia and reovirus mrna synthesis . in some molecules , additional 2'-o - ribose methylations are found at the penultimate and the antepenultimate nucleosides , forming the cap 1 ( mg(5')ppp(5')nm2'o ) and cap 2 ( mg(5')ppp(5')nm2'opnm2'o ) structures , respectively ( reviewed in ) . the capping apparatus differs significantly in fungi , metazoans , protozoa and viruses in respect of the evolutionary origin and structure of individual domains , and the arrangement of domains within subunits . hence , the capping enzymes encoded by viral , fungal and protozoal pathogens are attractive targets for specific inhibitors that would exert a limited effect on the host enzyme . reoviruses are non - enveloped icosahedrally symmetric viruses with two concentric protein capsids surrounding and protecting the multisegmented , double - stranded ( ds ) rna genome ; they replicate in the cytoplasm of the eukaryotic host cell ( reviewed in ) the reovirus core particle can produce mgpppgm2'opc(pn)n - oh plus - strand rna from each genomic double - stranded rna segment in vitro , indicating that it contains all of the enzymes necessary for de novo synthesis of capped mrna . on the basis of sequence analysis and the results of uv - crosslinking and site - directed mutagenesis it has been suggested that the 144-kda 2 protein possesses an s - adenosyl - l - methionine ( adomet)-dependent methyltransferase ( mtase ) domain ( or domains ) , which mediates both the cap 0 and cap 1 methylation activities in forming a 5 ' cap structure on reovirus mrna . nevertheless , the failure of isolated 2 to exhibit methylation activity limited studies to dissect its putative methyltransferase activities and domain structure at a molecular level . the structure of the reovirus core has been recently solved at 3.6 resolution by reinisch et al . ( entry 1ej6 in protein data bank ) , revealing a series of seven domains in the 2 monomer , of which one domain ( amino acids 1385 ) was identified as a guanylyltransferase and two domains ( 434691 and 8041,022 ) were found to share the three - dimensional fold with typical adomet - dependent mtases ( reviewed in ) . in the turret formed by 2 pentamers , the gtase site from one monomer is closer to the mtase 1 in the clockwise neighbor than to other mtase domains . hypothesized that a correlation may exist between the spatial arrangement of domains in 2 , and the order of reactions in a pathway ( guanylyl transfer n7 methylation 2'o methylation ) . thus , it has been proposed that mtase 1 is the cap 0 mtase and mtase 2 is the cap 1 mtase . however , no groove to guide the mrna from one active site to another could be identified and the problem of specificities of reactions carried out by the individual mtase domains was left open . in the course of a large - scale analysis of mtase sequences and structures we observed low similarity between various ribose 2'-o - mtases from eubacteria and negative - strand rna viruses and a fragment of the grass carp reovirus protein corresponding to the carboxy - terminal part of mtase 1 of human reovirus ( j.m.b . and l.r . disappointingly , sequence database searches carried out using psi - blast initiated with the 2 sequence and its fragments revealed significant similarities only between the human reovirus and grass carp reovirus proteins . nonetheless , vast searches of the protein data bank revealed high similarity of both putative mtase domains to numerous known mtase structures . detailed inspection of the coordinates superimposed using swiss - pdb viewer allowed us to predict which amino - acid residues might be involved in cap 0 and cap 1 methylation of reovirus mrna . according to vast , mtase 1 showed highest similarity to the rrmj ( 1ejo ; p - value = 10 ) and vp39 ( 1av6 ; p - value = 10 ) proteins . the mutual similarity of mtase 1 and mtase 2 , which can be regarded as a reference , was evaluated as high , albeit significantly lower ( p - value = 10 ) . rrmj and vp39 are both 2'-o - ribose mtases ; rrmj targets u2552 in eubacterial rrna , whereas vp39 is a bonafide cap 1 mtase of vaccinia virus mrna . remarkably , simultaneous superposition of the three structures revealed perfect conservation of the k - d - k - e tetrad of putative catalytic residues ( figures 1,2,3 ) , which has been observed by us in other families of genuine and putative 2'-o - mtases ( and j.m.b . and l.r . , unpublished data ) . these results strongly argue that mtase 1 is more likely to be a cap 1 mtase than a cap 0 mtase . in the mtase 1 structure we could identify no aromatic residues that would superimpose well with y22 and f180 from vp39 , which form enhanced stacking interactions with the n7-methylated cap . moreover , a region corresponding to the guanine - binding pocket in vp39 is blocked off by a differently positioned loop in the reovirus 2 protein structure . nevertheless , we identified a cluster of aromatic side chains ( y460/464 , f461/465 and f618/f622 ) that are invariant between the human and grass carp reovirus sequences and map in the vicinity of the methylated cap , if the mrna coordinates are copied from the superimposed vp39 structure ( figure 2 ) . it is tempting to speculate that a conformational change in this region occurring on mrna binding would , for instance , reallocate y460 and f618 residues of human reovirus to positions equivalent to y22 and f180 from vp39 . the sequence alignment ( figure 3 ) has been manually adjusted to illustrate this hypothesis . the mtase 2 structure showed highest similarity to the glycine n - mtase ( gnmt ; 1xva ; p - value = 10 ) . it is noteworthy that the mtase 2 domain showed lower similarity ( p - value = 10 ) to vp39 than to gnmt and the mtase 1 domain showed lower similarity ( p - value = 10 ) to gnmt than to the 2'-o - ribose mtases . figure 4 shows that in addition to the common catalytic domain , gnmt and mtase 2 share a topologically equivalent ' lid ' domain made of three antiparallel strands . in gnmt , the -lid domain forms a wall of a large ' molecular basket ' structure , which may accommodate a variety of small molecules , including adomet , tetrahydrofolate and polycyclic aromatic hydrocarbon molecules such as benzopyrene ( reviewed in ) . in mtase 2 , the strands forming the -lid domain are much shorter and the pocket is also smaller . however , a guanine moiety can be docked into that pocket in a way that the n7 nitrogen is presented for the methyl group donor ( data not shown ) . most of the amino acids that line up the putative guanine - binding pocket of mtase 2 ( for instance q925 , n927 , f951 , r956 , and e958 ) are conserved between the human and grass carp reovirus sequences ( figure 5 ) . to our knowledge , all structurally characterized mtases that modify bases in nucleic acids and do not employ covalent bond formation with the target use aromatic or aliphatic side chains to bind the base to be methylated and stabilize it in the active site . examples of such mtases , in which the structure of the active site was determined experimentally or predicted from sequence analysis , include enzymes generating n - methyladenine in dna and rna ( reviewed in ) , n - methylcytosine in dna , and n - guanine in rna . we hypothesize that the invariant f951 and l890 ( substituted by f897 in grass carp reovirus ) may be involved in van der waals interactions with an aromatic ring of guanine . but because of the low resolution of the original structure and lack of a precise docking model , the detailed contacts between the target and the enzyme could not be predicted . nevertheless , we believe that our model will be a useful guide for site - directed mutagenesis experiments that would allow to elucidate the role of individual side chains of the putative cap 0 mtase . the data presented suggest that mtase domains 1 and 2 of reovirus 2 protein function as cap 0 and cap 1 mtases , respectively ; that is , that the previous assignments should be swapped . on the basis of detailed comparison of the available mtase structures , we predicted the substrate - binding mode for both mtase domains and proposed which residues are likely to participate in catalysis we suggest that substitution of the predicted cap - binding or catalytic residues in the mtase 1 domain should result in accumulation of cap 0-methylated viral mrnas that nevertheless lack cap 1 modification . we also report for the first time a remarkable structural similarity between the predicted mg mtase domain and gnmt , suggesting a common evolutionary origin for the two n - mtase families . it is noteworthy that threading analysis and subsequent homology modeling suggest a similar structure for a family of cellular and poxviral cap 0 mtases that show no significant sequence similarity to predicted cap 0 mtases from the sindbis - like supergroup of positive - strand rna viruses nor to the predicted cap 0 mtase from reoviruses analyzed herein ( j.m.b . and l.r . on the other hand , structure prediction for the family of aminoglycoside - resistance 16s rrna : mg mtases suggests that it shares only a common catalytic domain , but not the -lid domain or predicted target - binding residues , with other mg mtases . it will be interesting to determine how many times the mechanism of guanine - n methy - lation appeared in the evolution of adomet - dependent mtases and what is the phylogenetic origin of the individual viral cap 0 mtase families . this work was supported by kbn ( grant 8t11f01019 to j.m.b . ) and bioinfobank . we would like to thank karen reinisch for comments and suggestions that helped to improve the manuscript . stereoview ribbon diagrams of superimposed structures of the mtase 1 domain ( green ) , vp39 ( blue ) and rrmj ( magenta ) . mtase 1 shows elaborations of the common fold similar to those of vp39 , which are altogether absent from the rrmj structure and which are predicted to take part in cap recognition and binding . adomet ( copied from the rrmj structure ) and the k - d - k - e tetrad of putative catalytic residues are shown in wireframe representation . sequence alignment of the predicted human reovirus cap 1 mtase with its counterpart from grass carp reovirus ( gcrv ) and vaccinia virus ( vp39 ) . residues that are invariant between the reoviruses and those predicted to participate in binding of the adomet , the cap moiety , and in catalysis in all cap 1 mtases , are highlighted in black ; other conserved residues are highlighted in gray . stereoview of superimposed structures of the mtase 1 domain ( ribbon in green , side chains and labels in red ) and vp39 ( ribbon and labels in blue , side chains in cyan ) , delineating the proposed binding sites for the guanine moiety and the target ribose 2'-oh . capped mrna is shown in orange and the methylated guanine ( black label ) is sandwiched between y22 and f180 of vp39 . a conformational change of both the protein and the rna is required for the cluster of conserved aromatic residues of the reovirus mtase to bind the cap structure in a similar manner . stereoview of superimposed structures of the mtase 2 domain ( green ) and gnmt ( blue ) showing extensive similarities of both domains . helicobacter pylori is the most common chronic bacterial infection in humans , affecting 50% of the world population . chronic colonization increases the risk of duodenal ulcer and gastric cancer , including adenocarcinoma and mucosa associated lymphoid tissue ( malt ) lymphoma . the relative risk of gastric carcinoma in infected persons is 2.3 to 8.3 times greater than that in normal population . according to the world health organization ( who ) classification , h. pylori has been classified as group i carcinogens . although family members are the most likely source of infection , the factors that make some people more susceptible to infection have not been determined . it has been suggested that infection with this organism starts in infancy , and most cases occur before the age of three . the mechanism of transmission of this organism is unknown , but the most likely route of transmission is fecal - oral or oral - oral . the prevalence of h. pylori infection is much higher in developing countries . in children , the risk factors for h. pylori infection include socioeconomic status , number of siblings , race / ethnicity , rural residence , institutional residence , lack of maternal education and infection status of family members . the protective effect of breast milk and its beneficial effects in preventing respiratory infections and diarrhea have long been known . several studies have suggested that breastfeeding can also prevent bacterial colonization of h. pylori during childhood . however , other studies had conflicting results and the importance of breastfeeding in the prevention of h. pylori is still under question . in fact , there are studies that have reported that breastfeeding may even increase the risk of h. pylori infection . breast milk may be infected by h. pylori and horizontal infection through breastfeeding may occur . we conducted the present study to evaluate the effects of breastfeeding on h. pylori infection in kurdish children in sanandaj , ir iran , by using the h. pylori stool antigen test ( hpsa ) . sanandaj is the center of kurdistan province , in west iran , with a population of about 450000 . a previous study has detected a high prevalence of h. pylori in children in this city . the study had a historical cohort design that was carried out from january 2011 through december 2012 in the pediatric department of besat tertiary hospital affiliated to kurdistan university of medical sciences . participants were selected from a list of healthy children in 12 primary healthcare centers across the city . we selected them from children who were going to attain 2 years old during the study period . the rationale for selection of these age groups was that the breastfeeding practice usually continues 2 years or even longer in our community . simple random sampling was used in breastfed children . however , in non - breastfed children , we had to enroll all of them due to paucity of cases . a computer based program generated random numbers in order to draw a selected group of children from the lists . the sample size for both breastfed and non - breastfed groups was calculated as at least 90 patients for each group . the sample size calculation was based on at least 20% prevalence in the community , 40% prevalence in studied children , 20% power , an odds ratio ( or ) of 2.5 , and 95% confidence interval ( ci ) , computed from previous epidemiological studies . all procedures and tests were carefully explained to the parents of children and informed consent was obtained . through interviews with the parents , the data regarding age , sex , parents education , duration of breastfeeding , family size , previous antibiotic usage , height and weight , were recorded for each child . breastfed child was defined as a child that has had at least 1 month duration of continuous breastfeeding . status of breastfeeding until the date of stool sampling ( at least age of 2 years or older ) was questioned and on the base of the breastfeeding status the children were divided into two groups , i.e. breastfed and non - breastfed . these two groups were compared in terms of the absolute breastfeeding ( no formula exclusively ) , those with mixed feeding with breast milk and formula for various durations of time and , those with no breastfeeding ( no breast milk exclusively ) . infants that have received antibiotics or proton pump inhibitors more than a week in the last 2 months were excluded from the study . a stool sample was collected from the enrolled children who attained at least 2 years old by parents with varying time after breastfeeding cessation . we used premier platinum hpsa plus ( meridian diagnostics , cincinnati , ohio , usa ) kits to detect infection in children . the stool assay kit was purchased from a local representative of meridian company , netherlands . it is cleared by the fda and ce - marked with high sensitivity and specificity in comparison to other stool antigen tests . the maastricht iv / florence consensus states that the diagnostic accuracy of the stool antigen test ( sat ) is equivalent to the urea breath test ( ubt ) if a validated laboratory - based monoclonal test is used . the sensitivity and specificity for the premier platinum hpsa plus test were reported as 92.2% and 94.4% , respectively , in a recent study . the test procedures are described in detail and available online ( http://www.meridianbioscience.com/diagnostic-products/h-pylori/premier/premier-platinum-hpsa-plus.aspx ) . according to the manufacturer s instructions , results are divided into two groups for h. pylori infection : negative ( the optical density at 450/630 nm < 0.100 ) and positive ( the optical density at 450/630 nm 0.100 ) . chicago , il , usa ) . by using descriptive statistics , frequency distribution tables and graphs were drawn and crude odds ors and 95% confidence interval ( ci ) were estimated . we calculated z scores for the indicators of the attained growth standards ( weight - for - age , height - for - age , weight - for - height ) based on who standards , using the respective spss macro ( syntax ) files . we used t test to compare z scores in infected and non - infected groups . a total of 221 children were selected and tested for h. pylori antigen in stool ( table 1 ) . a number of 130 children had a history of variable duration of breastfeeding time ( table 2 ) . age , gender and growth characteristics of studied children numbers in each gender group ; sd : standard deviation prevalence of h. pylori infection based on duration of breast milk feeding and gender a stool sample was tested for helicobacter infection after varying times of breast milk cessation . the age range of enrolled children at the time of stool was 2 to 4 years . we found no difference in the odds of infection between breastfed and non - breastfed groups ( or=0.809 , 95% ci [ 0.4531.444 ] ) . the studied children were divided into five groups as not - breastfed , 16 , 712 , 1318 , and 1824 months of breastfeeding . there was a marginal decrease in the odds of infection as the duration of breastfeeding increased ( p=0.07 ) . we compared the chance of acquiring infection for the two groups of children that lied at the two ends of the spectrum , the first group fed the longest time with breast milk ( 1824 months ) and the second group with no breastfeeding . there were no differences in the odds of infection of infection ( or=1.043 , 95% ci [ 0.5711.904 ] ) . there was no correlation between the prevalence of infection and either the type of feeding in the female gender group ( or=0.44 , 95% ci [ 0.181.056 ] ) nor the duration of breastfeeding among girls . similarly , there was no correlation between prevalence of infection and either the type of feeding in male gender group ( or=1.28 , 95%ci [ 0.5692.864 ] ) , nor the duration of breastfeeding among boys ( =8.16 , p=0.08 ) . when the frequency of h. pylori infection was calculated in various age groups ( table 3 ) , there was a statistically significant association ( =11.92 , p=0.008 ) . association of h. pylori infection with age , family size , home size , body weight , father education and mother education chi square test was used 7 ( 3% ) values for weight were missed 4 ( 2% ) values for weight were missed fisher s exact test was used p values assessing the association of infection with categorized z scores or : odds ratio ; ci : confidence interval ; hpsa : h. pylori stool antigen test the prevalence of infection marginally increased as the family size grew ( = 5.08 , p=0.07 ) . however , when we compared the family size in three member families with four and more member families , we found significant differences in odds of infection ( or=1.93 , 95%ci [ 1.043.6 ] ) ( table 3 ) . we corrected these numbers by dividing family size by home size , i.e. calculated housing density , and observed a significant increase in the odds of infection , as the density increased ( or=2.12 , 95% ci [ 1.104.10 ] ) . we calculated the z scores for weight - for - age , height - for - age , and weight - for - height for both non - breastfed and breastfed groups based on who standards and the prevalence of infection in respective groups ( table 3 ) . the mean z score for weight - for - age in infected and non - infected children was calculated as 0.071.45 and 0.541.16 , respectively , which was statistically a significant difference ( p=0.02 ) . the mean z score for height - for - age in infected and non - infected children was calculated as 0.141.73 and 0.42 1.85 , respectively , which was not statistically significant ( p=0.3 ) . the mean z score for weight - for - height in infected and non - infected children was calculated as 0.051.61 and 0.221.79 , respectively which similarly was not statistically significant ( p=0.5 ) . we were unable to confirm the protective association between breast milk feeding in prevention of h. pylori infection . we found higher odds of infection in the selected ages of the two groups , breastfed and non - breastfed . the prevalence of infection was higher in these age groups in comparison to previous studies . there are multiple research articles that have surveyed the effect of breast milk on infection by h. pylori . our study found no protective effect of breast milk against h. pylori infection . a systematic review by chak et al suggested that breastfeeding is protective against h. pylori infection . however , they reported that the studies conducted in developed countries did not achieve statistically significant enough or to conclude that breastfeeding has a protective effect against h. pylori infection . most of the studies with significant or were in the low and middle income countries . the prevalence of h. pylori infection in each community depends on two factors , the rate of acquisition and the rate of persistence of infection . in developing countries , most of the acquisition of this infection occurs in early childhood and continues as chronic infection to adult life . unhygienic behavior , especially during early childhood , frequent contacts with the sources of infection and frequency of other risk factors lead to persistence of infection . based on data from united nations secretariat , iran consequently , lack of protection by breast milk feeding against h. pylori in this country , may be more consistent with the results that have been obtained in developed countries . however , the high prevalence of h. pylori in many epidemiologic studies in iran is more compatible with the epidemiology of h. pylori in low income countries . the components of breast milk , such as antibodies , lactoferrin , lysosomes , and kappa - casein possess antibacterial and antiviral characteristics that protect infants against many infectious agents causing diarrhea and pneumonia . however , these types of immunity are mostly passive and are not long - lasting enough to protect the infant after cessation of breastfeeding . once breastfeeding has ended , it is likely that it is the better nutritional status and the well development fostered by breastfeeding that protect the child against infections , and not the direct effect of breastfeeding ( i.e. the immunity acquired passively by breastmilk ) . one assumption regarding the different results found in our study is that , in the low and middle income countries , malnutrition is very common . there is no large variation between nutritional status of infants fed with breast milk and formula fed infants as seen in the low income african countries such as gambia or egypt . unlike low income countries , many types of substitute feedings including various formulas for infants are readily available to even low income people in our country . as there are no significant differences in nutritional status of breastfed and non - breastfed children in our county , we may be unable to detect differences between the prevalence of helicobacter infection in these two groups . one study in gambian children reported a high prevalence of h. pylori infection in children with malnutrition or chronic diarrheal disease . in our study , the mean z scores for body weight in non - breastfed children in non - infected and infected groups was calculated as 0.00 and 0.29 , respectively ( p=0.4 ) . in other words , there was no association between the infection odds and lower z scores for body weight in non - breastfed children . however , in breastfed - children , the mean z scores for body weight in non - infected and infected groups were calculated as 0.12 and 0.69 , respectively ( p=0.02 ) . there was a significant correlation between odds of infection and lower z scores in breast milk fed children . in the absence of correlation of the other malnutrition indicators ( height - for - age and weight - for - height ) with the rate of infection , it seems that , in iran , the infection is more associated with a mild type of acute malnutrition . with other words , in the context of h. pylori infection , maternal infection with h. pylori plays a major role in the acquisition of this infection in the early years of life . long term and close contact with mother , despite presumptive passive protection by breast milk feeding , involve frequent exposure to infectious sources of h. pylori . non - breastfed infants have less contact with their mothers . in iran , on the other hand , many mothers avoiding breast milk feeding because of their employment are from high socioeconomic graduated class . these factors may explain the mechanism by which the infection rates increase in breast milk fed infants . moreover , breast milk feeding causes a decrease of other infectious diseases and less need for antibacterial drugs . less exposure to antibacterial drugs results in the persistence of h. pylori in the stomach , which is usually primarily eradicated by accidental administration of antibacterial drugs for respiratory or diarrheal diseases . high infection odds due to high carriage rate may be a reason for the lack of protective effect of breast milk feeding in our study . there may be a relative protective effect for breastfeeding that is overcasted by the high burden of h. pylori infection . in most of the studies that reported a protective effect from breast milk infection , the prevalence of infection was notably below 4050% of the study population in lower age groups . there is a general agreement that the protective role of breastfeeding against h. pylori infection is passive , and therefore is not long lasting . reinfection is possible after waning of the protective effect of maternal antibodies occurring with the cessation of breast milk feeding . in our study , the mean age of the studied children was 39 months and the mean interval between sampling time and the cessation of breastfeeding was calculated as 13 months . considering the high carriage rate in our community , this interval may be long enough for waning immunity , reinfection and surpassing the suspicious protective effect of breastfeeding . the prevalence of exclusive breastfeeding and the mean duration of breastfeeding in iran are above 75% and 23 months duration , respectively . the prevalence of continuing breastfeeding for a minimum of 1215 months is of 84% in iran . on the other hand , the prevalence of h. pylori infection in iran has consistently been reported as well above 50% in many studies conducted in this country . consideration of these facts challenges largely against the protective effects of breastfeeding in our community . lengthening breastfeeding duration to multiple months in the early years of age had no effect on the acquisition of infection . however , this is a contradiction in articles advocating for the protective effects of breastfeeding against h. pylori infection . if one assumes any protective effect for breast milk against h. pylori infection , it should be a dose - response relationship . moreover , in many of these articles , we found no case definition for breastfeeding based on duration . in one study , a prolonged time of breastfeeding was associated with an increased chance of acquiring infection . the majority of the studies regarding the protective effects of breast milk against h. pylori infection have used laboratory tests , other than stool antigen test , as diagnostic tools . this test has low sensitivity and specificity in comparison to the stool antigen test . these variables may cause confusing results in the study of the protective effect of breast milk against infection . it is a worthwhile way with high sensitivity and specificity , comparable to hpsa test . however due to the high cost , limited availability in developing countries and the complexity of the test , it is less applicable on large scale in healthy children . a recent meta - analysis discussed that the use of 13c labeled isotope ubt ( 13c - ubt ) tests are less accurate for the diagnosis of h. pylori infection in young children . our search for the epidemiologic studies that used hpsa as a diagnostic tool to evaluate effects of breast milk feeding have revealed only limited results . we were not able to identify the time of acquisition of infection and its relationship to breastfeeding to unveil any short term protection . another prospective cohort study may help to solve this problem and clarify the effects of breast milk feeding on h. pylori infection . the presented data suggest that breastfeeding in infancy does not protect against h. pylori infection for long duration among studied children in iran . the protective effect of breastfeeding , if any , is at most a transient one . j. soltani , b. nikkhoo , j. khormehr , p. ataee , f. gharibi : substantial contributions to conception and design , acquisition of data , analysis and interpretation of data . all authors participated in drafting the article and they critically reviewed the manuscript and approved the final manuscript as submitted .

backgroundthe reovirus 2 protein catalyzes mrna capping , that is , addition of a guanosine to the 5 ' end of each transcript in a 5'-to-5 ' orientation , as well as transfer of a methyl group from s - adenosyl - l - methionine ( adomet ) to the n7 atom of the added guanosyl moiety and subsequently to the ribose 2'-o atom of the first template - encoded nucleotide . the structure of the human reovirus core has been solved at 3.6 resolution , revealing a series of domains that include a putative guanylyltransferase domain and two putative methyltransferase ( mtase ) domains . it has been suggested that the order of domains in the 2 protein corresponds to the order of reactions in the pathway and that the m7 g ( cap 0 ) and the 2'-o - ribose ( cap 1 ) mtase activities may be exerted by the mtase 1 and the mtase 2 domains , respectively.resultswe show that the reovirus mtase 1 domain shares a putative active site with the structurally characterized 2'-o - ribose mtases , including vaccinia virus cap 1 mtase , whereas the mtase 2 domain is structurally similar to glycine n-mtase.conclusionson the basis of our analysis of the structural details we propose that the previously suggested functional assignments of the mtase 1 and mtase 2 domains should be swapped . objective : helicobacter pylori ( h. pylori ) is the most common chronic bacterial infection in humans . chronic colonization increases the risk of duodenal ulcer and gastric cancer . the risk factors for acquiring the infection have been extensively studied . however , there are conflicting results on the role of breastfeeding in the prevention of h. pylori infection . we conducted a study to evaluate the effects of breastfeeding on the h. pylori infection in kurdish children in sanandaj , ir iran.methods:a historical cohort study was carried out from january 2011 through december 2012 . totally 221 children who were going to attain 2 years old during the study period were randomly enrolled . they were divided into two groups , i.e. breastfed and non - breastfed . we used h. pylori stool antigen test to detect infection in the selected group of children after age of 2 years and cessation of breastfeeding . each group was subdivided into two subgroups , infected and non - infected . the associations of breastfeeding with h. pylori infection was assessed using statistical software.findings:we found no difference in the odds of infection between breastfed and non - breastfed groups ( or=0.809 , 95% ci [ 0.4531.444 ] ) . an association between age and the prevalence of infection was found ( p=0.008 ) . there was an increase in the odds of infection as the family size grew ( or=1.93 , 95% ci [ 1.043.6 ] ) as well as increasing housing density ( or=2.12 , 95% ci [ 1.104.10]).conclusion : the data suggests that breastfeeding in infancy does not protect against h. pylori infection for long duration among studied children in iran . the protective effects of breastfeeding , if any , are at most transient .

chronic sialadenitis is one of the major disorders that can cause salivary hypofunction and correct diagnosis and management is essential for its recovery . the classification of this pathological condition has changed in the past decade and nowadays was revised and modified , for new diagnostic ( high - resolution ultrasonography , ct and mr sialography and sonoelastography ) and therapeutic methods ( sialendoscopy ) were introduced . sialolithiasis is the most common cause of inflammatory diseases of large salivary glands and occurs in about 1.2 % of the population [ 9 , 21 ] mostly in the submandibular gland87 % . salivary gland stones are single or multiple , located in the efferent duct distally or proximally , rarely occur intraparenchymally , representing various shapes and sizes . the annual increase in size of salivary stones is estimated at 1 mm , and thus the duration of complaints history is crucial for treatment planning . contemporary achievement in endoscopy caused strong common belief that stones of up to 45 mm in diameter can be successfully removed through sialendoscopy ( se ) . this applies especially to stones which lie freely in the lumen of the duct and are mobile . in these cases , the stones can be extracted under endoscopic control in more than 80 % of cases [ 8 , 9 , 14 ] . larger sialoliths may , however , be fragmented in the lumen of the duct , either mechanically or using a laser beam . lithotripsy ( eswl ) is another possibility for the fragmentation of large sialoliths of any size and location ; although up to three sessions of lithotripsy may be required . thus , the introduction of sialendoscopy has significantly reduced the number of submandibular glands removal in the course of sialolithiasis [ 5 , 6 , 8 , 9 , 14 , 17 ] . according to literature data , the use of lithotripsy is effective in 75 % of cases , and in turn , allows for the complete retrieval of stones in half of the cases [ 2 , 3 , 6 , 7 , 22 ] . the number of successes in the use of lithotripsy clearly decreases with increase in the stone diameter . despite notable technological progress , 510 % of patients with sialolithiasis can not be successfully treated using minimally invasive techniques . the main cause appears to be the large size of the stones and long - standing history of recurrent inflammations , which lead to the impaction of the sialolith to the wall of the efferent duct . in these cases , the aim of the study was to analyze the trends in the treatment methods in submandibular sialolithiasis in the past decade , the effectiveness of particular methods and to present treatment schedule proposed by the authors . 112 patients with submandibular gland sialadenitis were treated in tertiary university centre ( otolaryngology , head and neck surgery department pozna medical university ) in the years 20042012 . data were analyzed retrospectively on the basis of medical documentation ( outpatient charts , operating protocols ) . the epidemiological data ( gender , age ) , duration of complaints , the treatment method and its effectiveness were analyzed and compared in both groups . in preoperative diagnosis , additionally , in the recent 3 years , ct was performed in 11 cases . during first period , patients were treated in outpatient department by incision of mucosa of floor of the mouth in local anesthesia . following the introduction of sialendoscopy , patients were admitted to the hospital for 1 day . during interventional sialendoscopy , 1.3 and 1.6 mm diameter endoscope ( karl storz tutlingen , germany ) was used . stones were removed with the help of the basket and forceps , introduced through the working canal . the se procedure was carried out under local anesthesia after premedication with ( midazolam , 7.5 mg ) . once the size of the stone localized in submandibular hilum was determined to be larger than 67 mm and endoscopic removal was deemed impossible , the decision of combined approach was made . the combined approach ( incision of the floor of the mouth at the level of submandibular hilum and sialendoscopy ) was performed also under local anesthesia . in case of failure of this treatment and when stone was primarily localized in gland parenchyma , the decision of eswl or total removal of the gland was undertaken . the open surgery was performed under general anesthesia with the help of facial nerve monitoring , with leads from area of marginal branch around the mouth . although the paper had a predominantly descriptive character , some statistical analysis was performed using spearman and kruskal wallis tests . there were 48 patients with submandibular sialolithiasis : 31 outpatient duct incisions with stone evacuation and 17 surgical excision of submandibular gland in the first 5 year period of time . there were 21 men and 27 women , aged from 20 to 73 , mean 43 . the length of complaints ranged from 3 months to 20 years , mean being 3.4 years . the number of treated patient ( in whom the removal of submandibular gland was undertaken ) in those years was as follows : 20041 patient , 20052 , 20063 , 20074 , 20082 , 20094 , 20100 , 20111 . in the second , 3.5-year period wharton duct stenosis was diagnosed in 33 patients ; 64 out of 97 patients with submandibular sialadenitis suffered from lithiasis . there were 38 men and 26 women , aged from 14 to 68 , mean 45 . the length of complaints ranged from 2 months to 17 years , median 2.9 years . endoscopic removal of stones was possible in 40 cases ( 62.5 % ) . in 37 patients , double approach ( incision of the floor of the mouth in hilar area and sialendoscopy ) in patients with refracted , big stones and long history of lithiasis was performed successfully in 21 out of 64 ( 32.8 % ) patients . surgical excision of the submandibular gland was performed in 3 out of 64 ( 4.7 % ) patients between 2009 and 12 . follow - up in this group of patients ranged from 48 to 6 months , mean being 19.6 months . comorbidities and patients age had no correlation with sialendosoppy stone removal rates ; these were , however , found to be statistically dependent from the duration of complaints and a history of more than 5 years doubled the risk of failure ( p < 0.005 ) . various methods of treatment through two distinct time intervals are depicted in table 1.table 1various methods of treatment throughout two time intervalssubmandibular gland calculiyears 20042008years 20082012removal of calculi through incision of mucous membrane of floor of the mouth in hilar area310sialoendoscopy064endoscopic removal of calculi040 ( 62.5 % ) double approach021 ( 32.8 % ) surgical removal of submandibular gland173total4864 various methods of treatment throughout two time intervals according to bigler , harrison , yoel , work , wang et al . , there are two particular subdivisions : chronic obstructive ( sialolithiasis , stenosis of the duct , inflammation of the glandular tissue with recurrent stenosis or enlargement of the duct ) and non - obstructive group of inflammations this agonizing experience may even occur without any component of mechanical obstruction , although presence of lithiasis is the main cause in 50 % of affected individuals . high - resolution ultrasonography could be utilized as an optional diagnostic method for visualization of calcified deposits or exclusion of tumor presence . the main feature of sialography is excision , constriction or enlargement of the excretory ducts . ultrasonographic confirmation of either ductal stricture or presence of inraparenchymal stones allows postponing the sialoendoscopy procedure until the acute state has subsided [ 6 , 8 , 13 ] . high - resolution computer tomography is still considered to be the most sensitive method for the determination of stones , whereas the ultrasonographic technique allows to view a sialolith > 2 mm . increased accumulation of inorganic calcified deposits within the center of the gland gives off a strong echo signal ; therefore , it is not uncommon to overlook miniscule remnants of the stones due to insufficient signal saturation . however , it is important to emphasize that false positive results could be obtained in case of excessive hyperemia caused by inflammation of the duct . in these particular situations , classic sialography , sialography performed by utilization of computer tomography or by magnetic resonance imaging , is an instrumental addition to the diagnostic evaluation ; however , many authors prefer high - resolution ultrasonography . the use of endoscopic and minimally invasive techniques allows for the greater preservation of the major salivary glands in cases of sialolithiasis . according to literature data , 8090 % of patients with parotid gland sialolithiasis can be treated using minimally invasive techniques such as sialendoscopy and eswl [ 5 , 6 , 8 , 9 , 14 , 15 , 17 , 19 , 27 ] . it should be remembered that stones larger than 6 mm in diameter and impacted in the wall of the duct limit the possibility of using sialendoscopy [ 8 , 9 , 14 , 15 , 19 ] . after performing eswl , larger stones ( larger than 810 mm in diameter ) our initial , 5-year emergency department data indicate that 48 patients who were admitted with the symptoms of sialolithiasis . in 31 patients , stones were removed through an incision in the mucous membrane . due to complications and persistent symptoms , the remaining 17 patients had their submandibular glands removed . following the introduction of sialendoscopy procedure to our department , 3.5 years worth of clinical data show 64 patients admitted with symptoms indicating submandibular sialolithiasis . double approach ( incision of the floor of the mouth in hilar area and sialendoscopy ) with the successful removal of stones was carried out in 21 out of 64 ( 32.8 % ) patients . three patients , however , had their submandibular glands totally removed due to several complications . it is valuable to include a diagnostic and therapeutic procedure developed by koch ( fig . 1 ) . sialendoscopy is considered as a significant diagnostic and therapeutic method of primary treatment . according to our data , 48 patients treated throughout the 5 year time interval without utilization of sialendoscopy , when compared with those 64 patients who were treated with sialoendoscopy procedure , showed mediocre outcomes . it is , therefore , imperative to point out that the introduction of sialendoscopy and superior results obtained throughout its treatment of chronic inflammation of glands affected by sialolithiasis allowed us to gradually adapt to the published literature [ 6 , 11 , 13 , 17 , 19 , 20 ] . in situation in which there is a limited access to eswl , a double approach could be used as an alternative method of treatment in both parotid and submandibular glands [ 10 , 13 , 16 , 18 , 23].fig . 1algorithm of salivary gland obstructive pathology treatment , according to koch et al algorithm of salivary gland obstructive pathology treatment , according to koch et al the most conservative method of treatment , as well as the one that provides us with the least amount of unfavorable outcomes , is the main objective for those affected by a chronic inflammatory process . according to the literature and our current data , in the vast majority of cases , surgical intervention could be replaced with a minimally invasive procedure by utilization of diagnostic and therapeutic sialendoscopy procedures [ 6 , 11 , 13 , 19 ] . according to data obtained at our center , the development of sialendoscopy , however , allowed us to treat our patients with a minimally invasive procedures . similarly , in the year 2004 through 2008 , 17 surgical removals of submandibular glands were performed due to lithiasis or advanced inflammatory states ; however , in the year 20092012 , only three were performed . with the advent of new diagnostic and therapeutic methods , it is imperative to verify current classification of chronic inflammation of large salivary glands . critical analysis of literature reviews and our own data indicate that continuous improvement of current methods and introduction of new ones , such as utilization of sialendoscopy are crucial in treatment of pathological obstructions of salivary glands . indication for a complete removal of the gland is becoming uncommon as a first line treatment although still indispensable in chosen cases . diabetes mellitus ( dm ) is one of the most common chronic metabolic diseases , which is on the rise globally.123 ) the dm prevalence for all age groups worldwide was estimated to be 2.8% in 2000 and projected to be 4.4% by 2030 . during this period , the total number of people with dm is expected to rise from 171 million to 366 million.4 ) several explanations have been provided for the increase in dm - prevalence , such as population growth , aging , urbanization , increasing obesity prevalence , and physical inactivity.5 ) diabetes poses a challenge not only to those living with the disease,6 ) but also to their families , communities , and the health care system.789 ) monitoring the dm prevalence and incidence trend is important for assessing the national burden of the disease to describe the impact of risk factors , develop interventions , and project needs for future health services.410 ) the states in the co - operation council for the arab states of the gulf ( gcc ) have some of the highest rates of type 2 dm in the world . five of the international diabetes federation 's top ten countries for dm prevalence in 2011 and projected prevalence in 2030 are in this region.11 ) the recent and rapid socioeconomic development of the gcc countries has been associated with this rising prevalence.12 ) according to the international diabetes federation , the number of people with dm in its middle east - north africa region will increase to 83% by 2030.11 ) in the united arab emirates ( uae ) , the dm prevalence has reached 24% and 17.4% among citizens and expatriates,13 ) respectively . the dm prevalence in the uae was ranked to be the 2nd highest in the world ( 19.5% ) in 2007 , and the expected prevalence for 2025 is 21.9%.12 ) the uae has developed a national diabetes care continuum program with guidelines to combat this disease.13 ) based on a population study conducted in al ain , in the uae , the age - standardized prevalence rates for dm ( diagnosed and undiagnosed ) and pre - diabetes among people aged 30 - 64 years were 29.0% and 24.2% , respectively.14 ) no published data are available about the age - standardized incidence rates of dm in the uae . such data are important , because information on incidence will help plan specific preventive and control strategies , and will enhance our knowledge about the profile of this disease in the uae . therefore , this study was conducted to determine the age and gender specific incidence rate of dm among nationals in ajman medical district in 2010 . the total population of ajman , uae as per 2005 census was 206,997 ( men , 131,684 ; women , 75,313 ) . among this , 39,231 were emiratis ( men , 20,155 ; women , 19,076 ) and the remaining were other nationals ( 11,529 males and 56,237 females ) . the age and gender distribution of the ajman population ( estimated for 2010 using the geometric growth method ) is shown in table 1 . health care services for the target population were mainly provided through 5 primary healthcare centers and 2 general hospitals ( sheikh khalifa hospital and gmc hospital , ajman ) in ajman during the study period , and most of the diabetic patients were presumed to be managed at these sites . the data were collected by using a proforma that was prepared by the team of investigators . every copy of the research proforma was allocated a code number to identify the primary health care center . the pro forma consisted of the domains , such as socio - demographic characteristics , clinical data , biochemical parameters , and mode of management . the emirates or health insurance identification number of the nationals and expatriates were recorded to eliminate duplication . approval from the ethics and research committees of gulf medical university and ministry of health was obtained before commencing this study , and a prior approval was sought from the director of ajman medical district to conduct the study at primary health care centers ( phc ) . participants were not identified by their names , and any information that could reveal the identity of the subjects was not recorded on the proforma . furthermore , confidentiality of the research data was maintained and anonymity was preserved . confidentiality was maintained by maintaining all research documents at the statistical support facility for 3 years , and only the researchers , ethics and research committee members , and statistical support facility members had access to the data with appropriate regulatory sight . any information that can be used to reveal the identity of a participant will not be included in any publications or presentations . cases of dm were documented retrospectively by studying the medical records from january 1 , 2010 to december 31 , 2010 . the cases diagnosed for the first time during the study period were included as incident cases . the following diagnostic criteria were used : ( 1 ) diabetes was defined as a fasting plasma glucose ( fpg ) concentration of 126 mg / dl or previous first diagnosis of dm within the same time period . impaired fasting glucose and normoglycemia were defined as a fpg concentration of 110 - 126 mg / dl and < 110 mg / dl , respectively;13 ) ( 2 ) when criterion ( 1 ) was not met , we relied on the notes written by doctors elsewhere given that the first diagnosis by the other physician was within the same time period . 2010 spreadsheet were transformed to ibm spss ver . 20.0 ( ibm co. , armonk , ny , usa ) for statistical analysis . the incidence rate was presented for different age groups ( i.e. , 5-year intervals ) , and confidence intervals for the proportions were estimated . the crude incidence rates were calculated by considering the population of ajman in 2010 and newly diagnosed dm cases . the dm cases and total population were arranged into different age groups with 5-year intervals to separately calculate age specific dm incidence rates for men and women . a total of 158 new dm cases were diagnosed during 2010 ; 102 of these cases were emiratis residents and the remaining cases were other nationalities . among the 102 emiratis cases , there was only 1 type 1 dm case , and the remaining 101 type 2 dm cases were included in the estimation of incidence rate . the overall incidence rate of dm cases observed among those aged 20 years was 4.8/1,000 person - years ( py ) . in the present study , 35 patients ( 34.7% ) were men and 66 patients ( 65.3% ) were women , indicating female predominance . the gender specific incidence rate was 3.3/1,000 py for men and 6.3/1,000 py for women . the incidence rates in men and women were almost similar until the age of 39 years . however , women who were 40 years showed a higher incidence rate than men . the highest incidence rate for both genders ( men , 23.4/1,000 py ; women , 32.4/1,000 py ) was observed in the 55 - 59 years age group , and then a downward trend was observed . in this study , we evaluated the incidence of dm among emirati residents because they constitute a more stable population than expatriates . future studies involving this population will reflect changes in the epidemiology of this problem that can be linked to lifestyle , socioeconomic , or health service factors . according to the present study , a similar type 2 dm incidence were observed both among emirati men and women in ajman , uae in the age group of 20 - 39 years , and a female preponderance in the incidence rate of dm in the later age groups . the incidence of dm was 4.4/1,000 py in 2005 according to a study from the united kingdom ( uk).15 ) the crude incidence rate of type 2 dm was 515/100,000 ( or 5.15/1,000 py ) in 2010 based on more recent data from the uk.16 ) according to this study , the incidence of dm increased with age , and was similar among men and women up to 40 years of age . after 40 years of age , men had a higher incidence rate of dm than women . the gender specific incidence rate for men and women was 4.86/1,000 py and 4.31/1,000 py , respectively.15 ) an overall dm incidence rate of 10/1,000 py with a male predominance was observed in a study from the united states of america ; using data from the new york city community health survey , the incidence rates were 11/1,000 py and 9/1,000 py in 2008 for men and women , respectively.17 ) the overall incidence of dm was 6.3/1,000 py in 2011 based on the report , " diabetes in canada , " and a male predominance was observed in the gender - specific rates ( 6.8/1,000 py in men compared to 5.7/1,000 py in women in the year 2009).18 ) a cohort study conducted over a period of 2000 - 2009 reported an overall incidence rate of dm in china was 9.5/1,000 py with a male predominance ( 11% men vs. 6.2% women).19 ) according to a previous study in the year 2000 - 2009 , the overall ( 20 - 79 years of age ) incidence rate of dm in taiwan was 11.6/1,000 py.20 ) in a report from sweden,21 ) the incidence rate ( 20 - 100 years of age ) in men and women were nearly similar ( 3.92/1,000 py and 3.64/1,000 py in men and women , respectively ) . the comparison of crude incidence rates in different countries is provided in figure 2 . an age - wise increasing trend in the age specific rates of dm however , only the 10-year age specific incidence rate was available , and the highest rate was observed in the 70 - 79 years age group ( 9.94/1,000 py and 7.77/1,000 py for men and women , respectively . the age specific incidence rates of dm were 0.20 , 0.54 , 2.37 , 4.68 , and 7.46/1,000 py for men and 0.29 , 0.70 , 1.71 , 3.57 , and 6.51/1,000 py for women in the 20 - 29 , 30 - 39 , 40 - 49 , 50 - 59 , and 60 - 69 years age groups , respectively.21 ) the age specific incidence rate of dm in canada was comparable with the present findings ( figures 3 , 4 ) . the rate of dm was almost similar at the age of 40 years in the present study . the peak incidence rate of dm was observed in the 55 - 59 years age group and then a downward trend in the incidence rate of dm was observed . in canada , a steady increase in the incidence rate of dm was observed with increasing age , and the incidence rate in women was much higher in ajman than in canada . a variation in the dm epidemiological pattern was observed in the available data in developing compared to developed countries . according to the present study , dm appears to affect mostly younger participants ( range , 44 - 64 years ) , while in developing countries , the majority of diabetic patients were > 64 years of age.4 ) the higher incidence rate of dm observed among women compared to men after the age of 40 years in this study was incompatible with data from the uk . a further study is needed to investigate the possible related factors ( i.e. , hormonal , dietary , lifestyle , and social factors ) that can restrict the physical activity of older women . both men and women have a nearly similar global prevalence of dm with slightly higher rates in men < 60 years of age and women at older age , greater than 60 years.4 ) it is worth noting that , according to the international diabetes federation,7 ) the estimated percentage of undiagnosed diabetic patients in middle - eastern high - income countries ( i.e. , uae ) was 40.6% in the year 2011 . therefore , the actual incidence of dm is about 40% higher than what was observed in this study . an important finding in this study was that the incidence of dm started to increase after the age of 30 years , which implies that earlier screening , rather than the currently recommended screening at 45 years of age for people without risk factors by the american diabetic association,22 ) is necessary for this disease in the uae . in this study , we have provided evidence to support recommendations by the uae national diabetes committee,13 ) which is included in the periodic health check up program23 ) to start screening for dm in all individuals in the uae at the age of 30 years . other researchers24 ) from the uae explored the need for such a modification to the current american diabetes association guidelines based on their finding of a low prevalence rate of undiagnosed dm ( 1.5% ) among low risk individuals ( age , < 45 years ; body mass index , < 25 kg / m ) . certainly , the final decision about the age to initiate screening for dm is affected by cost - effectiveness and health system capacity factors in addition to the incidence / prevalence of the disease.25 ) we recommend future studies to be conducted in these areas . the incident cases of dm were collected from 5 phcs at shaikh khalifa hospital and gulf medical college hospital in ajman , uae . it is possible that the incidence rate of dm was underestimated in this study because emirati residents at other hospitals were not considered . even with this limitation , we can conclude that the incidence rate of dm was higher than in the uk and sweden . the highest incidence rate of dm was observed in the 55 - 59 year age group ( men , 23.4/1,000 py ; women , 32.4/1,000 py ) . the incidence rate among men was much lower than in women in the > 59 years age groups . the incident cases of dm were collected from 5 phcs at shaikh khalifa hospital and gulf medical college hospital in ajman , uae . it is possible that the incidence rate of dm was underestimated in this study because emirati residents at other hospitals were not considered . even with this limitation , we can conclude that the incidence rate of dm was higher than in the uk and sweden . the highest incidence rate of dm was observed in the 55 - 59 year age group ( men , 23.4/1,000 py ; women , 32.4/1,000 py ) . the incidence rate among men was much lower than in women in the > 59 years age groups .

our research was conducted to determine the algorithm changes during the treatment of submandibular sialolithiasis . two time periods were compared between 20042008 and 20092012 . the turning point was december 2008 , when sialendoscopy procedure was introduced . in the first period , 48 patients were treated : 31 outpatient duct incisions with stone evacuation and 17 surgical excision of submandibular gland . in the second period , 207 sialendoscopy procedures were performed on 197 patients . out of this particular group , 158 patients were diagnosed with pathological obstruction of salivary glands and 64 of them were confirmed to have sialolithiasis of submandibular gland . deposits of calcifications in 40 individuals ( 62.5 % ) affected by sialolithiasis were removed endoscopically ; however , in 21 patients , due to the increased circumference of the stone , the intimate association of deposits within the wall of the duct along with its presence inside the deep portions of the gland , double approach ( incision of the floor of the mouth in hilar area and sialendoscopy ) was performed . three individuals had their salivary glands totally removed due to the presence of calcified deposits within the glandular parenchyma . our results allow us to affirm that sialendoscopy is the current treatment of choice for submandibular glands affected by sialoliths . indication for a complete removal of the gland is becoming uncommon as a first line treatment although still indispensable in chosen cases . backgrounddiabetes mellitus ( dm ) , particularly type-2 is a major public health concern worldwide . not much information is available with regard to the incidence of dm in united arab emirates ( uae ) . this study aimed at determining the incidence rate of diabetes mellitus among emirati population in ajman , uae . this is a retrospective cohort study.methodsthis study was conducted in all primary health care centers and shaikh khalifa and gmc hospitals , ajman , uae where the emirati population primarily go for diagnosis and treatment . the incident cases of diabetes mellitus were collected during the period 2010 january to december.resultsa total of 158 cases newly detected in 2010 , 54 were among non - emiratis and one was type-1 diabetes . eliminating these , the remaining 101 were included in the analysis . the age of the patients ranged from 23 years to 78 years ; 35 ( 34.7% ) males and 66 ( 65.3% ) were females . the overall incidence observed was 4.8/1,000 person - years ( py ) with a female predominance of 6.3/1,000 py against incidence among males of 3.3/1,000 py . with regard to age specific incidence rate among males , it increases with age till 60 years and then showed a decreasing trend . among females also the same trend was observed but not as similar to males.conclusionthe highest incidence rate was observed in the 55 - 59 age group among males , 23.4/1,000 py and females , 32.4/1,000 py . among males the incidence rate was much less compared to females in - the age groups older than 59 years .

core autism symptoms such as social communication deficits have proven difficult to change through social skills training ( rao et al . 2008 ) . although several large - scale interventions for young children with autism such as pivotal response treatment meet american psychological association criteria for probable efficacy , therapeutic treatment programs for school - aged children with an autism spectrum disorder ( asd ) have not achieved this level of empirical support . however , there are no known methodologically rigorous studies of cognitive behavioral therapy ( cbt ) for core autism symptoms in spite of the fact that cbt may well suited for this indication . this paper presents preliminary results on the impact of cbt on parental reports of core autism symptoms in 711-year - old children . a common therapeutic approach that has been used to address core autism symptoms in school - aged children with asd up to this point has been social skills training ( rao et al . 2008 ) . this approach often involves practicing specific social skills ( e.g. , greetings ) in regularly - meeting groups of children with asd using hypothetical or dramatized scenarios as the principle learning mechanism . studies of this approach have generally ( a ) relied on diagnostically nonspecific measures of social skills rather than measures of core autism symptoms that are typically used in the diagnosis of asd and ( b ) either have found little evidence of generalization of treatment effects or have used uncontrolled designs that preclude conclusions about efficacy ( rao et al . 2008 ) . in comparison to social skills training , cbt relies on a cognitive science - informed model of psychological change that aims to promote retrievable memories of adaptive responses that can successfully compete with and suppress memories of previously learned maladaptive responses evoked under real world conditions outside the therapy office ( e.g. , brewin 2006 ) . in developing cbt treatment methods for children with asd , target goals ( e.g. , social skill development and generalization ) need to be matched with procedures for enhancing memory retrieval . we have outlined cbt procedures for addressing numerous core autism symptoms elsewhere ( sze and wood 2007 , 2008 ; wood et al . given space limitations , two examples of such procedures are given : ( 1 ) to promote reciprocal conversation skills , the encoding specificity principle from basic memory research suggests that skill learning should occur in the actual settings where social deficits are exhibited , rather than in simulated social situations such as therapy settings , as is often done in traditional social skills training . teachers and parents can be trained on simple social coaching procedures that promote the child s use of reciprocal conversation skills ( e.g. , posing questions ) in real - world settings immediately preceding actual social interactions ( i.e. , moments before , rather than hours or days before , the interactions ) to address this clinical need . ( 2 ) research on levels of processing in human memory has demonstrated that deep semantic processing rather than rote memorization increases the chance of the retrieval of a target memory ( e.g. , for a social skill ) . to promote deep semantic processing of new concepts , socratic questions ( questions that incorporate hints of the correct answer ) can be posed by the therapist to encourage children to put accurate answers in their own words ( sze and wood 2008 ) . the combination of repeated in vivo rehearsal of social skills in real - world settings coupled with socratic discussions about the positive effects of such skills may promote deep semantic processing and increase the memory retrieval of the targeted skills in naturalistic contexts while helping to suppress memories of habitual maladaptive responses such as social avoidance . cbt has achieved large , clinically significant outcomes in other childhood disorders ( e.g. , walkup et al . 2008 ) and high - functioning children with asd are considered promising candidates for this treatment approach . studies of information processing and cognition in high - functioning children with asd show that contrary to common speculation , their capacity for abstract reasoning is varied rather than absent ( e.g. , minshew et al . it is therefore plausible that the cognitive aspects of cbt would be understandable for many high - functioning school - aged children with asd , as long as the nature of asd is taken into account in the presentation of skills and concepts ( anderson and morris 2006 ) . in a recent randomized , controlled trial , a cbt program was found to have a positive impact on parent- and diagnostician - reports of anxiety in children with asd ( wood et al . anxiety disorders are common among children with asd and are associated with greater impairments in social responsiveness and more repetitive behaviors ( sukhodolsky et al . ( 2007 ) have proposed that anxiety in asd may be an indicator of a meaningful subclass of children with a greater likelihood of benefitting from the intervention techniques used in cbt . in the present study , it was hypothesized that cbt would outperform a waitlist condition on these outcomes and that a reduction of anxiety symptoms during the trial would be associated with a reduction of autism symptoms . the sample included 19 children , ranging in age from 711 years ( m = 9.37 , sd = 1.42 ) , and their primary parent(s ) ( defined as parent(s ) who were responsible for overseeing the child s daily activities ) living in a major metropolitan area of the western united states ( see wood et al . the 19 children in this sample include the final 11 participants from the wood et al . ( 2009 ) trial as well as eight additional children subsequently randomized into the same treatment protocol : ( a ) beginning with the 29th child enrolled in the wood et al . ( 2009 ) trial , the social responsiveness scale ( srs ; constantino and gruber 2005 ) was added as an outcome measure . once the 40th child completed treatment , anxiety disorder outcomes were evaluated as planned and the paper ( wood et al . ( 2009 ) paper was submitted , we randomized eight additional children into the same study protocol and collected srs data from their parents . ( c ) hence , the final 19 of the 48 total participants who had been randomized to condition had srs data . the cbt intervention was identical for all 48 participants and there were no extra treatment components administered to children whose parents completed the srs . children were referred by a medical center - based autism clinic , regional centers , parent support groups , and school personnel such as inclusion specialists . children met research criteria for asd ( see below ) and at least one anxiety disorder . psychiatric medication , if used , was at a stable dose prior to intake and throughout the duration of the trial . children with verbal iqs less than 70 or who were in concurrent psychotherapy were excluded . most children were boys ( n = 16 ; 84% ) and most primary parents had a college degree ( n = 15 ; 79% ) . children s race / ethnicity varied : caucasian ( n = 10 ; 53% ) ; asian ( n = 4 ; 21% ) ; native american / caucasian ( n = 2 ; 11% ) ; latino ( n = 1 ; 5% ) ; latino / asian ( n = 1 ; 5% ) ; african american / caucasian ( n = 1 ; 5% ) . therapists included four doctoral students in clinical or educational psychology and one postdoctoral fellow in psychology . therapists received at least 8 h of initial training on the intervention , read the treatment manual and attended weekly hour - long supervision with the treatment developers . therapists worked with families for 16 weekly sessions , each lasting 90 min ( about 30 min with the child and 60 min with the parents / family ) , implementing a version of the building confidence cbt program ( wood and mcleod 2008 ) modified by the study authors for use with children with asd . as with other cbt programs for child anxiety disorders , the manual includes coping skills training followed by in vivo exposure . a hierarchy is created in which feared situations are ordered from least to most distressing . children work their way up the hierarchy and are rewarded as they attempt increasingly fearful activities . in asd - specific treatment modules , children and parents are taught friendship skills ( e.g. , giving compliments , acting like a good sport , hosting peer get - togethers successfully , etc . ) and children are given social coaching by the therapist , parents , and available school providers on appropriate ways to enter interactions and ( later in treatment ) maintain conversations with peers . unlike traditional social skills training , social coaching is provided on - site immediately before attempting to join a social activity at school or home or in public and discussed in terms of others thoughts and feelings . these skills are practiced in session , at school , and during play dates and are reinforced with a comprehensive reward system that relies on both daily privileges and longer - term incentives . to address the social isolation that many children with asd experience at school , peer buddy and mentoring programs are set up during the school meeting . two meetings are scheduled at the child s school to teach the social intervention techniques to relevant school providers ( e.g. , aides , teachers ) . all skill development and practice efforts are supported by guided conversations in which the therapist uses socratic questioning to promote conceptual development and particularly , perspective taking ( e.g. , immediately before entering a playground interaction : if you offered her a turn , can you think of a nice thought she would have about you ? children s circumscribed interests and stereotypies are incorporated into the intervention in two ways . to enhance rapport , therapeutic concepts ( e.g. , emotion recognition , cognitive restructuring ) are taught using children s special interests as examples ( e.g. , for a child primarily interested in a particular cartoon character , the character s feelings and thoughts in socially awkward situations could serve as the basis of discussion ) and as rewards ( e.g. , granting access to the preferred stimulus ) . later in treatment , after rapport has been established ( as evidenced by the therapist s and supervisor s observation of at least three consecutive sessions of high child involvement during therapy ) , a suppression approach is introduced , in which increasing amounts of time per day are devoted to consciously refraining from discussing or engaging in activities related to the circumscribed interest or engaging in stereotypies such as flapping . to help children understand the rationale for suppression , information about social expectations and acceptance is provided during these modules ( e.g. , that these behaviors are fine in private but tend to confuse peers and get in the way of friendship ) . asd diagnoses were assigned with an algorithm using the autism diagnosis interview - revised ( adi - r ; le couteur et al . 2002)module 3 , a checklist regarding circumscribed interests , and a review of previous assessment records ( also see wood et al . the adi - r and ados were administered by doctoral students and doctoral - level psychologists who received standardized training and assessment certification . the srs ( constantino and gruber 2005 ) is a standardized , 65-item , 4-point likert style parent - report form assessing children s autism - specific characteristics such as social communication deficits and repetitive behaviors . the srs provides a total score and five subscales , and has demonstrated robust reliability and validity . the anxiety disorders interview schedule : child and parent versions ( adis - c / p ) ( silverman and albano 1996 ) is a semi - structured diagnostic interview with favorable psychometric properties that generates diagnoses as well as clinician s rating scale ( crs ) scores ranging from 0 to 8 ( with higher scores representing more severe anxiety ) . parents gave written informed consent and children gave written assent to participate in the study . children who met all inclusion / exclusion criteria were block randomized by a research assistant to either immediate treatment ( it ) or a 3-month waitlist ( wl ) using a computer randomization program ( the randomization sequence was concealed from investigators until interventions were assigned ) . posttreatment assessments were completed on the final day of treatment or within 1 week of termination ; postwaitlist assessments were conducted 3 months after the baseline assessment but before initiating cbt . families in it also participated in a follow - up assessment 3 months after termination of treatment . families received $ 20 for participating in the assessments . the sample included 19 children , ranging in age from 711 years ( m = 9.37 , sd = 1.42 ) , and their primary parent(s ) ( defined as parent(s ) who were responsible for overseeing the child s daily activities ) living in a major metropolitan area of the western united states ( see wood et al . the 19 children in this sample include the final 11 participants from the wood et al . ( 2009 ) trial as well as eight additional children subsequently randomized into the same treatment protocol : ( a ) beginning with the 29th child enrolled in the wood et al . ( 2009 ) trial , the social responsiveness scale ( srs ; constantino and gruber 2005 ) was added as an outcome measure . once the 40th child completed treatment , anxiety disorder outcomes were evaluated as planned and the paper ( wood et al . ( 2009 ) paper was submitted , we randomized eight additional children into the same study protocol and collected srs data from their parents . ( c ) hence , the final 19 of the 48 total participants who had been randomized to condition had srs data . the cbt intervention was identical for all 48 participants and there were no extra treatment components administered to children whose parents completed the srs . children were referred by a medical center - based autism clinic , regional centers , parent support groups , and school personnel such as inclusion specialists . children met research criteria for asd ( see below ) and at least one anxiety disorder . psychiatric medication , if used , was at a stable dose prior to intake and throughout the duration of the trial . children with verbal iqs less than 70 or who were in concurrent psychotherapy were excluded . most children were boys ( n = 16 ; 84% ) and most primary parents had a college degree ( n = 15 ; 79% ) . children s race / ethnicity varied : caucasian ( n = 10 ; 53% ) ; asian ( n = 4 ; 21% ) ; native american / caucasian ( n = 2 ; 11% ) ; latino ( n = 1 ; 5% ) ; latino / asian ( n = 1 ; 5% ) ; african american / caucasian ( n = 1 ; 5% ) . therapists included four doctoral students in clinical or educational psychology and one postdoctoral fellow in psychology . therapists received at least 8 h of initial training on the intervention , read the treatment manual and attended weekly hour - long supervision with the treatment developers . therapists worked with families for 16 weekly sessions , each lasting 90 min ( about 30 min with the child and 60 min with the parents / family ) , implementing a version of the building confidence cbt program ( wood and mcleod 2008 ) modified by the study authors for use with children with asd . as with other cbt programs for child anxiety disorders , the manual includes coping skills training followed by in vivo exposure . a hierarchy is created in which feared situations are ordered from least to most distressing . children work their way up the hierarchy and are rewarded as they attempt increasingly fearful activities . in asd - specific treatment modules , children and parents are taught friendship skills ( e.g. , giving compliments , acting like a good sport , hosting peer get - togethers successfully , etc . ) and children are given social coaching by the therapist , parents , and available school providers on appropriate ways to enter interactions and ( later in treatment ) maintain conversations with peers . unlike traditional social skills training , social coaching is provided on - site immediately before attempting to join a social activity at school or home or in public and discussed in terms of others thoughts and feelings . these skills are practiced in session , at school , and during play dates and are reinforced with a comprehensive reward system that relies on both daily privileges and longer - term incentives . to address the social isolation that many children with asd experience at school , peer buddy and mentoring programs are set up during the school meeting . two meetings are scheduled at the child s school to teach the social intervention techniques to relevant school providers ( e.g. , aides , teachers ) . all skill development and practice efforts are supported by guided conversations in which the therapist uses socratic questioning to promote conceptual development and particularly , perspective taking ( e.g. , immediately before entering a playground interaction : if you offered her a turn , can you think of a nice thought she would have about you ? children s circumscribed interests and stereotypies are incorporated into the intervention in two ways . to enhance rapport , therapeutic concepts ( e.g. , emotion recognition , cognitive restructuring ) are taught using children s special interests as examples ( e.g. , for a child primarily interested in a particular cartoon character , the character s feelings and thoughts in socially awkward situations could serve as the basis of discussion ) and as rewards ( e.g. , granting access to the preferred stimulus ) . later in treatment , after rapport has been established ( as evidenced by the therapist s and supervisor s observation of at least three consecutive sessions of high child involvement during therapy ) , a suppression approach is introduced , in which increasing amounts of time per day are devoted to consciously refraining from discussing or engaging in activities related to the circumscribed interest or engaging in stereotypies such as flapping . to help children understand the rationale for suppression , information about social expectations and acceptance is provided during these modules ( e.g. , that these behaviors are fine in private but tend to confuse peers and get in the way of friendship ) . asd diagnoses were assigned with an algorithm using the autism diagnosis interview - revised ( adi - r ; le couteur et al . 2002)module 3 , a checklist regarding circumscribed interests , and a review of previous assessment records ( also see wood et al . the adi - r and ados were administered by doctoral students and doctoral - level psychologists who received standardized training and assessment certification . the srs ( constantino and gruber 2005 ) is a standardized , 65-item , 4-point likert style parent - report form assessing children s autism - specific characteristics such as social communication deficits and repetitive behaviors . the srs provides a total score and five subscales , and has demonstrated robust reliability and validity . the anxiety disorders interview schedule : child and parent versions ( adis - c / p ) ( silverman and albano 1996 ) is a semi - structured diagnostic interview with favorable psychometric properties that generates diagnoses as well as clinician s rating scale ( crs ) scores ranging from 0 to 8 ( with higher scores representing more severe anxiety ) . parents gave written informed consent and children gave written assent to participate in the study . children who met all inclusion / exclusion criteria were block randomized by a research assistant to either immediate treatment ( it ) or a 3-month waitlist ( wl ) using a computer randomization program ( the randomization sequence was concealed from investigators until interventions were assigned ) . posttreatment assessments were completed on the final day of treatment or within 1 week of termination ; postwaitlist assessments were conducted 3 months after the baseline assessment but before initiating cbt . families in it also participated in a follow - up assessment 3 months after termination of treatment . for the participating sample ( n = 19 ) , recruitment began in 3/2006 and ended in 8/2007 ; posttreatment assessments were completed by 12/2007 . there were no statistically significant pretreatment group differences on the demographic variables or srs scores . evidence of intervention adherence is excellent ( 94% of core elements of each session detailed in the treatment manual , as rated by a blind coder listening to audiotapes of sessions , were implemented per protocol ) and is described in detail elsewhere ( wood et al . ancova was used to test group differences at posttreatment / postwaitlist on srs scores , with the children s baseline srs scores included as a covariate . there was a statistically significant group difference , f ( 1 , 16 ) = 5.39 , p < .05 . posttreatment / postwaitlist total srs scores were lower in the it group than in the wl group ( d = .77 , approaching a large effect ; cohen 1988 ) ( see table 1 ) . in additional ancovas , posttreatment / postwaitlist group differences favoring cbt were found on three of five srs subscales : social communication , social motivation , and social awareness ( ps < .05 ) ; a trend favoring cbt was also found for social cognition ( p = .10 ) . no group difference was found on the autistic mannerisms subscale.table 1total social responsiveness scale scores for the it and wl groupsbaselineposttreatment / postwaitlistitwlitwlm113116.4089110.30sd18.2730.1926.3929.22range87145611515314053146for it , n = 9 ; for wl , n = 10 . raw scores are reported total social responsiveness scale scores for the it and wl groups for it , n = 9 ; for wl , n = 10 . raw scores are reported follow - up data was collected on four of the nine children in the it group ( the remaining five children were lost to follow - up ) . all four children had lower srs total scores at follow - up than they had at posttreatment , suggesting treatment gains had been maintained or augmented . a t - score of 70 or greater is considered a clinically significant level of autism symptoms . at baseline all but one child in the sample had a total srs t - score above 70 ( the exception was in wl , t = 63 ) . of the nine wl children who began the trial with a t - score in the clinical range , all remained at or above a t - score of 70 at postwaitlist . at immediate posttreatment , two of nine children in it had t - scores below 70 . at 3-month follow - up , three of four it children had t - scores below 70 ( as noted above , the remaining five it children were lost to follow - up ) . hence , using a last - observation - carried - forward ( locf ) approach , five of nine it children ( 56% ) were below a t - score of 70 at their last available assessment ( i.e. , posttreatment or follow - up ) . two ols linear regression models were fitted to evaluate the correspondence of anxiety reduction and autism symptom improvement . in the first model , predictors included adis - c / p crs change scores and baseline srs total scores . in the second model , srs change - scores and baseline adis - c / p scores both models had significant effects ( model 1 adis - c / p change score = .53 , and model 2 srs change score = .63 , ps < .01 , respectively ) , suggesting that when anxiety decreased over time , so did autism symptoms ( and vice versa ) . these results suggest that autism symptoms such as social communication deficits may be positively influenced by cbt . if this finding proves to be robust in larger - scale trials , it would represent a major advance in the treatment of core autism symptoms in high - functioning school - aged children . a key finding was that parent - reported autism symptoms were lower in the cbt group than the waitlist group at the post assessment , with a medium to large effect size . although reliance on parent report is in some ways a limitation , parent report does have the potential advantage of capitalizing on a parent s extensive observations of child behavior at home and in the community . generalization and maintenance of treatment effects in these settings has generally not been attained in social skills training ( rao et al . 2008 ) . furthermore , few if any clinical trials of non - pharmacological behavioral treatments for school - aged children with asd have used diagnostic measures of autism symptoms as an outcome measure ; social skill training studies have tended to use diagnostically nonspecific measures of social skill mastery , which may not be capable of documenting improvement in autism - specific social communication deficits . a second major finding in this study was that improvement in anxiety was associated with improvement in autism symptoms and vice versa , suggesting that for children with comorbid asd and anxiety disorders , the two symptom domains could be interconnected . the potential role of anxiety in exacerbating asd symptoms for some children has been documented in various descriptive studies ( e.g. , sukhodolsky et al . 2008 ) . hypothetically , after experiencing a reduction of the aversive affective experience of anxiety , children with asd may feel less need to engage in maladaptive coping behaviors such as social avoidance and repetitive behaviors to regulate their mood state , permitting interventions in the core autism symptom domain ( e.g. , social reciprocity ) to be more effective . this study was limited by a small sample and reliance on parent reports of symptomatology , which are vulnerable to bias . future research could expand upon these initial findings by using a larger sample of participants and by including objective measures of outcome based on independent evaluators ratings and direct observations of children s behavior . given the limited number of empirical studies on effective intervention modalities for core autism symptoms in this population , further investigation of cbt for the treatment of core autism symptoms appears indicated . this study was strengthened by its use of methods that are consistent with contemporary guidelines for clinical trials research ( chambless and hollon 1998 ) . if the present findings are representative of the effects of cbt on autism symptoms in high - functioning school - aged children , this intervention modality could represent an important step towards developing more efficacious practices for the treatment of asd . it is characterized by neuropsychiatric manifestations . in minimal hepatic encephalopathy ( mhe ) , patients have alterations in sleep pattern , and there is difficulty in performing even day - to - day routine activities like driving a vehicle , doing simple calculations , and so forth [ 13 ] . initially there is a mild impairment in cognition and psychomotor skills which then progress to a gross impairment in orientation and general consciousness . these manifestations are potentially reversible , especially , if the diagnosis is made early in the course of the disease [ 4 , 5 ] . the diagnosis of minimal hepatic encephalopathy ( mhe ) is made using a battery of neuropsychometric or neurophysiological tests . the existing neuropsychometric tests include paper - pencil tests like the number connection tests a and b , the figure connection test ( fct ) , trail tracing test , block design test , digit symbol test , and the comparatively recent inhibitory control test . inaccuracies in the paper - pencil tests have been widely reported [ 7 , 8 ] . also , there is a lack of consensus on the diagnostic criteria and a limited correction for educational level and age in these studies . the fct was designed for the indian population and has been widely accepted as a sensitive test to detect mhe in illiterate individuals . however , this test assesses only a few discrete domains of impairment ( attention and psychomotor skills ) . much of the tests hitherto mentioned are seldom used in day - to - day clinical practice . more recent objective neurophysiological tests include critical flicker frequency ( cff ) and , auditory and visual evoked potentials . all these tests involve expensive specialized equipment and trained experienced personnel [ 7 , 11 ] . there is therefore a need to design an inexpensive test which would measure all the impaired domains of the mind using the same metric . we designed a test that would examine the three important domains cognition , memory , and psychomotor skills and used this test in patients with chronic liver disease to detect mhe . the impaired domains of mental function in mhe have an impact on the quality of daily life . the mind and liver test ( malt ) was conceptualized on a scientific basis and was designed to measure the daily mental needs of an individual as a whole , and not for just one specific domain . malt was designed such that it served as a single metric measure for all impaired domains like cognition , memory , and psychometric skills . in order to design the specific tasks which make up the malt , a questionnaire investigating the specific daily mental demands encountered by the target population was administered to 100 individuals attending the out - patient liver clinic of institute of gastroenterology and hepatology , stanley hospital , chennai , india . except for 5 patients who were either professionals or businessmen , the rest belonged to lower socioeconomic status and were laborers , unskilled workers , or farmers ( kuppuswamy 's socioeconomic status scale ) . based upon the results of the questionnaire , the questions were designed to identify certain animals , ability to follow the sequence of a natural phenomenon ( e.g. , morning - noon - evening - night ) , and ability to perform simple mathematical calculations needed during daily transactions ( e.g. , transaction at the shop ) . for memory we also tested the ability to recall a visual as well as an auditory experience . the psycho - motor abilities were tested by asking the patient to copy certain figures on a calibrated graph paper , to build a three - dimensional structure of a wall using building blocks , to cut along a drawn line using a scissor , and to trace along a narrow path ( analogous to a line tracing test ) . all the three tests were designed using a colorful board , set of printed question cards , plastic building blocks , animal models , an audio device , and an instruction cum scoring sheet . for convenience , red steps tested memory , green steps tested cognition , and yellow steps tested psycho - motor skills . these tasks were grouped into three different sections : section 1 : figure the field , section 2 : home sweet home , section 3 : road not taken . section 1 : figure the field , section 2 : home sweet home , section 3 : road not taken . section 1 included steps like identification of animals and vegetables on the countryside / neighborhood , and identification of well - known sequences of events like sowing seeds , transplanting the saplings , harvesting , and finally transporting to the city . in this section the failure to perform this task was considered a sign of psycho - motor impairment . section 2 included steps like remembering the arrangement of things on the shelves at home and ability to narrate the steps of a task performed at home daily , such as washing a cloth or making tea . impairment in memory and organization reflected directly on the performance in these steps . in the same section we tested the ability of the patient to identify and remember sounds by using different stimuli , such as a church bell or an ambulance siren . examinees were also asked to cut paper using a pair of scissors , a measure of psychomotor abilities . section 3 included tasks that tested the numerical abilities of the patient ( like we asked the patient to perform small transactions at the shop ) and tasks that asked them to identify places that they may visit on a daily basis , like a temple , a water pump , or a market place . short term memory was tested by asking the patient to recall the short list of things to buy from the shop that was narrated to him in section 2 . we included a test analogous to the trail tracing test in section 3 where the patient had to negotiate a narrow path from the shop back to home , using his index finger along the trail . two other tests were performed on the malt board , which could affect the daily activities in mhe . these included ( a ) divided attention : ability to observe images on the board that were not included as a part of the task ; ( b ) judgment ability : to select the shortest of the three shown paths . table 1 shows the steps included in the three sections of the test that appear on the malt board ( figure 1 ) and the corresponding mental function that each step evaluates . based upon the performance of initial 10 controls in the test , we fixed an upper limit of 1 minute for each of steps 3 , 5 , and 6 . the maximum scores on the entire test was 60 and 20 each on cognition , memory , and psychomotor skills . informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the ethics committee of stanley medical college and hospital . forty proven cirrhotic patients were recruited as cases from among the out - patients and in - patients registered in our department . patients with neurological deficit , obvious overt encephalopathy , alcohol abuse 72 hours prior to the test , or those on sedatives or psycho - active drugs in the preceding 24 hours were excluded . patients with cardiac failure , renal disease , and chronic obstructive airway disease were also excluded . it was ensured that the patients had not received lactulose , lactitol , or antibiotics like rifaximin , neomycin , or quinolones in the past three months . all controls and patients had critical flicker frequency test using portable battery - powered hepatonorm analyzer , accelab gmbh , kusterdingen , germany . the analyzer evoked an intrafoveal light stimulus with defined pulses [ 7 , 16 ] . the patient was asked to press a hand held switch when the change was apparent . the procedure was repeated 5 times to make the subject familiar with the process and 9 readings were recorded . the mean for each patient was calculated . a value below the 38 hz mark was considered diagnostic of mhe [ 5 , 7 , 16 ] . the subjects ( cases and controls ) were then brought into a well lit room and were allowed a relaxation time of 15 minutes after which malt was administered by two trained medical professionals ( sd and sm ) . the patient and the tester are seated across a table with the test board , question cards , an audio device , and a table clock . the performance of the patient was noted on the instruction / scoring sheet , including time to completion . both tests were executed by two different persons ( sd and sm ) who were blinded to the other examiner 's results . the performers administering the tests were trained by experienced clinicians so as to minimize the intra- and interobserver variations . student 's t - test was used to compare the mean cff values and mean malt scores between the cases and the controls . preliminary analyses provided data for receiver - operating characteristic curves to determine the optimum cut - off value for malt score for the diagnosis of mhe . the best sensitivity and specificity was found at a cut - off score of 20 . using the cff performance as the reference standard , statistical analysis was performed using student 's t - test and the receiver - operating characteristic ( roc ) curve . cirrhotics with mhe and cirrhotics without mhe , based upon their cff readings . spss version 10 was used for all statistical analyses and in the generation of the plots . twenty subjects including 10 cases and 10 controls were administered malt twice at an interval of a six hours . it was ensured that patients being evaluated were not on lactulose , lactitol , neomycin , or any sedative meanwhile . the report was prepared in accordance with standards for reporting diagnostic accuracy , stard guidelines ( figure 2 ) . the study comprised of 40 patients with cirrhosis ( hereinafter referred to as cases ) and included 25 males ( 62.5% ) . there were 36 healthy controls ( hereinafter referred to as controls ) and included 16 males ( 44.44% ) . the mean age of the cases and controls was significantly different ( p = 0.04 ) . there was no difference in sex distribution ( p = 0.18 ) , educational status ( p = 0.93 ) , and monthly income ( p = 0.09 ) among the cases and the controls . the etiology of cirrhosis was alcohol in 15 patients ( 37.5% ) , hcv in 13 ( 32.5% ) , nonalcoholic fatty liver disease in 6 ( 17.5% ) , autoimmune in 1 ( 2.5% ) , and cryptogenic in 8 ( 20% ) . there were 22 patients ( 55% ) belonging to child - pugh class a and 18 ( 45% ) to class b. among the cirrhotics , there were 18 variceal bleeders ( 45% ) and 2 patients ( 5% ) had coagulopathy . 4 patients ( 10% ) had refractory ascites , 2 patients ( 5% ) had spontaneous bacterial peritonitis , and 8 others ( 20% ) had urosepsis . using 38 hz as cff cut - off for mhe , 16 out of the 40 cirrhotics had mhe ( 40% ) , 8 each ( 50% ) belonged to child class a and child class b. among the 24 cirrhotics without mhe , 14 ( 58.33% ) belonged to child a and 10 ( 41.66% ) to child b at the time of mhe testing . the mean cff for the cases was 39 4.2 hz compared to 42 1.9 hz among the controls ( p = 0.00 ) ( figure 3(a ) ) . the demographic characteristics and the lab parameters of the mhe and non - mhe groups among the cirrhotics or the cases are compared in table 4 . the controls scored 10 5 compared to the cases , who scored 18 8 ( figure 3(b ) ) . on plotting the roc curve ( figure 4(a ) ) , the sensitivity and the specificity were found to be 94% and 83% , respectively , for a cut - off value set at 20 . using roc with cff as the gold standard , the auc for diagnosis of mhe using malt score was 89% . figure 3(b ) compares the mean malt scores of mhe , non - mhe cirrhotics , and controls . mhe patients scored higher than the non - mhe and controls using the 20 mark as cut - off . the test - retest variability was assessed using the intraclass correlation coefficient , icc = 0.894 ( p = 0.000 ) . the bland - altman plot ( figure 4(b ) ) shows that the malt retest scores of the 10 cases and 10 controls ( who were subjected to the same test at an interval of 6 hours ) was in agreement with the initial test scores . the hepatonorm analyzer , manufactured in germany , is an electronic device which runs on a battery that needs to be regularly changed . the mean time of administration for both malt and cff was 10 2 minutes . the highlights of the study are ( a ) a significant correlation between cff and malt scores in the population under study ( p < 0.05 ) ; ( b ) the roc curve and the box - whisker plots indicate that malt is a highly sensitive and specific test considering cff as the standard ; and ( c ) the intraclass correlation coefficient was 0.894 on assessment of test - retest variability . the sensitivity and specificity of malt was found to be 94% and 83% , respectively , compared to earlier reported 72.4% and 77.2% for cff when the cut - off is set at 38 hz . though malt may be seen to be a time taking procedure , the mean time taken for malt and cff did not vary significantly . a number of neuropsychological tools have been used to diagnose cognitive deficits in patients with cirrhosis . the extended neuropsychological assessment is based upon the expert judgment and is difficult to validate . the two shorter batteries rbans ( repeatable battery for assessment of neuropsychological status ) and phes ( psychometric hepatic encephalopathy score ) are primarily paper - pencil tests . the performance on these tests is affected by the level of literacy and on the familiarity of the subject to paper and pencil among the literates . however , these tests have their own advantages . rbans is validated on a large normative data , it is available in multiple languages and has parallel versions available for repeated testing . however , most studies are in alzheimer 's patients and the studies in cirrhotics are only few . phes was originally validated in a series of nonalcoholic patients in germany and has normative data available from some countries . however , our knowledge of mhe has grown since those studies and , now , it is understood that an ideal diagnostic tool should reflect on those daily activities which affect the quality of life , the ability to drive , and so forth , which are often impaired in mhe [ 21 , 22 ] . to overcome the shortcoming of these short batteries for the use in resource - limited settings including their dependence on the literacy level , the figure connection test ( fct ) was reported by a group in india . however , fct only tests a discrete cognitive domain impaired in mhe . computerized neuropsychological tests include cognitive drug research ( cdr ) , inhibitory control test ( ict ) , and the scan test which have been validated in the uk , usa , and italy , respectively [ 2325 ] . the introduction of malt has been based on the fundamental assumption that the currently available tests for the diagnosis of mhe are designed to test for one or two domains of mental impairment , and are expensive or not widely available . malt was designed to include tasks performed on a day - to - day routine basis . in the population under study , more than 36% of the participants were illiterate and the remaining who were literate were not used to paper and pencil . this is in line with the demise of the pencil reported by iduru and mullen . moreover , age and education correction factors are absent for psychometric hepatic encephalopathy score ( phes ) in our population . it is in agreement with the prevalence of 41% reported in a recent study involving 200 cirrhotics from north india while on the higher side compared to 30% reported in the study from spain using phes [ 18 , 26 ] . it would either signify that malt included more true positives or that the prevalence is different in our population [ 27 , 28 ] . malt is a psychometric test which displays day - to - day activity on a colorful board and disagreement between administrators of the test is significantly less ( 85% agreement in interobserver studies ) . though the tasks on the game board remain the same , the elements of the tasks are not printed on the board but on multiple cards . malt includes steps like build the wall and trace the path which are analogous to block design and trail tracing test . in malt , the subject is not asked to connect numbers , but rather to trace the sequence of a day - to - day event from morning to night , which in true sense does not need a level of literacy . although the majority of surveyed hepatologists in spain and the united states agreed that mhe was a significant problem requiring testing during out - patient visits , only a few were able to actually perform the test during their day - to - day clinical practice [ 29 , 30 ] . when the test is available in the form of a touch screen audio - visual videogame , the test could be self - administered by the patient during the wait period in the clinic . . it might be interesting to note if administration of such a test has any effect on the mood and affect of the depressed cirrhotic . it will also be of interest to follow up malt scores of patients on therapy like lactulose for hepatic encephalopathy . also , to see a head to head analysis of malt compared to other psychometric tests in future . however , these were beyond the scope of the present study . the choice of cff as the gold standard in the current study was also to circumvent many problems known to be encountered with administering multiple psychometric tests [ 2 , 31 ] . this study largely aimed to design a psychometric battery to diagnose mhe in our population , which is useful for the illiterate patients and to check its reliability . the high sensitivity and specificity of malt , its ease of administration , reproducibility , and cost - effectiveness seen in this pilot beckon further validation studies involving larger sample sizes .

this pilot study tested the effect of cognitive behavioral therapy ( cbt ) on parent - reported autism symptoms . nineteen children with autism spectrum disorders and an anxiety disorder ( 711 years old ) were randomly assigned to 16 sessions of cbt or a waitlist condition . the cbt program emphasized in vivo exposure supported by parent training and school consultation to promote social communication and emotion regulation skills . parents completed a standardized autism symptom checklist at baseline and posttreatment / postwaitlist and 3-month follow - up assessments . cbt outperformed the waitlist condition at posttreatment / postwaitlist on total parent - reported autism symptoms ( cohen s d effect size = .77 ) . treatment gains were maintained at 3-month follow - up . further investigation of this intervention modality with larger samples and broader outcome measures appears to be indicated . background and aims . minimal hepatic encephalopathy ( mhe ) is diagnosed using neuropsychometric tests or neurophysiological tests that are either inapplicable to illiterate patient population in resource - poor settings or require sophisticated and expensive equipment . the available tests assess discrete domains of mental impairment . our aim was ( a ) to design a neuropsychometric test that measures all domains of mental impairment in mhe using one metric ; ( b ) to evaluate its sensitivity , specificity , and reproducibility . methods . the mind and liver test ( malt ) , a psychometric test assessing cognition , memory , and psychometric impairment , each on a scale of 20 , was designed keeping in mind the requirements of a universal test . 40 cirrhotics and 36 controls were subjected to critical flicker frequency ( cff ) and malt in same sitting . roc curve was plotted for malt using cff as gold standard . bland - altman plot was used to find test - retest agreement . results . cff values and malt scores varied significantly between the cases and the controls ( p < 0.05 ) . malt was 94% sensitive and 83% specific . using roc with cff as gold standard , the auc for diagnosis of mhe using malt score was 0.89 . test - retest agreement was high ( icc = 0.89 ) . conclusion . in this pilot study , malt proved to be highly sensitive , specific , inexpensive , and reproducible .

pseudocholinesterase ( pche ) is an enzyme with a complex molecular structure.1 it is synthesized in the liver and immediately released into the plasma.2 the plasma half - life has been estimated to be approximately 12 days.3 deficiency or reduced activity of this enzyme results in significant prolongation of mivacurium- or succinylcholine - induced neuromuscular blockade.4 in addition , a number of disease states or concomitant drug administration may reduce pche activity . mivacurium , which is a nondepolarizing neuromuscular blocking drug administered in doses of 0.1 to 0.2 mg / kg , also produces rapid onset of neuromuscular blockade lasting for 15 to 30 minutes.5 the rapid ester hydrolysis of mivacurium by pche results in the short duration of action of this drug , which is ideal for providing muscle relaxation for brief surgical procedures.6 but the duration of mivacurium of action in adults is inversely related to serum pche activity.7,8 sertraline is an antidepressant and antipanic agent that is a potent and selective inhibitor of serotonin reuptake into the presynaptic terminals . it has relatively weak effects on the cytochrome p450 system , a tolerability profile that is positive compared with antidepressants of other classes , and it is relatively safe in overdose . these findings contribute to sertraline being the first - line agent for the treatment of several psychiatric disorders.9 it was shown , however , that sertraline and other antidepressants inhibited acetylthiocholine hydrolysis by cholinesterases in human serum and erythrocyte membrane.10 in this article , we share our experience of a case with acquired pseudocholinesterase enzyme deficiency due to sertraline use , and provide a literature review . a 47-year - old turkish male , 83 kg in weight and 175 cm in height , was scheduled for laparoscopic cholecystectomy ( due to cholecystitis with an acute stone ) under general anesthesia . under general anesthesia , the case had an appendectomy 28 years ago and a right inguinal hernia operation 19 years ago . it was learned that the patient was administered succinylcholine during both these operations and that he did not have a history of prolonged recovery or postoperative apnea . except for alkaline phosphatase ( alp ) ( 176 u / l ) , alanine aminotransferase ( alt ) ( 52 u / l ) and a minimal increase in aspartate aminotransferase ( ast ) ( 61 u / l ) , and a slight leucocytosis ( 9.8 10/l ) , no other pathologies were identified during preoperative laboratory investigation . the patient had been using sertraline ( 100 mg / day ) for 3 years for major depressive disorder . after surgery , the inhalation agent was discontinued , the patient received 100% oxygen but there was no clinical evidence of neuromuscular function recovery and there was no response to train - of - four stimulation . anesthesia was therefore maintained with 0.5% isoflurane in 50% nitrous oxide and oxygen because there was no evidence of improved neuromuscular function 50 minutes after administration of mivacurium . five milligrams of midazolam was administered intravenously for its sedative and amnestic effects . later the patient was transferred to the post - anesthesia care unit ( pacu ) for observation and ventilator support . train - of - four response ratio was 75% 2 hours and 55 minutes after mivacurium administration . the patient regained sufficient motor function to meet extubation requirements 3 hours and 15 minutes after the initial use of mivacurium . the patient was extubated for 205 minutes following the administration of mivacurium and further recovery was uneventful . iu / l ) . the patient was transferred to a hospital ward for the evening and was discharged 4 days later . a recommended intravenous dose of 0.15 to 0.2 mg / kg provides tracheal intubating conditions within 2 to 2.5 minutes , with a predicted duration of action of 15 to 25 minutes , making it an ideal drug for short procedures requiring tracheal intubation.11 it is a structural relative of atracurium , but it does not undergo hofmann elimination and is rapidly hydrolyzed by pche . the activity of pche affects its duration of action.4 pche is a tetrameric glycoprotein enzyme produced by the liver , which hydrolyzes choline esters such as those found in succinylcholine , mivacurium , procaine , chloroprocaine , tetracaine , cocaine , and heroine.12 in patients with a normal genotype for pche , mivacurium s duration of action is inversely related to pche activity and duration of action is slightly prolonged if activity is low.13 when there is a deficiency of this enzyme due to the presence of one or more atypical alleles , mivacurium is not properly metabolized and thus muscle paralysis can last for several hours . the heterozygous atypical form affects from 1 in 25 , to 1 in 480 individuals ( depending on the severity of the condition).12 patients heterozygous for an abnormal enzyme may show up to 50% prolongation of block.13 in patients homozygous for an abnormal enzyme , duration may be significantly prolonged , and even a small dose ( eg , 0.03 mg / kg ) can result in complete paralysis for up to 128 minutes.14 there are now several case reports in the literature of prolonged block following mivacurium administration . physiological reductions may occur with extremes of age and during pregnancy.15 other acquired causes of decreased activity include renal and liver disease , malignancy , burns , chronic debilitation / malnutrition , myocardial infarction / cardiac failure , collagen diseases , myxedema , and organophosphate poisoning.16 plasma cholinesterase levels in human subjects with protein energy malnutrition have been investigated.17 drugs that inhibit the enzyme s activity include acetylcholinesterase inhibitors ( neostigmine , pyridostigmine , physostigmine , and edrophonium ) , anticholinesterases ( especially echothlophate ) , cytotoxic agents ( such as cyclophosphamide ) , steroids , ester - type local anesthetics , hexafluorenium , pancuronium , and oral contraceptives.18 our patient previously had 2 operations under general anesthesia with succinylcholine . however , a prolonged apnea and low enzyme activity ( 788 although the patient had not had any problems during the previous operations , absence of a medical history proving the existence of the same problem in close relatives led us to think that this was an acquired pche deficiency . at this stage , we at once started to implement an algorithm which is usually used in cases of pche deficiency . at this stage however , we noted that the medical conditions ( such as extremes of age , pregnancy ) and problems ( such as renal and liver disease , malnutrition ) reported in the literature that we have tried to summarize above did not match our case . therefore , we considered the drugs known to affect pche activity that the patient may have been taking . we discovered that the patient had been using sertraline ( 100 mg / day ) for 3 years due to depression . sertraline is a serotonin reuptake inhibitor ( ssri ) that is indicated for the treatment of depression . with therapeutic doses , sertraline has a wide therapeutic index and appears to be safer than the tricyclic antidepressants in overdose.19 the usual clinical antidepressants ( fluoxetine , sertraline , and amitriptyline ) are , however , inhibitors of the cholinesterases in human serum and erythrocyte membrane . antidepressants exhibit this inhibiting effect by reversibly attaching to the enzyme s bottom of the active site gorge , and the collapse of the product ( eg , mivacurium or succinylcholine ) being hydrolyzed by the enzyme decreases during this process of attachment.10 the order of inhibitory potency was sertraline > amitriptyline > > fluoxetine . the concentrations of antidepressants necessary to inhibit serum cholinesterase are relatively close to the clinical levels found after pharmacological use of sertraline and amitriptyline . consequently , undesirable pharmacological and toxicological side effects of these antidepressants can be related , at least in part , to inhibition of serum and erythrocyte cholinesterases.10 therefore , chronic enzyme inhibition which might have developed due to 3 years of use of sertraline in our patient led to a significant and marked prolongation in the activity time of the neuromuscular blocking agent metabolized by this enzyme . the results achieved by mller et al10 corroborated our suspicions about sertraline . also , the retested ( 3 months after the operation ) pche value ( 2762 iu / l ) was seen to have returned to normal after the patient gave up using sertraline under the supervision of a psychiatrist . we did not discover any case that implied an interaction between sertraline and pseudocholinesterase in our literature review , and we believe this is the first presentation of such a case . when we understood that we were dealing with a case of acquired pche deficiency , we made our treatment plan . our main aim was to administer a supportive treatment to the patient until enzyme activity returned to normal . therefore we did not consider a blood transfusion ( considering the risks of blood transfusion , such as allergic reactions , infection , fever reaction ) . we also avoided administering drugs ( such as calcium , neostigmine ) that might complicate the present condition . the clinician should be especially aware that administering neostigmine to a patient with prolonged mivacurium - induced block will inhibit plasma cholinesterase activity . therefore , blood should not be drawn for cholinesterase activity until the effect of neostigmine dissipates . in our case , when we suspected pche deficiency , together with pns findings , we sent the blood samples to the laboratory for enzyme analysis . consequently , we supported our case solely with sedation , mechanical ventilation , and pns monitoring , and without neostigmine application . prolonged blocks may be encountered as a result of pche enzyme deficiency when mivacurium is used . in our case , this situation was due to sertraline , which is commonly administered for various psychiatric disorders , particularly depression . in this context , we believe two issues should be highlighted . firstly , while examining the drugs used by patients during preoperative anesthesia anamnesis , next - generation antidepressants ( especially ssris ) should not be ignored . as in the case reported , this group of drugs , known to have a wide safety margin , may cause casual and unexpected block prolongation when used along with drugs ( mivacurium and succinylcholine ) that are hydrolyzed especially by pche activity during anesthesia . secondly , in these situations , attempted reversal of the block was at best only partially successful , and the patients were partially paralyzed and minimally sedated while attempts were made to have them breathe a very unpleasant experience . in cases of prolonged block the patients were kept sedated , and their lungs mechanically ventilated until the block recovered spontaneously , and then extubated without problem . six and 18monthold male c57 and mdx mice were euthanized , and the tibialis anterior ( ta ) muscle and the diaphragms were dissected . they were either flash frozen in liquid nitrogen for biochemical analysis or embedded in oct and frozen in melting isopentane for histological evaluation . portions of ta and diaphragm were collected from 1yearold golden retriever muscular dystrophy ( grmd ) dogs ( n = 3 ) and an unaffected dog ( n = 1 ) following unrelated terminal endpoints and were either flash frozen or frozen in oct . samples of human quadriceps muscle ( n = 3 ) were obtained from the wellstone muscular dystrophy cooperative research center , tissue and cell repository at the university of iowa , carver college of medicine ( iowa city , iowa ) . the dmd patients were between 5 and 8 years of age , and control patients between 8 and 13 years of age at the time of biopsy . total rna was isolated from murine and canine muscles using trizol reagent ( invitrogen ) treated with dnase ( promega ) and reverse transcribed to cdna using the geneamp rna polymerase chain reaction ( pcr ) kit ( applied biosystems ) using the manufacturer 's directions . realtime pcr ( model 7300 ; applied biosystems ) was conducted on the resulting cdna with power sybr green master mix ( applied biosystems ) . expression levels of lox ( accession numbers : mouse , nm_010728 ; dog , xm_538599 ) , loxl2 ( mouse , xm_006519744 ) , and col1a2 ( mouse , nm_007743 ; dog , nm_001003187 ) were determined with 18s expression serving as the reference housekeeping gene . flashfrozen murine and canine muscle tissue was finely crushed and homogenized in tissue protein extraction reagent ( tper ; thermo scientific ) supplemented with protease and phosphatase inhibitor cocktails ( thermo scientific ) . quadriceps biopsy samples from healthy males and dmd patients ( pooled from 25 10m frozen sections from each patient ) were lysed in radioimmunoprecipitation assay ( ripa ) buffer supplemented with protease and phosphatase inhibitor cocktails ( sigmaaldrich ) . polyacrylamide gel electrophoresis ( sdspage ) on trishclpolyacrylamide gels ( biorad ) and transferred to polyvinylidene fluoride ( pvdf ) membranes . membranes were blocked with 5% milk in trisbuffered saline and tween 20 ( tbst ) and incubated in primary antibody diluted in 2% milk tbst overnight at 4 c , using antilox ( abcam ab31238 ; 1:500 ) for human and canine samples and antilox ( novus nbp224877 ; 1:500 ) for mouse samples . the membranes were washed , incubated in antirabbit horseradish peroxidase ( hrp)conjugated secondary antibody ( cell signaling technologies ) , and visualized using ecl plus reagents ( thermo scientific ) and autoradiography film ( bioexpress ) . to provide loading controls , membranes were stripped and either reprobed for glyceraldehyde 3phosphate dehydrogenase ( gapdh ; sc25778 ; 1:2,000 ; santa cruz biotechnology ) or stained with coomassie blue ( biorad ) or ponceau s ( sigmaaldrich ) solution for visualization . a collagen solubility assay was adapted22 , 23 to quantify the proportion of mature crosslinked collagen from non crosslinked and immature crosslinked collagen . briefly , flashfrozen muscle tissue was ground using a mortar and pestle over dry ice and then weighed . the tissue was washed in 1 ml of pbs and stirred for 30 minutes at 4 c before centrifuging at 16,000 g for 30 minutes at 4 c . crosslinked collagen was digested in a 1:6 ( weight : volume ) solution of 0.5 m acetic acid with 1 mg / ml pepsin , stirring overnight at 4 c . after centrifuging at 16,000 g for 30 minutes at 4 c , the supernatant was collected as the pepsinsoluble fraction ( psf ) , and the pellet was kept as the pepsininsoluble fraction ( pif ) . for the psf , a 1:1 volume of 4 m nacl was added and stirred for 30 minutes at 4 c . the psf was then centrifuged at 16,000 g for 30 minutes at 4 c , and the supernatant was discarded . the collagen contents of the psf and pif were then determined by a hydroxyproline assay . a hydroxyproline assay was performed to quantify collagen content in both the psf and pif , as described in previous studies.12 , 24 the samples were hydrolyzed overnight in 0.5 ml of 6 m hydrochloric acid at 105 c . 10 l of hydrolysate were mixed with 150 l isopropanol followed by 75 l of 1.4% chloraminet ( sigma , st . louis , missouri ) in citrate buffer and oxidized at room temperature for 10 minutes . the samples were then mixed with 1 ml of a 3:13 solution of ehrlich reagent [ 1.5 g of 4(dimethylamino)benzaldehyde ( sigma ) , 5 ml ethanol , and 337 l sulfuric acid ] to isopropanol and incubated for 45 minutes at 55 c . a standard curve ( 01,000 m , trans4hydroxylproline ; sigma ) was included in each assay . frozen octembedded grmd samples were sectioned at 10 m , and picosirius red staining was performed as previously described.12 briefly , sections were fixed in 4% paraformaldehyde for 10 minutes , rinsed , air dried , stained for 1 hour in 0.1% ( wt / vol ) sirius red ( sigmaaldrich ) , dissolved in saturated aqueous picric acid ( sigmaaldrich ) , washed in 2 changes of 0.5% acetic acid , dehydrated in 3 changes of 100% ethanol , cleared with citra solv , and mounted with cytoseal . the resulting slides were viewed under circularly polarized light scope ( dmlp ; leica ) , rotating polarizer , rotating analyzer , and dual quarter wave plates on a camera system ( micropublisher 5.0 ; q imaging ) . acetonefixed 10m sections of mouse , dog , and patient muscle were incubated overnight at 4 c with antilox ( abcam ab31238 ; 1:500 ) and antivimentin ( novus mb300 ; 1:800 ) primary antibodies using 5% bovine serum albumin ( bsa)pbs with 0.1% triton x100 as a blocking solution / diluent . alexa 568conjugated antirabbit igg ( molecular probes ; 1:500 ) and alexa 488conjugated antichicken igy ( jackson ; 1:500 ) secondary antibodies were used . slides were mounted using vectashield with 4,6diamidino2phenylindole ( dapi ; vector labs ) and imaged using the confocal microscope ( model tscsp8 ; leica ) . quantification of human lox immunoreactivity was achieved by analysis of 5 independent fields of view ( 20 ) from control ( n = 3 ) and dmd patient ( n = 3 ) quadriceps sections using smash muscle image analysis software.25 all results are presented as mean sem unless otherwise stated . statistical analysis of the mouse data was performed by 2way analysis of variance using genotype and muscle followed by a bonferroni post hoc test comparing genotypes within muscles and muscles within a genotype . for the human samples , a student ttest was used to compare muscles from unaffected to dmd subjects . for the dog samples there was only a single control dog , so only a paired student ttest was performed between muscles of grmd dogs . six and 18monthold male c57 and mdx mice were euthanized , and the tibialis anterior ( ta ) muscle and the diaphragms were dissected . they were either flash frozen in liquid nitrogen for biochemical analysis or embedded in oct and frozen in melting isopentane for histological evaluation . portions of ta and diaphragm were collected from 1yearold golden retriever muscular dystrophy ( grmd ) dogs ( n = 3 ) and an unaffected dog ( n = 1 ) following unrelated terminal endpoints and were either flash frozen or frozen in oct . samples of human quadriceps muscle ( n = 3 ) were obtained from the wellstone muscular dystrophy cooperative research center , tissue and cell repository at the university of iowa , carver college of medicine ( iowa city , iowa ) . the dmd patients were between 5 and 8 years of age , and control patients between 8 and 13 years of age at the time of biopsy . total rna was isolated from murine and canine muscles using trizol reagent ( invitrogen ) treated with dnase ( promega ) and reverse transcribed to cdna using the geneamp rna polymerase chain reaction ( pcr ) kit ( applied biosystems ) using the manufacturer 's directions . realtime pcr ( model 7300 ; applied biosystems ) was conducted on the resulting cdna with power sybr green master mix ( applied biosystems ) . expression levels of lox ( accession numbers : mouse , nm_010728 ; dog , xm_538599 ) , loxl2 ( mouse , xm_006519744 ) , and col1a2 ( mouse , nm_007743 ; dog , nm_001003187 ) were determined with 18s expression serving as the reference housekeeping gene . flashfrozen murine and canine muscle tissue was finely crushed and homogenized in tissue protein extraction reagent ( tper ; thermo scientific ) supplemented with protease and phosphatase inhibitor cocktails ( thermo scientific ) . quadriceps biopsy samples from healthy males and dmd patients ( pooled from 25 10m frozen sections from each patient ) were lysed in radioimmunoprecipitation assay ( ripa ) buffer supplemented with protease and phosphatase inhibitor cocktails ( sigmaaldrich ) . polyacrylamide gel electrophoresis ( sdspage ) on trishclpolyacrylamide gels ( biorad ) and transferred to polyvinylidene fluoride ( pvdf ) membranes . membranes were blocked with 5% milk in trisbuffered saline and tween 20 ( tbst ) and incubated in primary antibody diluted in 2% milk tbst overnight at 4 c , using antilox ( abcam ab31238 ; 1:500 ) for human and canine samples and antilox ( novus nbp224877 ; 1:500 ) for mouse samples . the membranes were washed , incubated in antirabbit horseradish peroxidase ( hrp)conjugated secondary antibody ( cell signaling technologies ) , and visualized using ecl plus reagents ( thermo scientific ) and autoradiography film ( bioexpress ) . to provide loading controls , membranes were stripped and either reprobed for glyceraldehyde 3phosphate dehydrogenase ( gapdh ; sc25778 ; 1:2,000 ; santa cruz biotechnology ) or stained with coomassie blue ( biorad ) or ponceau s ( sigmaaldrich ) solution for visualization . a collagen solubility assay was adapted22 , 23 to quantify the proportion of mature crosslinked collagen from non crosslinked and immature crosslinked collagen . briefly , flashfrozen muscle tissue was ground using a mortar and pestle over dry ice and then weighed . the tissue was washed in 1 ml of pbs and stirred for 30 minutes at 4 c before centrifuging at 16,000 g for 30 minutes at 4 c . crosslinked collagen was digested in a 1:6 ( weight : volume ) solution of 0.5 m acetic acid with 1 mg / ml pepsin , stirring overnight at 4 c . after centrifuging at 16,000 g for 30 minutes at 4 c , the supernatant was collected as the pepsinsoluble fraction ( psf ) , and the pellet was kept as the pepsininsoluble fraction ( pif ) . for the psf , a 1:1 volume of 4 m nacl was added and stirred for 30 minutes at 4 c . the psf was then centrifuged at 16,000 g for 30 minutes at 4 c , and the supernatant was discarded . the collagen contents of the psf and pif were then determined by a hydroxyproline assay . a hydroxyproline assay was performed to quantify collagen content in both the psf and pif , as described in previous studies.12 , 24 the samples were hydrolyzed overnight in 0.5 ml of 6 m hydrochloric acid at 105 c . 10 l of hydrolysate were mixed with 150 l isopropanol followed by 75 l of 1.4% chloraminet ( sigma , st . louis , missouri ) in citrate buffer and oxidized at room temperature for 10 minutes . the samples were then mixed with 1 ml of a 3:13 solution of ehrlich reagent [ 1.5 g of 4(dimethylamino)benzaldehyde ( sigma ) , 5 ml ethanol , and 337 l sulfuric acid ] to isopropanol and incubated for 45 minutes at 55 c . a standard curve ( 01,000 m , trans4hydroxylproline ; sigma ) was included in each assay . frozen octembedded grmd samples were sectioned at 10 m , and picosirius red staining was performed as previously described.12 briefly , sections were fixed in 4% paraformaldehyde for 10 minutes , rinsed , air dried , stained for 1 hour in 0.1% ( wt / vol ) sirius red ( sigmaaldrich ) , dissolved in saturated aqueous picric acid ( sigmaaldrich ) , washed in 2 changes of 0.5% acetic acid , dehydrated in 3 changes of 100% ethanol , cleared with citra solv , and mounted with cytoseal . the resulting slides were viewed under circularly polarized light scope ( dmlp ; leica ) , rotating polarizer , rotating analyzer , and dual quarter wave plates on a camera system ( micropublisher 5.0 ; q imaging ) . acetonefixed 10m sections of mouse , dog , and patient muscle were incubated overnight at 4 c with antilox ( abcam ab31238 ; 1:500 ) and antivimentin ( novus mb300 ; 1:800 ) primary antibodies using 5% bovine serum albumin ( bsa)pbs with 0.1% triton x100 as a blocking solution / diluent . alexa 568conjugated antirabbit igg ( molecular probes ; 1:500 ) and alexa 488conjugated antichicken igy ( jackson ; 1:500 ) secondary antibodies were used . slides were mounted using vectashield with 4,6diamidino2phenylindole ( dapi ; vector labs ) and imaged using the confocal microscope ( model tscsp8 ; leica ) . quantification of human lox immunoreactivity was achieved by analysis of 5 independent fields of view ( 20 ) from control ( n = 3 ) and dmd patient ( n = 3 ) quadriceps sections using smash muscle image analysis software.25 . statistical analysis of the mouse data was performed by 2way analysis of variance using genotype and muscle followed by a bonferroni post hoc test comparing genotypes within muscles and muscles within a genotype . for the human samples , a student ttest was used to compare muscles from unaffected to dmd subjects . for the dog samples there was only a single control dog , so only a paired student ttest was performed between muscles of grmd dogs . gene expression of the collagen crosslinking enzyme lox was evaluated at the ends of the mouse phenotypic spectrum by comparing a less affected limb muscle ( ta ) and the more severely affected diaphragm muscle of mdx mice at age 6 months to agematched c57 mice ( fig . although lox expression was not significantly different between the wildtype and dystrophic ta , it was significantly elevated in the diaphragms of mdx mice . accordingly , col1a2 expression was also highly upregulated in the mdx diaphragm ; however , expression of the lox family member , loxl2 , was unchanged across the samples . at the protein level ( fig . 1b ) , lox abundance was similarly increased in the mdx diaphragms at age 6 months compared with those of c57 mice and was also significantly higher in the dystrophic ta . ( a ) lox transcript level is increased in mdx muscle ( n = 7 ) over control ( n = 6 ) . col1a2 is increased in mdx muscles ( n = 7 ) over control ( n = 6 ) , and in mdx diaphragm over ta . loxl2 tends to be upregulated in mdx muscles ( n = 3 ) over control ( n = 3 ) . ( b ) representative portion of immunoblot for lox ( 32 kda ) with ponceau stain for loading control . lox protein is increased in mdx ( n = 3 ) over control ( n = 3 ) , with mdx diaphragm higher than ta . ( c ) soluble hydroxyproline content , representing non crosslinked or immature crosslinked collagen , of mdx ( n = 6 ) muscles are not different from c57 ( n = 6 ) . insoluble hydroxyproline content , representing crosslinked collagen , and total collagen are increased in mdx , with mdx diaphragm higher than control diaphragm or mdx ta for both insoluble and total collagen . ( d ) immunoblot of mdx diaphragm for lox shows no additional increase at 18 months ( n = 2 ) compared with 6 months . insoluble crosslinked collagen is higher at 18 months ( n = 2 ) compared with 6 months in mdx diaphragm . significantly different posthoc test , indicated by * p < 0.05 and * * p < 0.01 , between genotypes of same muscle ; p < 0.05 and p < 0.01 , between muscles of the same genotype ; and p < 0.05 , between ages of mdx diaphragm . the consequence of higher levels of lox was evaluated with a collagen solubility assay , where the soluble , insoluble , and total collagen levels were determined in mdx and control muscles ( fig . the amount of soluble collagen , representing non crosslinked and immature crosslinked collagen , was similar between all muscles analyzed and represented a minor proportion of the total collagen pool . in contrast , 6month mdx diaphragms had significantly more mature crosslinked insoluble collagen and total collagen than the mdx ta and agematched c57 diaphragms . to determine whether there was an agedependent increase in either lox or collagen , muscles from 18monthold mdx mice lox levels were similar between the 2 ages , but the mdx diaphragm continued to accrue insoluble collagen ( fig . 1d ) , whereas the ta insoluble content remained unchanged ( data not shown ) . these data demonstrate that lox is chronically elevated in the dystrophic mouse diaphragm , which results in progressive accumulation of crosslinked collagen . due to the relatively mild phenotype of the mdx mouse compared with dmd patients , we also investigated lox levels and collagen crosslinking in the more severe grmd dog model of dmd . like the mdx mouse , grmd exhibits differential disease severity between limb muscles and the diaphragm . similar to our findings in the mdx mouse , grmd diaphragm displayed high upregulation of both lox and col1a2 gene expression over control dog levels , with no change in the ta ( fig . 2a ) . lox protein , although not detectable in control dog samples by western blotting , was observed in grmd samples , where its abundance among grmd diaphragms was consistently higher than the dogmatched ta ( fig . accordingly , grmd diaphragms also contained more insoluble and total collagen than all other samples ( fig . 2c ) , in agreement with the findings in mice described above . lox expression and collagen crosslinking in the grmd dog model of dmd in limb and diaphragm muscle . ( a ) transcriptional level of lox and col1a2 ( b ) immunoblot for lox ( 32 kda ) using gapdh ( 37 kda ) as a loading control from wildtype and grmd dogs . protein level of lox is not observable in wildtype dogs , but is present in grmd muscles , with diaphragm consistently higher than dog matched ta . crosslinked collagen or immature crosslinked collagen , does not show a consistent difference between wildtype and grmd . ( d ) representative images of grmd dog ta and diaphragm muscles viewed under circularly polarized light . birefringent fibrillar collagen is visible with loosely ( green ) , moderately ( yellow ) , and densely ( red ) packed collagen . as previously performed on mdx mouse muscle,12 the collagen density of grmd muscle sections was visualized under circularly polarized light ( fig . in accordance with the biochemical data described above , the grmd ta contained mostly loosely packed collagen bundles ( visualized as green ) , with an extensive perimysial layer , whereas the diaphragm collagen bundles were largely intermediate ( yellow ) or densely ( orange to red ) packed , verifying that collagen density is increased in grmd diaphragm compared with limb muscle . it is also notable that , in the grmd diaphragm , unlike the ta , the robust endomysial expansion resulted in a lack of distinction between endomysial and perimysial stromal regions . thus , the canine model of dmd also displays heightened indices of collagen crosslinking in the diaphragm as compared with less affected limb muscle . lox crosslinking activity occurs in the ecm , but many different cell types can produce this enzyme , including fibroblasts and muscle fibers.19 , 26 , 27 , 28 to determine the distribution of excess lox in dystrophic muscle , immunofluorescence of lox and vimentin , a marker of mesenchemyl cells , including fibroblasts , was performed . in healthy muscle , lox was evident in sparse puncta associated with the ecm . for the mouse diaphragm , these areas were observed along the epimysium and also in small foci in the perimysium and perivascular areas ( fig . 3a and b ) . the same infrequent lox locations were observed in the wildtype mouse ta ( data not shown ) . for the diaphragm of an unaffected dog ( fig . of particular interest were muscle fibers that were positive for lox both inside and outside of the sarcolemma , suggesting that the muscle fiber itself was producing lox ( fig . hot spots , but these were more extensive both in number and in size ( fig . 3d ) . this included more pronounced distribution of lox along the endomysium , evident in the mdx ta muscle . taken together , the expanded distribution of lox in mdx and grmd muscle sections was consistent with the increased lox transcript and protein levels found in dystrophic muscles . confocal microscopic immunofluorescent images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections . ( a ) c57 mouse diaphragm shows epimysial focal regions of lox as well as ( b ) perimysial regions of focal lox , shown in insets . ( c ) unaffected dog diaphragm demonstrates similar focal expression of lox as c57 mice . ( d ) diaphragm and tibialis anterior ( ta ) samples from mdx mice and grmd dogs depict increased frequency of focal lox regions and endomysial lox . higher magnification outsets of ( b ) and ( c ) are indicated by dotted square regions of the lower magnification image . scale bars = 25 m , unless indicated otherwise . to substantiate the translational value of our animal model data , we measured lox protein in quadriceps biopsy samples from unaffected control subjects and dmd patients by western blotting ( fig . similar to the mouse and dog tissue , immunofluorescence of unaffected control human muscle demonstrated infrequent loxpositive loci , which were primarily located near the perimysial regions ( fig . , lox again had a broader distribution that included more frequent foci observed in both endomysial and perimysial regions . quantification of the loxpositive area fraction confirmed our western blotting results , with a 3fold increase compared with control subjects . these data demonstrate that the muscles of dmd patients have increased lox content and distribution , likely contributing to the progressive fibrotic replacement of muscle . ( a ) immunoblot for lox ( 32 kda ) using coomassie staining as a loading control from quadriceps biopsies of control ( n = 3 ) and dmd ( n = 3 ) patients . all lanes are on the same blot with intervening lanes excluded . quantification of lox blots from human patients indicates a significant increase in lox expression of dmd patients compared with unaffected controls . ( b ) immunofluorescent confocal microscopy images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections from human quadriceps muscle . area fractions are quantified showing 5 regions of interest ( circles ) from each subject biopsy with averages ( lines ) from dmd ( n = 3 ) and control ( n = 3 ) patients , demonstrating significantly increased lox staining in dmd muscle . significant difference indicated by * p < 0.05 for lox expression between human patient groups gene expression of the collagen crosslinking enzyme lox was evaluated at the ends of the mouse phenotypic spectrum by comparing a less affected limb muscle ( ta ) and the more severely affected diaphragm muscle of mdx mice at age 6 months to agematched c57 mice ( fig . although lox expression was not significantly different between the wildtype and dystrophic ta , it was significantly elevated in the diaphragms of mdx mice . accordingly , col1a2 expression was also highly upregulated in the mdx diaphragm ; however , expression of the lox family member , loxl2 , was unchanged across the samples . at the protein level ( fig . 1b ) , lox abundance was similarly increased in the mdx diaphragms at age 6 months compared with those of c57 mice and was also significantly higher in the dystrophic ta . ( a ) lox transcript level is increased in mdx muscle ( n = 7 ) over control ( n = 6 ) . col1a2 is increased in mdx muscles ( n = 7 ) over control ( n = 6 ) , and in mdx diaphragm over ta . loxl2 tends to be upregulated in mdx muscles ( n = 3 ) over control ( n = 3 ) . ( b ) representative portion of immunoblot for lox ( 32 kda ) with ponceau stain for loading control . lox protein is increased in mdx ( n = 3 ) over control ( n = 3 ) , with mdx diaphragm higher than ta . ( c ) soluble hydroxyproline content , representing non crosslinked or immature crosslinked collagen , of mdx ( n = 6 ) muscles are not different from c57 ( n = 6 ) . insoluble hydroxyproline content , representing crosslinked collagen , and total collagen are increased in mdx , with mdx diaphragm higher than control diaphragm or mdx ta for both insoluble and total collagen . ( d ) immunoblot of mdx diaphragm for lox shows no additional increase at 18 months ( n = 2 ) compared with 6 months . insoluble crosslinked collagen is higher at 18 months ( n = 2 ) compared with 6 months in mdx diaphragm . significantly different posthoc test , indicated by * p < 0.05 and * * p < 0.01 , between genotypes of same muscle ; p < 0.05 and p < 0.01 , between muscles of the same genotype ; and p < 0.05 , between ages of mdx diaphragm . the consequence of higher levels of lox was evaluated with a collagen solubility assay , where the soluble , insoluble , and total collagen levels were determined in mdx and control muscles ( fig . the amount of soluble collagen , representing non crosslinked and immature crosslinked collagen , was similar between all muscles analyzed and represented a minor proportion of the total collagen pool . in contrast , 6month mdx diaphragms had significantly more mature crosslinked insoluble collagen and total collagen than the mdx ta and agematched c57 diaphragms . to determine whether there was an agedependent increase in either lox or collagen , muscles from 18monthold mdx mice lox levels were similar between the 2 ages , but the mdx diaphragm continued to accrue insoluble collagen ( fig . 1d ) , whereas the ta insoluble content remained unchanged ( data not shown ) . these data demonstrate that lox is chronically elevated in the dystrophic mouse diaphragm , which results in progressive accumulation of crosslinked collagen . due to the relatively mild phenotype of the mdx mouse compared with dmd patients , we also investigated lox levels and collagen crosslinking in the more severe grmd dog model of dmd . like the mdx mouse , grmd exhibits differential disease severity between limb muscles and the diaphragm . similar to our findings in the mdx mouse , grmd diaphragm displayed high upregulation of both lox and col1a2 gene expression over control dog levels , with no change in the ta ( fig . 2a ) . lox protein , although not detectable in control dog samples by western blotting , was observed in grmd samples , where its abundance among grmd diaphragms was consistently higher than the dogmatched ta ( fig . accordingly , grmd diaphragms also contained more insoluble and total collagen than all other samples ( fig . 2c ) , in agreement with the findings in mice described above . lox expression and collagen crosslinking in the grmd dog model of dmd in limb and diaphragm muscle . ( a ) transcriptional level of lox and col1a2 ( b ) immunoblot for lox ( 32 kda ) using gapdh ( 37 kda ) as a loading control from wildtype and grmd dogs . protein level of lox is not observable in wildtype dogs , but is present in grmd muscles , with diaphragm consistently higher than dog matched ta . crosslinked collagen or immature crosslinked collagen , does not show a consistent difference between wildtype and grmd . ( d ) representative images of grmd dog ta and diaphragm muscles viewed under circularly polarized light . birefringent fibrillar collagen is visible with loosely ( green ) , moderately ( yellow ) , and densely ( red ) packed collagen . scale bar = 50 m . as previously performed on mdx mouse muscle,12 the collagen density of grmd muscle sections was visualized under circularly polarized light ( fig . in accordance with the biochemical data described above , the grmd ta contained mostly loosely packed collagen bundles ( visualized as green ) , with an extensive perimysial layer , whereas the diaphragm collagen bundles were largely intermediate ( yellow ) or densely ( orange to red ) packed , verifying that collagen density is increased in grmd diaphragm compared with limb muscle . it is also notable that , in the grmd diaphragm , unlike the ta , the robust endomysial expansion resulted in a lack of distinction between endomysial and perimysial stromal regions . thus , the canine model of dmd also displays heightened indices of collagen crosslinking in the diaphragm as compared with less affected limb muscle . lox crosslinking activity occurs in the ecm , but many different cell types can produce this enzyme , including fibroblasts and muscle fibers.19 , 26 , 27 , 28 to determine the distribution of excess lox in dystrophic muscle , immunofluorescence of lox and vimentin , a marker of mesenchemyl cells , including fibroblasts , was performed . in healthy muscle , lox was evident in sparse puncta associated with the ecm . for the mouse diaphragm , these areas were observed along the epimysium and also in small foci in the perimysium and perivascular areas ( fig . 3a and b ) . the same infrequent lox locations were observed in the wildtype mouse ta ( data not shown ) . for the diaphragm of an unaffected dog ( fig . of particular interest were muscle fibers that were positive for lox both inside and outside of the sarcolemma , suggesting that the muscle fiber itself was producing lox ( fig . hot spots , but these were more extensive both in number and in size ( fig . this included more pronounced distribution of lox along the endomysium , evident in the mdx ta muscle . taken together , the expanded distribution of lox in mdx and grmd muscle sections was consistent with the increased lox transcript and protein levels found in dystrophic muscles . confocal microscopic immunofluorescent images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections . ( a ) c57 mouse diaphragm shows epimysial focal regions of lox as well as ( b ) perimysial regions of focal lox , shown in insets . ( c ) unaffected dog diaphragm demonstrates similar focal expression of lox as c57 mice . ( d ) diaphragm and tibialis anterior ( ta ) samples from mdx mice and grmd dogs depict increased frequency of focal lox regions and endomysial lox . higher magnification outsets of ( b ) and ( c ) are indicated by dotted square regions of the lower magnification image . to substantiate the translational value of our animal model data , we measured lox protein in quadriceps biopsy samples from unaffected control subjects and dmd patients by western blotting ( fig . similar to the mouse and dog tissue , immunofluorescence of unaffected control human muscle demonstrated infrequent loxpositive loci , which were primarily located near the perimysial regions ( fig . , lox again had a broader distribution that included more frequent foci observed in both endomysial and perimysial regions . quantification of the loxpositive area fraction confirmed our western blotting results , with a 3fold increase compared with control subjects . these data demonstrate that the muscles of dmd patients have increased lox content and distribution , likely contributing to the progressive fibrotic replacement of muscle . ( a ) immunoblot for lox ( 32 kda ) using coomassie staining as a loading control from quadriceps biopsies of control ( n = 3 ) and dmd ( n = 3 ) patients . quantification of lox blots from human patients indicates a significant increase in lox expression of dmd patients compared with unaffected controls . ( b ) immunofluorescent confocal microscopy images of lysyl oxidase ( lox ; red ) , vimentin ( green ) , and dapi ( blue ) on 10mthick cryosections from human quadriceps muscle . area fractions are quantified showing 5 regions of interest ( circles ) from each subject biopsy with averages ( lines ) from dmd ( n = 3 ) and control ( n = 3 ) patients , demonstrating significantly increased lox staining in dmd muscle . significant difference indicated by * p < 0.05 for lox expression between human patient groups . skeletal muscle fibrosis is an excessive deposition of ecm components and accompanies the chronic damage that occurs in muscular dystrophy . little is known about how this aberrant ecm is organized in fibrotic dystrophic muscle , but there is evidence that posttranslational modification of the ecm may be part of the dystrophic pathology . previous measurements of a rarely used chicken model of muscular dystrophy have shown increased proportions of highmolecularweight collagen suggestive of increased crosslinking.29 in addition , studies using mdx mice demonstrated increased lox expression in heart and limb muscle20 and histological association between lox and fibrosis.17 we have now extended these observations to show that lox protein is increased in dmd patient muscle , as well as in mouse and dog dmd models . in dystrophic mouse and dog diaphragm , where pathology is the most severe,10 there is also more collagen crosslinking , as demonstrated by the increased insoluble fraction of collagen . insoluble collagen content increased progressively in the mdx diaphragm , demonstrating the negative implications of chronic lox elevation . we also demonstrated that the increase in lox is conserved across species with muscular dystrophy . taken together , we conclude that increased lox leads to extensive collagen crosslinking , which is an important feature of dmd muscle . although the mdx mouse is the most commonly studied model of dmd , it does not typically develop fibrosis in the limbs at a young age , and it has several differences from the human disease.4 , 30 lifespan is only moderately affected in the mdx mouse , and , although limb muscles undergo cycles of degeneration and regeneration , there is relatively little fibrosis . the mdx diaphragm most accurately reflects the muscle pathology observed in dmd patients,10 thus it is not surprising that our measurements of crosslinking were also most prominent in the mdx diaphragm compared with the ta muscles . this is consistent with our previous observations of increased collagen packing density in the mdx diaphragm.12 to determine whether our results were consistent in a model that more accurately exhibits features of dmd , we used the grmd dog model.31 grmd dogs also exhibit increased crosslinked collagen and increased lox levels , supporting collagen crosslinking as a major feature of the disease that is maintained across species . part of the reason we could not detect this difference , as one would expect for larger mammals , is the presence of a very thick perimysium layer that is found in both unaffected healthy and dystrophic dog ta muscles . this layer is an important structural component of limb muscles in dogs and is crosslinked to a greater extent than the rest of the ecm in this muscle . consistent with this is the localization of more densely packed collagen within the ta perimysium compared with the endomysium . as such , the collagen solubility measurements of muscle homogenates where the perimysium is the overwhelmingly dominant source of matrix likely masks the changes that may have occurred in the endomysial layer around the fibers . in contrast , the grmd diaphragm possessed more densely packed collagen fibrils throughout the muscle , which was accompanied by an increase in collagen insolubility . to verify that the elevation of lox observed in animal models is also a feature of dmd , human biopies were examined . in agreement with the animal models , lox expression was shown to be elevated in quantity as well as in frequency of hot spots with high focal expression . this is also consistent with 3 published microarray studies in the gene expression omnibus database,32 which showed significant upregulation of lox in dmd ( p = 4.9 10 , gds21433 ; p = 6.8 10 , gds302734 ; p = 4.7 10 , gds56335 . although we were not able to measure collagen crosslinking in human samples directly , the increased lox levels suggest that muscle in dmd is more extensively crosslinked as observed in animal models . collagen crosslinking has been described as a major factor in fibrosis of multiple tissues,36 , 37 including the heart.38 the role of collagen crosslinking in the heart is of great clinical significance in patients with dmd , as it also undergoes a fibrotic response . whereas lox mrna is known to be increased in mdx cardiac muscle,20 lox protein levels and collagen crosslinking have not been studied but should be a target of future investigation . increased collagen crosslinking is thought to lead to a stiffer ecm and tissue , as demonstrated in the left ventricle of the fibrotic heart.38 however , recent reports of muscle stiffness in mdx mice have shown relatively more stiffening of limb muscle as opposed to the diaphragm,12 even though crosslinking is most prevalent in the diaphragm . collagen content shows little correlation with passive stiffness,12 but collagen crosslinking does not account for additional variability in muscle fiber bundle stiffness.39 this suggests that , although collagen may be stiffer at the molecular level , other parameters of collagen organization dictate muscle stiffness at the tissue level . however , this does not preclude the microstiffness of the crosslinked collagen fibrils from playing a critical role in the cellular response to collagen crosslinking in fibrosis . lox is critical to the integrity of the developing diaphragm , as mice that lack lox often die perinatally from diaphragmatic hernias.40 further , skeletal muscle development is disrupted in lox knockout mice independent of skeletal or tendon defects , demonstrating a role for lox in this process.28 loxl1loxl4 are also capable of crosslinking collagen , but little is known about their expression in muscle . the loxl1loxl4 family members have a conserved domain responsible for catalyzing the initial reaction in collagen crosslinking . their distinct roles are not well understood , but loxl2 has been shown to be upregulated in fibrotic tissue and responsible for collagen crosslinking in fibrosis.37 our initial investigation showed that loxl2 expression is not significantly altered in dystrophic muscle , providing supporting evidence for lox being the primary enzyme responsible for crosslinking changes in dystrophic muscle . critically , it is well established that crosslinked collagen is much less susceptible to turnover,21 thus implicating collagen crosslinking in the development of progressive scar tissue formation that predominates in muscles from dmd patients . lox is known to be produced by vascular cells , including endothelial cells,26 smooth muscle,19 and fibroblasts,27 which are all muscle resident cells ; however , a recent report by kutchuk et al . suggested skeletal muscle is also a major contributor of lox expression , in which myofiber lox secretion is a negative feedback switch for stromal collagen production and tgf activity.28 although evidence of lox production from a specific cell type was not conclusive in our study , we observed scattered stromal foci of lox immunoreactivity throughout muscle crosssections , as well as evidence of staining within muscle fibers . in healthy muscle , these foci are typically localized to perimysial and epimysial regions , which are points of high mechanical stress in contracting muscle . the increased frequency and intrafascicular presence of lox staining in dystrophic muscle could be due to the increased mechanical burden experienced by dystrophindeficient muscle fibers from reduced attachment to the ecm . taken together , these lines of evidence suggest lox secretion into the ecm region is likely regulated by myofiber mechanical sensing , which leads to appropriate ecm adaptation to stress stimuli in nonpathological situations . unfortunately , dystrophic muscle fibers are impaired in their attachment to the ecm independent of the extent of ecm accumulation and/or crosslinking , thereby preventing the shutdown of collagen and lox production . this scenario potentially results in continuous endomysial ecm accumulation , which leads to replacement of functional muscle with fibrosis , as seen in dmd patients.41 these studies raise important questions regarding the best therapeutic targets for fibrosis . if lox is critical for reducing tgf actions , then targeting it may cause rampant collagen production and lead to increased fibrosis . however , if excessive lox activity on ecm collagen crosslinking is detrimental to muscle function , then lox inhibition may be beneficial to diseased muscles where reduced stiffness may also lessen structural damage to muscle fibers . clinically , therapies that target lox and lox family member activity show promise in preventing lung and liver fibrosis37 and set a precedent for trials to counter muscle fibrosis using this strategy . however , the balance between the extent of fibrosis and the proportion of crosslinking is a major consideration with respect to the therapeutic window for treatments . because crosslinked collagen is relatively stable and refractory to remodeling,18 antifibrotic therapies may not remove crosslinked collagen , yet they may gradually reduce overall levels of fibrosis . this could be important if antifibrotic therapies are applied to latestage disease patients in whom considerable cellular loss has occurred in the heart and diaphragm . at that disease stage , removal of all fibrosis could lead to herniation of the diaphragm and aneurysms in the heart , both lethal consequences . in that case , the crosslinked collagen would safeguard against these potentially lifethreatening events . min liu and zuozhen tian from the physiological assessment core at the university of pennsylvania . steven moore from the wellstone muscular dystrophy cooperative research center for providing human biopsies , and dr . margaret sleeper from the department of small animal clinical sciences , university of florida college of veterinary medicine , for providing dog tissue .

a 47-year - old turkish male was scheduled for laparoscopic cholecystectomy under general anesthesia . the patient had 2 operations 28 and 19 years ago under general anesthesia . it was learned that the patient was administered succinylcholine during both of these previous operations and that he did not have a history of prolonged recovery or postoperative apnea . the patient had been using sertraline for 3 years before the operation . pseudocholinesterase is a drug - metabolizing enzyme responsible for hydrolysis of the muscle - relaxant drugs mivacurium and succinylcholine . deficiency of this enzyme from any cause can lead to prolonged apnea and paralysis following administration of mivacurium and succinylcholine . the diagnosis of pseudocholinesterase enzyme deficiency can be made after careful clinic supervision and peripheral nerve stimulator monitoring . a decrease in the activity of pseudocholinesterase enzyme and a decline in the block effect over time will help verify the diagnosis . our patient s plasma cholinesterase was found to have low activity . instead of pharmacological interventions that may further complicate the situation in such cases , the preferred course of action should be to wait until the block effect declines with the help of sedation and mechanical ventilation . in our case , the prolonged block deteriorated in the course of time before any complications developed . abstractintroductioncollagen crosslinking is a key parameter in extracellular matrix ( ecm ) maturation , turnover , and stiffness . we examined aspects of collagen crosslinking in dystrophindeficient murine , canine , and human skeletal muscle.methodsdmd patient biopsies and samples from mdx mice and golden retriever muscular dystrophy dog samples ( with appropriate controls ) were analyzed . collagen crosslinking was evaluated using solubility and hydroxyproline assays . expression of the crosslinking enzyme lysyl oxidase ( lox ) was determined by realtime polymerase chain reaction , immunoblotting , and immunofluorescence.resultslox protein levels are increased in dystrophic muscle from all species evaluated . dystrophic mice and dogs had significantly higher crosslinked collagen than controls , especially in the diaphragm . distribution of intramuscular lox was heterogeneous in all samples , but it increased in frequency and intensity in dystrophic muscle.conclusionthese findings implicate elevated collagen crosslinking as an important component of the disrupted ecm in dystrophic muscles , and heightened crosslinking is evident in mouse , dog , and man . muscle nerve 54 : 7178 , 2016

with the aging of the population worldwide , dementia is a real public health priority . in 2000 , the estimated number of dementia cases was 25.5 million people , representing 0.4% of the worldwide population . nearly two thirds of all people with dementia lived in low- and middle - income countries . the other most important risk factors are low level of education , heredity , low early - life economic conditions , smoking , alcohol , weak social network , hypertension , cardiovascular diseases , stroke , epilepsy , diabetes , anemia , parkinson 's disease , and head trauma . although several population - based studies were conducted in developed countries to better understand the epidemiology of this new epidemic , only a few studies have been done in africa . in senegal , the estimated number of elderly aged 65 years was 421,305 in 2008 and 420,795 in 2009 . considering the economic cost of dementia care , senegal is not able to afford such cost . it is important to have reliable information on the prevalence of dementia to plan for more accurate provision of social and medical services for the elderly population . studies on dementia in the senegalese elderly population are rare [ 20 , 21 ] . thus , a study was conducted in senegal to determine the epidemiology of dementia in an elderly population utilizing a primary health care service for retirees in dakar , senegal . the aim of the present study was to identify risk factors associated with dementia in this population of retirees utilizing the social and medical center ( smc ) of ipres ( institution de prevoyance retraite du senegal ) , dakar , senegal . this is a health center for the retired senegalese elderly population affiliated to ipres ensuring medical and social services . as a primary care center with diverse health personnel , the study population was composed of senegalese elderly patients aged 65 years who came to the smc of ipres for health problems . those patients who were either < 65 years old or were not able to fulfill the interview ( due to aphasia , delirium , coma , extreme visual and auditory impairment , or cancer at terminal phase ) were excluded . 507 elderly patients aged 65 years or their relatives who consulted a doctor for a medical problem at the smc of ipres were first assessed with a screening interview questionnaire . those who were cognitively impaired , with a sex - matched sample , underwent a clinical exam with neuropsychological testing . this study was the first step of a research on the validation of a screening tool to screen for dementia in a senegalese elderly population called the test of senegal . two screening instruments were used : the interview questionnaire called aging in senegal and the clinical assessment instrument . the screening interview questionnaire aging in senegal contained the following : sociodemographic variables ( age , sex , marital status , education ) , medical history [ vascular diseases ( hypertension , heart diseases , vascular peripheral disease , stroke , diabetes ) , respiratory diseases , arthritis , cancer / benign tumor , parkinson 's disease , epilepsy , genitourinary disease , cataract , glaucoma , hearing impairment , digestive disease ( gastritis , constipation ) , anemia , thyroid disease , head trauma , bone fracture ] , familial history of memory impairment , lifestyle ( smoking , alcohol consumption , walking ) , social network ( social ties with spouse , children , brothers / sisters , friends ; frequency of weekly contact with children , brothers / sisters , friends ; members of community association , member of religious association ) , the patient 's functional autonomy , and the neuropsychological tests with the abbreviated mental test and the test of senegal . the clinical assessment instrument had four components : ( 1 ) a historical review of the patient 's cognitive function , i.e. , the onset and progression of any reported symptoms of cognitive impairment ; ( 2 ) a review of the patient 's medical , surgical , and familial history , exposure to toxic products and medications ; ( 3 ) a review of the patient 's functional autonomy , and ( 4 ) a review of the patient 's clinical exam and neuropsychological testing with the mini - mental state examination ( mmse ) . each patient underwent a screening interview with the questionnaire aging in senegal by four medical students at the smc who were trained for this issue . it aimed to collect data related to sociodemographics , lifestyle , past medical history , and social network and to screen for neuropsychological performance with the abbreviated mental test . after the interview , each patient who had a score of 5 on the abbreviated mental test was referred for clinical assessment to the principal investigator ( k.t . ) . at the same time , a sex - matched normal subject was also referred for clinical assessment . the clinical assessment consisted of a complete physical exam followed by a neuropsychological testing with the mmse . if a patient was suspected to have depression , the ces - d scale was administered to him / her to confirm the diagnosis . all clinical assessments were made without knowledge of the screening status of the patient . at the end of the consultation , the team members met in a room to confirm the diagnosis of the patient . on the basis of the examination , appropriate laboratory exams and computerized tomography of the head were ordered and treatment of associated medical conditions proposed . sociodemographic variables [ age in 4 categories ( 6569 , 7074 , 7579 , and 80 years ) , sex , marital status , education ] were collected with the medical history and familial history of cognitive impairment . lifestyle was divided into smoking habit ( yes , no ) , alcohol consumption ( yes , no ) , and walking ( yes , no ) . for the social network , we computed two indexes : diversities of social ties ( score 04 ) and frequency of weekly contact with relatives ( score 06 ) . diversity of social ties was computed by summing having a spouse or husband , children , brothers / sisters , and friends and categorized into 3 levels : 02 ties , 3 ties , and 4 ties . frequency of weekly contact with relatives was obtained by summing the frequency of weekly contact with children , brothers / sisters , and friends and categorized into 4 levels : 03 weekly contacts , 4 weekly contacts , 5 weekly contacts , and 6 weekly contacts . the medical conditions related to medical variables were dichotomized into yes or no. dementia was defined according to the dsm - iv - r criteria [ 27 , 28 ] . this study was approved by the ethical committee of the senegalese ministry of health and university of montreal , que . , this is a health center for the retired senegalese elderly population affiliated to ipres ensuring medical and social services . as a primary care center with diverse health personnel , the study population was composed of senegalese elderly patients aged 65 years who came to the smc of ipres for health problems . those patients who were either < 65 years old or were not able to fulfill the interview ( due to aphasia , delirium , coma , extreme visual and auditory impairment , or cancer at terminal phase ) were excluded . 507 elderly patients aged 65 years or their relatives who consulted a doctor for a medical problem at the smc of ipres were first assessed with a screening interview questionnaire . those who were cognitively impaired , with a sex - matched sample , underwent a clinical exam with neuropsychological testing . this study was the first step of a research on the validation of a screening tool to screen for dementia in a senegalese elderly population called the test of senegal . two screening instruments were used : the interview questionnaire called aging in senegal and the clinical assessment instrument . the screening interview questionnaire aging in senegal contained the following : sociodemographic variables ( age , sex , marital status , education ) , medical history [ vascular diseases ( hypertension , heart diseases , vascular peripheral disease , stroke , diabetes ) , respiratory diseases , arthritis , cancer / benign tumor , parkinson 's disease , epilepsy , genitourinary disease , cataract , glaucoma , hearing impairment , digestive disease ( gastritis , constipation ) , anemia , thyroid disease , head trauma , bone fracture ] , familial history of memory impairment , lifestyle ( smoking , alcohol consumption , walking ) , social network ( social ties with spouse , children , brothers / sisters , friends ; frequency of weekly contact with children , brothers / sisters , friends ; members of community association , member of religious association ) , the patient 's functional autonomy , and the neuropsychological tests with the abbreviated mental test and the test of senegal . the clinical assessment instrument had four components : ( 1 ) a historical review of the patient 's cognitive function , i.e. , the onset and progression of any reported symptoms of cognitive impairment ; ( 2 ) a review of the patient 's medical , surgical , and familial history , exposure to toxic products and medications ; ( 3 ) a review of the patient 's functional autonomy , and ( 4 ) a review of the patient 's clinical exam and neuropsychological testing with the mini - mental state examination ( mmse ) . each patient underwent a screening interview with the questionnaire aging in senegal by four medical students at the smc who were trained for this issue . it aimed to collect data related to sociodemographics , lifestyle , past medical history , and social network and to screen for neuropsychological performance with the abbreviated mental test . after the interview , each patient who had a score of 5 on the abbreviated mental test was referred for clinical assessment to the principal investigator ( k.t . ) . at the same time , a sex - matched normal subject was also referred for clinical assessment . the clinical assessment consisted of a complete physical exam followed by a neuropsychological testing with the mmse . if a patient was suspected to have depression , the ces - d scale was administered to him / her to confirm the diagnosis . all clinical assessments were made without knowledge of the screening status of the patient . at the end of the consultation , the team members met in a room to confirm the diagnosis of the patient . on the basis of the examination , patients with dementia were followed by the principal investigator ( k.t . ) . appropriate laboratory exams and computerized tomography of the head were ordered and treatment of associated medical conditions proposed . sociodemographic variables [ age in 4 categories ( 6569 , 7074 , 7579 , and 80 years ) , sex , marital status , education ] were collected with the medical history and familial history of cognitive impairment . lifestyle was divided into smoking habit ( yes , no ) , alcohol consumption ( yes , no ) , and walking ( yes , no ) . for the social network , we computed two indexes : diversities of social ties ( score 04 ) and frequency of weekly contact with relatives ( score 06 ) . diversity of social ties was computed by summing having a spouse or husband , children , brothers / sisters , and friends and categorized into 3 levels : 02 ties , 3 ties , and 4 ties . frequency of weekly contact with relatives was obtained by summing the frequency of weekly contact with children , brothers / sisters , and friends and categorized into 4 levels : 03 weekly contacts , 4 weekly contacts , 5 weekly contacts , and 6 weekly contacts . the medical conditions related to medical variables were dichotomized into yes or no. dementia was defined according to the dsm - iv - r criteria [ 27 , 28 ] . this study was approved by the ethical committee of the senegalese ministry of health and university of montreal , que . , univariate , bivariate , and multivariate stepwise logistic regression analyses were computed . the odd ratios of the different risk factors were computed with a 95% ci . forty - five patients ( 8.87% ; 95% ci 7.6110.13 ) had dementia . the whole population with a mean age of 72.4 years ( 5.2 ) was mostly male , married , and illiterate . the elderly population had a high diversity of ties and frequency of contact with relatives and friends ( table 1 ) . hypertension ( 58.6% ) , arthritis ( 49.5% ) , gastrointestinal diseases ( 24.1% ) , respiratory diseases ( 14.7% ) , and cataract ( 14.4% ) were the main health conditions reported in the past medical history ( table 2 ) . in the bivariate analysis , age ( p < 0.045 ) , education ( p < 0.02 ) , diversity of ties with relatives ( p < 0.001 ) , frequency of contact with relatives ( p < 0.0001 ) , stroke ( p < 0.002 ) , epilepsy ( p < 0.002 ) , and family history of dementia ( p < 0.0001 ) were associated with dementia . in the multivariate model , advanced age ( 80 years ; or 4.26 ; 95% ci 1.3613.3 ) , illiteracy ( or 2.76 ; 95% ci 1.236.20 ) , epilepsy ( or 11.72 ; 95% ci 2.1065.23 ) , and family history of dementia ( or 7.56 ; 95% ci 3.4216.76 ) were independently associated with dementia ( table 3 ) . in our study , prevalence of dementia was 8.87% with advanced age , illiteracy , epilepsy , and family history of dementia as independent risk factors . these risk factors were either self - reported by the patient or informant - reported . the role of age as a risk factor for dementia has been highlighted in several studies . in fact , the prevalence of dementia is increasing importantly with age as observed in a clinical setting and during population studies . thus , the result we observed confirms the role of ageing ( especially advanced age ) in the occurrence of dementia as described elsewhere in europe [ 3 , 29 , 30 , 31 ] , america [ 32 , 33 , 34 ] , asia [ 35 , 36 , 37 ] , and africa [ 21 , 38 , 39 , 40 , 41 ] . illiteracy was frequent ( 57% ) and was associated with dementia in our elderly population . this result confirms the role of illiteracy as a risk factor for dementia in an elderly population as already seen in studies realized worldwide [ 42 , 43 , 44 , 45 , 46 ] . epilepsy in the elderly is frequent , with an estimated prevalence of 1% after 60 years of age and an incidence increasing with age . in our study population , history of epilepsy this finding has been observed in several studies conducted worldwide , enhancing the association between a history of epilepsy and dementia in an elderly population [ 13 , 49 , 50 , 51 , 52 ] . this association could be related to the disease itself , its cause or risk factor , but also to the treatment prescribed , especially in our country , where most of the epileptic patients are on a first - generation anti - epileptic regimen , which is harmful for cognition . familial history of dementia , which was observed in 9.1% of our study population , has been associated with the occurrence of dementia . in fact , studies in australia , africa [ 55 , 56 ] , europe [ 57 , 58 ] , and the usa have demonstrated that familial history of dementia is a main risk factor for dementia in an elderly population . also , apoe 4 has been associated with alzheimer 's disease in the developed world [ 59 , 60 ] . however , this association is absent in an african study . genetic mutations are also observed in demented persons at chromosome 12 and chromosomes 6 , 9 , 10 , 12 , 19 , and 21 [ 63 , 64 ] . limitations of the study include the underestimation of the prevalence because all the demented patients were not recruited during the study period . medical illnesses were based upon self - report or informant report rather than on direct examination . moreover , memory bias could underestimate risk factors and , thus , their role in the occurrence of dementia . also , this study is a prevalence study , not an incidence study , characterized by temporality of the association between risk factors and dementia . however , it highlights and confirms the role of these risk factors on dementia occurrence in an elderly population . this study has shown that illiteracy , epilepsy , and familial history of dementia were associated with dementia in this study population . additionally , it confirms the role of these risk factors in the occurrence of dementia in an elderly population as described elsewhere . as the senegalese population is aging , it is necessary to take dementia into consideration in the social , political , and medical practice and to sensitize political leaders , the health personnel , and the population for the prevention of this disease . see detailed information on materials , virus construction , quantitative pcr , and adipocyte area measurement in the supplementary methods . adipoq mice were created as previously described ( 5 ) on the 129/svev genetic background and had been back - crossed to c57bl/6 for five generations . the experiments using mouse models were carried out under the association for assessment and accreditation of laboratory animal care guidelines with approval of the animal care and use committee . for some animal studies , 1 10 pfu of purified adenovirus vectors was injected into the mouse tail vein ( 6 ) . 3t3-l1 and 3t3-l1car1 cells and induction of adipocyte differentiation were described in a previous publication ( 7 ) . op9 adipocyte differentiation was induced using knockout sr ( 8) . although both 3t3-l1 and op9 adipocytes secrete adiponectin into the medium , adiponectin in overnight cultured medium accumulated only to 0.1% of mouse serum adiponectin . therefore , co - culture or conditioned medium was used to increase adiponectin protein levels . for co - culture , adipocytes were cultured in the outside well of the bd biosciences ( franklin lakes , nj ) transwell co - culture system , and fao cells were grown on the protein and cytokine - permeable membrane of the insert well . fao cells were transduced with adenovirus encoding mouse adiponectin ( ad - acrp30 ) or green fluorescent protein ( gfp ) for 12 h in a regular 6-well plate , and then the inserts with transduced fao cells were washed and transferred to the co - culture plates with differentiated adipocytes in the bottom wells . this system allows increase of medium adiponectin levels ( 40% of mouse serum adiponectin ) without physical interaction between 3t3-l1 adipocytes and fao cells . in a separate setting , conditioned medium from ad - acrp30transduced fao cells was collected and used for increasing adiponectin levels in some of the studies , as indicated in figure legends . for hormone - stimulated lipolysis , bt2-camp ( 100 mol / l ) was added to culture medium and cells were incubated for 1 h. total protein was used for certain protein or protein phosphorylation measurements using western blots . for pka rii protein half - life measurement , cycloheximide ( 100 g / ml ) was used to inhibit protein synthesis . for the in vivo lipolysis assays , the selective 3-adrenergic agonist brl37344 was injected i.p . into mice at 5 g / g body blood samples were collected 0 , 10 , and 20 min after the injection . for the lipolysis assay of adipose explants , 20 mg of epididymal adipose tissue explants were cultured in dulbecco s modified eagle s medium with 0.5% fatty acid brl37344 was added to the medium at 50 ng / ml . the culture tubes were shaken at 400 rpm . the medium samples were collected 30 , 60 , 120 , and 180 min after adding brl37344 . the level of free fatty acid ( ffa ) or glycerol was normalized to the weight of adipose explants . for primary adipocyte lipolysis assays , adipocytes were prepared from epididymal fat using collagenase with an optimized procedure ( 11,12 ) . briefly , epididymal fat tissue was minced and digested in a krebs - ringer bicarbonate ( krb ) buffer ( ph 7.4 ) supplemented with 3% fatty acid free bsa fraction v , 0.5 mmol / l adenosine , and 1 mg / ml type i collagenase . after 40-min digestion and three washes , isolated adipocytes were resuspended in krb supplemented with 5 mmol / l glucose and 3% fatty acid . suspended adipocytes ( 6,000 cells ) were used for a lipolysis assay in 400 l krb buffer plus 3% fatty acid free bsa , 1 unit / ml adenosine deaminase , and 100 nmol / l ( -)-n-(2-phenyl - isopropyl)-adenosine ( basal only ) with or without brl37344 ( 50 ng / ml ) . after 1-h incubation at 37c , 200 l of infranatant was removed and stored at 20c . serum free glycerol and glycerol released from fat explants were measured using a free - glycerol kit ( sigma - aldrich co. , st . glycerol in the assay media of primary and 3t3-l1 adipocytes was measured using bioluminescence , which is very sensitive and especially well adapted when only small amounts of adipose tissue or cells are available ( 13,14 ) . statistical analysis was performed using the student t test or anova , followed by contrast test . adipoq mice were created as previously described ( 5 ) on the 129/svev genetic background and had been back - crossed to c57bl/6 for five generations . the experiments using mouse models were carried out under the association for assessment and accreditation of laboratory animal care guidelines with approval of the animal care and use committee . for some animal studies , 1 10 pfu of purified adenovirus vectors was injected into the mouse tail vein ( 6 ) . 3t3-l1 and 3t3-l1car1 cells and induction of adipocyte differentiation were described in a previous publication ( 7 ) . op9 adipocyte differentiation was induced using knockout sr ( 8) . although both 3t3-l1 and op9 adipocytes secrete adiponectin into the medium , adiponectin in overnight cultured medium accumulated only to 0.1% of mouse serum adiponectin . therefore , co - culture or conditioned medium was used to increase adiponectin protein levels . for co - culture , adipocytes were cultured in the outside well of the bd biosciences ( franklin lakes , nj ) transwell co - culture system , and fao cells were grown on the protein and cytokine - permeable membrane of the insert well . fao cells were transduced with adenovirus encoding mouse adiponectin ( ad - acrp30 ) or green fluorescent protein ( gfp ) for 12 h in a regular 6-well plate , and then the inserts with transduced fao cells were washed and transferred to the co - culture plates with differentiated adipocytes in the bottom wells . this system allows increase of medium adiponectin levels ( 40% of mouse serum adiponectin ) without physical interaction between 3t3-l1 adipocytes and fao cells . in a separate setting , conditioned medium from ad - acrp30transduced fao cells was collected and used for increasing adiponectin levels in some of the studies , as indicated in figure legends . for hormone - stimulated lipolysis , bt2-camp ( 100 mol / l ) was added to culture medium and cells were incubated for 1 h. total protein was used for certain protein or protein phosphorylation measurements using western blots . for pka rii protein half - life measurement for the in vivo lipolysis assays , the selective 3-adrenergic agonist brl37344 was injected i.p . into mice at 5 g / g body wt ( 9 ) . blood samples were collected 0 , 10 , and 20 min after the injection . for the lipolysis assay of adipose explants , 20 mg of epididymal adipose tissue explants were cultured in dulbecco s modified eagle s medium with 0.5% fatty acid the medium samples were collected 30 , 60 , 120 , and 180 min after adding brl37344 . the level of free fatty acid ( ffa ) or glycerol was normalized to the weight of adipose explants . for primary adipocyte lipolysis assays , adipocytes were prepared from epididymal fat using collagenase with an optimized procedure ( 11,12 ) . briefly , epididymal fat tissue was minced and digested in a krebs - ringer bicarbonate ( krb ) buffer ( ph 7.4 ) supplemented with 3% fatty acid free bsa fraction v , 0.5 mmol / l adenosine , and 1 mg / ml type i collagenase . after 40-min digestion and three washes , isolated adipocytes were resuspended in krb supplemented with 5 mmol / l glucose and 3% fatty acid free bsa . suspended adipocytes ( 6,000 cells ) were used for a lipolysis assay in 400 l krb buffer plus 3% fatty acid free bsa , 1 unit / ml adenosine deaminase , and 100 nmol / l ( -)-n-(2-phenyl - isopropyl)-adenosine ( basal only ) with or without brl37344 ( 50 ng / ml ) . after 1-h incubation at 37c , 200 l of infranatant was removed and stored at 20c . serum free glycerol and glycerol released from fat explants were measured using a free - glycerol kit ( sigma - aldrich co. , st . glycerol in the assay media of primary and 3t3-l1 adipocytes was measured using bioluminescence , which is very sensitive and especially well adapted when only small amounts of adipose tissue or cells are available ( 13,14 ) . statistical analysis was performed using the student t test or anova , followed by contrast test . to study the effects of adiponectin on lipid metabolism in adipocytes , adipoq mice were used . consistent with previous studies ( 5,15 ) , there were no significant differences of body weights between adipoq and wild - type ( wt ) littermate controls when the mice were fed normal chow ( data not shown ) . however , body fat percentage of adipoq mice was significantly lower than that of wt mice ( fig . epididymal fat mass was also significantly smaller in adipoq male mice ( 0.26 0.02 vs. 0.42 0.04 g ; vs. wt , p < 0.05 , n = 10 ) . adipocyte cross - sectional areas of fixed epididymal fat of adipoq mice were 20% smaller than those of wt control mice ( supplementary fig . when mice were fed a 60% ( calorie ) high - fat diet for 8 weeks , adipoq mice gained remarkably less body weight and adipose tissue mass and had smaller adipocyte area ( fig . similar to previous studies ( 5,16 ) , high - fat diet induced more severe insulin resistance in adipoq mice ( data not shown ) . therefore , these studies clearly show that adipoq mice have less fat mass and are resistant to high - fat diet induced obesity . similar phenotypes have been reported by a group using an independent line of adipoq mice ( 17 ) . adiponectin knockout mice have less body fat and are resistant to high - fat diet induced obesity . body composition of chow - fed 2-month - old male adipoq and wt mice was scanned using echomri ( a ) . then the mice were fed a high - fat diet ( 60% of calories from fat ) . body fat was measured after 8 weeks of high - fat feeding ( c ) . our study shows that neither adiponectin gene knockout nor overexpression has any significant effect on gene expression of the key enzymes of triglyceride synthesis in epididymal fat ( supplementary fig . 2 ) , which suggests that adiponectin may regulate lipid metabolism in adipocytes through a pathway other than de novo lipogenesis . although serum ffa levels at the fed state were similar between chow - fed adipoq and wt control mice , prolonged fasting - induced increase of ffa was significantly higher in adipoq mice ( supplementary fig . in addition , high - fat diet further increased fasting blood ffa in adipoq mice ( supplementary fig . these results imply that adiponectin might suppress ffa release and prompted us to study the effects of adiponectin on lipolysis in adipocytes . initially , we used the selective 3-adrenergic receptor agonist brl37344 and adipoq mice to carry out an in vivo lipolysis study , in which increases of serum free glycerol were measured to represent hormone - stimulated lipolysis . as expected , brl37344 treatment robustly increased serum free glycerol and ffa levels in all mice . the magnitudes of increase of serum glycerol in adipoq mice were significantly higher than those in wt mice 10 or 20 min after brl37344 injection ( p < 0.05 , fig . second , to avoid influence from other systems or hormones that may be rapidly altered by brl37344 administration , in vitro lipolysis assays were conducted using epididymal adipose explants . results showed that there were no significant differences in glycerol release between adipose explants from adipoq and wt control in a basal condition ( fig . consistent with the in vivo lipolysis study , brl37344-stimulated glycerol releases were remarkably higher in adipose explants from adipoq mice compared with wt control mice ( p < 0.01 , fig . 2b ) . in the third study , lipolysis was assayed in primary mature adipocytes from adipoq and wt control mice . as shown in fig . 2c , basal medium free glycerol levels were similar between adipocytes from wt control and adipoq mice . however , in bt2-camp treated cells , free glycerol levels in the medium of adipoq adipocytes were twofold higher than those of adipocytes from wt mice ( fig . mouse embryonic fibroblast - derived adipocytes from adipoq mice were treated with or without adiponectin using a transwell co - culture system . results showed that camp - stimulated release of glycerol from adiponectin - treated adipocytes was significantly less than from control adipocytes ( supplementary fig . a : 3-adrenergic receptor agonist brl37344 was injected i.p . into 8-week - old male mice , which had been fasted overnight . n = 6 , * p < 0.05 vs. wt at the same time point . b : epididymal adipose explants were incubated in dulbecco s modified eagle s medium with or without brl37344 stimulation . n = 8 , * p < 0.05 vs. wt adipose explants with brl37344 stimulation . c : primary adipocytes were prepared from epididymal fat of adipoq or wt control mice . adipocytes were treated with or without bt2-camp ( 100 mol / l ) for 1 h. glycerol levels in medium were measured . however , there was no significant difference in serum glucagon and insulin concentrations between chow - fed adipoq and wt mice ( data not shown ) , which prompted us to look at the expression and activation state of the main lipases in wat of adipoq mice . hsl and adipose triglyceride lipase ( atgl ) are two major lipases in adipocytes ( 21,22 ) . we examined the expression and activation status of these lipolytic proteins in epididymal adipose tissues from mice with adiponectin gene deletion or overexpression and from cultured adipocytes . for some animal studies , ad - acrp30 was used to reconstitute serum adiponectin in adipoq mice or to elevate serum adiponectin in c57bl/6 mice . as we previously reported ( 18 ) , 3 days after virus injection , total serum adiponectin protein levels were increased 12-fold in c57bl/6 mice , whereas fasting ffa levels were significantly reduced ( supplementary fig . serum multimeric adiponectin levels were proportionally elevated , and there were no significant changes in insulin sensitivity , as measured by an insulin challenge test ( data not shown ) ( 18 ) . 3a , there were no significant differences of total protein levels of hsl , atgl , or cgi-58 in epididymal fat from adipoq , adiponectin - overexpressing , or adiponectin - reconstituted mice compared with their corresponding controls . in addition , adiponectin treatment did not alter protein levels of hsl , atgl , or cgi-58 in 3t3-l1 adipocytes ( fig . these results suggest that adiponectin - inhibited lipolysis is not through altering hsl , atgl , or cgi-58 protein levels in adipocytes . a : protein samples were prepared from epididymal fat of mice with indicated genotypes or treatment . relative protein levels of hsl , atgl , and cgi-58 , and serine phosphorylation levels of hsl were measured by western blot using specific antibodies . for adiponectin overexpression or reconstitution , ad - acrp30 or ad - gfp adenovirus b : mature 3t3-l1 adipocytes were co - cultured overnight with fao cells transduced with ad - acrp30 or ad - gfp . the insert wells with fao cells were removed , and adipocytes were treated with bt2-camp for 1 h. n = 6 . c : differentiated 3t3-l1 adipocytes were treated with cc ( 10 mol / l ) and co - cultured with ad - acrp30 or ad - gfp transduced fao cells overnight . d : perilipin a protein levels in epididymal fat from adipoq and wt mice were measured using western blotting . e : for measuring pka - phosphorylated perilipin a , perilipin a proteins were immunoprecipitated and then probed with an antibody against phospho - pka substrate . f : after overnight co - culture with adiponectin secreting fao cells and wortmannin ( 200 nmol / l ) , 3t3-l1 adipocytes were stimulated with bt2-camp and insulin ( 25 nmol / l ) for 1 h. glycerol in medium was measured . hsl is the first lipase that was found to respond to lipolytic hormones and hydrolyze triglycerides in adipocytes . pka plays an essential role in mediating hormone - stimulated hsl activation and lipolysis in adipocytes . however , only ser660 phosphorylation activates hsl , whereas ser563 phosphorylation does not correlate with hsl activation ( 24 ) . by using phospho - hsl specific antibodies , our study showed that the levels of hsl ser660 phosphorylation in epididymal fat from adipoq mice were strikingly elevated compared with those in wt control mice ( p < 0.001 , fig . in contrast , phosphorylation of hsl ser660 was significantly suppressed in epididymal fat of the mice with adenovirus - mediated adiponectin overexpression and adiponectin reconstitution ( p < 0.05 , fig . 3a ) , whereas a significantly reduced brl - stimulated glycerol release was observed in epididymal adipose explants of mice with adiponectin overexpression ( supplementary fig . 5a ) . in addition , significantly increased hsl ser660 phosphorylation was observed in the epididymal fat of adipoq mice fed a high - fat diet ( supplementary fig . these data indicate that hsl activity in wat is increased in adipoq mice and suppressed in mice with elevated adiponectin . by using 3t3-l1 adipocytes and co - culture system , our study further revealed that overnight adiponectin treatment significantly decreased both basal and camp - stimulated hsl ser660 phosphorylation ( p < 0.05 , fig . 3b ) . together , these results demonstrate that adiponectin suppresses hsl activation by reducing ser660 phosphorylation . it has been reported that ser565 in hsl can be phosphorylated by amp - activated protein kinase ( ampk ) , resulting in deactivation of hsl ( 21,25 ) . we postulated that adiponectin might increase hsl ser565 phosphorylation via ampk to inhibit hsl . to our surprise , hsl ser565 phosphorylation levels neither decreased in adipoq mice nor increased in adiponectin - overexpressing or -reconstituted mice compared with their controls ( fig . these results exclude the possibility that adiponectin inhibits hsl activation by increasing hsl ser565 phosphorylation . to further determine the role of ampk in adiponectin - suppressed lipolysis however , cc treatment did not abolish the inhibitory effects of adiponectin on glycerol release ( fig . similar results were obtained in 3t3-l1car1 adipocytes transduced with adenovirus encoding dominant negative ampk ( data did shown ) . these results do not support any key role of ampk in adiponectin - suppressed lipolysis . however , our study does not completely exclude the involvement of ampk in adiponectin - suppressed lipolysis . apparently , the underlying mechanisms through which ampk modulates lipolysis are far more complicated than a single serine site phosphorylation in hsl . studies have shown that pka or lipolysis itself induces ampk activation in adipocytes ( 26,27 ) . furthermore , a recent study reported that ampk not only suppresses but also increases lipolysis and that the effects are dependent on duration of ampk activation ( 28 ) . perilipin a is a lipid droplet - binding protein that plays an important role in lipolysis in adipocytes ( 29,30 ) . upon lipolytic hormone stimulation , pka - phosphorylated perilipin a recruits hsl to the lipid droplet surface ( 31 ) . our results showed that perilipin a protein levels in epididymal fat of adipoq mice were similar to those of wt control mice ( fig . these results suggest that pka - mediated perilipin a phosphorylation may be not responsible for adiponectin - inhibited lipolysis in adipocytes . adiponectin sensitizes insulin and insulin inhibits lipolysis ( 32 ) . this raises the question whether adiponectin inhibits lipolysis through insulin and whether there is a synergistic effect . we used the insulin signaling blocker wortmannin , which abolishes the inhibitory effects of insulin on lipolysis in adipocytes ( 32 ) . as expected , wortmannin treatment attenuated the inhibitory effects of insulin on glycerol release 3f ) . in addition , adipocytes treated with both adiponectin and insulin did not exhibit a synergistic reduction in glycerol release . adipor1 is ubiquitously expressed and is the main adiponectin receptor in white adipocytes ( 33 ) . to verify whether adiponectin inhibits hsl activation through the adiponectin receptor , adipor1 or sirna oligo against adipor1 was expressed using adenovirus vectors in 3t3-l1car1 adipocytes , which express adenovirus receptor to facilitate adenovirus - mediated gene transduction in mature adipocytes ( 35 ) . phosphorylation levels of hsl ser660 at both basal level and after camp stimulation were significantly reduced in ad - adipor1treated cells but were remarkably increased in adipor1 sirna treated adipocytes ( supplementary fig . the coordinate changes of hsl ser660 phosphorylation in adipoq mice and adiponectin - treated adipocytes suggest that adiponectin might suppress hsl activation in adipocytes through reducing pka activity . with the use of an antibody that recognizes the consensus of phosphorylated pka substrate , our study showed that there are significant increases of pka substrate phosphorylation of proteins in epididymal fat from adipoq mice ( supplementary fig . 7a ) , which further supports the notion that adiponectin inhibits pka activation in adipocytes . however , adiponectin gene deletion has no significant impact on total camp levels in wat ( p > 0.05 , supplementary fig . pka type ii regulatory subunit ( rii ) has two distinct isoforms , rii and rii. rii is ubiquitously expressed , but rii is predominantly expressed in adipose , brain , and adrenal tissues . rii gene knockout mice are lean and resistant to high - fat diet induced obesity ( 36 ) . we measured the gene expression levels of pka catalytic subunits and regulatory subunits ri and rii at both mrna and protein levels in epididymal fat from adipoq and adiponectin - restored mice . there were no significant changes of mrna levels of the two catalytic and four regulatory subunits of pka in adipoq mice ( data not shown ) . protein levels of two catalytic subunits , pka c and c , were also similar between adipoq and wt controls ( fig . 4a ) . rii but not rii protein levels were significantly elevated in epididymal fat of adipoq mice ( p < 0.05 , fig . 4a ) . in contrast , adiponectin reconstitution in adipoq mice remarkably reduced rii in the epididymal fat ( p < 0.05 , fig . a similar inhibitory effect of adiponectin on rii protein level was observed in 3t3-l1 adipocytes ( p < 0.05 , fig . 4b ) . these results led us to hypothesize that reduced rii mediates the inhibitory effect of adiponectin on hsl activation . a : total protein samples were prepared using epididymal fat from adipoq or ad - acrp30treated mice . b : fully differentiated 3t3-l1 adipocytes were co - cultured with ad - acrp30transduced fao cells , which secrete adiponectin . data are expressed as means sem . to verify this hypothesis , we overexpressed rii in differentiated mature adipocytes . the levels of hsl ser660 phosphorylation were significantly increased in ad - pka riitreated 3t3-l1car1 adipocytes ( p < 0.05 , fig . overexpression of rii even slightly decreased hsl ser660 phosphorylation without statistical significance ( p > 0.05 , supplementary fig . most important , overexpression of rii abolished adiponectin - induced inhibition of hsl ser660 phosphorylation in 3t3-l1car1 adipocytes ( fig . 5a ) . furthermore , knocking down rii in 3t3-l1car1 adipocytes significantly reduced both basal and camp - stimulated hsl ser660 phosphorylation ( fig . these results indicate that adiponectin suppresses hsl activation by reducing rii. pka rii overexpression attenuates the inhibitory effects of adiponectin on hsl ser660 phosphorylation . adenovirus vectors encoding gfp , pka rii ( a ) , or sirna against pka rii ( b ) were added to the medium . a : cells were co - cultured 12 h later with ad - acrp30transduced fao cells , which secrete adiponectin . b : some cells were treated with bt2-camp for 1 h. protein samples were prepared , and the phosphorylation levels of hsl ser660 and other proteins were detected using specific antibodies . * p < 0.05 vs. ad - lacz sirna treated cells at basal state . the above studies show that adiponectin reduces rii protein levels without altering rii mrna level . therefore , we compared the protein half - life of rii between 3t3-l1 adipocytes treated with or without adiponectin . as shown in fig . rii protein levels were reduced by 30% after just 1 h of adiponectin treatment in the presence of cycloheximide ( fig . cycloheximide ( 100 g / ml ) was added to the medium , and the insert wells with fao cells were removed . b : 3t3-l1 adipocytes were treated with adiponectin - enriched conditioned medium from ad - acrp30transduced fao cells . c : proteasome inhibitor mg132 ( 25 mol / l ) was added to the medium 30 min before adiponectin treatment . d : mature 3t3-l1 adipocytes were co - cultured with adiponectin - expressing fao cells for 4 h. the pierce crosslink ip kit ( thermo scientific pierce protein research products , rockford , il ) was used for immunoprecipitation to eliminate nonspecific bands . * p < 0.05 vs. control cells at the same time point ( b ) or without mg132 treatment ( c ) . to further investigate how adiponectin reduces rii , the proteasome - specific inhibitor mg-132 was added into medium 30 min before adiponectin treatment . 6c ) , which indicates that adiponectin enhances rii protein degradation through the ubiquitin - proteasome system . then , we measured ubiquitin levels of immunoprecipated rii protein from adiponectin - treated and control 3t3-l1 adipocytes . 6d shows , the levels of ubiquitinated rii protein were significantly elevated in adiponectin - treated 3t3-l1 adipocytes . together , these results indicate that adiponectin reduces rii protein levels by enhancing ubiquitin - proteasome mediated protein degradation . it should be noted that overall protein ubiquitin levels were not clearly increased in adiponectin - treated cells ( supplementary fig . similar effects of adiponectin on overall protein ubiquitination have also been observed in adiponectin - treated c2c12 myotubes ( data not shown ) . these results suggest that adiponectin - enhanced rii ubiquitination and protein degradation occur in a protein - specific manner . to study the effects of adiponectin on lipid metabolism in adipocytes , adipoq mice were used . consistent with previous studies ( 5,15 ) , there were no significant differences of body weights between adipoq and wild - type ( wt ) littermate controls when the mice were fed normal chow ( data not shown ) . however , body fat percentage of adipoq mice was significantly lower than that of wt mice ( fig . epididymal fat mass was also significantly smaller in adipoq male mice ( 0.26 0.02 vs. 0.42 0.04 g ; vs. wt , p < 0.05 , n = 10 ) . adipocyte cross - sectional areas of fixed epididymal fat of adipoq mice were 20% smaller than those of wt control mice ( supplementary fig . when mice were fed a 60% ( calorie ) high - fat diet for 8 weeks , adipoq mice gained remarkably less body weight and adipose tissue mass and had smaller adipocyte area ( fig . similar to previous studies ( 5,16 ) , high - fat diet induced more severe insulin resistance in adipoq mice ( data not shown ) . therefore , these studies clearly show that adipoq mice have less fat mass and are resistant to high - fat diet induced obesity . similar phenotypes have been reported by a group using an independent line of adipoq mice ( 17 ) . adiponectin knockout mice have less body fat and are resistant to high - fat diet induced obesity . body composition of chow - fed 2-month - old male adipoq and wt mice was scanned using echomri ( a ) . then the mice were fed a high - fat diet ( 60% of calories from fat ) . body fat was measured after 8 weeks of high - fat feeding ( c ) . our study shows that neither adiponectin gene knockout nor overexpression has any significant effect on gene expression of the key enzymes of triglyceride synthesis in epididymal fat ( supplementary fig . 2 ) , which suggests that adiponectin may regulate lipid metabolism in adipocytes through a pathway other than de novo lipogenesis . previous animal studies have clearly demonstrated that adiponectin reduces circulating ffas ( 16,1820 ) . although serum ffa levels at the fed state were similar between chow - fed adipoq and wt control mice , prolonged fasting - induced increase of ffa was significantly higher in adipoq mice ( supplementary fig . in addition , high - fat diet further increased fasting blood ffa in adipoq mice ( supplementary fig . these results imply that adiponectin might suppress ffa release and prompted us to study the effects of adiponectin on lipolysis in adipocytes . initially , we used the selective 3-adrenergic receptor agonist brl37344 and adipoq mice to carry out an in vivo lipolysis study , in which increases of serum free glycerol were measured to represent hormone - stimulated lipolysis . as expected , brl37344 treatment robustly increased serum free glycerol and ffa levels in all mice . the magnitudes of increase of serum glycerol in adipoq mice were significantly higher than those in wt mice 10 or 20 min after brl37344 injection ( p < 0.05 , fig . second , to avoid influence from other systems or hormones that may be rapidly altered by brl37344 administration , in vitro lipolysis assays were conducted using epididymal adipose explants . results showed that there were no significant differences in glycerol release between adipose explants from adipoq and wt control in a basal condition ( fig . consistent with the in vivo lipolysis study , brl37344-stimulated glycerol releases were remarkably higher in adipose explants from adipoq mice compared with wt control mice ( p < 0.01 , fig . 2b ) . in the third study , lipolysis was assayed in primary mature adipocytes from adipoq and wt control mice . as shown in fig . 2c , basal medium free glycerol levels were similar between adipocytes from wt control and adipoq mice . however , in bt2-camp treated cells , free glycerol levels in the medium of adipoq adipocytes were twofold higher than those of adipocytes from wt mice ( fig . 2c ) . to further verify the effects of adiponectin on lipolysis , mouse embryonic fibroblast - derived adipocytes from adipoq mice were treated with or without adiponectin using a transwell co - culture system . results showed that camp - stimulated release of glycerol from adiponectin - treated adipocytes was significantly less than from control adipocytes ( supplementary fig . a : 3-adrenergic receptor agonist brl37344 was injected i.p . into 8-week - old male mice , which had been fasted overnight . n = 6 , * p < 0.05 vs. wt at the same time point . b : epididymal adipose explants were incubated in dulbecco s modified eagle s medium with or without brl37344 stimulation . n = 8 , * p < 0.05 vs. wt adipose explants with brl37344 stimulation . c : primary adipocytes were prepared from epididymal fat of adipoq or wt control mice . however , there was no significant difference in serum glucagon and insulin concentrations between chow - fed adipoq and wt mice ( data not shown ) , which prompted us to look at the expression and activation state of the main lipases in wat of adipoq mice . hsl and adipose triglyceride lipase ( atgl ) are two major lipases in adipocytes ( 21,22 ) . we examined the expression and activation status of these lipolytic proteins in epididymal adipose tissues from mice with adiponectin gene deletion or overexpression and from cultured adipocytes . for some animal studies , ad - acrp30 was used to reconstitute serum adiponectin in adipoq mice or to elevate serum adiponectin in c57bl/6 mice . as we previously reported ( 18 ) , 3 days after virus injection , total serum adiponectin protein levels were increased 12-fold in c57bl/6 mice , whereas fasting ffa levels were significantly reduced ( supplementary fig . serum multimeric adiponectin levels were proportionally elevated , and there were no significant changes in insulin sensitivity , as measured by an insulin challenge test ( data not shown ) ( 18 ) . as shown in fig . 3a , there were no significant differences of total protein levels of hsl , atgl , or cgi-58 in epididymal fat from adipoq , adiponectin - overexpressing , or adiponectin - reconstituted mice compared with their corresponding controls . in addition , adiponectin treatment did not alter protein levels of hsl , atgl , or cgi-58 in 3t3-l1 adipocytes ( fig . these results suggest that adiponectin - inhibited lipolysis is not through altering hsl , atgl , or cgi-58 protein levels in adipocytes . a : protein samples were prepared from epididymal fat of mice with indicated genotypes or treatment . relative protein levels of hsl , atgl , and cgi-58 , and serine phosphorylation levels of hsl were measured by western blot using specific antibodies . for adiponectin overexpression or reconstitution , ad - acrp30 or ad - gfp adenovirus b : mature 3t3-l1 adipocytes were co - cultured overnight with fao cells transduced with ad - acrp30 or ad - gfp . the insert wells with fao cells were removed , and adipocytes were treated with bt2-camp for 1 h. n = 6 . c : differentiated 3t3-l1 adipocytes were treated with cc ( 10 mol / l ) and co - cultured with ad - acrp30 or ad - gfp transduced fao cells overnight . d : perilipin a protein levels in epididymal fat from adipoq and wt mice were measured using western blotting . e : for measuring pka - phosphorylated perilipin a , perilipin a proteins were immunoprecipitated and then probed with an antibody against phospho - pka substrate . f : after overnight co - culture with adiponectin secreting fao cells and wortmannin ( 200 nmol / l ) , 3t3-l1 adipocytes were stimulated with bt2-camp and insulin ( 25 hsl is the first lipase that was found to respond to lipolytic hormones and hydrolyze triglycerides in adipocytes . pka plays an essential role in mediating hormone - stimulated hsl activation and lipolysis in adipocytes . however , only ser660 phosphorylation activates hsl , whereas ser563 phosphorylation does not correlate with hsl activation ( 24 ) . by using phospho - hsl specific antibodies , our study showed that the levels of hsl ser660 phosphorylation in epididymal fat from adipoq mice were strikingly elevated compared with those in wt control mice ( p < 0.001 , fig . 3a , bottom quantified graphs ) . in contrast , phosphorylation of hsl ser660 was significantly suppressed in epididymal fat of the mice with adenovirus - mediated adiponectin overexpression and adiponectin reconstitution ( p < 0.05 , fig . 3a ) , whereas a significantly reduced brl - stimulated glycerol release was observed in epididymal adipose explants of mice with adiponectin overexpression ( supplementary fig . 5a ) . in addition , significantly increased hsl ser660 phosphorylation was observed in the epididymal fat of adipoq mice fed a high - fat diet ( supplementary fig . these data indicate that hsl activity in wat is increased in adipoq mice and suppressed in mice with elevated adiponectin . by using 3t3-l1 adipocytes and co - culture system , our study further revealed that overnight adiponectin treatment significantly decreased both basal and camp - stimulated hsl ser660 phosphorylation ( p < 0.05 , fig . 3b ) . together , these results demonstrate that adiponectin suppresses hsl activation by reducing ser660 phosphorylation . it has been reported that ser565 in hsl can be phosphorylated by amp - activated protein kinase ( ampk ) , resulting in deactivation of hsl ( 21,25 ) . to our surprise , hsl ser565 phosphorylation levels neither decreased in adipoq mice nor increased in adiponectin - overexpressing or -reconstituted mice compared with their controls ( fig . these results exclude the possibility that adiponectin inhibits hsl activation by increasing hsl ser565 phosphorylation . to further determine the role of ampk in adiponectin - suppressed lipolysis , selective ampk inhibitor compound c ( cc ) was used . however , cc treatment did not abolish the inhibitory effects of adiponectin on glycerol release ( fig . similar results were obtained in 3t3-l1car1 adipocytes transduced with adenovirus encoding dominant negative ampk ( data did shown ) . these results do not support any key role of ampk in adiponectin - suppressed lipolysis . however , our study does not completely exclude the involvement of ampk in adiponectin - suppressed lipolysis . apparently , the underlying mechanisms through which ampk modulates lipolysis are far more complicated than a single serine site phosphorylation in hsl . studies have shown that pka or lipolysis itself induces ampk activation in adipocytes ( 26,27 ) . furthermore , a recent study reported that ampk not only suppresses but also increases lipolysis and that the effects are dependent on duration of ampk activation ( 28 ) . perilipin a is a lipid droplet - binding protein that plays an important role in lipolysis in adipocytes ( 29,30 ) . upon lipolytic hormone stimulation , pka - phosphorylated perilipin a recruits hsl to the lipid droplet surface ( 31 ) . our results showed that perilipin a protein levels in epididymal fat of adipoq mice were similar to those of wt control mice ( fig . these results suggest that pka - mediated perilipin a phosphorylation may be not responsible for adiponectin - inhibited lipolysis in adipocytes . adiponectin sensitizes insulin and insulin inhibits lipolysis ( 32 ) . this raises the question whether adiponectin inhibits lipolysis through insulin and whether there is a synergistic effect . we used the insulin signaling blocker wortmannin , which abolishes the inhibitory effects of insulin on lipolysis in adipocytes ( 32 ) . 3f ) . in addition , adipocytes treated with both adiponectin and insulin did not exhibit a synergistic reduction in glycerol release . adipor1 is ubiquitously expressed and is the main adiponectin receptor in white adipocytes ( 33 ) . to verify whether adiponectin inhibits hsl activation through the adiponectin receptor , adipor1 or sirna oligo against adipor1 was expressed using adenovirus vectors in 3t3-l1car1 adipocytes , which express adenovirus receptor to facilitate adenovirus - mediated gene transduction in mature adipocytes ( 35 ) . phosphorylation levels of hsl ser660 at both basal level and after camp stimulation were significantly reduced in ad - adipor1treated cells but were remarkably increased in adipor1 sirna treated adipocytes ( supplementary fig . the coordinate changes of hsl ser660 phosphorylation in adipoq mice and adiponectin - treated adipocytes suggest that adiponectin might suppress hsl activation in adipocytes through reducing pka activity . with the use of an antibody that recognizes the consensus of phosphorylated pka substrate , our study showed that there are significant increases of pka substrate phosphorylation of proteins in epididymal fat from adipoq mice ( supplementary fig . 7a ) , which further supports the notion that adiponectin inhibits pka activation in adipocytes . however , adiponectin gene deletion has no significant impact on total camp levels in wat ( p > 0.05 , supplementary fig . pka type ii regulatory subunit ( rii ) has two distinct isoforms , rii and rii. rii is ubiquitously expressed , but rii is predominantly expressed in adipose , brain , and adrenal tissues . rii gene knockout mice are lean and resistant to high - fat diet induced obesity ( 36 ) . we measured the gene expression levels of pka catalytic subunits and regulatory subunits ri and rii at both mrna and protein levels in epididymal fat from adipoq and adiponectin - restored mice . there were no significant changes of mrna levels of the two catalytic and four regulatory subunits of pka in adipoq mice ( data not shown ) . protein levels of two catalytic subunits , pka c and c , were also similar between adipoq and wt controls ( fig . 4a ) . rii but not rii protein levels were significantly elevated in epididymal fat of adipoq mice ( p < 0.05 , fig . 4a ) . in contrast , adiponectin reconstitution in adipoq mice remarkably reduced rii in the epididymal fat ( p < 0.05 , fig . a similar inhibitory effect of adiponectin on rii protein level was observed in 3t3-l1 adipocytes ( p < 0.05 , fig . 4b ) . these results led us to hypothesize that reduced rii mediates the inhibitory effect of adiponectin on hsl activation . a : total protein samples were prepared using epididymal fat from adipoq or ad - acrp30treated mice . b : fully differentiated 3t3-l1 adipocytes were co - cultured with ad - acrp30transduced fao cells , which secrete adiponectin . data are expressed as means sem . to verify this hypothesis , we overexpressed rii in differentiated mature adipocytes . the levels of hsl ser660 phosphorylation were significantly increased in ad - pka riitreated 3t3-l1car1 adipocytes ( p < 0.05 , fig . overexpression of rii even slightly decreased hsl ser660 phosphorylation without statistical significance ( p > 0.05 , supplementary fig . most important , overexpression of rii abolished adiponectin - induced inhibition of hsl ser660 phosphorylation in 3t3-l1car1 adipocytes ( fig . knocking down rii in 3t3-l1car1 adipocytes significantly reduced both basal and camp - stimulated hsl ser660 phosphorylation ( fig . these results indicate that adiponectin suppresses hsl activation by reducing rii. pka rii overexpression attenuates the inhibitory effects of adiponectin on hsl ser660 phosphorylation . adenovirus vectors encoding gfp , pka rii ( a ) , or sirna against pka rii ( b ) were added to the medium . a : cells were co - cultured 12 h later with ad - acrp30transduced fao cells , which secrete adiponectin . b : some cells were treated with bt2-camp for 1 h. protein samples were prepared , and the phosphorylation levels of hsl ser660 and other proteins were detected using specific antibodies . * p < 0.05 vs. ad - lacz sirna treated cells at basal state . the above studies show that adiponectin reduces rii protein levels without altering rii mrna level . therefore , we compared the protein half - life of rii between 3t3-l1 adipocytes treated with or without adiponectin . as shown in fig . rii protein levels were reduced by 30% after just 1 h of adiponectin treatment in the presence of cycloheximide ( fig . cycloheximide ( 100 g / ml ) was added to the medium , and the insert wells with fao cells were removed . b : 3t3-l1 adipocytes were treated with adiponectin - enriched conditioned medium from ad - acrp30transduced fao cells . c : proteasome inhibitor mg132 ( 25 mol / l ) was added to the medium 30 min before adiponectin treatment . d : mature 3t3-l1 adipocytes were co - cultured with adiponectin - expressing fao cells for 4 h. the pierce crosslink ip kit ( thermo scientific pierce protein research products , rockford , il ) was used for immunoprecipitation to eliminate nonspecific bands . * p < 0.05 vs. control cells at the same time point ( b ) or without mg132 treatment ( c ) . the ubiquitin - dependent proteasome is the most common system for protein degradation . to further investigate how adiponectin reduces rii , the proteasome - specific inhibitor mg-132 was added into medium 30 min before adiponectin treatment . 6c ) , which indicates that adiponectin enhances rii protein degradation through the ubiquitin - proteasome system . then , we measured ubiquitin levels of immunoprecipated rii protein from adiponectin - treated and control 3t3-l1 adipocytes . 6d shows , the levels of ubiquitinated rii protein were significantly elevated in adiponectin - treated 3t3-l1 adipocytes . together , these results indicate that adiponectin reduces rii protein levels by enhancing ubiquitin - proteasome mediated protein degradation . it should be noted that overall protein ubiquitin levels were not clearly increased in adiponectin - treated cells ( supplementary fig similar effects of adiponectin on overall protein ubiquitination have also been observed in adiponectin - treated c2c12 myotubes ( data not shown ) . these results suggest that adiponectin - enhanced rii ubiquitination and protein degradation occur in a protein - specific manner . adipose tissue is the main energy depot in humans and some animals . during fasting and exercise , triglycerides are hydrolyzed to release ffas , which are oxidized by skeletal muscle , heart , and other tissues as fuel . with the discovery of adipocyte - derived hormones , the regulatory role of adipose - derived hormones on lipid metabolism has emerged as an important system in maintaining energy homeostasis . our current study provides strong evidence demonstrating that adiponectin suppresses hormone - stimulated lipolysis in mouse adipocytes . during the revision of this article , the same effects of adiponectin on lipolysis were reported in human adipocytes ( 37 ) . therefore , we propose that adiponectin plays an important role in maintaining energy homeostasis by suppressing lipolysis in wat . it has been reported that leptin , another adipose - derived hormone , directly increases lipolysis and enhances lipid depletion from adipocytes ( 38,39 ) . although it is uncertain whether leptin and adiponectin regulate lipolysis through the same pathway , these studies suggest that adiponectin and leptin exert opposite regulatory effects on lipolysis in adipocytes . in addition , the results from cultured adipocytes clearly indicate that adiponectin suppresses lipolysis through an autocrine loop . the discovery of the inhibitory effects of adiponectin on lipolysis will help us to understand how adiponectin improves circulating lipid profiles . however , it is premature to claim the importance of adiponectin - inhibited lipolysis in the development of obesity . there is a decrease of adiponectin gene expression in obesity ( 2 ) , which should attenuate the inhibitory effect of adiponectin on lipolysis , resulting in less lipid storage in fat . thus , obesity - associated low adiponectin expression may be beneficial in the context of obesity . in addition , despite lipolytic defects that have been observed in obese human and animal models ( 10,40 ) , the role of lipolytic defects in obesity is still uncertain . therefore , more studies are required to elucidate the role of adiponectin - suppressed lipolysis in the development of obesity and in energy metabolism under physiologic conditions . plasma ffa inversely associates with insulin sensitivity , especially in obese subjects ( 41,42 ) . our current study demonstrates that adiponectin inhibits lipolysis in wat , which should decrease the level of circulating ffa . therefore , we postulate that inhibition of lipolysis and reduction of ffa is a mechanism through which adiponectin enhances insulin sensitivity . on the other hand , the insulin - sensitizing effect of adiponectin however , our study demonstrated that insulin signaling is not responsible for adiponectin - inhibited lipolysis in adipocytes . furthermore , although high - fat feeding induces more severe insulin resistance in adipoq mice compared with wt control mice ( 16 ) , the inhibitory effects of adiponectin on lipolysis are still well preserved . therefore , these studies indicate that adiponectin inhibits hormone - stimulated adipocyte lipolysis through a mechanism(s ) other than insulin sensitizing . the essential role of pka in mediating hormone - stimulated lipolysis in adipocytes has been well established . it has been reported that rii gene knockout mice are lean and resistant to high - fat diet induced obesity because of increased locomotor activity , energy expenditure , and lipolysis in wat ( 4345 ) . our study showed that adiponectin has no effect on rii expression and that rii overexpression even slightly reduces hsl ser660 phosphorylation in adipocytes . the current study does not provide any evidence to support the involvement of rii in adiponectin - inhibited lipolysis in adipocytes . in contrast , we found that the protein level of rii was elevated in adiponectin gene knockout mice . ectopic expression of rii abolished the inhibitory effects of adiponectin on hsl ser660 phosphorylation and lipolysis . conversely , knocking down rii reduced both basal and camp - stimulated hsl ser660 phosphorylation in adipocytes . these results led us to conclude that reduced rii mediates the inhibitory effects of adiponectin on lipolysis in adipocytes . it should be pointed out that by comparing the pka substrate phosphorylation profile and pka protein levels between adipoq and wt mice in different tissues ( data not shown ) , we found that the effects of adiponectin on pka subunit expression and pka activation occur in a tissue - specific manner . the mechanism by which rii mediates the inhibitory effect of adiponectin on lipolysis remains unknown . the large number of substrates and diverse downstream signaling pathways of pka , precise localization of pka to its specific substrate by a kinase - anchoring proteins ( akaps ) has been demonstrated to be an important mechanism to restrict pka action ( 46 ) . recent studies revealed that rii binds tightly with some akaps and plays an important role in compartmentalizing camp / pka signal transduction ( 4648 ) . we postulate that adiponectin suppresses rii expression , which may shift akaps / pka away from the compartment where hsl localizes , resulting in decreased hsl phosphorylation and activation . the absence of effect of adiponectin on perilipin a phosphorylation also supports this postulation . in conclusion , our study demonstrates that adiponectin inhibits lipolysis and directly regulates lipid metabolism in adipocytes . our data also suggest that adiponectin suppresses triglyceride hydrolysis by inhibiting pka - induced hsl activation .

backgroundwith the aging of the population , dementia is increasing worldwide . the objective of this study was to identify risk factors for dementia in an elderly population utilizing a primary health care service in dakar , senegal.methodsthrough a cross - sectional study conducted from march 2004 to december 31 , 2005 , 507 elderly patients aged 65 years who came to the social and medical center of ipres , dakar , senegal , were first screened with the screening interview questionnaire aging in senegal. those who were cognitively impaired underwent a clinical examination to detect dementia . univariate , bivariate , and multivariate logistic regression analyses were done.resultsthe whole population had a mean age of 72.4 years ( 5.2 ) and was mostly male , married , and non - educated . hypertension , arthritis , and gastrointestinal diseases were the main health conditions reported in the past medical history . smoking was important while alcohol consumption was rare . social network was high . forty - five patients ( 8.87% ) had dementia . in the multivariate model , only advanced age , education , epilepsy , and family history of dementia were independently associated with dementia.conclusionthe risk factors identified are also found in developed countries confirming their role in dementia . it is important to take dementia into consideration in senegal and to sensitize the community for prevention . objectiveadiponectin is an adipocyte - derived hormone that sensitizes insulin and improves energy metabolism in tissues . this study was designed to investigate the direct regulatory effects of adiponectin on lipid metabolism in adipocytes.research design and methodsbasal and hormone - stimulated lipolysis were comparatively analyzed using white adipose tissues or primary adipocytes from adiponectin gene knockout and control mice . to further study the underlying mechanisms through which adiponectin suppresses lipolysis , cultured 3t3-l1 adipocytes and adenovirus - mediated gene transduction were used.resultssignificantly increased lipolysis was observed in both adiponectin gene knockout mice and primary adipocytes from these mice . hormone - stimulated glycerol release was inhibited in adiponectin - treated adipocytes . adiponectin suppressed hormone - sensitive lipase activation without altering adipose triglyceride lipase and cgi-58 expression in adipocytes . moreover , adiponectin reduced protein levels of the type 2 regulatory subunit rii of protein kinase a by reducing its protein stability . ectopic expression of rii abolished the inhibitory effects of adiponectin on lipolysis in adipocytes.conclusionsthis study demonstrates that adiponectin inhibits lipolysis in adipocytes and reveals a novel function of adiponectin in lipid metabolism in adipocytes .

the national library of medicine 's ( nlm ) medline / pubmed biomedical literature database ( ) is rapidly expanding and as of the 2006 edition , the baseline database contains more than 15 million citations . a number of papers have introduced tools to perform searches more advanced than those available with the current entrez - pubmed user interface to mine the abstracts ( 14 ) . to have more control in conducting searches , investigators have also started to reorganize the medline data into structured relational systems ( 5 ) or to exploit different data formats and create new databases ( ) . these nlm citations are available in several formats ( e.g. medline , asn.1 , and so on ) . importing these citation data into a traditional , relational database limits the query capability to sql ( structured query language ) techniques . the extensible markup language ( xml ) format , however , is emerging as a de facto standard for the hierarchical organization of data in a highly granular structure , and provides a much richer query capability beyond what is available through traditional sql . oracle xml db , available in oracle 9i and 10 g , recognizes this new format . it supports both the necessary massive storage requirements and the extensions to traditional sql that enable a different kind of query which is hierarchical and highly granular . an early study using oracle xml db involved the evaluation of data - form standards and database technologies for medical informatics systems ( 6 ) ( ) . as described by wang et al . ( 6 ) , the set of utilities associated with xml db permits data to be represented both as xml elements from xml documents and as cells within relational tables . a recently developed website that includes a specialized , oracle - based resource for the clostridial neurotoxins , botdb ( 7 ) ( ) , has been extended to conduct literature searches of medline / pubmed using botxminer . the application of xml db for botxminer was based , in large measure , on the documentation and thorough analysis of an original implementation using xml - formatted medline citations ( ) . an advantage offered by botxminer over pubmed is that it can make certain queries possible that can not be performed using the entrez - pubmed interface . the practical example herein illustrates how citations , containing user - supplied search terms , selected medline search fields and terms , are grouped and displayed as hyperlinked tabulated or graphic results . a more detailed technical description of the architecture associated with botxminer and of the more than 15 million medline xml records created through the 2005 production year that were loaded into oracle xml db , a limited reference subset was generated for botxminer using the search words tetanus. this subset has 26 563 citations , a value that represents < 1% of the total number that are available . the search option ( data not shown ) allows the user to query the standard fields of a medline output [ author , terms in the title or abstract , pubmed identification number ( pmid ) and so on ] by entering desired words or terms in the appropriate boxes and by selecting an optional range of publication years . initially , the user - provided search words or terms are searched for within each medline xml file . these results are then grouped by botxminer with terms contained in a select list of medline fields : the medical subject headings ( mesh ) qualifier or descriptor names , author , chemical , keyword and gene symbol . advanced search feature is also available that allows the user to search the common medline fields of journal title , mesh and chemical in addition to the title and abstract . the option to specify a range of publication years is also applicable to the group articles and the advanced search. queries may contain common logical operators for literature searches that are supported by oracle text : and ( & ) , not ( ) , or ( ) and accum ( , ) ( meaning either term is acceptable but both are preferred ) . other operators use context grammar : near ( ;) for proximity searches , and minus ( ) for a lower preference . output graphics can presently be viewed in png , html or svg formats and have so far been tested using microsoft internet explorer ( ie ) v.6 , netscape v.8 and the mozilla firefox v.1 browsers . the html format offers the advantage of being able to hyperlink the graphic points ( rectangles and circles ) to the citation summary information . label information is presented as a pop - up label when moving the mouse over the rectangles and circles . for the svg option , an interactive graphic view of the results is dynamically produced using the aisee graphics package ( ) . the svg format also has interactive hyperlinks while its associated plug - in ( available for ie and netscape ) provides the user with a drop - down feature menu to zoom in and zoom out , pan across the image , save the image , and other graph features . in the svg format under ie , positioning the mouse over the rectangles and circles produces the label information within the window status bar. at present , the firefox browser does support the svg format by using the aisee software but does not support the drop - down feature menu . for these reasons , it is recommended that , at this time , the botxminer graphics be viewed in the svg format using either the ie or the netscape browsers . as a practical example , a search was conducted to find potential interactions between the snare proteins ( three of which are substrates for the proteolytic botulinum neurotoxins ) and other proteins . the substrates of interest [ syntaxin-1a , synaptobrevin ( vamp ) and snap-25 ] are intimately involved in the evoked release of neurotransmitter from synaptic vesicles . protein interactions may , thus , help to predict what other cellular functions might be affected in addition to the toxin - induced blockade of neurotransmission . group articles advanced search was used with the query : near ( ( snare , interact% ) , 5 ) ( figure 1 , back panel ) . snare and interact% ( or interacts , interacting , etc . ) that are separated by no more than five words . this query resulted in a tabulated listing of 83 group ( chemical ) terms that appeared in 18 medline xml files ( figure 1 , middle panel ) . 18 produces a list of all the retrieved citations ( figure 1 , front panel ) . values for the number of articles for a given term are hyperlinked to a second table that lists citations associated with the selected term ( data not shown ) . the pmid numbers are hyperlinked to their corresponding medline pages , which are presented in a modified format , while article titles in this table are hyperlinked to their pubmed abstracts ( data not shown ) . another way to visualize these data is in a network graph format ( figure 2 , upper panel ) with a method analogous to that used in chilibot ( 2 ) and pubnet ( 3 ) . before a graph , there are 83 group terms ( chemical names ) and 859 connections ( circles ) between these terms . all pairs of chemical terms are connected by color - coded lines and circles that show the relative number of articles for two co - occurring chemical terms . each rectangle is hyperlinked to a list of citations containing the group ( chemical ) term , whereas each circle is hyperlinked to a list of citations that contain the pair of terms associated with that connection ( data not shown ) . these graphical views also allow the user to quickly focus on and recognize patterns for the most frequent co - occurring terms . in this example , the cluster of terms near the top of the graph is examined more closely with the zoom in option ( accessed through the drop - down menu with the right - hand mouse button ) that is used with svg ( figure 2 , lower panel ) . in this panel , the pairs are only associated with a single paper ( gray lines and circles ) . protein interactions between the snares and other proteins including calmodulin , neurotransporters and other synaptic proteins were readily identified using this approach . one goal in the development of botxminer was to mine the maximum amount of information from the medline xml files without doing any natural language processing . this development began with l. c. wang 's original documentation ( ) that described how the whole collection of medline data can be stored inside oracle xml db in object - relational tables . since entire medline xml files are stored in this database , the structure of these files is maintained , in contrast to writing new code to parse the data and to subsequently store them into relational tables . the schema - based , structured type data storage strategy is made more efficient in botxminer with oracle 's text indexing capability which makes proximity searches with operators such as near , a context grammar operator , and wildcard searches feasible . the group articles feature of botxminer allows the user to look at a single , tabulated list of relevant grouped terms and to quickly retrieve citations of interest . finally , botxminer has the first user interface for medline xml files that are stored in oracle xml db . in comparison to other publicly available pubmed search applications , the differences exhibited by botxminer provide an opportunity for its descendants to enhance searches of the continually growing biomedical literature . in the tools described by oliver et al . ( 5 ) , medline xml files that have been downloaded are subsequently parsed and stored in schema - defined relational tables . devising appropriate schema to optimally in contrast , pubfinder ( 4 ) stores abstracts in its database and creates a reference dictionary of commonly used words . the user needs to provide pmids of abstracts of interest which are processed for word frequencies and compared to the reference dictionary . applications , such as chilibot ( 1 ) , pubgene ( 8) and medminer ( 9 ) , require , as a minimal user - supplied input , a recognized gene name to conduct its pubmed search . textpresso ( 10 ) , as part of its knowledge retrieval strategy , uses a text - to - xml converter to systematically mark up sentences by its specialized ontology ( that is presently based on caenorhabditis elegans ) from full - length , searchable articles . the example query provided here illustrates the power of botxminer to help the user to efficiently and selectively search for co - occurrences of grouped terms from medline xml files and to focus on the general problem of protein protein interactions . since we have created a database from the entire set of medline xml files , it is anticipated that a variety of other , specialized citation subsets ( e.g. cytoskeletal proteins , signal transduction pathways and diseases such as cancer ) will be developed that are similar in design to botxminer . it is further anticipated that botxminer will serve as a template for future applications that will mine the entire medline and other very large sources of biomedical information . literature search conducted in botdb using botxminer and the group articles ( advanced search option ) . the end - user types in a desired word or term ( words separated by logical or other operators ) and selects one or more of the medline search fields and one of the six presently available terms . middle panel : tabulated results are shown for the query near ( ( snare , interact% ) , 5) , the the resulting list of chemical terms are tallied in the form of an alphabetically sorted table that can be expanded by clicking on a number . 18 associated with the total number of articles ( middle panel ) is hyperlinked to these citations . graphical view link ( figure 1 , middle panel ) produces a dynamic network graph ( upper panel ) that shows the interconnectedness of the chemical terms which , in turn , are linked to references ( color - coded lines ) with the number of co - occurrences labeled in the small circles . this graph displays all the terms from the table ( figure 1 , middle panel ) that are connected by the number of articles in which they co - occur . the lines and circles are color coded depending on the number of articles : gray , the terms co - occur in 1 article ; yellow , co - occurrence in 25 articles ; pink , co - occurrence in 610 articles ; green , co - occurrence in > 10 articles . lymphangioma circumscriptum ( lc ) or microcystic lymphatic malformation is a hamartomatous malformation of the lymphatic channels of the skin . treatment of lc is difficult because of the persistent nature of the disease due to deeper subcutaneous cisternal connections . we report here a case of lc treated effectively with radiofrequency ablation without any recurrence . a 16-year - old male with fitzpatrick skin type v presented to our clinic with partially fluid - filled lesions on left upper arm with slight local swelling . the patient had these lesions since the age of 2 years and had been repeatedly treated with antibiotics for local infection . examination showed a well - defined plaque on left upper arm with grouped vesicular lesions , giving the typical appearance of a a biopsy was taken from the edge of the lesion and histopathology showed multiple thin - walled , dilated lymphatic spaces in the papillary dermis and these spaces contained lymph and few erythrocytes . the overlying epidermis was thin , and there were elongated rete ridges that appeared to surround the lymphatic channels . the patient was given a systemic antiobiotic , cefadroxil 500 mg twice daily for 5 days , to treat the infection including oozing from the lesions and to decrease the inflammation . radiofrequency ablation was done a week later ( basco radiofrequency device , model rf - b2 , high frequency 2 mhz , power < 150 w ) , in cut and coagulate mode with wire loop electrode , under local anesthesia . the patient was advised to clean the area with betadine lotion and apply topical antibiotic mupirocin on the lesions . three sessions were required at weekly intervals for the lesions to clear as some deeper components were left untreated at the first two sessions . one week after last treatment , the area had partially healed with slight depigmentation . at 1 month , the lesions had completely healed and swelling had subsided [ figure 1 ] . no recurrence has occurred in the past 1 year and the patient is still under surveillance . ( a ) lc lesion on the left upper arm ; ( b ) lesions being treated with radiofrequency ablation ; ( c ) immediate post treatment red erythematous area ; ( d ) complete healing of the lesions at 1 month lc is benign ectasia with two components : the clinically obvious , dermal vesicular component , visible on the skin , and the deeper subcutaneous cisternal element . whimster described the pathogenesis and said that lc arises from the subcutaneous muscle - coated lymphatic cisterns which receive lymphatic flow from the surrounding tissue , but this is not drained to the normal lymphatic system . these dilated cisterns conduct the lymph through communicating channels into the dermal thin lymphatics , which balloon out into the epidermis . treatment modalities include surgical excision , lasers and sclerotherapy with varying success.[35 ] we tried the radiofrequency ablation to treat lc , which produced near - complete clinical ablation with coagulation of lesional and perilesional skin leading to fibrosis of the perivesicular lymphatics . the follow up was longer in our case , i.e. , 1 year and we are encouraged to do a case series to determine the efficacy of radiofrequency in this notorious condition .

this paper outlines botxminer , a publicly available application to search xml - formatted medline data in a complete , object - relational schema implemented in oracle xml db . an advantage offered by botxminer is that it can generate quantitative results with certain queries that are not feasible through the entrez - pubmed interface . after retrieving citations associated with user - supplied search terms , medline fields ( title , abstract , journal , mesh and chemical ) and terms ( mesh qualifiers and descriptors , keywords , author , gene symbol and chemical ) , these citations are grouped and displayed as tabulated or graphic results . this work represents an extension of previous research for integrating these citations with relational systems . botxminer has a user - friendly , intuitive interface that can be freely accessed at . lymphangioma circumscriptum ( lc ) , a hamartomatous lymphatic malformation , is a therapeutic challenge for the dermatologist . various modalities like surgical excision , lasers , and sclerotherapy have been used in the past to treat this notorious skin condition . we report the efficacy of a radiofrequency ablation in a patient with lc . the treatment efficacy of radiofrequency was satisfactory in our patient with no recurrence during 1 year follow - up period . the radiofrequency technique is a safe and economic treatment for management of lc .

a patient was immediately referred to our department after epidural endoscopy with severe bilateral visual impairment and underwent complete ophthalmologic examination . a 45-year old man suffered from low back pain unresponsive to pharmacologic treatment , radiating to the left hip and left lower extremity , due to a back injury 4 years ago . other medical history was noncontributory and he was otherwise healthy without hypertension , diabetes mellitus , cardiovascular disease , or blood clotting abnormalities . the patient underwent an epidural endoscopy and endoscopic adhesiolysis in the level of l4l5 and o5s1 by infusion of 120 cc of normal saline . at the end of the procedure the patient received an epidural injection of 80 mg methylprednisolone . after the operation the general condition of the patient was good without clinical signs of elevated cerebrospinal pressure and blood pressure and heart rate remained stable . however , a few hours later , he became aware of a major decrease of vision in both eyes and was immediately admitted to the department of ophthalmology . at presentation , best corrected visual acuity ( bcva ) was 20/400 re and counting fingers le . the ophthalmic examination revealed the presence of extensive scattered preretinal , subhyaloid , and subretinal hemorrhages in both eyes and especially in the le ( figure 1 ) . ocular history before the operation did not reveal any ocular disease and bcva was 20/20 in both eyes . examination of the fundus showed mild vitreous hemorrhage with extensive intraretinal and subretinal hemorrhages involving the macula especially of the le . four months after the operation , bcva was 20/30 in both eyes . despite the improved vision fundus examination revealed a remarkable resolution of the retinal and subretinal hemorrhages bilaterally with mild pigmentary changes of the macula especially of the le ( figure 2 ) . intraocular hemorrhages often occur in association with acute subarachnoid hemorrhage and secondary aneurysms of the anterior communicating and internal carotid arteries ( terson 1900 ) . the present case represents an extremely rare incidence of intraocular hemorrhage following epiduroscopy and endoscopic adhesiolysis ( amirikia et al 2000 ; brian et al 2005 ) . several mechanisms have been proposed to explain the occurrence of intraocular hemorrhage ( purdy et al 1998 ) . it seems that a sudden increase of the epidural pressure after the epiduroscopy causes cerebrospinal fluid to effuse through the communication of the subarachnoid space within the optic nerve sheath and subsequently compresses the optic nerve and its vasculature . specifically , the retinochoroidal anastomosis and the central retinal vein are occluded resulting in venous stasis allowing extravasation of blood through the vessels causing subhyaloid , retinal , and subretinal hemorrhages . the different types of hemorrhages suggest an acute generalized extravasation of blood within the various layers of the posterior segment opposed to the direct tracking of blood from within the optic nerve sheath . more particularly , intracranial pressure can be directly affected if the dura is punctured and fluid is added to the subarachnoid space . the visual prognosis is considered to be good , with recovery occurring within 6 months ( tabandeh 2000 ) . the present case is rare and it represents a rare complication of a rather common operation . however , as endoscopic spinal procedures become widely established , it is anticipated that similar cases will be recognized more frequently in the future . therefore , surgeons should be aware of the possibility of possible vision loss after this procedure . the human genome is laden with repetitive sequences in the form of transposable elements ( tes ) and tandem repeats , which comprise 45% and 3% of the human genome , respectively . using new methods capable of annotating repetitive sequences that have substantially diverged from known te sequences , recent analyses indicate that up to 6669% of the human genome may be composed of repetitive sequences , predominately in the form of tes . tes , as their name implies , are able to move about in the genome and create interspersed repeats . tes include dna transposons , long - terminal repeat ( ltr ) retrotransposons and non - ltr retrotransposons . in this review , we will focus on non - ltr retrotransposons , which are the only active tes in the human genome . non - ltr retrotransposons include long interspersed elements ( lines ) and short interspersed elements ( sines ) . both mobilize via a copy and paste mechanism , which requires the transcription of a donor element into an rna intermediate and the subsequent reverse transcription and incorporation of the rna intermediate into the genome as a new dna copy ( i.e. , the retrotransposition process ) . among them , line-1s ( l1s ) are autonomous ; full - length l1s encode two proteins , orf1 and orf2 , which are essential for their mobilization ( fig . in contrast , sines are non - autonomous and their mobilization relies on l1 proteins . 1b ) and mouse b1 , both of which are derived from 7sl rna ; the second class is typified by mouse b2 elements , which are derived from trnas . the quantity and mobility of non - ltr retrotransposons have made them important drivers of genomic diversity and evolution . ( a ) the locations of mononucleotide microsatellite seed sequences ( i.e. , 57 bp proto - microsatellites ) are diagramed along the length of the consensus human l1 sequence . five proto - microsatellites within the sequence are 7 units long , only one nucleotide below the threshold for mononucleotide microsatellites . a long poly(a ) tail is highlighted at the 3 end of the l1 element ( in bold font ; only 7 a s shown due to space limitation ) . ( b ) the locations of 36 bp mononucleotide proto - microsatellites are diagramed along the length of the consensus alu sequence . unlike l1 , alu is g / c rich and only two poly(a ) proto - microsatellites are found in the middle linker region between left and right monomers . over time a long poly(a ) tail is highlighted at the 3 end of the alu element ( in bold font ; only 7 a s shown due to space limitation ) . in contrast to interspersed repeats , tandem repeats consist of sequences that are sequentially repeated . tandem repeats are classified by the size of their repeating unit : those with a repeating unit length of 16 base pairs ( bp ) are commonly referred to as microsatellites ( fig . however , the boundary between microsatellites and minisatellites is often disputed , where the latter typically refer to tandem repeats of greater unit sizes . as they are highly polymorphic , both microsatellites and minisatellites have been instrumental in genetic mapping , evolutionary and phylogenetic studies as well as dna forensics . this review focuses on microsatellites , which encompass mono- , di- , tri- , tetra- , penta- and hexa - nucleotide repeats . notably , mononucleotide a repeats ( i.e. , poly(a ) dna tracts ) represent the most abundant microsatellite class with an overall length of 12 bp in the human genome . due to the repeating nature of these dna tracts , they are prone to rapid and variable contraction and expansion of repeat length ( fig . ( a ) microsatellite anatomy and terminology . a mononucleotide microsatellite and a trinucleotide microsatellite are depicted . the repeat unit length , the number of repeating units , and the overall length are noted for each microsatellite . both microsatellites have the same number of repeating units , but the unit length and the overall length vary among them . the first involves line / sine retrotransposition ; a microsatellite , typically a mature poly(a ) repeat , is born into the genome concurrently upon line / sine integration . the second pathway involves birth from random sequences ( not shown ) and/or proto - microsatellites via base substitution , indel and replication slippage . when a poly(a ) tract reaches 89 a s , it is considered a mature microsatellite . as the length increases , length contractions outweigh expansions , or vice versa , forming an indefinite loop ( thus , bypassing the path to death ) . the interrupted microsatellites may recover and continue expanding ( not shown ) or may be further mutated and eventually die . collectively , the dynamics of these two entities are a major source of genomic variation and serve as the architectural framework for protein - coding genes . recent work has revealed an intimate relationship between non - ltr retrotransposons and microsatellites . on one hand , non - ltr retrotransposons are a major source of new microsatellites as they give birth to microsatellites both concurrent and subsequent to their integration into the genome . on the other hand , the inherent instability of microsatellite repeats has a direct impact on the fitness of their non - ltr retrotransposon parent . this review will focus on the interactions and mutation dynamics between non - ltr retrotransposons and microsatellites as well as the variability and rates of mutation of microsatellite repeats that make them instigators of genomic evolution . it is proposed that each microsatellite locus possesses a lifecycle that begins with its birth into the genome and ends with its death phase , a period that is punctuated by contractions and expansions , which dynamically shorten and lengthen the overall microsatellite length . the most commonly proposed mechanism for microsatellite contractions and expansions is replication slippage by dna polymerases . two other mutation mechanisms , base substitution and indel slippage , are also thought to play an important role in microsatellite birth and death . one is through de novo retrotransposition by non - ltr retrotransposons , while the other involves the birth of proto - microsatellites from random sequences . it is the goal of this review to integrate these two pathways and highlight the role of non - ltr retrotransposons in microsatellite genesis . non - ltr retrotransposons can give birth to microsatellites concurrently to their integration into the genome ( i.e. , the first pathway ; fig . the association between a - rich microsatellites and non - ltr retrotransposons has been repeatedly observed by many independent studies . a recent analysis of microsatellites at orthologous loci in three primate genomes estimated that upon retrotransposition , alus and l1s contribute to 36% of mono- , di , tri- and tetranucleotide microsatellites in human , chimpanzee and orangutan genomes . this pathway is best illustrated by the birth of poly(a ) microsatellites . to mobilize , non - ltr retrotransposons require a poly(a ) tail at the 3 end of their sequence ( discussed later ) . thus , a common feature of alu and l1 insertions that have integrated into the genome is the presence of a 3 poly(a ) dna tail ( fig . 1 ) . these a - rich sequences are born into the genome as mature microsatellites , far above the threshold size ( fig . 3 ) . in humans , 90% of these poly(a ) repeats are derived from poly(a ) tails of l1 and alu sequences . even this may be an underestimate because l1 can integrate a pure poly(a ) dna tract into the genome if the insertion is severely 5 truncated ; such events would typically be undetected . the abundance of l1 and alu sequences in the primate genomes likely accounts for the overrepresentation of poly(a)/poly(t ) repeats compared with poly(c)/poly(g ) repeats . in contrast , dinucleotide microsatellites are most abundant in rodents , and ac repeats are the most frequent dinucleotide repeat in all vertebrates . not surprisingly , 15% of ac repeats are derived from the ac - rich tails of b2 sine elements found in the mouse genome . non - ltr retrotransposons can give birth to microsatellites both concurrently and subsequently to their integration in the genome . ( a ) concurrent birth occurs via the integration of a long poly(a ) tract , which is part of the alu or l1 element . additionally , the integrated element carries proto - microsatellites , depicted by the middle a stretch . here , the waved line represents genomic dna target , and the gray box represents the body of an alu or l1 element . ( b ) subsequent to integration , both the poly(a ) tail microsatellite and the internal proto - microsatellites may expand or contract due to dna polymerase slippage . ( c ) subsequent to integration , the poly(a ) tail microsatellite may also be mutated via base substitution or indel to form a new repeating unit , here depicted as ta at the end of the poly(a ) tract . the combination of mutational forces may eventually lead to the formation a new dinucleotide microsatellite at the 3 end of the retrotransposon . the second pathway posits that microsatellites initially arise from random sequences via mutation as proto - microsatellites , small tandem repeated sequences that are below the length threshold for mature microsatellites ( fig . theoretically , these initiating loci can be found everywhere in the genome . however , the overwhelming abundance of tes in the human genome made it the perfect hotbed for microsatellite births . it has been suggested that the conversion of aaa to naa is a major mechanism for the generation of all a - rich trinucleotide repeats in the human genome as over 60% of gaa , caa and taa repeats with repeat number of 8 or higher are located within alu poly(a ) tails . furthermore , poly(a ) tails have also been implicated in the birth of dinucleotide and tetranucleotide microsatellites . aside from their 3 tails , both alu and l1 have internal proto - microsatellite sequences that can birth new microsatellites subsequent to their integration ( fig . 1 ; fig . 3 ) . an analysis of microsatellites at orthologous loci in three primate genomes indicates that 26% of microsatellite births and it is noteworthy that microsatellites are found in distinct distributions over the length of l1 and alu elements , likely reflecting differences in their sequence compositions . l1 sequences are at - rich and give rise to microsatellites during and after their insertion along their entire length ( fig . in contrast , alus are gc - rich and microsatellite births and deaths are enriched at the 3 poly(a ) tail and the middle a stretch that connects the left and the right monomers ( fig . the middle a - rich region of alu elements tends to expand and mature into poly(a ) microsatellites and can also give rise to gaa repeats . approximately 2% of gaa repeats with a repeat number of 8 or longer are located in the alu middle a tract ; one of such gaa microsatellite is positioned in intron 1 of the frda gene and its expansion is responsible for the neuromuscular disorder friedreich s ataxia . classic studies on microsatellite mutation rates utilize pedigree analysis , usually in cancer patients with microsatellite instability . several model systems , including e. coli , yeast , human and mouse cells have also been described and used to obtain in vivo mutation rates . in addition , computational and mathematical models have been utilized to describe the behavior of microsatellites by comparing genomic sequences from large scale sequencing efforts and from evolutionarily related species . the majority of computational studies and in vivo studies focus exclusively on pure or perfect repeats , which contain only nucleotides pertaining to the repeating unit . imperfect repeats , in contrast , have additional nucleotides that do not belong to the repeating units . overall , the rate of microsatellite mutation is highly variable , and it is influenced by several factors , including the number of repeating units , unit repeat length , sequence composition , genomic loation and the secondary structures of the repeat . specifically , microsatellites become highly mutable when the number of repeating units exceeds a threshold value . although there is disagreement on the exact threshold value , the consensus is that the threshold represents the size that allows the sequence to acquire slippage mutations at a higher rate than the genomic average . the threshold values for mono- , di- , tri- and tetranucleotide repeats are estimated to be 89 , 45 , 3.44 and 4 respectively . in fact , the distinct mutational behaviors manifested by microsatellite before and after they reach the threshold have prompted some researchers to consider microsatellites only as those that exceed their respective threshold . once a tandem repeat has reached its threshold , experimental studies with human and mouse models have shown that the mutation rate increases exponentially as the number of repeating units increases ( table 1 ) . however , due to the differences in the experimental systems used , it is difficult to compare across these studies . the exponential increase in mutation rates is not seen in mismatch repair deficient cells , indicating that mismatch repair proteins are implicated in the expansion process ( table 1 ) . contraction rate ; number in parenthesis is the contraction rate for the same repeat ; trinucleotide studies focus primarily on mismatch repair deficient cells . trinucleotide repeats that cause disease tend to be more unstable in the germline than in somatic cell lines . sequence composition also affects mutability . for similar sized repeats , ga repeats are more mutable than ca repeats and sequences with higher gc content have lower rates of mutation ( table 1 ) . stability increases with the complexity of the repeating pattern , as demonstrated in tetranucleotide repeat studies . despite a doubling of repeat unit length , di- and tetranucleotide repeats have similar stabilities ( table 1;tc vs. ttcc ) . in contrast , mononucleotide microsatellites , such as those associated with retrotransposons , are highly unstable and hypermutable in the germline . for example , the human bat-40 locus is an intronic poly(a ) tract of 40 a s in the 3--hydroxysteroid dehydrogenase gene ( hsd3b1 ) . . five - fold more mutant alleles are found in sperm than in leukocytes of the same individual . the bat-40 poly(a ) microsatellite has not been identified to be associated with non - ltr retrotransposons ; here , we report that it is in fact the poly(a ) tail of an alujb element . despite experimental focus on the expansion dynamics of microsatellites , the mutation dynamics for very long ( > 50 bp ) mononucleotide repeats had been frequently overlooked because of their rarity in the genome . using a transgenic l1 mouse model , we characterized the contraction dynamics of long poly(a ) tracts associated with nascent l1 insertions . these poly(a ) microsatellites show rapid and variable contraction to lengths below 50 bp in both somatic and germ cells at frequencies of 14.8% and 13.3% , respectively . of note , these values represent the highest mutation frequencies hitherto reported for all microsatellite repeats , highlighting the extraordinary mutability of long poly(a ) tracts ( table 1 ) . long poly(a ) microsatellites birthed into the genome via retrotransposition are predicted to shorten in length rapidly within the first few generations . this prediction is consistent with observations from surveys of genomic resident alu and l1 sequences , which demonstrated an inverse correlation of poly(a ) tract length with the evolutionary age of the elements . one prominent form of microsatellite death is interruption by a non - repeat base , which can arise from base substitutions or short indel mutations . in particular , a comparison of human , chimpanzee and orangutan genomes suggests that substitutions are the primary cause of deaths for microsatellites of all lengths . although not widely recognized , non - ltr retrotransposons may also act as a mechanism for microsatellite interruption . such interruption can occur when new copies of non - ltr retrotransposons insert into pre - existing microsatellites . mammalian l1s are members of a diverse and widely distributed group of autonomous non - ltr retrotransposons that encode an endonuclease with homology to apurinic / apyrimidinic endonucleases ( ape ) . a small subset of these ape - type elements insert in a sequence - specific manner into microsatellites : ( 1 ) acay microsatellites are targeted by three waldo elements ( waldoag1,waldoag2 in the african malaria mosquito a. gambiae and walsofs1 in the earwig f. scudderi ) ; ( 2 ) ac microsatellites are targeted by a mino element in a. gambiae ( minoag1 ) ; ( 3 ) tc microsatellites are targeted by four kibi elements ( kibidr1 and kibidr2 in zebrafish d. rerio , kibifr1 in the torafugu f. rubripes , and kibitn1 in the green spotted pufferfish t. nigroviridis ) ; ( 4 ) ttc microsatellites are targeted by two koshi elements ( koshifr1 in f. rubripes and koshitn1 in t. nigroviridis ) ; ( 5 ) taa microsatellites are targeted by three dong elements ( dong in the silkworm b. mori , dongag in a. gambiae and dongbg in the freshwater snail b. glabrata ) . the molecular mechanisms underlying microsatellite targeting by these ape - type non - ltr retrotransposons are not well understood . it is believed that the sequence - specific cleavage by the endonuclease plays a major role in active targeting . in addition , two ape - type elements ( waldo - a and waldo - b in the fruitfly d. melanogaster ) are frequently found within or close to ca microsatellites , most likely as a result of passive accumulation in these low recombination regions . microsatellites may also be targeted by dna transposons ; examples include mine-1 helitrons and micron mites . the former insert at gaaa microsatellites in the genomes of the european corn borer o. nubilalis , the silkworm b. mori and the pink bollworm p. gossypiella , while the latter insert into ta microsatellites in the rice genome . in contrast , mammalian l1s have a weak target site preference with a consensus sequence 5-ttaaaa-3. although they may fortuitously land in or near certain microsatellite sites , they are unlikely to serve as a major factor in disrupting microsatellites . the relationship between microsatellites and non - ltr retrotransposons is not unidirectional . while both l1s and alus give birth to microsatellites , especially poly(a ) mononucleotide microsatellites , these microsatellite sequences can also affect the fitness of their parent due to their unusually high mutation rates . the impact of microsatellite instability on non - ltr retrotransposons depends on the location of the microsatellite , i.e. , whether it is internal or at the 3 terminal of the element . the effect of variation in microsatellites internal to non - ltr retrotransposons is less understood . newly inserted l1 and alu copies carry many mononucleotide proto - microsatellites ( fig . 1 ) . as microsatellites are predicted to mutate faster than the genomic average , expansion and contraction of microsatellite loci internal to l1s may introduce frameshift mutations , abolishing l1 coding capacity ( fig . ( a ) effect of internal microsatellite loci . expansions or contractions of proto - microsatellite loci inside an l1 element can cause frameshift mutations , leading to loss of orf1 and/or ofr2 function . the mutagenized l1 element would be unable to mobilize itself and to support alu retrotransposition . the contraction or expansion of an alu s poly(a ) tail diminishes or stimulates its retrotransposition , respectively , presumably because the length of its poly(a ) rna tail is positively correlated with the efficiency of target - primed reverse transcription ( tprt ) . ( c ) effect of the 3 poly(a ) microsatellite on l1 elements . due to the random nature of integration , an l1 sequence may not have a gt rich region downstream to its polyadenylation signal . however , there is evidence that the l1 poly(a ) tail exerts a crucial role in ensuring proper polyadenylation of l1 mrna in the absence of a strong gt - rich downstream sequence . the relative efficiency in mobilization is depicted by a difference in size of l1 mrna and protein products . the 3 poly(a ) tail of an l1 or alu element is a crucial component of the retrotransposition process and therefore , its length directly affects their retrotransposition potential . distinct cellular processes are responsible for the poly(a ) tail formation in l1 and alu elements . l1 elements are transcribed by rna polymerase ii and poly(a ) polymerases produce a poly(a ) rna tail as the 3 end of an l1 mrna . in contrast , alu elements are transcribed by rna polymerase iii and the resulting transcripts are not polyadenylated . however , active alu elements have a poly(a ) dna tract , which is transcribed as part of the alu rna . the 3 poly(a ) rna tail is predicted to serve two important roles during retrotransposition . first , the initial base pairing of these a s with the t - rich dna sequence at the target site may be required for efficient first - strand cdna synthesis during target - primed reverse transcription ( tprt ) . the l1 orf1 protein is an rna - binding protein with nucleic acid chaperone activity and it may facilitate this strand transfer and annealing process . second , increasing evidence indicates that these poly(a ) rna tails are bound by poly(a ) binding proteins ( pabps ) and that this interaction is critical for the formation of the ribonucleoprotein complex between l1 proteins and l1/alu rnas . indeed , retrotransposition assays demonstrate that the poly(a ) tail is strictly required for alu mobilization and that its retrotransposition activity is positively correlated with the length of poly(a ) tails . in parallel , poly(a ) tail shortening in expressed l1 mrnas impairs rnp formation and retrotransposition . therefore , the high mutability of poly(a ) tails has direct impact on l1 and alu retrotransposition . once captured in genomic dna , the initial long 3 poly(a ) tract undergoes rapid shortening in the first few generations . in fact , genome - wide , the length of the poly(a ) tract is inversely correlated with the evolutionary age of alu and l1 subfamilies . as the length of the poly(a ) tail is a key determinant of alu activity , a - tail expansion due to microsatellite instability is predicted to promote its retrotransposition and , conversely , its contraction would predict the extinction of an alu s ability to mobilize ( fig . indeed , newly generated alu copies typically have longer poly(a ) tails than the original donor elements . additionally , a nascent retrotransposition - competent alu element is able to propagate its newfound mobility to progeny copies through poly(a ) tail expansion during reverse transcription . it is generally accepted that the l1 poly(a ) tail is regenerated during each round of retrotransposition via reverse transcription of the polyadenylated l1 mrna . thus , the length of the poly(a ) tail in the donor l1 element may affect l1 retrotransposition only if it alters transcription and polyadenylation . a canonical polyadenylation signal contains a conserved aataaa hexamer and a downstream gt - rich region . the l1 polyadenylation signal is atypical in that a poly(a ) tail separates the aataaa hexamer from any downstream sequence ; the sequence composition of the latter is determined by chance depending on where the donor element is inserted during the previous round of retrotransposition . such a configuration may predispose an l1 element to bypass its own polyadenylation signal and instead to use a stronger polyadenylation signal fortuitously situated further downstream . nevertheless , the poly(a ) tail appears to exert a critical role in proper polyadenylation of l1 mrna in the absence of a strong gt - rich downstream sequence ( fig . although full length transcription is possible in the context of a weak polyadenylation context , the presence of a string of 17 a s brings about significant increases in the transcriptional efficiency . although its presence and length is not as critical for l1 mobility as for alu mobility , the poly(a ) tail s dynamics still subtly influence the efficiency of its parent in a disadvantageous genomic neighborhood . the availability of complete genome sequences for many organisms has enabled genome - wide analyses of repeat distribution . microsatellites with overall repeat length of 12 bp or longer make up 1.3% of the reference human genome ( i.e. , 13.3 kb per mb genome , excluding hexameric repeats at telomeres ) . although the overall density of microsatellites is relatively uniform in all human chromosomes , the distribution of each repeat class ( mono- , di- , tri- etc . ) varies : ( 1 ) mononucleotide microsatellites show a uniform distribution among exonic , intronic and intergenic regions ( note exonic regions herein include not only protein - coding sequences but also 5 and 3 untranslated regions ) ; ( 2 ) di- , tetra- and pentanucleotide microsatellites are relatively depleted in exonic regions ( 74% , 75% and 87% of the genome average , respectively ) ; ( 3 ) tri- and hexanucleotide microsatellites are enriched in exonic regions ( 191% and 146% of the genome average , respectively ) . the vast majority of human microsatellites that are conserved during vertebrate evolution are found in intergenic and intronic regions , consistent with the prediction that microsatellites in exonic regions are more disruptive to gene function . in comparison , the two major types of non - ltr retrotransposons , l1s and alus , display contrasting distribution patterns relative to genes . alus typically are overrepresented inside genes ( i.e. , residing anywhere in a transcription unit ) as well as within 30 kb from annotated genes . in addition , alus display no orientation bias relative to genes . in contrast , l1s show prominent orientation bias within genes : while antisense l1s are close to the predicted density , sense l1s are strongly depleted within genes . in addition , both sense and antisense l1s are significantly underrepresented within 5 kb from annotated genes . the differential distribution of alus and l1s relative to genes is not a result of target preference because both are retrotransposed by l1 proteins . rather it is consistent with the predicted impact of sense - oriented l1s on premature polyadenylation , cryptic splicing and/or inhibition of transcriptional elongation . this idea is further supported by the random chromosomal distribution of recent endogenous l1 and alu copies in the human genome and de novo insertions from l1 transgenes in mice . furthermore , the large catalog of polymorphic alu and l1 insertions clearly shows that exons can be targeted in proportion to their abundance in the genome . due to their dynamic nature , microsatellite loci are highly polymorphic among individuals . by comparing the reference human genome to a second genome , overall , 2.7% of the microsatellite loci are polymorphic , a frequency that is 45-fold higher than single nucleotide polymorphism ( 0.06% ) . exonic , especially the coding exonic sequences , show the least amount of microsatellite variation as compared with intergenic and intronic sequences , reflecting increased selective constraints on exonic sequences . polymorphism incidence increases with repeat number , an observation that is reproduced by surveying polymorphic microsatellite loci among individual genomes from the 1000-genome project . there is remarkable heterogeneity in the levels of variation at specific microsatellite loci . for example , the trinucleotide repeat in the 5utr of the fragile x mental retardation 1 ( fmr1 ) gene is one of the most studied microsatellite loci with respect to polymorphism in ethnically diverse , healthy individuals . the range of repeat number varies from population to population ; the overall range is 6 to 87 in surveyed populations . a repeat number of > 200 is considered a full mutation , which is typically associated with intellectual and developmental disabilities . whole - genome as well as targeted sequencing has also started to reveal the extent of genetic variations caused by non - ltr retrotransposons . polymorphisms in non - ltr retrotransposons are manifested as indels ( i.e. , insertions in one genome and deletions in another genome ) , a common form of structural variations ( other forms include tandem duplications , inversions and translocations ) . recent whole - genome sequencing efforts have cataloged a large number of structural variations among diverse human populations . detailed analyses of mutational mechanisms indicate 2030% of svs are caused by non - ltr retrotransposons . on average , each human genome contains 10002000 polymorphic non - ltr retrotransposons , with 7985% alu , 1217% l1s and 3% svas . based on the number of polymorphic insertions and the estimated genetic distance between individual genomes , the frequency of retrotransposition has been calculated as one alu insertion in 21 births and one l1 insertion in 108 to 212 births . most intriguingly , an analysis of the 1000 genomes project pilot data set suggests a recent increase in retrotransposition rates in humans . these insertions continuously generate new germline polymorphisms that can be passed across generations ( fig . 5 ) . as compared with humans , tes play an even bigger role in generating structural variations in the mouse genome . this discrepancy can be largely explained by the presence of a large number of active ltr retrotransposons in the mouse genome besides non - ltr retrotransposons . together , retrotransposon insertions contribute to of structural variations between two inbred mouse lines , with 52% , 43% and 5% contribution from ltr retrotransposons , l1s and sines , respectively . in addition to retrotransposition in the germline , non - ltr retrotransposons are active in somatic tissues , including normal brain tissues and most epithelial cancers ( further discussed in the next section ) . despite the significant progress made thus far , it should be noted that the discovery of structural variations mediated by tes remains a challenge because of the technical difficulty in de novo assembling repetitive sequences from short sequence reads as well as the high sequence depth that is required for detecting low - frequency alleles . the schematic illustrates the mutation dynamics of poly(a ) microsatellites across generations , beginning with an individual who has a newly acquired long poly(a ) microsatellite from l1 or alu retrotransposition . color is used to distinguish the size and mutability of the microsatellite , where darker color indicates longer poly(a ) tails that are more mutable . a long poly(a ) microsatellite can expand or contract between generations and give rise to variations in the offspring . however , when a poly(a ) microsatellite mutates below the threshold repeat number , the rate of mutation decreases to the average genomic mutation rate . the impact of a microsatellite or a transposable element and its associated variability is dependent on its genomic location . the most direct phenotypic impact of microsatellites and retrotransposons is manifested as disease causing mutations . microsatellite instability has been implicated in a variety of human diseases with over 40 neurological , neurodegenerative and neuromuscular disorders associated with trinucleotide repeat instability . well known examples include huntington s disease , fragile x syndrome and friedreich s ataxia . unlike static mutations , which can be stably passed from one generation to the other , alleles with expanded microsatellites may continue to expand over the course of the individual s life or through germline transmission . depending on the genomic neighborhood , variation in microsatellite length these mechanisms have been best characterized in trinucleotide repeat expansion disorders and typically consist of one ( or the combination ) of three following three pathways : the loss of function at the protein level , the gain of function at the protein level , or the gain of function at the rna level . the latter is gaining increased attention due to the recognized role of non - coding rna in epigenetic pathways . a noticeable absence among these molecular mechanisms is a loss of function at the rna level , a potential mechanism that should be investigated in future studies . likewise , de novo retrotransposition has been implicated in a variety of sporadic human diseases , including hemophilia , apert syndrome , neurofibromatosis type 1 and congenital muscular dystrophy . a multitude of molecular mechanisms have been proposed to account for the mutagenesis effect for intronic or exonic insertions . a less emphasized impact is post - insertional genome instability mediated by non - ltr retrotransposons , which likely poses even greater hazard to the human genome . for example , nonallelic homologous recombination ( nahr ) between two separate copies of alu repeats may generate structural variations , such as indel , inversion and translocation mutations . in addition , a hallmark of non - ltr retrotransposition is the formation of a pair of target site duplications ( tsds ) that flank the new insertion . these tsds represent pre - insertion l1 endonuclease cleavage sites and may be targeted for subsequent cleavage by other l1 endonucleases . it is proposed that new double strand breaks in tsds may trigger rearrangement events due to their adjacency to non - ltr retrotransposons with abundant homologous copies . these mechanisms highlight the dynamic nature of l1 and alu sequences in the human genome . furthermore , as has been discussed earlier , non - ltr retrotransposons are not static entities after insertion ; they are a significant source for microsatellite genesis . high - frequency microsatellite instability ( msi - h ) is a molecular feature of tumors associated with hereditary nonpolyposis colorectal cancers ( hnpcc ; lynch syndrome ) and has been seen in other sporadic cancers , usually of the gastrointestinal system . these tumors generally arise from defects in the mismatch repair system , leaving dna vulnerable to a high degree of mutation within microsatellites . detection of msi - h tumors is often achieved with a reference panel of five microsatellites , including two mononucleotide and three dinucleotide markers . the two mononucleotide markers , bat26 and bat25 , contain ( a)26 and ( a)25 repeats , respectively ; their length changes can be easily discerned even in the absence of normal tissue . it is thought that msi - h contributes to tumorigenesis mainly through mutating genes with coding microsatellites . a recent genome - wide survey yields the initial glimpse of microsatellite mutational landscapes in msi - h gastric cancers . there are four to 6-fold higher microsatellite mutations in genes in msi - h gastric cancers than microsatellite stable ( mss ) cancers . only a very small fraction of microsatellite mutations ( 0.1% ) are located in protein - coding exonic regions ; the remaining are found in 5utr , 3utr , intronic and intergenic regions of the genome . importantly , genes with significant changes in mrna expression between msi - h and mss samples are enriched for microsatellite mutations in their utrs , indicating a role of non - coding microsatellite instability in regulating gene expression . a role of non - ltr retrotransposons in tumorigenesis several studies have used nextgen sequencing approaches to detect genome - wide structural variations or specifically new retrotransposon insertions in tumor samples . the first study identified 9 tumor - specific ( i.e. , somatic ) l1 insertions in 6 of the 20 lung tumors but none in 10 brain tumors by targeted sequencing . a subsequent study characterized retrotransposon insertions in 43 human tumors of different origins by whole - genome sequencing . tumor - specific somatic insertions were found in all tumors of epithelial origin ( colorectal , prostate and ovarian ) , but not in blood or brain cancers . among epithelial cancers , colorectal cancers had the highest incidence of l1 insertions at an average of 9 insertions per tumor among 4 colorectal samples ; the fifth colorectal tumor sample alone had an astonishingly high number of 102 l1 insertions . interestingly , this tumor was the only colorectal sample that manifested msi - h with altered dna repair pathways , suggesting a common origin to both sources of genomic instability . an independent study mapped tumor - specific l1 insertions in 16 colorectal tumors by targeted l1 sequencing . intriguingly , although two samples with the highest number of somatic insertions showed microsatellite instability , no correlation was found between the number of insertions and the microsatellite instability status . future studies with higher coverage and/or larger sample size may be needed to establish a connection between microsatellite instability and retrotransposon mutagenesis . it should be emphasized that , in addition to somatic insertions , germline insertions may also be an important etiological factor in predisposing the carriers to cancers , as has been recently shown for hepatocellular carcinoma patients . the precise role of retrotransposon insertions in cancer remains to be determined but they can have a significant impact on gene function . consistent with sense - oriented l1 insertions being more disruptive to gene expression , genes targeted by sense insertions , but not those targeted by antisense insertions , showed statistically significant decrease in transcription . although usually highlighted for their negative roles , microsatellites and retrotransposons have evolutionary importance due to their ability to provide genetic novelties in human populations . a salient example is sequence shuffling through 3 transduction . due to the weak nature of the l1 poly(a ) signal , it is possible for l1 to bypass the signal during transcription and use an alternative poly(a ) signal downstream . the latter involves a 3 transduction event that is severely 5 truncated , which leaves behind no l1 sequence to be associated with the mobilized sequence . thus , 3 transduction has the potential to generate new genes and bring new regulatory features to the vicinity of other genes . although no cases have been reported , it is conceivable that l1s could also mobilize microsatellites by the same mechanism . another way of introducing evolutionary novelties is through trans - mobilization of cellular rnas , which form processed pseudogenes . lastly , since the exciting discovery of somatic retrotransposition in the brain , it has been postulated that l1-induced neuronal diversity may be an important source for behavioral phenotypes and confer evolutionary advantage . beyond genome sequence changes , the importance of other epigenetic mechanism , including dna methylation , histone modification and nucleosome occupancy , is coming to light . therefore , the insertion of an l1 into a genomic region may alter the previously existing epigenetic state ( and our unpublished data ) . due to their unique sequence composition , microsatellites can change the physical forms of dna where they occur , which can have implications for gene expression and genome stability . though not all microsatellites meet the criteria to do so , many repeats form non - b dna structures . these non - b forms of dna are often associated with locations of chromosomal breakage . an inverse correlation exists between a repeat s ability to form non - b form dna and its abundance in the genome , suggesting that these unstable structures are selected against due to reduced evolutionary fitness . the presence of poly(a ) tracts within promoter sequences also has implications for nucleosome organization . poly(a ) repeats form shorter and more rigid helix structures and have a narrower minor groove , altering nucleosome positioning in manners which increase the binding ability of transcription factors . thus , the mobilization of retroelements and their associated poly(a ) tails present a direct manner for l1 , alu and sva elements to affect the genome compaction and expression dynamics of the local regions where they land . in summary , microsatellites and non - ltr retrotransposons are dynamic dna sequences that rapidly change from generation to generation . expansion and mutation of microsatellites within l1 and alu sequences may change the size and sequence identity of these non - ltr retrotransposons , perhaps modulating their mobility . future studies to compare the sequence of full length l1s may reveal how the dynamics of microsatellites affect the retrotransposons from which they are born .

purposeto report a case of acute visual loss after endoscopic spinal surgery.methodsa patient was immediately referred to our department after epidural endoscopy with severe bilateral visual impairment and underwent complete ophthalmologic examination.resultsvisual acuity was decreased in both eyes . fundus examination revealed the presence of retinal and vitreous hemorrhages bilaterally . four months later , visual acuity increased and the hemorrhages were remarkably resolved.conclusionthe present case represents an extremely rare incidence of intraocular hemorrhage following epiduroscopy . the human genome is laden with both non - ltr ( long - terminal repeat ) retrotransposons and microsatellite repeats . both types of sequences are able to , either actively or passively , mutagenize the genomes of human individuals and are therefore poised to dynamically alter the human genomic landscape across generations . non - ltr retrotransposons , such as l1 and alu , are a major source of new microsatellites , which are born both concurrently and subsequently to l1 and alu integration into the genome . likewise , the mutation dynamics of microsatellite repeats have a direct impact on the fitness of their non - ltr retrotransposon parent owing to microsatellite expansion and contraction . this review explores the interactions and dynamics between non - ltr retrotransposons and microsatellites in the context of genomic variation and evolution .

one important and interesting problem in genome research is the prediction of transcription factor binding sites ( tfbss ) . binding of transcription factors to their dna binding sites in the regulatory region of a gene is a prerequisite for its activation or repression . the combinatorial presence of tfbss controls gene regulation at least partially , and the prediction of tfbss is of importance for unraveling the underlying molecular mechanisms . wet - lab experiments allow the identification of tfbss , but these experiments are expensive and time consuming . computational methods , which are often less accurate but easy to conduct with available resources , are a welcome complementation to wet - lab experiments . many tfbs prediction algorithms employ statistical models for scoring a given sequence as tfbs or non - tfbs . for these algorithms , the family of markov models ( 13 ) is chosen in many tfbs prediction algorithms as well as for a variety of other classification problems . well - known examples are the position weight matrix ( pwm ) model ( 4 ) , which is an inhomogeneous markov model of order 0 , and the weight array matrix ( wam ) model ( 5 ) , which is an inhomogeneous markov model of order 1 . from a statistical point of view , markov models employ assumptions about the statistical independence of nucleotides at different positions of the binding site . the strongest independence assumption is realized by the pwm model , where each position is assumed to be statistically independent of all other positions . as indicated e.g. in ( 6 ) , it is an open question whether this strong independence assumption is reasonable in view of recent results indicating the presence of statistical dependences between positions ( 7,8 ) . markov models of higher order take into account statistical dependences from previous positions , called the context , where the length of the context is equal to the order of the markov model . despite the often unjustified independence assumption , pwm models are often found to outperform markov models of higher order in the prediction of tfbss . this is probably caused by the rather limited amount of training data available from experimentally verified tfbss . as the number of model parameters grows exponentially with the order of the markov model , markov models of higher order tend to be over - fitted , resulting in poor performance . one possibility to circumvent this problem is provided by variable order markov models , which do not require the contexts to be of a fixed length , but allow contexts with variable lengths ( 912 ) . the power of variable order markov models stems from the freedom to include only those contexts into the model for which there are strong statistical dependences . another possibility to capture statistical dependences between non - adjacent positions without the burden of increasing the number of model parameters exponentially are provided by bayesian trees ( bts ) ( 1316 ) . in bts the contexts are not restricted to the previous positions within the binding site , and it could be demonstrated that bts outperform pwm models and wam models in the prediction of splice sites ( 17,18 ) and tfbss ( 6,19 ) . as an extension of variable order markov models and bts , we introduce variable order bts ( 19 ) . the vombat web server is designed to train variable order markov models and variable order bts on user - supplied data and to apply the resulting models to the prediction of tfbss . in addition , the vombat web server provides a cross - validation platform , allowing advanced users to compare different model combinations with different parameter settings on problem - specific datasets . the vombat web server is primarily designed for the prediction of tfbss , but applicable to other types of fixed - length motifs as well . the prediction of tfbss is based on one statistical model for tfbss and one for non - tfbs sequences constituting the background . each statistical model assigns a probability p(x1xl ) to a given dna sequence x1xl of l nucleotides . for any value of m , 1 m l , p(x1xl ) markov models of order m assume that the conditional probabilities pl(xlx1xl1 ) do not depend on all previous nucleotides x1xl1 , but only on the m previous nucleotides xlmxl1 , which are called the context of position l. hence , a markov model of order m is defined by p(x1xl)=p0(x1xm)l = m+1lpl(xlxlmxl1 ) . if the conditional probabilities pl(xlxlmxl1 ) are identical at all positions , the markov model is called homogeneous , otherwise it is called inhomogeneous . in contrast to a markov model of order 1 , where the conditional probabilities at position l depend only on the previous nucleotide xl1 , a bt allows that the conditional probabilities at position l may depend on the nucleotide xpa(l ) at a possibly non - adjacent position pa(l ) , which is called the parent of position l. in a bt , arbitrarily remote positions may be chosen as parents as long as no cycles between positions are induced . this implies that there is one position , called the root position , which must not depend on any other position , and that the statistical dependences of a bt may be graphically represented by a rooted tree , where the positions are represented by nodes and the statistical dependences are represented by edges ( 1316 ) . the probability distribution defined by a bt with root position r decomposes as p(x1xl)=pr(xr)lrpl(xlxpa(l ) ) . in the vombat web server , the so - called motif model pmotif ( used for modeling tfbss ) and the so - called background model pbg ( used for modeling background dna sequences ) can be chosen by the user . an inhomogeneous markov model or a bt should be chosen if statistical dependences are assumed to vary from position to position within the sequence . a homogeneous markov model should be chosen if statistical dependences are assumed to be the same at all positions within the sequence . the appropriate order of a markov model depends on the expected range of statistical dependences and on the amount of available training data . for markov models of fixed order , the number of model parameters grows exponentially with the order , resulting in a sharp transition from under - fitted to over - fitted models . the idea of variable order markov models is to shorten the context in those cases where extending the context does not yield mathematically this is formulated by measuring to which degree the conditional probabilities change when extending or reducing the context . the kullback leibler divergence ( 20 ) is used as a measure of change of the conditional probability distributions . it measures the degree of dissimilarity of the conditional probability distribution given the extended context and the conditional probability distribution given the reduced context . leibler divergence is always non - negative , but small positive values of the kullback the threshold above which the kullback leibler divergences are considered important can be set by an external parameter , c , called pruning constant . for c = 0 , any extension of the context is considered important , and the resulting variable order markov model becomes a traditional markov model of order m. for c , any extensions of the context is considered unimportant , and the resulting variable order markov model becomes a traditional markov model of order 0 , i.e. a pwm model . as c grows from 0 to , more and more contexts are considered unimportant , and the resulting variable order markov models become smaller and smaller , interpolating between a markov model of order m and a pwm model . vombat uses a maximum likelihood estimator with optional pseudo counts , which can be specified by the user , for smoothing the conditional probability distributions and to compensate for zero occurrences of nucleotides and contexts . the motif model pmotif is learned from a user - supplied training set of tfbss , and the background model pbg is learned from a user - supplied training set of background sequences . after training pmotif and pbg , vombat can be used to compute genome - wide predictions of the learned motif in a set of user - supplied sequences . for each position of a sliding window of length l , the log - likelihood ratio of the oligonucleotide y1yl occurring at that position is computed , and y1yl is predicted as tfbs if the log - likelihood ratio is greater than a given threshold t , i.e. if log2pmotif(y1yl)pbg(y1yl)t . specific knowledge , such as the number of tfbss expected in the genome , can be taken into account to adjust the threshold t to achieve tfbs predictions with the desired balance of sensitivity and specificity . for advanced users , vombat allows a cross - validation of the prediction accuracy of a user - defined combination of pmotif and pbg based on a user - supplied set of tfbss and a user - supplied set of background sequences . from each of the datasets , 10% of the sequences are randomly excluded , and the models pmotif and pbg are trained on the remaining 90% . subsequently , both models are used for the prediction of the withheld data , and the sensitivity given a user - defined value of the specificity is computed . this procedure is repeated k times , where k can be specified by the user , and the mean sensitivity and its standard error are reported . vombat is based on a variant of the three - tier architecture , where the presentation layer , the management layer and the execution framework are logically separated and physically located on different servers . the web front - end of vombat is based on standard technologies like javaserver faces for the web forms and servlets for displaying images and providing downloads of results . the user - supplied parameters and input files are stored in a mysql - database . this database is queried by the execution framework . if the execution framework detects a job to be executed , it requests the necessary parameters and input files and submits the corresponding job to the scheduler of the attached linux cluster . after the job is finished , the results are written to the database and can be displayed by the web front - end , which loads numerical results , model descriptions and images from the result - tables of the mysql - database . imagine a user interested in a genome - wide prediction of binding sites of the transcription factor sp1 in gc - poor upstream regions of human refseq ( 21 ) genes . if the user were interested in a prediction based on pre - trained pwm models stored in the transfac database ( 22 ) , he could use the tfbs prediction program match ( 3 ) . if the user were interested in a prediction based on variable order markov models or variable order bts , he could use the vombat web server . the two main functions of the vombat web server are training variable order markov models or variable order bts based on user - supplied sequences and predicting putative tfbss based on user - supplied variable order markov models or variable order bts . the training function allows the user to choose an optimal combination of a motif model and a background model and train these models on problem - specific datasets . for example , the user might choose a variable order bt as motif model and a variable order markov model of order 5 as background model , he might choose that subset of sp1 binding sites available from transfac that are located in gc - poor promoters as motif dataset , and he might choose a representative set of gc - poor promoter regions as background dataset . for training a model from user - supplied input data , the user first selects train a model from data from the selection of tasks . the user can enter a comment on the model to be trained in order to make it easier to identify the corresponding results in a list of all results . the rest of the parameters defines the type of model to be trained as well as the pruning constant and the pseudo count . the default value of the pseudo count is 1 , and we recommend the user to always specify a pseudo count greater than 0 to compensate for zero occurrences . if the model is defined to be inhomogeneous , the user can select between a markov model and a bt as a third parameter . based on ( 6,19 ) and based on systematic cross - validation analyses of different model combinations applied to different sets of tfbss and background sequences , we generally recommend to use a bt as motif model and a homogeneous markov model with an initial order of at least 3 as background model . the user - supplied input file to train an inhomogeneous model must consist of aligned sequences of identical length , separated by line breaks . the vombat web server is designed for , but not restricted to , the prediction of motifs in dna sequences . hence , it is possible to specify the alphabet of the sequences . in case of dna sequences the alphabet is acgt. as a last option the user may select a checkbox that determines if a graphical representation of the trained model shall be displayed as supplementary data . if the job of training a model could be successfully submitted to the scheduler of the attached linux cluster , the subsequently displayed page reports jobs success, and vombat gives the user the possibility to go either to the list of tasks for starting another job or to the job overview . the job overview displays a table of all jobs with the following columns : the time at which the job was started , the comment that was entered by the user , the type of the job ( train , classify , crossval ) , the current state of the job , a button for the already available results and a button to stop a running job . the possible states of a job are view results in the same row to inspect the results of this job . the results of training a model are a link to an xml representation of the trained model , which can be saved and used as input for one of the other tasks presented in the following . if the user selected to obtain a graphical representation of the trained model , this representation is also displayed as a series of images , which may be saved using the corresponding browser command . after a motif model and a background model have been trained and saved , they can be combined to predict putative tfbss in a user - supplied set of input sequences . for this purpose , the first input field of the subsequently displayed form , which is shown in figure 1 , allows to enter a user - defined comment on the job . next , the user uploads two files containing the xml representations of the desired motif and background models , which have been trained using the train a model from data task described above . the classification threshold must be specified , and the file containing the set of sequences in which the motif is to be predicted must be uploaded . after a classification job has been started , the user can monitor the current state of the job in the job overview of vombat . if the job is finished , the predictions can be inspected by clicking on the corresponding view results button . the html file contains a textual description of the putative motifs , which contains for each predicted motif its position , its sequence , its probabilities returned by the two models , and the corresponding log - likelihood ratio . additionally , a profile of the log - likelihoods and the log - likelihood ratio is plotted for each sequence of the input file . the chosen classification threshold is also plotted as a reference for the log - likelihood ratio , allowing the user to judge the influence of the classification threshold on the prediction results at a glance . as an auxiliary function for advanced users , vombat provides a platform for cross - validation analyses of different combinations of user - defined models based on problem - specific data . selecting cross validation from the list of tasks brings up the cross - validation input form . the first input field allows to enter a user - defined comment on the cross - validation job . next , the number of iterations of the cross - validation job can be entered . two files containing xml representations of the motif model and the background model must be uploaded by the user . these files can be obtained by a standard training on the datasets to be used for cross - validation . next , the user must specify the desired specificity for which the mean sensitivity is to be computed . the last option in the form allows to plot a histogram of the log - likelihood ratios for the motif sequences and background sequences , which allows further analyses of the cross - validation results , such as judging the effect of a shifted threshold or inspecting the separation of the samples of both classes . the presented results are the mean ( and standard error ) of the sensitivity for the fixed specificity entered in the cross - validation form , the mean ( and standard error ) of the maximum correlation coefficient , and the corresponding thresholds and optionally the histogram of the log - likelihood ratios as shown in figure 3 . the following prototypical example illustrates how the three functions of vombat may be utilized for the prediction of putative tfbss . typically , the user has no a priori knowledge of which model combination and which model parameters could provide an accurate prediction of these binding sites in gc - poor upstream regions . hence , he would start a series of different cross - validation experiments for different model combinations and different parameter settings using the cross - validation function of vombat . after having established an optimal model combination ( including optimal parameters ) for his specificity requirements and based on his problem - specific datasets , e.g. based on already annotated sp1 binding sites from transfac as motif dataset and representative gc - poor upstream regions as background dataset , he would then use these models to run the training and prediction functions of vombat as outlined above . for training a model from user - supplied input data , the user first selects train a model from data from the selection of tasks . the user can enter a comment on the model to be trained in order to make it easier to identify the corresponding results in a list of all results . the rest of the parameters defines the type of model to be trained as well as the pruning constant and the pseudo count . the default value of the pseudo count is 1 , and we recommend the user to always specify a pseudo count greater than 0 to compensate for zero occurrences . if the model is defined to be inhomogeneous , the user can select between a markov model and a bt as a third parameter . based on ( 6,19 ) and based on systematic cross - validation analyses of different model combinations applied to different sets of tfbss and background sequences , we generally recommend to use a bt as motif model and a homogeneous markov model with an initial order of at least 3 as background model . the user - supplied input file to train an inhomogeneous model must consist of aligned sequences of identical length , separated by line breaks . for homogeneous models the length of the sequences may differ . the vombat web server is designed for , but not restricted to , the prediction of motifs in dna sequences . hence , it is possible to specify the alphabet of the sequences . in case of dna sequences the alphabet is acgt. as a last option the user may select a checkbox that determines if a graphical representation of the trained model shall be displayed as supplementary data . if the job of training a model could be successfully submitted to the scheduler of the attached linux cluster , the subsequently displayed page reports jobs success, and vombat gives the user the possibility to go either to the list of tasks for starting another job or to the job overview . the job overview displays a table of all jobs with the following columns : the time at which the job was started , the comment that was entered by the user , the type of the job ( train , crossval ) , the current state of the job , a button for the already available results and a button to stop a running job . the possible states of a job are if the state of a job is finished, the user can click on the button view results in the same row to inspect the results of this job . the results of training a model are a link to an xml representation of the trained model , which can be saved and used as input for one of the other tasks presented in the following . if the user selected to obtain a graphical representation of the trained model , this representation is also displayed as a series of images , which may be saved using the corresponding browser command . after a motif model and a background model have been trained and saved , they can be combined to predict putative tfbss in a user - supplied set of input sequences . for this purpose , the first input field of the subsequently displayed form , which is shown in figure 1 , allows to enter a user - defined comment on the job . next , the user uploads two files containing the xml representations of the desired motif and background models , which have been trained using the train a model from data task described above . the classification threshold must be specified , and the file containing the set of sequences in which the motif is to be predicted must be uploaded . after a classification job has been started , the user can monitor the current state of the job in the job overview of vombat . if the job is finished , the predictions can be inspected by clicking on the corresponding view results button . the html file contains a textual description of the putative motifs , which contains for each predicted motif its position , its sequence , its probabilities returned by the two models , and the corresponding log - likelihood ratio . additionally , a profile of the log - likelihoods and the log - likelihood ratio is plotted for each sequence of the input file . the chosen classification threshold is also plotted as a reference for the log - likelihood ratio , allowing the user to judge the influence of the classification threshold on the prediction results at a glance . as an auxiliary function for advanced users , vombat provides a platform for cross - validation analyses of different combinations of user - defined models based on problem - specific data . selecting cross validation from the list of tasks brings up the cross - validation input form . the first input field allows to enter a user - defined comment on the cross - validation job . next , the number of iterations of the cross - validation job can be entered . a higher number of iterations produces more reliable results . on the other hand , two files containing xml representations of the motif model and the background model must be uploaded by the user . these files can be obtained by a standard training on the datasets to be used for cross - validation . next , the user must specify the desired specificity for which the mean sensitivity is to be computed . the last option in the form allows to plot a histogram of the log - likelihood ratios for the motif sequences and background sequences , which allows further analyses of the cross - validation results , such as judging the effect of a shifted threshold or inspecting the separation of the samples of both classes . the presented results are the mean ( and standard error ) of the sensitivity for the fixed specificity entered in the cross - validation form , the mean ( and standard error ) of the maximum correlation coefficient , and the corresponding thresholds and optionally the histogram of the log - likelihood ratios as shown in figure 3 . the following prototypical example illustrates how the three functions of vombat may be utilized for the prediction of putative tfbss . typically , the user has no a priori knowledge of which model combination and which model parameters could provide an accurate prediction of these binding sites in gc - poor upstream regions . hence , he would start a series of different cross - validation experiments for different model combinations and different parameter settings using the cross - validation function of vombat . after having established an optimal model combination ( including optimal parameters ) for his specificity requirements and based on his problem - specific datasets , e.g. based on already annotated sp1 binding sites from transfac as motif dataset and representative gc - poor upstream regions as background dataset , he would then use these models to run the training and prediction functions of vombat as outlined above . the form requesting the parameters for a tfbs prediction . the results of a tfbs prediction . the list of putative tfbss are displayed as a link to sites.html, and the profiles for the sequences are plotted . the results of a cross - validation . spinal epidural hemangiomas have been reported in the literature , but most of them were cavernous type hemangiomas that enable a preoperative differential diagnosis with relative ease . the limited number of spinal epidural hemangiomas and few radiological findings make an exact diagnosis difficult prior to surgery . a high vascularization of spinal epidural hemangiomas may result in an unexpected surgical situation in the case of preoperative misinterpretation . in the current case , a 51-year - old woman presented with a 3-week history of lower back pain with right anterior thigh numbness . the patient had a magnetic resonance imaging ( mri ) at a local hospital which revealed a lesion at the l3 level , located in the ventral epidural space and connected with l3/4 protruded disc material that demonstrated a heterogeneous signal at t2 weighted images and an iso- to low signal at t1 weighted image ( t1-wi ) . 1 ) . based on a presumed diagnosis of a ruptured disc with possible sequestration or granulation tissue formation , the patient underwent surgery . a right hemilamincetomy of l3 was performed , and a retracting thecal sac revealed a highly engorged vascular structure . near - infrared indocyanine green videoangiography ( icg - va ) showed a delayed mass filling . histological examination revealed a vascular lesion composed of small to medium sized veins with irregular calibers , which is consistent with an arteriovenous hemangioma ( fig . the typical symptoms of epidural lesions other than disc herniation of the lumbar region are low back pain or radiculopathy which are indistinguishable from the clinical symptoms of disc herniation diseases . moreover , spinal epidural hemangiomas are very rare . also , spinal epidural hemangiomas constitute approximately 4% of all epidural tumors and 12% of all intraspinal hemangiomas4 ) . the differential diagnosis for spinal epidural hemangiomas before surgery included schwannoma , lymphoma , meningioma , angiolipoma , disk herniation , synovial cysts , granulomatous infection , pure epidural hematoma , and extramedullary hematopoiesis9,12 ) . the cavernous type displays histologically with large number of sinusoidal channels in collagenous tissue7 ) , whereas the arteriovenous type shows with a cluster of abnormal arteries and veins and vessel walls containing elastin , and smooth muscle5 ) . a complete surgical en bloc removal is the treatment of choice for spinal epidural hemangiomas with mass effect because of the excessive vascularity of hemangioma , piece to piece resection should be avoided6 ) . an indocyanine green videoangiography can help surgeons understand the vasculature surrounding the mass and facilitate the en bloc removal of the hemangioma . fluorescence angiography with indocyanine green provides real - time information regarding the patency of vessels . an icg - va enhances the flow direction delineation capability , flow velocity and sequence of dye filling in different components of complex spinal vascular lesions3 ) . due to the high vascularization of hemangiomas , the incomplete surgical removal of a spinal hemagioma because of diffuse bleeding or minimal exposure during disk surgery might result in the persistence of clinical symptoms or recurrence . reoperation for remnant or recurrent spinal hemangioma is very difficult due to peridural or periradicular adhesion and unclear margins ; as a result , complete resection can not be guaranteed . therefore , proper preoperative planning and complete resection during the operation is essential . for this , but , like this case , an angiography may not always confirm the diagnosis of an artriovenous hemangioma . clinicians should be aware that an angiography can not provide conclusive evidence of the presence of an antriovenous hemangioma . if spinal epidural hemangiomas are unexpected encountered during surgery , an icg - va can be helpful to diagnose and surgery . this technique provides accurate information about the flow dynamics through the anatomy of vascular lesions in real time10,11,13,14 ) . further study is required to recognize and to provide a differential diagnosis of spinal epidural hemangiomas . if the lesion is like a ruptured disc in mri , we should consider spinal epidural hemangioma as one of differential diagnosis .

variable order markov models and variable order bayesian trees have been proposed for the recognition of transcription factor binding sites , and it could be demonstrated that they outperform traditional models , such as position weight matrices , markov models and bayesian trees . we develop a web server for the recognition of dna binding sites based on variable order markov models and variable order bayesian trees offering the following functionality : ( i ) given datasets with annotated binding sites and genomic background sequences , variable order markov models and variable order bayesian trees can be trained ; ( ii ) given a set of trained models , putative dna binding sites can be predicted in a given set of genomic sequences and ( iii ) given a dataset with annotated binding sites and a dataset with genomic background sequences , cross - validation experiments for different model combinations with different parameter settings can be performed . several of the offered services are computationally demanding , such as genome - wide predictions of dna binding sites in mammalian genomes or sets of 104-fold cross - validation experiments for different model combinations based on problem - specific data sets . in order to execute these jobs , and in order to serve multiple users at the same time , the web server is attached to a linux cluster with 150 processors . vombat is available at . a spinal epidural hemangioma is rare . in this case , a 51 year - old female patient had low back pain and right thigh numbness . she was initially misdiagnosed as having a ruptured disc with possible sequestration of granulation tissue formation due to the limited number of spinal epidural hemangiomas and little - known radiological findings . because there are no effective diagnostic tools to verify the hemangioma , more effort should be put into preoperative imaging tests to avoid misdiagnosis and poor decisions ) .

numerous investigators have reported improved function , reduced pain , and high rates of implant survivorship at up to 20 years followup [ 4 , 29 ] . however , concerns about limitations in implant longevity related to the osteolysis associated with metal - on - polyethylene ( m - pe ) bearing surface wear have led to innovation in tribology [ 10 , 15 , 16 ] and the introduction of new tha bearing couples over the past decade . in particular , second - generation hard - on - hard bearings , including metal - on - metal ( m - m ) and ceramic - on - ceramic ( c - c ) , have been introduced into the us market based on laboratory studies showing lower wear rates when compared with m - pe bearings [ 9 , 10 , 15 ] . lower wear rates associated with hard - on - hard bearings are likely to have the biggest impact on revision rates in younger , more active patients , who are at higher risk for wear - related failures [ 3 , 12 , 17 , 18 , 27 ] . the long - term benefits of more costly hard - on - hard bearings are less obvious in older patients , who are at lower risk for wear - related failures . however , a potential benefit of m - m bearings is the ability to use large diameter femoral heads , which theoretically could result in a lower short - term risk of dislocation when compared with m - pe bearings . this potential benefit could be particularly advantageous in older patients who are reportedly at higher risk for tha dislocation [ 5 , 25 , 28 ] . however , that study employed a cross - sectional study design , and due to the nature of the database used ( the nationwide inpatient sample ) , we were unable to compare longitudinal patient outcomes and differences in the risks of complication and revision tha among patients with different tha bearings . the purpose of this study was to compare the short - term risk of complication and revision tha among medicare patients having a primary tha with m - pe , m - m , and c - c tha bearings . the 2005 to 2007 100% medicare inpatient claim files were used to perform a matched cohort analysis in three separate cohorts of tha patients ( m - pe , m - m , c - c ) . patients in each tha cohort were matched by age , gender , and us census region to the comparison or m - m hip resurfacing arthroplasty procedures ( which have a separate icd-9-cm procedure code ) were excluded from the analysis . each m - m patient and c - c patient ( case ) were matched to three m - pe patients ( control ) , while each c - c patient ( case ) was matched to three m - m patients ( control ) . case patient , the eligible pool of controls consisted of those patients who were within 3 years in age of the case patient and who were at least 65 years old . if more eligible controls were available than the three required per case , controls closest in age were selected first . eligible controls were also of the same gender and resided in the same us census region . all cases and controls had to be enrolled in both part a and part b of medicare . patients who received their medicare health benefits through a health maintenance organization were excluded because their healthcare expenses were not submitted to the centers for medicare and medicaid services ( cms ) for payment and , therefore , claims from these beneficiaries were not available from the database . primary tha patients were identified using international classification of diseases , 9th rev , clinical modification ( icd-9-cm ) procedure code 81.51 from the inpatient claims records and grouped into three bearing cohorts based on their corresponding icd-9-cm optional procedure modifier bearing codes ( 00.74 for m - pe , 00.75 for m - m , 00.76 for c - c ) . using each patient s unique de - identified beneficiary i d , the patients were tracked longitudinally with revision tha or selected complications as end points . the selected complications included deep venous thrombosis ( dvt ) , dislocation , infection , and mechanical loosening . dvt was identified using the icd-9-cm diagnosis codes 451.1 , 451.2 , 451.81 , 451.9 , 453.1 , 453.2 , 453.4 , 453.8 , and 453.9 , while tha dislocation was identified using any occurrence of icd-9 diagnosis codes 718.35 , 835 , or 996.42 . infection and mechanical loosening were identified using icd-9 diagnosis codes 996.66 and 996.41 , respectively . an overall cohort of 36,423 tha patients with m - pe bearings were identified , along with 17,789 tha patients with m - m bearings and 2,835 tha patients with c - c bearings , from which the matched cohorts were derived . the mean age of all tha recipients included in the study was 74.5 years ( sd = 6.4 years ) . multivariate cox proportional - hazards models were constructed to compare complication and revision tha risk among cohorts , adjusting for medical comorbidities , race , socioeconomic status , and hospital factors . the medicare buy - in status was used as an identifier of patients whose medicare premiums and deductibles were subsidized by the state ( eg , medicaid ) and were used as a proxy for the patient s socioeconomic status . to account for the health status of each patient , the charlson comorbidity index was used as a measure of comorbid illness . for this analysis , the charlson index values were grouped into the following previously validated categories : 0 ( none ) , 1 to 2 ( low ) , 3 to 4 ( moderate ) , and 5 or more ( high ) . among younger medicare patients ( aged 6569 years ) , m - m bearings accounted for 35% of tha bearings and c - c bearings accounted for 8% . among older medicare patients ( older than 80 years ) , among female patients , 67% had m - pe bearings , while 29% had m - m bearings , compared to male patients , among whom 60% had m - pe and 35% had m - m bearings ( fig . 2 ) . the highest frequency of m - pe bearing surface use was in the northeast ( 72% ) , and the highest frequency of m - m bearing usage was in the south ( 37% ) , while c - c bearing usage was fairly consistent across the four us census regions ( 4%5% ) ( fig . 1the bearing surface frequency in medicare tha patients by age is shown.fig . 2the bearing surface frequency in medicare 3the bearing surface frequency in medicare tha patients by us census region is shown . the bearing surface frequency in medicare tha patients by age the bearing surface frequency in medicare tha patients by us census region is shown . when comparing the unadjusted risk of complication using the kaplan - meier method , m - m bearings showed an apparent trend toward higher overall risk of dvt ( fig . 5 ) than c - c bearings . however , these differences were not present after adjusting for other covariates in these matched cohorts ( table 1 ) . after controlling for patient and hospital factors , m - m bearings were associated with a higher risk of periprosthetic joint infection ( hazard ratio , 3.03 ; confidence interval , 1.029.09 ) when compared with c - c bearings . there were no other differences among cohorts in the adjusted risk of revision tha or any other complication , including dvt , dislocation , or mechanical loosening ( table 1).fig . 4this graph shows trends of higher overall rates of dvt for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( dvt ) at the given time points.fig . 5this graph shows trends of higher overall rates of revision for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( revision ) at the given time points.table 1risk - adjusted comparison of risk of complication and revision tha between m - pe and m - m bearings , m - pe and c - c bearings , and m - m and c - c bearingscomplicationm - pe ( n = 54,431)m - m ( n = 17,789)hazard risk ratio ( compared to m - pe)p valuem - pe ( n = 8073)c - c ( n = 2835)hazard risk ratio ( compared to m - pe)p valuem - m ( n = 7853)c - c ( n = 2835)hazard risk ratio ( compared to m - m)p valuedvt1.05%1.03%1.010.881.08%0.88%0.590.070.90%0.88%0.900.73dislocation0.80%0.74%0.920.490.68%0.78%1.080.800.57%0.78%1.210.57infection0.49%0.53%0.980.890.37%0.32%0.620.360.59%0.32%0.330.045mechanical loosening0.22%0.20%0.870.560.25%0.39%0.970.970.23%0.39%1.100.89revision1.16%1.12%0.940.511.04%1.02%0.670.141.34%1.02%0.670.09m - pe = metal - on - polyethylene ; m - m = metal - on - metal ; c - c = ceramic - on - ceramic ; dvt = deep vein thrombosis . this graph shows trends of higher overall rates of dvt for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( dvt ) at the given time points . this graph shows trends of higher overall rates of revision for m - m versus c - c bearings ( unadjusted ) . ( t ) = percentage of patients without the complication ( revision ) at the given time points . risk - adjusted comparison of risk of complication and revision tha between m - pe and m - m bearings , m - pe and c - c bearings , and m - m and c - c bearings m - pe = metal - on - polyethylene ; m - m = metal - on - metal ; c - c = ceramic - on - ceramic ; dvt = deep vein thrombosis . tha has become one of the most commonly performed operations in medicare patients , with over 160,000 primary and revision tha procedures reported in medicare patients in 2007 . successful tha allows elderly patients who suffer from disabling hip disease to regain their function and maintain a more active , healthy lifestyle . nevertheless , increased utilization and rising costs have led to concerns regarding the appropriateness of using hard - on - hard bearings are associated with lower wear rates in the laboratory setting [ 15 , 20 ] . however , wear - related failures , such as bearing surface wear , osteolysis , and mechanical loosening , may be less of a concern among medicare patients when compared with younger , more active patients who undergo primary tha . therefore , the higher cost associated with hard - on - hard bearings may not be justifiable in terms of improved patient outcomes and lower reoperation rates in medicare patients , unless they are associated with fewer short - term complications , such as dislocation . one example of a potential short - term benefit of m - m bearings that is often cited is the ability to use large diameter femoral heads , which theoretically could result in a lower risk of dislocation when compared with m - pe bearings . we therefore compared the short - term risks of complication and revision tha among medicare patients having a primary tha with m - pe , m - m , and c - c tha bearings . our findings are limited by the use of an administrative database , where bearing surface type is an optional modifier code that can be reported in conjunction with the primary procedure code ( primary or revision total hip arthroplasty ) , which introduces a potential source of bias into our study . however , this limitation is somewhat mitigated by the matched cohort study design , where patients with known bearing types were matched by age , gender , and u.s . second , other variables of interest which may influence patient outcomes , including surgical approach , surgeon experience , and implant design are not accessible from administrative claims , and we were only able to evaluate complications which are captured in administrative claims data , such as dvt , dislocation , infection , mechanical loosening , and revision surgery , rather than pain and patient reported functional outcomes . third , since the optional bearing surface modifier administrative codes were not introduced until october , 2005 , our study was limited to outcomes and complications that occurred within two years of the index procedure . however , the rationale for our study was that presumably the reason for using alternative bearings in medicare patients is to reduce short - term complication rates ( eg , dislocation ) , rather than long - term complications ( eg , wear / osteolysis ) , which are more of a concern in younger patients than in medicare patients . furthermore , the australian hip registry and other reports in the literature [ 6 , 11 , 19 , 22 , 23 , 26 ] have raised concerns regarding higher than expected short - term complication and revision rates in patients with hard - on - hard bearings . nevertheless , further study is necessary to evaluate differences in long - term clinical outcomes and revision rates as additional data becomes available . we found the adjusted risk of short - term complication and revision tha among medicare tha patients was similar , regardless of bearing surface , with the exception of patients who had m - m bearings , who had a slightly higher risk of periprosthetic joint infection than patients who had c - c bearings . the reasons underlying the increased risk of infection for m - m bearings compared with c - c bearings , even after adjusting for medical comorbidities and hospital factors , remain unclear . it is possible that some of these patients may have been misdiagnosed as having a periprosthetic joint infection when in fact they had a local soft tissue inflammatory reaction related to the m - m articulation [ 6 , 11 ] . the findings in some patients with such reactions mimic periprosthetic joint infection , as reported previously by mikhael et al . . although the higher risk of infection in the m - m cohort compared with the c - c cohort was significant ( hazard ratio 3.03 , ci = 1.02 9.09 ) , the clinical importance of this difference is unclear , especially given the relatively low incidence of infection ( 0.59% versus 0.32% , respectively ) . the relatively high incidence of m - m bearings reported in the medicare population is somewhat surprising , given m - m bearings are thought by some to be primarily indicated for younger , more active patients who are at higher risk for bearing surface wear and osteolysis with m - pe bearings [ 14 , 17 , 20 ] . however , one theoretical advantage of m - m bearings in older patients is improved stability ( eg , lower risk of dislocation ) due to the ability to use larger - diameter femoral heads . our data do not demonstrate an advantage of m - m bearings in terms of lower short - term risk of dislocation when compared with either m - pe or c - c bearings . this could be related to the increasing trend of using 32- and 36-mm heads in m - pe bearings , which may be sufficient to substantially reduce the risk for dislocation . although the database used in this study did not include information about femoral head size , according to industry sources , the proportion of femoral heads implanted in the us sized 32 mm or greater has increased from approximately 12% to 79% between 1998 and 2008 . in summary , we found medicare tha patients with hard - on - hard ( m - m , c - c ) bearings had a similar risk of complications and revision tha compared to patients who had m - pe bearings during the first 2 years after primary tha . these findings provide a basis for additional analyses of the comparative effectiveness of tha bearing surfaces in the medicare population . out of the 10,807 patients enrolled for the original survey , access survival data were available for 7058 ( 65% ) patients . these patients resided in portugal , the united kingdom , ireland , italy , turkey , romania , slovenia , poland , and spain . the mean age was 63.515.0 years , 38.5% were female , 27.1% were diabetic , 90.6% had a native fistula , and 9.4% had a graft . median dialysis vintage was 43.2 months ( minimum : 0.1 months ; maximum : 419.6 months ) . access location was lower arm for 51.2% of patients . during the follow - up , prevalent needle sizes were 15 and 16 g for 63.7% and 32.2% of the patients , respectively ( 14 g : 2.7% 17 g : 1.4% ) . in spain , 98% of patients were treated with 15-g needles , and in romania 75% of patients were treated with 16-g needles . cannulation technique was area for 65.8% , rope - ladder for 28.2% , and buttonhole for 6% of patients , with some country preferences clearly visible : area technique was applied in as much as 77% of patients in romania , and rope - ladder was more common in poland than in the total study population ( 44% ) . the direction of arterial puncture was antegrade for 57.3% of patients ; this was the preference for 99% of patients in poland . the bevel orientation was upward for 70.2% of the patients , peaking in poland with 95% . the practice of needle rotation after insertion was practiced for 42% of patients , with a much higher percentage in italy ( 82% ) . the prevalent combination between arterial needle puncturing and bevel direction was antegrade with bevel upward ( 43.1% ) , followed by retrograde with bevel up ( 27.1% ) . the proportion of the two other combinations , that is , antegrade and retrograde with bevel downward , was 14.2% and 15.6% , respectively . the 15.6% with retrograde and bevel down were mainly treated in two countries ( spain and portugal ) . median blood flow was 350400 ml / min . in italy and spain , 40% and 38% of patients conversely , in slovenia and in poland 5455% of patients were treated with blood flows below 300 ml / min . figure 3 shows the distribution of patients according to the prescribed needle size , blood flow , and venous pressure levels . the primary outcome event ( i.e. , surgery for a new va during the follow - up period ) was observed in 1485 patients ( 21% ) . univariate survival analysis revealed a significant benefit for access survival for patients who are younger , nondiabetic , male , have lower body mass index , do not take platelet antiaggregants , do not have heart failure , and are able to assist with compression . a significant benefit was also seen for patients with fistula ( vs. graft ) , smaller needles , distal location of the access , and low venous pressure . with regard to cannulation technique , positive effects were observed for antegrade needle direction ( vs. retrograde ) , bevel up ( vs. down ) , nonalcohol - based disinfection , and application of local anesthesia . although not statistically significant , a potential survival benefit was indicated for higher blood flow ( p=0.056 ) and buttonhole technique ( vs. rope - ladder and area , p=0.11 ) . needle rotation did not affect the access survival ( p=0.81 ) , neither did access vintage ( age < 1 month before baseline vs. 1 month before baseline ; p=0.29 ) . meier access survival curves according to blood flows , venous pressures , needle sizes , and cannulation techniques are presented in figure 4 . in a second step , after adjustment for age , gender , diabetes , va type , access location ( proximal vs. distal ) , dialysis vintage and heart failure , and incorporation of country differences , the use of a 16-g needle was associated with a significantly higher risk of access failure ( hazard ratio ( hr ) 1.21 ) compared with the use of a 15-g needle . very few ( 1.4% ) patients were treated with the even smaller 17-g needles , but the direction of the results is the same , that is , increased hr for smaller needle size . using a blood flow of 300350 ml / min as a reference , the hr tended to decrease as the blood flow increased . with regard to cannulation technique , both rope - ladder and buttonhole techniques performed significantly better than the area technique . considering antegrade with the bevel up as reference , the retrograde direction of the arterial needle with bevel down is associated with a significant increase of access failure risk of 18% . all other options , that is , antegrade direction with bevel down or retrograde direction with bevel up , were not associated with a hr significantly different from 1.00 . with regard to venous pressure , using as reference the range between 100 and 150 mm hg , the hrs increased proportionally to 1.4 , 1.87 , and 2.09 with the increase of venous pressure from 150 to 200 mm hg , 200 to 300 mm hg , and > 300 mm hg , respectively ( all p0.008 ) . of note , venous pressures of > 300 mm hg are extreme cases and were only recorded in 0.6% of the patients . in addition , a venous pressure of < 100 mm hg was associated with a significantly higher hr of 1.51 . to investigate this further , we also looked for interaction effects between blood flow and venous pressure , as well as between arterial and venous pressures ; no significant associations were found . finally , the use of a tourniquet and not applying any pressure at the time of cannulation were associated with hrs of 1.30 and 1.25 ( p<0.008 and < 0.02 ) , respectively , compared with exertion of arm compression by the patient at the time of cannulation ( labeled patient assistance ' in table 1 ) . in summary , this study revealed that area cannulation technique , albeit being identified as the most commonly used technique in this population of over 7000 patients , was inferior to rope - ladder and to buttonhole for maintenance of va functionality . with regard to the effect of needle and bevel direction , the combination of antegrade positioning of the arterial needle with bevel - up orientation was significantly associated with better access survival than retrograde positioning with bevel down . the use of larger needles tended to favor access patency , with 15 g being superior to 16 or 17 g. the application of arm pressure by the patient at the time of cannulation had a favorable effect on access longevity compared with not applying pressure or using a tourniquet . results pertaining to the type and location of the access and the technical parameters ( i.e. , blood flow and venous pressure ) were as follows : there was an increased risk for access failure for grafts vs. fistulas , proximal location vs. distal , right arm vs. left arm , blood flows below 300 ml / min vs. those in the range of 300350 ml / min , and for the presence of a venous pressure > 150 mm hg vs. pressures between 100 and 150 mm hg . tissue reparative processes triggered by cannulation procedures may cause enlargement of the fistula and the formation of aneurisms and scars that , in turn , can favor the development of stenotic lesions and ultimately impact fistula survival . repetitive punctures at the va site cause vessel wall defects that are initially filled by thrombi before finally healing . of the three cannulation techniques , the buttonhole approach has the theoretical advantage of limiting the process of dilatation and fibrosis because the thrombus is displaced while being formed , favoring the formation of a cylindrical scar from the subcutaneous and vessel wall tissues . the rope - ladder technique may have the initial advantage of favoring progressive maturation along the entire length of the fistula , but it requires fistula with sufficiently long segments suitable for cannulation . the area puncture technique weakens the fistula wall and is associated with the least favorable consequences , that is , localized dilation , disruption of the vessel wall , and subsequent development of ( pseudo)aneurysms and strictures . despite this and the fact that area cannulation has been discouraged for over two decades , it was disheartening to observe that this was the predominant practice in almost two - thirds of patients . according to the ebpg and the clinical practice guidelines for va , the rope - ladder technique should be used for cannulation of grafts . specifically , according to the latter , this study showed a 22% lower risk for va failure in those patients whose va was cannulated with the buttonhole technique as opposed to area , confirming the results of a recently published randomized controlled clinical trial . although the buttonhole technique is associated with good results , one should also take into consideration that it is a practice performed in centers with highly trained personnel that work with strict protocols and that it may also be used for fistulas with only short segments available for cannulation . in our study , this practice is used in 22 centers , mainly in portugal , turkey , the united kingdom , and italy . research questions that arise from current guidelines address the effectiveness of structured cannulation training , increased remuneration for expert cannulators , and whether self - cannulation can lead to better outcomes . indeed , as buttonhole cannulation requires the designation of a reference nurse , especially for the initial 46 weeks , it is likely that this technique benefits from its association with centers offering the necessary training ( i.e. , centers capable of stemming the increased organizational effort and assigning the right cannulator to the right patient ) . in addition , once the tunnel is created , cannulation can be performed directly by patients . however , irrespective of the influence of cannulator training and center organizational issues , the underlying question to be addressed , optimally in a well - designed clinical study , is which cannulation techniques can be recommended to ensure long - term va functionality . this study showed that retrograde direction of arterial needle with bevel down is associated with the least favorable outcome . this is consistent with the findings of woodson and shapiro who reported that retrograde puncturing may be associated for an increased risk of hematoma formation , possibly owing to the related venous return of the blood ( i.e. , retrograde filling ) . antegrade puncturing , on the other hand , may be considered fistula - protective by the same reasoning , that is , tract closure through flow force . therefore , retrograde direction of the arterial needle is more likely to be associated with a higher risk for aneurism . despite recommendations by kdoqi to rotate the needle during insertion , the univariate analysis performed here found no evidence of any benefit of this practice . on the contrary , the authors share the opinion of many cannulators that the 180 rotation of the needle is unnecessary and may constitute an additional trauma to the va . further studies are needed to clarify whether rotation of the va needle during cannulation should be recommended or not . there are a number of possible reasons for the association of the higher failure risk with smaller needle sizes . while increased trauma and prolonged bleeding time are generally associated with the use of large needles , the use of small needles at the same blood flow results in a higher speed of the blood returning to the vasculature , possibly damaging the intima of the avfs . for example , at an operative blood flow of 350 ml / min , the maximum speed of the injected blood will be 8.79 m / s with a 17-g needle and 5.80 m / s with a 15-g needle ( presented by ralf jungmann at vascular access coursestockholm , 1112 october 2012 , stockholm , sweden ) . furthermore , the shear forces created by returning blood can have a role in inflammation and stenosis formation . stenotic fistula and graft lesions are associated with the induction of the expression of profibrotic cytokines , local inflammation , and neointimal proliferation . however , we can not exclude that this association may be a consequence of bias by indication . needles of smaller inner dimension are generally prescribed not only for a new va but also for problematic avfs , that is , those likely to fail in the following months . therefore , it is difficult to derive a conclusion from this association , but on the basis of figure 3 , 17-g needles are clearly linked to blood flow levels below 300 ml / min and , on the contrary , 14-g needles are mainly prescribed to patients with 350400 ml / min or greater blood flows . it is also of interest to underline that higher venous pressure is mainly associated with the 16-g needles , which have a wider distribution of different blood flows . measurement of venous pressure during dialysis is currently used as a surveillance tool within the dialysis session , and not as a standard monitoring strategy . this study showed a significant and proportionally increasing risk of va failure with venous pressures higher than 150 mm hg . an increased hr was also detected for venous pressure below 100 mm hg . as shown in figure 3 , an association between needle size , blood flow , and venous pressure is indicated in that for needle gauges 15 , 16 , and 17 low venous pressures appear to be associated with low blood flows . such an association could be an indication of stenosis in the artery . venous pressure is crucially dependent on the characteristics of the needle ( e.g. , the needle gauge , the length of the metallic portion , and the length and the thickness of the needle shaft ) , which vary among manufacturers . in this network , at the time of the study , the vast majority of the needles ( 85% ) were from a single producer and the length of the needle was 25 mm . the unexpectedly high hr associated with a venous pressure of under 100 mm hg compared with 150200 mm hg should motivate reflection on the currently accepted limits . one could consider integration of venous pressure monitoring into an algorithm for the detection of increased risk of access failure . this study has certain limitations over and beyond those inherent to observational studies , for example , that residual confounding can not be completely ruled out . being a retrospective study , patient data for those patients on dialysis before admission to the nephrocare clinic were not collectable , and thus robust information on the number of prior vaes , on their respective lengths , and on first cannulation was not available . particularly , the missing information on the length of the va , its depth , and the access flow constitute a major weakness because a particular cannulation technique could have been chosen on the basis of what is possible with the given access characteristics . in addition , the length of the access can influence the way in which the needles are placed . despite these missing data , we feel that this study has its merits , as it shows that traditional local practices have a significant influence on procedures exercised . a further limitation is that the va practice was surveyed in april 2009 and was assumed not to have been changed during the follow - up ( 31 march 2012 ) . however , as nursing practices in this field are strongly related to the clinic culture and experience , we have reason to believe that it is should not constitute a significant bias . of course , some cannulation particulars , such as needle size and arterial blood flow , may vary over time , in that smaller needle sizes and low blood flow rate are used for initial access use and that large needles are taken for mature accesses . however , we feel that the model selected here is also justified because it is an explanatory model , based on the association of baseline characteristics with access survival . other limitations are that we had follow - up of 65% of the patients and that most countries were in europe ( owing to deployment of the electronic reporting system ) . as reported , an association between clinical practice patterns and country has been detected , and consequently not all different practices were covered by our model . however , according to the results of this analysis , each country has a combination of practices that positively and negatively influence the va survival . for example , in romania , positive influences were the puncture direction being antegrade ( 82% ) , bevel orientation being predominantly upward ( 95% ) , and needle not rotated ( 84% ) ; negative associations were the use of area technique ( 77% ) , preferred needle size ( 75% with 16 g ) , and the use of blood flows < 300 ml / min ( 47% ) . for this reason , intracountry correlations were considered using a sandwich estimator in the multivariate model . to assess the influence of individual center practices , we also performed a sensitivity analysis by applying the sandwich estimator at the center level . there were only negligible differences to the results obtained with the original model at the country level , raising our confidence that there is no severe confounding of the model by center practice effects . given the relevant impact of the investigated variables on the survival of the va , itself a key driver of hemodialysis patient survival , we believe it is time to organize a large - scale randomized clinical trial to facilitate the formulation of practical and comprehensive cannulation practice guidelines . as the associations between practice patterns and va survival reported here are mainly related to national procedures and only partially related to actual patient limitations , they offer some promising indications for improving clinical practice . in april 2009 , a cross - sectional survey was conducted in 171 dialysis units located in europe , the middle east , and africa to collect details on va cannulation practices on a clinic by clinic level . all patients who were on double - needle hemodialysis or online hemodiafiltration during the week of the survey were selected for analysis , as long as a fistula or graft was used for va , survey data were complete , and follow - up data were available in our clinical database . primary outcome was time until the first surgical access intervention resulting in the generation of a new access ( i.e. , as opposed to any surgical intervention done just for revision , thrombectomy , etc . , or any endovascular intervention ) . patients were censored for transplantation , death , loss of follow - up , or end of the follow - up period ( 31 march 2012 ) . information on cannulation retrieved from the survey comprised fistula type and location , cannulation technique , needle size , needle and bevel direction , needle rotation , blood flow , arterial and venous pressure , use of disinfectants , use of local anesthesia , and application of arm compression at the time of cannulation . to adjust for individual patient characteristics , the following information was extracted from the clinical database : patient age , gender and body mass index , prevalence of diabetes , and the use of ace inhibitors , platelet antiaggregants , and anticoagulants . in addition , the median blood flow prescription was documented at a center level at the time of the survey . for univariate analysis , kaplan meier curves were calculated and comparisons were performed using the log - rank test . by combining univariate results with medical and statistical experience , a set of variables for multivariable analysis was determined . in particular , specific interaction terms ( e.g. , bevel vs. arterial needle direction ) were defined for statistical examination , and decisions were made regarding their inclusion or omission in the cox model depending on their significance or collinearity , respectively . a final cox model based on these variables was calculated , using the sandwich estimator to account for within - country correlation . step by step , the final model was reduced , setting a p - value of 0.1 for variable inclusion . all analyses were performed with sas v9.2 ( sas institute , cary , nc ) .

backgroundto address the long - term problems of bearing surface wear and osteolysis associated with conventional metal - polyethylene ( m - pe ) total hip arthroplasty ( tha ) , metal - metal ( m - m ) , and ceramic - ceramic ( c - c ) bearings have been introduced . these bearing surfaces are associated with unique risks and benefits and higher costs . however the relative risks of these three bearings in an older population is unknown.questions/purposeswe compared the short - term risk of complication and revision tha among medicare patients having a primary tha with metal - polyethylene ( m - pe ) , metal - metal ( m - m ) , and ceramic - ceramic ( c - c ) bearings.methodswe used the 2005 to 2007 100% medicare inpatient claim files to perform a matched cohort analysis in three separate cohorts of tha patients ( m - pe , m - m , and c - c ) who were matched by age , gender , and us census region . multivariate cox proportional - hazards models were constructed to compare complication and revision tha risk among cohorts , adjusting for medical comorbidities , race , socioeconomic status , and hospital factors.resultsafter adjusting for patient and hospital factors , m - m bearings were associated with a higher risk of periprosthetic joint infection ( hazard ratio , 3.03 ; confidence interval , 1.029.09 ) when compared with c - c bearings ( 0.59% versus 0.32% , respectively ) . there were no other differences among bearing cohorts in the adjusted risk of revision tha or any other complication.conclusionsthe risk of short - term complication ( including dislocation ) and revision tha were similar among appropriately matched medicare tha patients regardless of bearing surface . hard - on - hard tha bearings are of questionable value in medicare patients , given the higher cost associated with their use and uncertain long - term benefits in older patients.level of evidencelevel ii , prognostic study . see guidelines for authors for a complete description of levels of evidence . hemodialysis patient survival is dependent on the availability of a reliable vascular access . in clinical practice , procedures for vascular access cannulation vary from clinic to clinic . we investigated the impact of cannulation technique on arteriovenous fistula and graft survival . based on an april 2009 cross - sectional survey of vascular access cannulation practices in 171 dialysis units , a cohort of patients with corresponding vascular access survival information was selected for follow - up ending march 2012 . of the 10,807 patients enrolled in the original survey , access survival data were available for 7058 patients from nine countries . of these , 90.6% had an arteriovenous fistula and 9.4% arteriovenous graft . access needling was by area technique for 65.8% , rope - ladder for 28.2% , and buttonhole for 6% . the most common direction of puncture was antegrade with bevel up ( 43.1% ) . a cox regression model was applied , adjusted for within - country effects , and defining as events the need for creation of a new vascular access . area cannulation was associated with a significantly higher risk of access failure than rope - ladder or buttonhole . retrograde direction of the arterial needle with bevel down was also associated with an increased failure risk . patient application of pressure during cannulation appeared more favorable for vascular access longevity than not applying pressure or using a tourniquet . the higher risk of failure associated with venous pressures under 100 or over 150 mm hg should open a discussion on limits currently considered acceptable .

the developing vertebrate heart tube contains an outer layer of myocardium and an inner layer of endocardium separated by an extracellular matrix ( ecm ) referred to as the cardiac jelly . during heart valve formation , a subset of endocardial cells in the atrioventricular ( av ) canal ( avc ) region is specified to delaminate , differentiate , and migrate into the cardiac jelly in a process called endocardial - mesenchymal transition ( emt ) ( armstrong and bischoff , 2004 ; combs and yutzey , 2009 ) . for av valve formation , locally swollen cardiac jelly together with mesenchymal cells derived from avc endocardial cells form the endocardial cushion ; however the extensive remodeling process that occurs subsequently is not clearly understood ( armstrong and bischoff , 2004 ; combs and yutzey , 2009 ) . signal molecules produced from both the myocardium and endocardium in the avc region are necessary for proper endocardial cushion formation and emt of endocardial cells ( krug et al . , 1985 ) . in the initial step of endocardial cushion formation , a bone morphogenetic protein ( bmp ) signal from avc myocardium inhibits the expression of chamber - specific genes in the avc region , while the endocardial cushion is enlarged by increased deposition of ecm or cardiac jelly between the myocardium and endocardium layers in the avc . upon receiving a myocardially derived bmp signal , a subset of avc endocardium cells undergo the emt process by producing multiple signals such as transforming growth factor , notch , and wnt/-catenin , and then migrate into the cardiac jelly where they transition into mesenchymal cells that form valve precursors during later development ( armstrong and bischoff , 2004 ; combs and yutzey , 2009 ; dlot , 2003 ; nakajima et al . , 2000 ) . therefore , deficiencies in bmp or notch signal - related genes inhibit or disrupt normal endocardial cushion formation and/or failure of avc endocardium emt in the mouse , chick , and zebrafish ( jiao et al . , 2003 ; ma et al . , 2005 ; in addition , fgfs , vegfs , nfat , and hdac are also prerequisites for normal heart valve development ( combs and yutzey , 2009 ; kim et al . , 2012b ) . glycosaminoglycan hyaluronan exists as a hydrated gel that expands the extracellular space , regulates ligand availability , and interacts with numerous ecm components including versican , a major constituent of cardiac jelly ( armstrong and bischoff , 2004 ) . disruption of hyaluronan synthase 2 ( has2 ) or versican in mice and zebrafish is reported to abrogate avc endocardial cushion formation and emt - associated mesenchymal cell migration , suggesting that an appropriate ecm environment is important for both emt of avc endocardium development and formation of the endocardial cushion ( camenisch et al . , 2000 ; although there is some debate on whether emt is a prerequisite for the migration of avc endocardial cells into the cardiac jelly during endocardial cushion formation in zebrafish , the mature av valve of the 2-chambered zebrafish adult heart and 4-chambered mammalian valves are structurally similar . in addition , cellular and molecular signaling events occurring during heart valve development are largely conserved between zebrafish and mammals ( beis et al . cysteine - rich protein 2 ( crip2 ) is a crip - type subgroup of the lim domain protein family and has two conserved cysteine - rich motifs that contain cysteine and histidine residues with zinc - binding properties ( karim et al . , 1996 ; tsui et al . mouse crip2 is reported to be highly expressed in the heart , brain , testis , kidney , and lung , and is involved in the dynamics of the cortical actin cytoskeleton by binding ptp - bl , a protein tyrosine phosphatase ( van ham et al . , 2003 ) . in zebrafish , crip2 is reported to be a target of wnt3a signaling and regulates smooth muscle cell differentiation ( kihara et al . , 2011 ) and the development of cardiac neural crest cells during early embryogenesis ( sun et al . , 2008 ) . however , the direct role of crip2 in the av valve formation has not yet been elucidated . zebrafish is a useful animal model for studying development of the heart and other circulation systems , including blood and lymphatic vessels ( kim and kim , 2014 , kim et al . , 2012a ; 2013a ; 2013b ; ) because the embryos are transparent and a multitude of transgenic lines that express fluorescent proteins in tissue- and cell - specific manners are readily available . in zebra - fish , the heart can be observed under a dissecting microscope soon after formation of the primitive heart tube . the heart starts beating at 24 h post - fertilization ( hpf ) , heart looping begins at 36 hpf , functional valves are formed by 48 hpf , and heart valve development is complete by approximately 55 hpf ( stainier et al . , 2002 ) . in this study , we found that crip2 expression in the avc endocardial cells , but not in the avc myocardial cells , in late heart development is critical for heart valve development in zebrafish embryos . furthermore , crip2-deficient embryos showed markedly increased expression of the has2 and versican a genes , which are responsible for the synthesis of versican a and hyaluronan . we provide data that crip2 expressed in the avc endothelial cells plays an important role in cardiac valve formation by suppressing genes involved in the synthesis of versican a and hyaluronan , which are essential ecm components in the cardiac jelly and endocardial cushion . zebrafish ( danio rerio ) were raised and maintained at 28.5c , and embryos were staged as described in a previous study ( kimmel et al . , 1995 ) . embryos were obtained from spontaneous spawnings and the embryos were manually dechorionated at appropriate stages by using watchmaker s forceps and then fixed using 4% paraformaldehyde in phosphate - buffered saline . tg ( cmlc2:egfp ) ( huang et al . , 2003 ) and tg ( flk1:egfp ) ( jin et al . , 2005 ) embryos were used for phenotypic analyses and immunostaining . antisense digoxigenin - labeled rna probes for crip2 , vmhc , cmlc2 , cdh5 , bmp4 , tbx2b , notch1b , versican a , and has2 were generated using in vitro transcription with t3 , t7 , or sp6 rna polymerase , according to the manufacturer s instructions ( roche , germany ) . whole - mount in situ hybridization was performed at the desired embryonic stages , as described by jowett and lettice ( 1994 ) . for fluorescent immunostaining after in situ hybridization , transgenic embryos were immunostained by anti - gfp antibody ( santa cruz biotechnology , inc . processed embryos were photographed using a microscope ( axioskop , zeiss , germany ) outfitted with a nikon coolpix 4500 digital camera system and a laser - scanning confocal microscope ( lsm 5 pascal ) . crip2 antisense morpholino oligonucleotide ( crip2 mo ) was designed and ontained from gene tools llc . ( usa ) to block the splicing of crip2 exon 2 and intron 2 and had the following sequence : 5-catgccgaggtgagtaaaacagaga-3. to generate crip2 sense mrna , the full open reading frame of crip2 ( nm_213497 ) was cloned into the pcs2 + expression vector . the construct was linearized using noti digestion and capped rna was synthesized using mg ( 5 ) ppp ( 5 ) g ( roche ) and sp6 rna polymerase ( roche ) . the rna was diluted to 100 ng/l and phenol red was added to a final concentration of 0.2% immediately before injection . the synthesized crip2 mo and sense mrna were injected into one- or two - cell stage embryos by using a gas driven microinjector ( world precision instruments , usa ) . zebrafish ( danio rerio ) were raised and maintained at 28.5c , and embryos were staged as described in a previous study ( kimmel et al . , 1995 ) . embryos were obtained from spontaneous spawnings and the embryos were manually dechorionated at appropriate stages by using watchmaker s forceps and then fixed using 4% paraformaldehyde in phosphate - buffered saline . tg ( cmlc2:egfp ) ( huang et al . , 2003 ) and tg ( flk1:egfp ) ( jin et al . , 2005 ) embryos were used for phenotypic analyses and immunostaining . antisense digoxigenin - labeled rna probes for crip2 , vmhc , cmlc2 , cdh5 , bmp4 , tbx2b , notch1b , versican a , and has2 were generated using in vitro transcription with t3 , t7 , or sp6 rna polymerase , according to the manufacturer s instructions ( roche , germany ) . whole - mount in situ hybridization was performed at the desired embryonic stages , as described by jowett and lettice ( 1994 ) . for fluorescent immunostaining after in situ hybridization , transgenic embryos were immunostained by anti - gfp antibody ( santa cruz biotechnology , inc . processed embryos were photographed using a microscope ( axioskop , zeiss , germany ) outfitted with a nikon coolpix 4500 digital camera system and a laser - scanning confocal microscope ( lsm 5 pascal ) . crip2 antisense morpholino oligonucleotide ( crip2 mo ) was designed and ontained from gene tools llc . ( usa ) to block the splicing of crip2 exon 2 and intron 2 and had the following sequence : 5-catgccgaggtgagtaaaacagaga-3. to generate crip2 sense mrna , the full open reading frame of crip2 ( nm_213497 ) was cloned into the pcs2 + expression vector . the construct was linearized using noti digestion and capped rna was synthesized using mg ( 5 ) ppp ( 5 ) g ( roche ) and sp6 rna polymerase ( roche ) . the rna was diluted to 100 ng/l and phenol red was added to a final concentration of 0.2% immediately before injection . the synthesized crip2 mo and sense mrna were injected into one- or two - cell stage embryos by using a gas driven microinjector ( world precision instruments , usa ) . in an in situ hybridization - based screen for candidate genes that regulate heart valve development , we found that crip2 expression was restricted to the avc region in 48-hpf zebrafish embryos ( fig . 1 and data not shown ) . detailed analysis of the gene expression profile showed that crip2 mrna was detected in zebrafish embryos at the one - cell stage , indicating that crip2 was maternally expressed ( fig . at the shield stage , crip2 was ubiquitously expressed in the entire embryonic cell ( fig . 1b ) , but its expression became restricted to the mesodermal region along the anterior - posterior axis at the 10 somite - stage ( ss ) ( fig . crip2 expression became further restricted to the head and trunk mesoderm , including the somites , lateral plate mesoderm , and notochord posterior edge ( fig . transverse sections of the posterior trunk region further confirmed its expression in the somitic muscle ( fig . 1h ) , and new expression appeared in the pharyngeal arch blood vessels ( fig . these gene expression profiling data suggest a function of zygotic crip2 in mesoderm - derived tissue formation during early embryogenesis . during heart development , crip2 expression was observed in the linear heart tube in 24 hpf embryos ( fig . 1k ) and its strong expression persisted in the ventricle and av canal regions at 36 hpf , but was barely detected in the atrium ( fig . crip2 expression was decreased in the ventricle and remained only at the avc and outflow tract ( figs . when heart valve development was completed at 55 hpf , crip2 expression at the avc and outflow tract had started to decrease ( fig . 1o ) . in tg(flk1:egfp ) transgenic zebrafish embryos , green fluorescent protein ( gfp ) is expressed in all endothelial cells ( jin et al . , 2005 ) . to verify which cells express crip2 , 48 hpf tg ( flk1:egfp ) embryos were immunostained with gfp antibodies and then whole - mount in situ hybridization was performed using a fluorescent crip2 riboprobe . endocardial cells in the ventricle , atrium , and avc region were visualized using the gfp antibody ( fig . 1p ) , and expression of crip2 was detected in cells at the avc and outflow tract ( fig . 1p and 1q , we confirmed that crip2 is expressed specifically in the endocardium of the avc and outflow tract and not in the heart chamber myocardium or endocardium during the late stage of heart development ( figs . to evaluate the contribution of crip2 to late heart development , we designed a crip2 splice morpholino ( crip2 mo ) ( fig . the crip2 mo effectively blocked splicing between exon 2 and intron 2 and resulted in a decrease of crip2 mrna transcript ( fig . 2b , * * ) and an increase in the aberrant transcript containing intron 2 ( fig . 2b , * ) in 24 hpf embryos . similarly , the abundance of the crip2 mrna transcripts was clearly reduced by the crip2 mo ( figs . 2d and 2d ) in 24 hpf embryos when compared to non - injection control embryos ( nics ; figs . 2c and 2c ) examined using whole - mount in situ hybridization . to analyze the effect of crip2 knockdown on heart tube formation , we used tg ( cmlc2:egfp ) transgenic embryos that express gfp in myocardial cells in both ventricular and atrial portions of the heart tube ( huang et al . , 2003 ) . depending on the amount of crip2 mo injected into one - cell stage embryos , three variable phenotypes were induced in 48 hpf embryos : normal ( fig . 2g ) and typical ring structures in the avc region ( arrow ) were observed ( fig . 2 g ) . however , typical ring structures were not formed in the type 1 and type 2 embryos because heart tubes failed to undergo looping ( figs . 2 h , 2h , 2j , and 2j ) . because type 2 morphant embryos ( fig . 2i ) showed more severe developmental defects than type 1 morphants , type 1 embryos were selected for further analysis of crip2 function in heart development . the ratios of the dose - dependent phenotypes were calculated and these data are summarized in fig . 2k . collectively , our data demonstrating that morphological heart valve defects occur in crip2 morphant embryos and that crip2 expression becomes spatiotemporally restricted to the endocardium of the avc and outflow tract strongly suggest an important role for crip2 in heart valve development . to further assess the function of crip2 in late heart development , including heart valve formation cardiac myosin light chain 2 ( cmlc2 ) is expressed in myocardia throughout both ventricular and atrial portions of the heart tube , whereas ventricular myosin heavy chain ( vmhc ) is restricted to the myocardia in the ventricular portion ( yelon et al . , 1999 ) . ve - cadherin ( cdh5 ) , on the other hand , is specifically expressed in the endocardium during heart development ( larson et al . , 2004 ) . in zebrafish embryos , the total abundance of cdh5 mrna was also not changed by crip2 mo ( figs . 3e and 3f ) , suggesting that crip2 is not involved in the development of myocardium and endocardium comprising the outer layers of heart chambers . next , we examined the expression of genes required for the development of the heart valve . bmp4 is known to be highly expressed specifically in the avc myocardium in zebrafish embryos ( larson et al . , 2004 ) . in the crip2 morphant embryos , bmp4 was expressed in the avc region ( fig . 3h , arrows ) , in a manner similar to that of the control embryos ( fig . 3 g ) . in agreement with this , expression of tbx2b , a marker of the avc myocardium ( walsh and stainier , 2001 ) , was also not altered ( figs . 3i and 3j , arrows ) . in mice , notch promotes emt during heart valve development and inhibition of notch activity prevents valve formation in mice and zebrafish ( timmerman et al . , 2004 ) . notch1b , a mouse notch1 ortholog , was expressed in the avc endocardium of 48 hpf zebrafish embryos ( hurlstone et al . , 2003 ) , thus we tested the expression of notch1b in crip2 morphant embryos . we found that the expression level of notch1b detected in the avc region of morphant embryos was comparable to that of control embryos at 48 hpf ( figs . 3k and 3l ) . this observation indicated that crip2 is not involved in regulating the emt process in the avc endocardium . an appropriate ecm environment in the endocardial cushion is reported to be required for normal heart valve formation ( camenisch et al . in zebrafish , it is well established that versican a and has2 synthesize versican a and hyaluronan , respectively , and that these genes have restricted expression in the avc region in 48 hpf embryos ( hurlstone et al . , 2003 ) . to further investigate whether crip2 regulates synthesis of ecm components , we analyzed the expression of versican a and has2 in crip2 morphant embryos . the expression of versican a in the avc region was significantly increased in the morphant embryos ( fig . 4a ) . similar to versican a , the expression of has2 in the avc region was markedly upregulated by the knockdown of crip2 ( figs . 4c and 4d ) . these results imply that crip2 functions as a negative regulator of ecm - related gene expression during av valve development . we also tested whether overexpression of crip2 could down - regulate versican a and has2 expression . in wild - type embryos , overexpression of crip2 did not decrease the expression of versican a and has 2 , although severe developmental defects , including curled body and cardiac edema , were observed ( supplementary fig . these results suggested that the expression level of crip2 in wild - type embryos is sufficient to suppress the expression of versican a and has 2 ; thus , further suppression of these ecm genes could not be achieved by the overexpression of crip2 . alternatively , crip2 might require a cofactor to suppress versican a and has 2 expression . therefore , overexpression of crip2 alone can not further suppress the expression of versican a and has 2 . adenomatous polyposis coli ( apc ) , a well - known tumor suppressor protein , negatively regulates wnt/-catenin signaling ( hurlstone et al . , 2003 ) . moreover , heart valve defects in apc null mice are reported to be caused by increased versican a and has2 expression in the avc region , indicating that versican a and has2 are transcriptional targets of the wnt/-catenin pathway ( hurlstone et al . according to our crip2 knockdown data , expression of versican a and has2 at the avc was markedly upregulated , suggesting an antagonistic role of crip2 towards wnt/-catenin signaling that activates versican a and has2 expression . our results , together with that of previous reports , indicate that either deficiency or over - production of versican and has2 causes heart valve defects ( hurlstone et al . , 2003 ; schroeder et al . , , we report a novel function of crip2 in suppressing the expression of versican a and has2 in the avc region , which is essential for normal heart valve development . through its spatiotemporal regulation of gene expression , crip2 likely contributes to the formation of an appropriate ecm environment in the endocardial cushion region during multiple stages of heart valve development . schwannoma may arise from any peripheral nerve and is often found in the chest wall and posterior mediastinum . however , case reports of intrapulmonary schwannoma are extremely rare . a 38-year - old man was referred to our institution because of an abnormal shadow found incidentally on the chest x - ray . the plain chest x - ray showed a mass lesion in the left middle zone . chest computed tomography ( ct ) showed a round and homogeneous mass 25 18 mm in size with a well - defined margin in the lingular segment of the left upper lobe ( fig . 18-fluorodeoxy - glucose ( fdg ) positron - emission tomography ( pet ) showed no accumulation in the tumor ( fig . the ct and fdg - pet findings suggested the possibility of a benign tumor or a low - grade malignancy . as a malignant tumor could not be definitively ruled out , we performed lingular segmentectomy.fig . 1 a computed tomography shows a mass with well - defined margins in the lingular segment of the left upper lobe . b 18-fluorodeoxy - glucose ( fdg ) positron - emission tomography ( pet ) showed no accumulation in the tumor a computed tomography shows a mass with well - defined margins in the lingular segment of the left upper lobe . b 18-fluorodeoxy - glucose ( fdg ) positron - emission tomography ( pet ) showed no accumulation in the tumor macroscopic examination of the resected specimen showed a well - demarcated round tumor in the lung , without any evidence of invasion of the surrounding tissues . microscopic examination revealed proliferation of elongated tumor cells having spindle - shaped nuclei , with cellular palisading ( fig . immunohistochemical staining demonstrated positive staining of the tumor cells for s-100 protein and bcl2 , but negative staining for cd34 and desmin . the histopathological diagnosis was intrapulmonary schwannoma.fig . 2 a macroscopic examination of the specimen showed a round , well - demarcated tumor in the lung , without invasion of the surrounding tissues . b microscopic examination revealed elongated tumor cells with spindle - shaped nuclei a macroscopic examination of the specimen showed a round , well - demarcated tumor in the lung , without invasion of the surrounding tissues . schwannoma is one of the few truly encapsulated neoplasms of the human body , and is almost always solitary . it most commonly occurs on the flexor surfaces of the extremities , neck , mediastinum , retroperitoneum , posterior spinal roots , and cerebellopontine angle . however , schwannomas are extremely rare in the lung , regardless of the patient age . ohtsuka and colleagues reported 62 patients ( 28 men , 34 women ) aged 583 years , with intrapulmonary or bronchial schwannomas , and stated that this neoplasm accounted for about 0.2 % of all pulmonary neoplasms . in 55 % of these patients , the tumor originated within the tissues in proximity to the terminal segmental bronchus . on radiographic images , peripheral intrapulmonary schwannoma appears as a round mass with well - defined margins . in patients with the tumor located proximal to a lobar bronchus , atelectasis or pneumonia about half of the patients have some symptoms ; however , most patients with peripheral intrapulmonary schwannomas , as in the present case , have no symptoms . today , fdg - pet is a useful approach to differentiate malignant solitary pulmonary nodules from benign nodules . beaulieu et al . reported the maximum standard uptake values ( suvmax ) of the 10 schwannomas ranged from 1.9 to 7.2 , and the variation in the suvmax could be explained by the variation in cellularity . however , the reason why high fdg accumulation is found in benign tumors such as schwannoma remains unclear , and a diagnostic value of fdg - pet with intrapulmonary schwannoma is not established . it is reported that in rare instances , schwannomas may undergo malignant transformation . the expression level of ki67 , a tumor cell proliferation marker , has been reported to be useful for determining the malignant potential of these tumors . kindblom and colleagues compared the ki67 expression levels in 26 malignant peripheral nerve sheath tumors and 24 benign nerve sheath tumors ( schwannomas ) , and reported that significantly high density of nuclear staining was found in malignant peripheral nerve sheath tumors . for the treatment of primary intrapulmonary schwannoma , surgical resection , intrabronchial resection with endoscopy , and yttrium aluminium garnet ( yag ) laser resection have been employed [ 57 ] . because of the low malignant potential of these tumors , tumor enucleation or partial lung resection is thought to be adequate , with lobectomy not being necessary . in the present case , we performed segmentectomy to accomplish complete resection , because we did not obtain the pathological diagnosis before the operation and the tumor was located in the central part of the superior lingular segment . in conclusion ,

the initial step of atrioventricular ( av ) valve development involves the deposition of extracellular matrix ( ecm ) components of the endocardial cushion and the endocardialmesenchymal transition . while the appropriately regulated expression of the major ecm components , versican and hyaluronan , that form the endocardial cushion is important for heart valve development , the underlying mechanism that regulates ecm gene expression remains unclear . we found that zebrafish crip2 expression is restricted to a subset of cells in the av canal ( avc ) endocardium at 55 hours post - fertilization ( hpf ) . knockdown of crip2 induced a heart - looping defect in zebrafish embryos , although the development of cardiac chambers appeared to be normal . in the avc of crip2-deficient embryos , the expression of both versican a and hyaluronan synthase 2 ( has2 ) was highly upregulated , but the expression of bone morphogenetic protein 4 ( bmp4 ) and t - box 2b ( tbx2b ) in the myocardium and of notch1b in the endocardium in the avc did not change . taken together , these results indicate that crip2 plays an important role in av valve development by downregulating the expression of ecm components in the endocardial cushion . a 38-year - old man without any symptoms was admitted to our institution because of an abnormal shadow found incidentally on a chest x - ray . chest computed tomography showed a round mass in the lingular segment of the left upper lobe . lingular segmentectomy was performed , and the histopathological diagnosis was intrapulmonary schwannoma . immunohistochemical staining revealed a positive result for s-100 protein and negative results for cd34 and desmin . we report this case of intrapulmonary schwannoma , which is extremely rare .

a pulmonary nodule is defined as a spherical well - circumscribed radiographic opacity that is surrounded completely by aerated lung . lesions that are larger than 3 cm are described as lung masses . as size has been shown to be important for classification of nodules ( see below ) the term micronodules is usually applied to very small nodules ( < 7 mm , < 5 mm ) which are almost always multiple and diffuse . if only one pulmonary nodule is detected , it is described as a solitary pulmonary nodule ( spn ) . if a nodule is detected in an examination performed for other reasons than a search for pulmonary nodules , it is called an incidental nodule , . most of the information on the aetiology and the natural course of incidentally found small pulmonary nodules is derived from studies on lung cancer screening with unenhanced low - radiation dose computed tomography ( low - dose ct ) in asymptomatic smokers and to a lesser extent non - smokers and workers exposed to asbestos . in these studies , at least one non - calcified pulmonary nodule was found in up to 66% of subjects , depending on the imaging technique ( e.g. multidetector - computed tomography ( ct ) versus single - detector ct ) . it was also shown that > 95% of these nodules were 10 mm in diameter and of these > 95% were benign . therefore , in order to avoid a high rate of invasive procedures for benign lesions in small nodules , algorithms were proposed that are based on mostly non - invasive strategies and , in particular , follow - up with low - dose ct . during follow - up changes in size ( or even better volume ) are recorded . nodules that decrease in size or resolve are obviously regarded as benign and no further follow - up is required . nodules that increase in size are likely malignant , particularly , if the growth rate is typical of malignant growth . most malignant tumors have been shown to double their volume ( i.e. increase their diameter in spherical lesions by 26% ) between 30 and 400 days . if malignancy is suspected , invasive procedures are usually performed to obtain a histological diagnosis ( bronchoscopic , percutaneous or thoracoscopic biopsy ) . in addition , positron emission tomography ( pet)-ct may be useful for small nodules of intermediate size ( 610 mm ) , as many malignant nodules show increased uptake of [ f]fluorodeoxyglucose ( fdg ) , whereas most benign nodules do not . pet - ct , however , is often negative in well - differentiated adenocarcinoma and bronchioloalveolar carcinoma ( bac ) and may be positive in inflammatory nodules ( e.g. sarcoid , tuberculosis ) . figure 1three non - solid nodules ( ground glass opacities ) in the posterior segment of the right upper lobe . three non - solid nodules ( ground glass opacities ) in the posterior segment of the right upper lobe . if the density of a nodule is lower than soft tissue attenuation , it does not obscure adjacent or transgressing vessels . part - solid nodules are characterised by a mixture of ground glass attenuation and solid components . benign non - solid nodules are due to inflammation ( eosinophilic pneumonia , cryptogenic organizing pneumonia ) , focal haemorrhage ( pulmonary endometriosis , pulmonary trauma , post biopsy ) , and focal interstitial fibrosis . persistent non - solid lesions , in contrast , mostly represent neoplasms , predominantly malignant ones . in a study by nakata et al . 54% of persistent ggos represented bronchioloalveolar carcinoma , 26% adenocarcinoma with mixed bronchioloalveolar carcinoma components and 21% atypical adenomatous hyperplasia . the proportion of malignant lesions differs between solid , part - solid and non - solid lesions . in a study by henschke et al . 7% of solid , 63% of part - solid and 18% of non - solid lesions were malignant . the majority of part - solid and non - solid malignant lesions were bronchioloalveolar carcinomas and adenocarcinomas with bronchioloalveolar features . noguchi et al . have described a classification of adenocarcinomas based on their histology which is reflected by their prognosis and this is also reflected by their thin - section ct morphology . types a and b represent bronchioloalveolar carcinomas , are characterised by a 100% 5-year survival rate and appear as non - solid lesions at thin - section ct , whereas types c , d , e and f represent more aggressive bacs or adenocarcinomas , exhibit part - solid or solid appearances with a less favourable prognosis . peripheral ill - defined lesion with mixed density ; the more peripheral component exhibits ground glass attenuation , whereas the more central part exhibits soft tissue ( solid ) attenuation . peripheral ill - defined lesion with mixed density ; the more peripheral component exhibits ground glass attenuation , whereas the more central part exhibits soft tissue ( solid ) attenuation . another non - solid lesion which was just recently defined is atypical adenomatous hyperplasia ( aah ) . this lesion is recognized as a separate entity which in itself is not malignant but may be a precursor of adenocarcinoma . it is characterised by proliferation of atypical cuboidal or columnar epithelial cells along the alveoli and respiratory bronchioles without invasion of the stroma or adjacent structures . at ct most of the recommendations for the management of solid nodules , also apply to non - solid and part - solid lesions . for example , the risk of malignancy should be estimated , previous imaging should be reviewed , and if growth is detected biopsy should be performed . there are , however , some recommendations unique to non - solid or part - solid nodules : if such a nodule is malignant the underlying lesion is likely to be bronchioloalveolar carcinoma or adenocarcinoma with bronchioloalveolar features. these may grow more slowly than other malignant tumors . therefore , the recommendation of follow - up after 3 , 6 , 12 and 24 months and no further follow - up when no growth is demonstrated in 2 years should not be applied to non - solid and part - solid lesions . these require longer follow - up to exclude growth for several years . because of the cumulative radiation exposure , dose levels during follow - up should be as low as possible. as bronchioloalveolar carcinoma and adenocarcinoma with bronchioloalveolar features may be negative at pet , examination with pet - ct should not be performed routinely as a negative result does not exclude malignancy . if such a nodule is malignant the underlying lesion is likely to be bronchioloalveolar carcinoma or adenocarcinoma with bronchioloalveolar features . therefore , the recommendation of follow - up after 3 , 6 , 12 and 24 months and no further follow - up when no growth is demonstrated in 2 years should not be applied to non - solid and part - solid lesions . these require longer follow - up to exclude growth for several years . because of the cumulative radiation exposure , dose levels during follow - up should be as low as possible . as bronchioloalveolar carcinoma and adenocarcinoma with bronchioloalveolar features may be negative at pet , examination with pet - ct should not be performed routinely as a negative result does not exclude malignancy . non - solid pulmonary nodules are more likely malignant than solid nodules . if malignant they are usually due to atypical adenomatous hyperplasia and bronchioloalveolar carcinoma . these lesions tend to grow more slowly than other malignant pulmonary lesions and are often not hypermetabolic at fdg - pet . therefore , follow - up to exclude growth in non - solid nodules needs to be longer than in solid lesions and pet - ct is not appropriate to rule out malignancy in non - solid nodules .

the application of a 32p - postlabeling assay for 7-methylguanines in dna was studied either by labeling the imidazole ring - opened dinucleotide derivatives or by using strong - anion - exchange column chromatography for the adduct enrichment from normal nucleotides . data showed that 7-methylguanines can be efficiently labeled as dinucleotides when in vitro methylated dna was first imidazole ring - opened and then digested to the dinucleotide level with deoxyribonuclease i , snake venom phosphodiesterase , and prostatic acid phosphatase . when using ion exchange chromatography for the adduct enrichment , dna was digested with micrococcal nuclease and spleen phosphodiesterase . anion exchange chromatography was applied for 7-methylguanine measurements in white blood cell dna of healthy nonsmokers ( n = 17 ) and patients ( n = 4 ) treated with the methylating drugs procarbazine and decarbazine . we found that the mean level of 7-methylguanine residues in nonsmokers was 2.5 per 10(7 ) nucleotides . the corresponding level in the patient samples immediately after the drug treatment was 57 per 10(7 ) nucleotides.imagesfigure 2 . abstractthis article describes the aetiology , epidemiology and clinical significance of incidental non - solid pulmonary nodules . non - solid nodules are more likely malignant . if malignant , they are mostly due to atypical adenomatous hyperplasia and bronchioloalveolar carcinoma . as these may be negative on positron emission tomography and slow growing , the diagnostic algorithms that are used for solid nodules have to be modified for non - solid nodules .

stressful life experiences have the amazing ability to synchronize organism - wide physiological responses toward the common goal of surviving threat . in this way , the major stress - responsive systems are often regarded by evolutionary biologists as essential adaptive mechanisms that ultimately promote survival . however , as stress challenges become greater in magnitude or more protracted in length , the toll on the host organism can be quite severe , and the adaptive value of mobilizing physiological processes toward survival exacts a long - term cost . this essential framework for understanding the relationship between stress challenges and their impact on organism health was articulated by hans selye , who noted that nearly all stress challenges were followed by a syndrome of being sick . unbeknownst to selye , this prescient view of the relationship between stressful experiences and their sickness - like outcomes , in today 's vernacular , implies that immune processes are probably essential mediators of the link between stress and adverse health outcomes . with this in mind , the present review takes the position that neuroimmune mechanisms of stress are highly conserved across species and serve as a likely mechanistic bridge between classic stress - responsive systems and stress - related pathologies ( figure 1 ) . to support this position , we ( i ) provide a succinct overview of neuroimmune mechanisms of stress that have been largely established in preclinical ( mouse and rat ) models ; ( ii ) extend these findings to other mammalian systems ( guinea pigs and nonhuman primates ) ; and ( iii ) translate these findings to the human condition . the intent here is not to provide a comprehensive summary , but rather to succinctly link together commonalities across models / species that argue in favor of viewing neuroimmune consequences of stress as highly conserved across taxonomic orders . at the same time ( and perhaps ironically ) , we argue that key features of the stress challenge ( nature , intensity , and duration of threat ) , as well as individual subject characteristics ( sex , age , stress history ) , will be critical for delineating individualized therapeutic approaches for the future . in doing so , we hope to provide guidance on significant gaps in our knowledge that remain to be filled and some possible pathways for the future . although we focus largely on the link between stress and negative affective states , such as depression and anxiety , the same processes are probably involved in risk for other forms of psychopathology as well . the first step in understanding neuroimmune mechanisms of stress and their eventual role in stress - related health outcomes is to define what is meant by , most studies have focused on intercellular signaling factors , such as cytokines , chemokines , and the many species of prostaglandins , whose primary roles were initially defined within an immunologically relevant context . demonstration that such intercellular mediators are often invoked by stress challenges in which there is no apparent tissue damage or other immunological insult suggests a natural role for these agents in mediating stress outcomes . however , the identification and interpretation of such findings can be quite difficult because there are literally dozens of inflammatory signaling factors that can be simultaneously regulated ; cytokines often have redundant biological action with one another ; and nearly all cytokines have pleiotropic functions . even more troublesome is the fact that actions of cytokines can manifest as generally pro- or anti - inflammatory , depending upon the presence of other signaling molecules and the context in which they are induced . in addition to these conceptual issues surrounding the identification and interpretation of cytokine action , there are also technical considerations surrounding the assessments of cytokines and other immune mediators . because immune - related factors are expressed at appreciably low quantities in the normal central nervous system ( cns ) , the techniques to detect and measure such cytokines , particularly in early studies , have often been inadequate to yield precise results . for instance , many studies have utilized gross tissue dissections ( or micropunches ) that aggregate cytokine measures over large anatomical areas and across cell types within the dissected tissue , which can significantly influence outcomes and interpretation . a second major technical consideration lies in the use of quantification procedures that allow for signal amplification ( reverse transcription polymerase chain reaction ) versus those that do not ( in situ hybridization ) , and the more general juxtaposition of studies that measure protein versus messenger rna ( mrna ) . the availability of well - validated antibodies and the use of appropriate immunohistochemical controls for target specificity and cell type have also been a historical problem . finally , deploying standard neuroscientific approaches ( cannulation , lesions , etc ) for manipulation of inflammatory signaling in the cns can significantly alter sensitivity to later stress challenges . thus , one must carefully consider the technical approach employed by studies as a key constraint in interpreting experimental outcomes . with that said , several inflammatory mediators have emerged as highly stress - responsive , and their physiological impact has been delineated clearly . for instance , numerous studies have indicated that interleukin 1 ip is rapidly increased in key limbic structures ( paraventricular nucleus [ pvn ] ; amygdala ) in response to stress challenges that involve application of an aversive / noxious stimulus such as footshock , but not in response to social stress challenges . in contrast , social stressors appear to increase release of another proinflammatory cytokine , il-6 , in both plasma and brain of mice , thereby contributing to stress phenotypes . importantly , recent evidence suggests that early exposure to maternal separation in male rats may change cytokine reactivity to later social stress challenges incurred during adulthood . moving beyond some of the classic cytokines ( such as il-1 , il-6 , tumor necrosis factor a [ tnf- ] ) , recent evidence suggests that chemokines are also dynamically altered by stress challenges . as a separate class of immune - related molecules , these stress - induced chemokines probably play a key role in structural changes to the cytoskeleton of microglia that allows for the motility and retraction of processes , in recruitment of other intrinsic microglia to the site of release , and in potentially inducing passage of monocytes across the blood - brain barrier . indeed , genetic ablation of the chemokine cx3cl1 ( fractalkine ) was recently shown to prevent microglial activation associated with chronic unpredictable stress . in addition , prostaglandins have emerged as rapid , stress - sensitive inflammatory mediators , particularly within the cortex . given the role that prostaglandins play as final common mediators of the febrile response , prostaglandins are also likely mediators of stress - induced fever responses . thus , a multitude of inflammatory signaling families are mobilized by stressful experiences and significantly impact cns function . a growing number of studies have also examined cellular manifestations of neuroinflammation , with microglia emerging as highly reactive to stress challenges . early studies examining microglial activation established that administration of minocycline , a putative microglial inhibitor , blocked the induction of central , but not peripheral , il-1 by footshock . other studies have shown that chronic stress exposure drives proliferation of microglia and increases both the density and activational state of microglia within certain brain structures . stress challenges also alter the expression of receptors expressed on the cell surface of microglia that are both indicative of microglial activation state and positively coupled to cytokine expression within microglia . indeed , dynamic alterations in cell surface receptors on microglia probably accounts , at least in part , for certain priming / sensitization effects incurred by stress , including more rapid cytokine responses produced by later injection of lipopolysaccharide ( lps ) . together , these findings provide multiple avenues by which stress challenges impact microglia and strongly support the notion that microglial ( re)activity may be a key culprit in mediating stress - dependent changes in behavior , cognition , and potentially multiple forms of psychopathology . the importance of inflammatory phenotype to behavioral and mood - related changes incurred by stress has been strengthened in recent years through the use of adoptive transfer studies . these clever studies show that repeated exposure to various forms of repeated social stress in mice increases microglial activation and expression / release of cytokines . intriguingly , when circulating monocytes are then extracted from previously stressed mice and transferred to other mice in which the existing monocytes / lymphocytes have been depleted , the recipient mice display behavioral and mood tendencies that reflect those of the original stressed host . these findings , combined with recent evidence showing that circulating monocytes may transit into the cns as a result of stressor exposure and actively influence mood state , add a new dimension to our understanding of bidirectional communication between the brain and the peripheral immune system . though this provocative area of research is still in its infancy , the emerging body of evidence provides , for the first time , causal evidence for the notion that non - neuronal cells ( microglia ) may be responsible for encoding stress - dependent changes in mood regulation , particularly for negative affective states like depression and anxiety . a key issue that must be considered in understanding the relation between stress challenges and their neuroimmune consequences is timing . in laboratory models , acute stress challenges or the individual bouts of daily , intermittent chronic stress procedures are typically elaborated across a 30-min to 3-hour window of stress exposure ( figure 2 ) . the central questions requiring consideration here are ( i ) how rapidly are neuroimmune signaling agents induced ? ( ii ) how long do such changes persist ? ( iii ) are there downstream neuroimmune effects that cascade or coalesce into subsequent neuroimmune alterations ? and ( iv ) what are the functional outcomes that can be tied to individual components of such neuroimmune cascades ? although the discussion above did not address these issues by indicating the time point at which individual changes were observed , recent studies examining the time course for neuroimmune responses provide some guidance . for instance , induction of il-1 gene expression is prevalent and significant within 30 min of stress onset , with il-1 protein responses peaking shortly thereafter ( 60 to 120 minutes after stress onset ) . in contrast , most studies examining microglial morphology , proliferation , or activational state tend to show changes that are prevalent 24 hours or more after stress termination , by which point many cytokine changes have largely resolved . though perhaps correlational at this stage , one interpretation of the timing of these events is that the expression and release of intercellular mediators ( cytokines , chemokines , prostaglandins ) have a nearly immediate impact upon behavioral changes observed in the post - stress recuperative period , while at the same time eliciting parallel changes in other aspects of neuroimmune function that persist for days to weeks after cessation of stress . thus , it will be critical for future studies to conceptually discriminate between those neuroimmune changes that represent acute , activational responses to an individual bout of stress , versus those neuroimmune changes that reflect the aggregate influence of a repeated history of stress across a more protracted period of time . the multitude of inflammatory signaling pathways that appear to be activated by stress challenges raises questions regarding the mechanisms that control such neuroimmune changes . here , we suggest that multiple upstream signaling pathways converge upon regulatory elements associated with neuroimmune signaling , thereby propagating various features of the neuroimmune response to stress . for ease of description , we will describe these pathways in three functional clusters ( figure 3 ) , with full recognition that these pathways are not independent of one another . for instance , norepinephrine release during stress - associated activation of the sympathetic nervous system has long been regarded as a primary driver of neuroimmune consequences of stress . a wide range of pharmacological studies lend support to this notion , showing that administration of -adrenergic blockers ( eg , propranolol ) block the induction of il-1 incurred by stress , whereas -adrenergic agonists ( eg , isoproterenol ) potently induce il-1 expression . consistent with this , previous treatment with desipramine ( a norepinephrine - reuptake inhibitor ) potentiated both basal and stress - induced il-1 expression . gross neurotoxic lesions of norepinephrine - containing cells via intracerebroventricular injection of the selective neurotoxin n-(2-chloroethyl)-n - ethyl-2 bromobenzylamine ( dsp-4 ) blocked the induction of il-1 in the hippocampus , whereas more focal lesions of the ventral noradrenergic bundle produced modest effects on il-1 induction by stress in the pvn . together , these studies underscore the importance of norepinephrine signaling as a key driver of neuroimmune consequences of stress . however , other studies have clearly tied microglial proliferative responses incurred by chronic stress to glutamate signaling , since chronic administration of mk801 effectively blocked this proliferative response , and n - methyl - d - aspartate ( nmda)-receptor activation is positively coupled to inflammatory signaling pathways . interestingly , considering that adenosine triphosphate ( atp ) is co - packaged and released with many classic neurotransmitters ( including norepinephrine , in particular ) , other groups have recently posited purinergic signaling as a putative mechanism controlling functional release of il-1 ( and potentially other neuroimmune factors ) in response to stress . another emerging class of neuroimmune intermediaries falls into the category of what we describe as danger , damage , and disease signals ( figure 3 ) . the basic premise is that our innate immune defense network is constructed to respond to general molecular motifs associated with pathogens or self - injury . damage - associated molecular patterns are host - derived molecules that are released in response to tissue trauma or other insults and are thus capable of activating inflammatory signaling pathways . a variety of studies have convincingly demonstrated several families of damps that are activated by exposure to intense stress challenges , including heat shock proteins ( hsp72 , in particular ) and high - mobility group box 1 ( hmgb1 ) . when released , these damps interact with cognate receptors that are positively coupled to inflammatory signaling pathways . this family of receptors is often referred to as pathogen recognition receptors ( prrs ) because they recognize and bind to general molecular motifs associated with classes of bacteria and viruses , as well as to certain damps that may be activated by stress and/or damage . yet another intriguing twist is the recent notion that bacteria from the gut or other areas of the microbiome may be stress - sensitive , and perhaps even mobile in response to stress challenges , thereby incurring a mild form of endogenous infection . thus , the damp->prr pathways have emerged as important alternative pathways by which neuroimmune consequences of stress are primed and/or activated . the propagation of neuroimmune signaling cascades in response to stress is also constrained by other features of physiology . perhaps the most logical of these counter - regulatory elements is the release of glucocorticoid ( gc ) hormone ( corticosterone in rat and mouse ; cortisol in guinea pigs and primates ) as a result of hypothalamic - pituitary - adrenal - axis ( hpa ) activation that accompanies stress exposure . the anti - inflammatory action of gcs has been established for decades , with great progress being made in recent years in the cis- and trans regulatory elements that give rise to anti - inflammatory effects of gcs . within stressful contexts , this was particularly evident in early studies where induction of central il-1 was massively potentiated in adrenalectomized subjects , and normalized by replacement of corticosterone . similar effects have been observed after pretreatment with metyrapone ( a gc synthesis inhibitor ) . however , there appear to be certain instances in which gcs prime or modestly stimulate inflammation , a notion which is now challenging some of the central dogma on the relation between gc hormone signaling and inflammation . these latter , proinfiammatory - like effects of gcs probably represent a small proportion of the overall gc effects on inflammation and relate to very specific parameters of low gc dose , the timing of gc hormone exposure , and the specific tissue in which the gcs act . gene expression profiling studies predict such effects , and so it is quite interesting to see these effects bearing out on functional responsiveness within the neuroimmune system . furthermore , a recent study found that microglial activation was more profound after several weeks of repeated restraint , but not after an equivalent exposure to chronic variable stress . although the reason for this difference has yet to be determined , the authors propose that hpa - axis habituation , and thus the lack of circulating gc hormone that accompanies repeated restraint , led to a disinhibition of stress - dependent microglial activation . additional studies showed that pharmacological activation of the hpa axis ( in the absence of an overt stress challenge ) via corticotropin - releasing hormone or corticotropin injection led to an expected inhibition of cytokine expression in the pvn , though paradoxically , both peptides stimulated inflammatory signaling at the level of the adrenal gland . yet another added complexity is that gcs are not the only hormone system engaged by stress exposure . recent studies demonstrated that progesterone is also rapidly released in response to stress , even in male rats . this makes sense as progesterone is a precursor molecule in the biosynthesis of corticosterone , yet the possibility that progesterone may contribute to the anti - inflammatory influence of gcs has not been systematically tested . one major area of neglect in the literature has been the elaboration of sex differences in neuroimmune responses to stress . in a recent set of studies from our lab , we examined fluctuations in the il-1 response to stress as a function of estrous stage in female rats . these studies reported relatively uniform il-1 induction at all phases of the cycle except during metestrus where basal il-1 was moderately increased , and the il-1 response to stress appeared to be blunted in the pvn . however , this effect requires further study to generalize the effects from mrna to protein , to expand the analysis from the pvn to other stress - sensitive sites , to incorporate additional neuroimmune measures , and to replicate what was originally a slightly underpowered effect . nevertheless , subsequent studies demonstrated that stress - induced il-1 was potentiated in ovariectomized rats and restored by estradiol - replacement injections . however , because endogenous progesterone evinced a stress - induced surge , we can not at this time attribute the rescue effect of estradiol replacement exclusively- to estradiol ( versus an estradiol plus progesterone effect ) . what we do know , however , is that progesterone replacement alone did not recapitulate the effect of estrogen replacement . nevertheless , few studies , if any , have examined how sex - specific gonadal steroids differentially affect sex differences in neuroimmune responses to stress in rodents . thus , future studies need to give serious consideration to potential additive , cooperative , and synergistic effects of steroid hormones on stress - dependent changes in neuroimmune function . although the vast majority of studies on stress and inflammation have focused on rats and mice ( as indicated in the above discussion ) , investigators have sometimes turned to alternative rodent species . guinea pigs in particular offer a number of advantages , especially in developmental studies . unlike rats and mice , brain regions are well defined , and the skull is calcified even in the first few days , which facilitates procedures such as implanting indwelling cannula and immunohistochemistry . thermoregulation matures rapidly , so lps- or stress - induced fever can be investigated early in preweaning pups . but for studies of stress - induced inflammatory responses during infancy , perhaps the greatest advantage stems indirectly from the fact that the pups are precocial . guinea pigs are capable of coordinated behavior , including locomotion and ingestion , from shortly after birth . licking of the pups , the primary active maternal behavior , is infrequent , particularly after about a week of age . there is no retrieval , no nest , and mothers simply accommodate nursing attempts initiated by the pups . as a result , mother - young proximity is maintained almost exclusively by the strong attraction or attachment that pups display for the mother . indeed , pups exhibit evidence of the classic markers of attachment commonly used in primate studies namely , approach , recognition , and preference for the attachment object ; use of the attachment object as a secure base for exploration ; and distressful responses to separation . maternal separation in guinea pigs thus affords a compelling translational model for the effects of attachment disruption , a class of early stressors frequently linked to later psychopathology in humans as well as altered inflammatory activity . there are many similarities in the responses to maternal separation shown by young guinea pigs and infant monkeys . these include , for instance , increased hpa and sympathetic activity and central catecholamine turnover associated with stress . as is seen in some species of macaques , as well as in human infants , guinea pig pups also display a two - stage , active / passive behavioral response to separation . when placed alone in a novel cage , pups initially show protest by vocalizing in an apparent attempt to re - establish contact with the mother . but after about an hour of separation , the pups typically quiet and adopt a characteristic crouched stance , with closed eyes , extensive piloerection , and apparent disinterest in their surroundings ( figure 1 ) . neither the active nor passive stages are observed if the mother is placed in the novel cage with the pup , establishing that the mother 's absence , and not just the novelty of the environment , is responsible for these responses . the passive , second stage of separation in guinea pig infants is reminiscent of the despair shown by monkeys separated for much longer periods and even the although the nature of the passive stage of separated guinea pig pups can suggest depression to a comparative psychologist , it may just as readily suggest cytokine - induced sickness behavior to the psychoneuroimmunologist . each of the components of the response moreover , direct stimulation of sickness with lps results in the same behavioral constellation of these three behaviors . it appears then that the stressor of separation in a novel environment initiates an inflammatory reaction that mediates the behavioral response . this claim is bolstered by findings that the separation procedure elicits a transitory fever and increased expression of the proinflammatory cytokine tnf- in spleen , two physiological indicators of a sickness response . behaviors of the second stage of separation can also be reduced by a variety of anti - inflammatory agents , including -melanocyte - stimulating hormone ( -msh ) , indomethacin , il-10 , and naproxen . although other systems are also probably involved , it is clear that inflammatory mechanisms play a fundamental role in the depressive - like response of separated guinea pig pups . in the adult human literature , there is now overwhelming evidence for involvement of inflammation in depressive illness ( as described below ) . the guinea pig results suggest that the particular form of depressive response shown by separated nonhuman and perhaps human primate infants may also be mediated , at least in part , by inflammatory factors . most current research on attachment disruption and depression in humans focuses not on immediate effects , but rather on long - term vulnerability for developing depression engendered by early abuse , neglect , or prolonged separation . whereas increased risk for later depressive illness has long been suspected for infants exposed to such forms of maltreatment , recent research has solidified this link and begun to provide glimpses of potential neurobiological mechanisms . that is , the early stress of attachment disruption is seen as sensitizing underlying stress - responsive machinery so that , in later life , exposure to stressors with which other individuals would be able to effectively cope elicit disproportional , protracted , and inadequately regulated stress responses that lead to , or constitute the underlying basis of , the depressive episode . these diathesis - stress or two - hit models have most often emphasized effects on elements of the hpa axis and its control , including increases in central corticotropin - releasing factor secretion , amygdala activity , and gc resistance , as well as a reduction in hpa inhibition by the frontal cortex . however , recently , there also has been a proliferation of findings implicating inflammatory mechanisms in the sensitization process . attachment disruption and other forms of early stress have repeatedly been found to be associated with increased markers of inflammation at later ages . from an evolutionary perspective , the increased inflammation may represent a remnant of an ontogenetic adaptation originating from a time when persistent early - life stress was predictive of a hazardous adult environment , in which injury was common and a robust innate immune system was adaptive . the process by which early stress enhances later inflammation remains unclear but may involve sensitization of resident microglia or increased transport of peripheral monocytes to the cns , resulting in heightened central release of inflammatory mediators . the augmentation of inflammatory processes , in turn , may be driven by activation of , and alterations in , other stress - responsive systems , such as increased resistance to the suppressive action of gcs and elevated sympathetic activity . results of guinea pig studies implicate inflammatory factors in long - term effects of early stress . repeated separation increases , ie , sensitizes , both the depressive - like behavior and febrile response to later separations during both the preweaning period and beyond . moreover , administration of the cyclooxygenase ( cox)-inhibitor naproxen before the initial separation suppressed the sensitization response not only to the initial separation experience but also to separations that followed 1 and 10 days later . the sensitization that occurs appears to be related to some broader , depressive - like state rather than just the separation response , as previously separated guinea pigs also showed more immobility in the forced swim test , a measure and paradigm that is selectively sensitive to antidepressant medications . in these studies , as is the case in the field more generally , sex differences have yet to be a major focal point . although males and females have typically both been included in the guinea pig work , it has been in numbers too small to sufficiently power examination of male - female differences . however , as rodent models for the role of inflammation in stress - related disease are nowbecoming established , it is imperative that male - female differences be taken into account . this is particularly important for disorders in which sex differences are profound , such as major depression , for which women are about twice as likely as males to be afflicted . the above results argue that the guinea pig model has strong internal validity in that there is good evidence that attachment disruption in the form of maternal separation results in depressive - like behavior that sensitizes with repeated separation . the evidence also indicates that the sensitization involves inflammatory processes and reflects changes in an underlying state that is manifested in more than one depressive - like response . the external validity , ie , the generality of the results to humans , would , however , be bolstered if we could demonstrate that a similar experimental procedure produced similar results in a primate . indeed , the guinea pig work was always intended as a complement to primate research ; that is , as a way of doing the investigative work necessary to ultimately generate hypotheses that might then be tested in primate models . the challenge has been how this might be accomplished without proposing to repeat the prolonged separation of monkey infants from their mothers that was common in experiments of the 1950s and 1960s . a possible solution arose from observations of the behavioral management unit and others at the california national primate research center . it was noted that when adult monkeys were brought from large outdoor social groups ( figure 1 ) to restricted indoor housing , as is commonplace for the beginning of experiments or veterinary care , a small proportion exhibited a hunched posture with apparent disinterest in their surroundings , a response that both is widely regarded as indicating a depressive - like reaction in macaques and which mimics the reaction observed in separated monkey infants . furthermore , when recordings of adult male monkeys introduced to the restricted indoor housing with no human observer in the room were analyzed , an even larger percentage of animals were found to exhibit the depressive - like hunched posture . these findings suggest that presence of a human may evoke a defensive reaction incompatible with the depressive - like response . these findings are compatible with interpretation of these depressive - like consequences as a manifestation of sickness , since expression of sickness behaviors are easily perturbed in the face of threat . from a modeling standpoint , however , the observations suggested that the relatively simple procedure of transferring adult monkeys from outdoor social groups to indoor housing may provide a means of evaluating the general relevance of the guinea pig results for nonhuman primates . therefore a new model was developed in which adult male rhesus macaques were brought from half - acre outdoor social groups to standard indoor housing , either alone or together with an affiliative juvenile partner for 8 days on two occasions at an approximate 2-week interval . behavior was filmed without a human observer present and blood samples were taken for cytokine analysis at the end of each 8-day period of indoor housing , as well as when the males were residing in the outdoor field cages . during the first separation , all monkeys of both groups exhibited the hunched posture , with an average of about a third of all observation time spent in this posture . during the second separation , time spent in the hunched posture increased ( ie , sensitized ) , but the increase in time spent in the hunched posture was accompanied by a decline in activity and environmental exploration . the monkeys housed alone showed a relative decline from the first to the second period of indoor housing in lps - induced expression of the anti - inflammatory cytokine il-10 , whereas those monkeys housed with a partner showed a relative increase . furthermore , regardless of whether monkeys were alone or with a partner , indoor housing reduced sensitivity- of the proinflammatory cytokines il-1 and tnf- to the suppressive action of gcs . it should be noted that both of these measures response to lps stimulation and gc resistance were chosen because they appear responsive to early - life stress in humans . finally , evidence for a coupling of the behavioral and cytokine findings was also observed . for monkeys tested alone , a large and significant positive correlation was observed between the number of seconds spent in the hunched posture during each period indoors and circulating levels of each of the three cytokines measured . although many particulars of the experimental design and measures in the guinea pig and monkey studies differed , the broad similarity of results suggests some cross - species commonality or conservation of basic relations between social separation , inflammatory activity , and depressive - like behavior and its sensitization . the findings also suggest relatively simple rodent and monkey models that might be used to continue to disentangle the way in which earlylife stress and inflammation contribute to depressive illness and other negative affective states . the above results argue that the guinea pig model has strong internal validity in that there is good evidence that attachment disruption in the form of maternal separation results in depressive - like behavior that sensitizes with repeated separation . the evidence also indicates that the sensitization involves inflammatory processes and reflects changes in an underlying state that is manifested in more than one depressive - like response . the external validity , ie , the generality of the results to humans , would , however , be bolstered if we could demonstrate that a similar experimental procedure produced similar results in a primate . indeed , the guinea pig work was always intended as a complement to primate research ; that is , as a way of doing the investigative work necessary to ultimately generate hypotheses that might then be tested in primate models . the challenge has been how this might be accomplished without proposing to repeat the prolonged separation of monkey infants from their mothers that was common in experiments of the 1950s and 1960s . a possible solution arose from observations of the behavioral management unit and others at the california national primate research center . it was noted that when adult monkeys were brought from large outdoor social groups ( figure 1 ) to restricted indoor housing , as is commonplace for the beginning of experiments or veterinary care , a small proportion exhibited a hunched posture with apparent disinterest in their surroundings , a response that both is widely regarded as indicating a depressive - like reaction in macaques and which mimics the reaction observed in separated monkey infants . furthermore , when recordings of adult male monkeys introduced to the restricted indoor housing with no human observer in the room were analyzed , an even larger percentage of animals were found to exhibit the depressive - like hunched posture . these findings suggest that presence of a human may evoke a defensive reaction incompatible with the depressive - like response . these findings are compatible with interpretation of these depressive - like consequences as a manifestation of sickness , since expression of sickness behaviors are easily perturbed in the face of threat . from a modeling standpoint , however , the observations suggested that the relatively simple procedure of transferring adult monkeys from outdoor social groups to indoor housing may provide a means of evaluating the general relevance of the guinea pig results for nonhuman primates . therefore a new model was developed in which adult male rhesus macaques were brought from half - acre outdoor social groups to standard indoor housing , either alone or together with an affiliative juvenile partner for 8 days on two occasions at an approximate 2-week interval . behavior was filmed without a human observer present and blood samples were taken for cytokine analysis at the end of each 8-day period of indoor housing , as well as when the males were residing in the outdoor field cages . during the first separation , all monkeys of both groups exhibited the hunched posture , with an average of about a third of all observation time spent in this posture . during the second separation , time spent in the hunched posture increased ( ie , sensitized ) , but the increase in time spent in the hunched posture was accompanied by a decline in activity and environmental exploration . the monkeys housed alone showed a relative decline from the first to the second period of indoor housing in lps - induced expression of the anti - inflammatory cytokine il-10 , whereas those monkeys housed with a partner showed a relative increase . furthermore , regardless of whether monkeys were alone or with a partner , indoor housing reduced sensitivity- of the proinflammatory cytokines il-1 and tnf- to the suppressive action of gcs . it should be noted that both of these measures response to lps stimulation and gc resistance were chosen because they appear responsive to early - life stress in humans . finally , evidence for a coupling of the behavioral and cytokine findings was also observed . for monkeys tested alone , a large and significant positive correlation was observed between the number of seconds spent in the hunched posture during each period indoors and circulating levels of each of the three cytokines measured . although many particulars of the experimental design and measures in the guinea pig and monkey studies differed , the broad similarity of results suggests some cross - species commonality or conservation of basic relations between social separation , inflammatory activity , and depressive - like behavior and its sensitization . the findings also suggest relatively simple rodent and monkey models that might be used to continue to disentangle the way in which earlylife stress and inflammation contribute to depressive illness and other negative affective states . the key question , of course , is how well these animal models translate to humans and help us better understand the human stress response and risk for psychopathology . psychosocial stress is a well - established risk factor for the development of various forms of psycho - pathology in humans , including major depressive disorder ( mdd ) , posttraumatic stress disorder ( ptsd ) , and other anxiety disorders . similar to findings from animal models of stress , evidence from human studies shows that exposure to stressors provokes neurochemical changes , including changes in levels of inflammation , that are frequently associated with psychopathology , and which may help to explain the mechanisms by which stress increases risk for psychiatric disorders , including mdd . neuroimmune effects of exposure to stressful events can persist beyond the immediate impact and potentiate an individual 's response to future stressors , thus increasing risk for future psychopathology . indeed , among individuals with mdd , those who experienced childhood adversity had higher circulating levels of c - reactive protein ( crp ) than individuals with no history of childhood trauma . additionally , adults who experienced childhood adversity exhibited higher levels of circulating il-6 levels in response to an acute stressor . finally , there is preliminary evidence that a haplotype in il-33 moderated the link between women 's history of childhood abuse and their risk for depression in adulthood . across a variety of stressors , ranging from low socioeconomic status to traumatic events , mammalian immune cells show an immediate conserved transcriptional response to adversity , which involves increased expression of inflammatory genes and decreases in the genes underlying antiviral responses . interestingly the inflammatory component of the leukocyte conserved transcriptional response to adversity may be driven by stress - induced upregulation of myelopoiesis and could contribute to development of negative health outcomes associated with adverse social conditions , including psychiatric disorders . overall , one of the pathways for the effects of exposure to stressors on psychopathology risk seems to be an increase in immediate and long - term expression of immune -related genes , outcomes that appear to recapitulate effects observed in preclinical ( animal ) models . immune - related transcripts involved in the cellular stress response have been shown to be upregulated in the prefrontal cortex ( pfc ) of individuals with schizophrenia , indicating that these genes may play an important role in chronic psychopathology . importantly , these observed transcriptome changes were not a result of acute immune system activation , as there were no differences in markers of acute inflammation or responses to infections between individuals with schizophrenia and controls . similarly , in the hippocampus , general upregulation of inflammatory transcripts was found in patients with mdd , bipolar disorder , and schizophrenia , though the authors reported no overlap in specific genes across disorders . this suggests that there are common immune - related gene changes in psychiatric disorders , but that there are also changes in expression that are differentially regulated between diseases . for example , circulating cytokines and upregulation of immune - related genes have been found to occur in patients undergoing a first episode of psychosis , whereas those with comorbid depression displayed a unique expression profile , suggesting separable transcriptional phenotypes . additionally , increased mrna levels of chemokine ( c - c motif ) ligand 24 ( ccl24 ) were found in circulating leukocytes among participants with mdd compared with individuals with bipolar personality disorder and healthy controls . in contrast , c - c chemokine receptor type 6 ( ccr6 ) was expressed consistently less among mdd patients than in controls . another example can be found in common variants in long - range enhancer elements that modulate transcriptional response to activation of gc receptors in human blood cells , thereby increasing the risk for later psychopathology , including mdd and schizophrenia . moreover , those functional genetic variants were associated with overgeneralized amygdala reactivity , suggesting that individual differences in the leukocyte 's immediate transcriptional reactivity to stress may influence an individual 's neurophysiological response to stress . epigenetic alterations in genes related to immune function are one of the plausible mechanisms underlying the lasting neuroimmune effects of stress exposure and increased risk for psychopathology . one of the first epigenome - wide association studies ( ewas ) that examined dna methylation among participants with a history of mdd identified tryptophan metabolism - related genes as one of the top three functional clusters in individuals with no history of mdd , one of which was a cytokine - induced reduction in tryptophan , the primary serotonin precursor often implicated in mdd . there is also evidence that methylation levels of the il-6 and crp genes are inversely related to circulating levels of il-6 and crp among individuals with a history of mdd . similarly , immune - related genes were shown to be over - represented among unmethylated genes among individuals with ptsd . furthermore , hypomethylation of immune - related genes among ptsd - affected individuals was linked to increased peripheral levels of inflammatory cytokines , which were , in turn , related to history of childhood abuse and life stress . overall , these findings suggest that exposure to stressors , perhaps particularly early - life stress , could result in immune - associated epigenetic changes that increase an individual 's susceptibility to psychiatric disorders . complementing these findings , researchers have recently begun exploring neural mechanisms underlying immune activation after stress exposure in humans via structural and functional imaging . for example , a functional magnetic resonance imaging study that examined changes in mood and neural activity after an in vivo immune challenge found that immune - induced mood decline was associated with increased activity in the subgenual anterior cingulate cortex ( sacc ) during emotional face processing . inflammation - induced mood deterioration was also associated with decreased functional connectivity of the sacc with the amygdala , medial pfc , nucleus accumbens , and superior temporal sulcus . additionally , exposure to an acute social stressor has been associated with increased circulating levels of il-6 and tnf- , along with increased activity in the dorsal acc ( dacc ) during a social rejection task . these findings suggest that stress - induced effects on inflammation may increase activity in brain regions associated with emotion processing , while decreasing connectivity with regions involved in emotion regulation . this increased sensitivity to social stressors and decreased emotion regulation may , in turn , increase the risk for various psychiatric disorders , including mdd . there is growing evidence that inflammation may also impact corticostriatal reward circuitry , which underlies symptoms of anhedonia that are common in various psychiatric disorders . specifically , there is evidence that increased circulating levels of crp , a common inflammatory marker , were associated with decreased connectivity between ventral striatum and ventromedial pfc and decreased dorsal striatal to ventromedial pfc connectivity among participants with mdd . notably , the association between the differences in connectivity and symptoms of anhedonia and motor slowing were significantly predicted by participants peripheral crp levels . similarly , depressive symptoms after exposure to an in vivo immune challenge was contingent , at least in part , on a reduction in ventral striatum activity in response to anticipated rewards . these findings parallel the results of neuroimaging studies showing that administration of interferon- , a potent proinflammatory cytokine , led to reduced activity in the ventral striatum during a hedonic reward task . moreover , positron emission tomography was utilized to show an interferon--induced increase in f - dopa ( radiolabeled dopamine precursor ) uptake , but decreased f - dopa turnover , in the basal ganglia , which correlated with increased depressive symptoms . together , these findings suggest that inflammation may adversely impact motivation and goal - directed behavior by decreasing activation and connectivity of brain regions involved in processing of rewarding stimuli and psychomotor speed , plausibly though the modulation of dopamine function . the recent understanding of the above interactions between stress and inflammation has given rise to research that applied these findings to both new and longstanding treatment approaches . for instance , there is a growing body of research that suggests that cognitive behavior therapy ( cbt ) reduces inflammation in the context of improving disturbed sleep and depressed mood . other treatment approaches , such as mindfulness meditation and yoga , are also associated with decreased stress - induced inflammation . finally , there is evidence in human clinical populations that targeting inflammation directly may help to alleviate symptoms of psychopathology , including mdd , ptsd , and schizophrenia , but only in a subgroup of patients who exhibit increased initial levels of inflammatory markers . specifically , meta - analyses have supported the beneficial use of anti - inflammatory medication in schizophrenia - affected individuals , particularly those who are in the early stages of this disorder . although preliminary results of the anti - inflammatory therapy in the treatment of psychiatric disorders are promising , it is important to identify specific subgroups that would benefit the most from such treatment . for example , one study examined the potential antidepressant effect of the tnf- inhibitor infliximab in patients with treatment - resistant depression who were otherwise healthy . infliximab is a monoclonal antibodydirected against tnf- that prevents this cytokine from binding to its receptor via immunoneutralization . interestingly , the anti - inflammatory therapy outperformed placebo , but only in patients with high peripheral levels of crp before treatment ( > 5 mg / l ) . baseline levels of inflammation could , therefore , serve as a biomarker of an individual 's likelihood of responding to anti - inflammatory therapies . intriguingly , among participants with a baseline crp value greater than 5 mg / l , anti - inflammatory therapy led to a reduction in a variety of symptoms , including sad mood , psychomotor retardation , anhedonia , anxiety , and suicidal ideation , all of which are linked to the neurocircuits typically targeted by inflammatory cytokines . overall , therefore , anti - inflammatory therapy may be a promising treatment for specific subgroups of patients with a variety of psychiatric disorders , such as those with elevated circulating inflammatory markers . the identification of , and ability to detect , specific biomarkers that can identify individuals who would benefit from anti - inflammatory therapy is a critical step in delivering individualized therapy . despite recent advances made in understanding the role of inflammatory processes in human psychopathology , direct evidence examining potential sex differences historically , men and women have been known to display pronounced biological and psychological differences in responses to stress , with females typically displaying nearly double the gc response relative to males . stress reactivity is also not constant across developmental epoch or across hormonal cycles ; women who are between the ages of puberty and menopause typically exhibit lower hpa - axis and autonomic nervous system responses to acute psychosocial stressors than older women . what is striking is that women have higher rates of stress - related psychiatric disorders that have been strongly linked to inflammation , including mdd and anxiety disorders . the sex - specific epidemiological pattern of psychiatric disorders highlights the role of sex hormones in stress reactivity , since many of these sex differences emerge in puberty . interestingly , there is also evidence of sexual dimorphism in the susceptibility of women and men to immune - related disorders . for instance , the prevalence of many inflammatory conditions , including autoimmune diseases is significantly higher in women , whereas men are more likely to suffer from infectious diseases . notably , young women were reported to exhibit higher peripheral levels of il-6 than men after mental or physical stress . clearly , more research is needed in this area , as it may be that sex differences in stress reactivity , including inflammatory and , potentially , neuroimmune responses to stress , could provide much needed insight into the vast sex differences in the rate of stress - related psychiatric disorders . the goal of this review was to highlight the neuroimmune mechanisms underlying the response to stress with an emphasis on extending findings from animal models toward the human experience . research on rodents has served as a starting point for understanding the molecular and cellular responses to stress , allowing for a greater understanding of how inflammatory factors , such as cytokines , chemokines , and prostaglandins , ultimately influence brain function . importantly , microglia have emerged as a key interface between stress - related signals and neuroimmune consequences of stress . the guinea pig in particular serves as a useful model of early - life stress with excellent early - life translatability that recapitulates findings from nonhuman primates and humans . further validation of the guinea pig model , and expansion of genetic tools and antibodies directed toward guinea pig - specific proteins , will allow for bridging the findings in rats and mice with a complementary , tractable model system . the use of nonhuman primates confers significant advantages not only in being able to bridge findings in rodent models to humans , but also in that they lead highly social lives allowing for examination of stress and social interactions ( eg , buffering effect of a cage partner on social separation discussed earlier in the review . ) . through studying conserved transcriptional responses to stress that have been examined in circulating leukocytes in humans , researchers have made connections between clinically relevant psychological disorders and expression levels of immune and inflammatory factors . the finding that epigenetic modulation of cytokines differs in individuals with stress - related disorders , such as mdd and ptsd , provides an intriguing avenue for animal model research in examining how stress can affect future gene expression . thus , viewing neuroimmune consequences of stress through a multispecies lens provides a compelling argument for the highly conserved nature of the relationship between cytokines , stress , and multiple forms of psychopathology . our challenge for the future , therefore , will be to dive deeper into individual differences in stress reactivity , using a combination of highly tractable animal models from different species to better discriminate between those neuroimmune consequences of stress that are constant within and across species , relative to those that differ as a function of the individual ( sex , age , recent stress history ) or that are species - specific . in doing so , we can exploit the strengths of individual model systems while at the same time circumventing their limitations , with the hope of defining novel therapeutic strategies to ameliorate adverse health consequences of stress . hemorrhage from pelvic blood vessels after trauma or obstetric delivery are major health care problems . trauma is a leading cause of death worldwide and a major cause of lost quality - adjusted life years ( qalys ) . treatment of pelvic trauma is difficult since hemorrhage can be massive and it is difficult to apply direct pressure to the bleeding vessels . postpartum hemorrhage is the leading cause of obstetric deaths and causes an estimated 150,000 maternal deaths per year . definitive control of severe pelvic bleeding from any cause often requires aggressive measures such as arterial embolization , laparotomy , internal fixation , or pelvic packing . these procedures are difficult to perform because they require immediate access to sophisticated medical and surgical expertise and equipment . blood volume must be maintained by transfusion of red blood cells , platelets , and clotting factors . this could provide time to mobilize more sophisticated resources and might decrease transfusion requirements and the need for aggressive therapy . pneumatic anti - shock garments ( pasg ) , also called medical or military anti - shock trousers ( mast ) , have been shown to greatly decrease pelvic blood flow [ 2 , 3 ] , and case reports imply that they are effective in patients with pelvic injuries and postpartum hemorrhage . however , most of the patients enrolled in clinical trials had thoracic or abdominal trauma and had ready access to definitive surgery . they only autotransfuse about 250 cc of blood from the lower body into the central circulation and have little or no effect on cardiac output . these findings would explain differing outcomes for upper body versus pelvic hemorrhage . in their favor pasg unfortunately pasg are complicated , expensive , and rarely available in the low resource areas where they would seem most useful . a simple and less expensive alternative to pasg this non - pneumatic anti - shock garment ( nasg ) is made of neoprene and is tightly applied to the patient using velcro straps . it has been commercialized as the zoex non - inflatable anti - shock garment ( zoex , ashland , or , usa ) . the nasg has been shown to decrease postpartum blood loss by 50% [ 8 , 9 ] and improve survival rates from postpartum hemorrhage . we measured pelvic blood flow in volunteers before and after being placed in the nasg and pasg . subjects ranged in age from 21 to 60 years of age and weighed 5282 kg . an acuson sequoia 512 ultrasound and 4vi probe operating at 23 mhz ( siemens , mountain view , ca , usa ) was used to measure heart rate , apparent artery diameter , doppler - to - vessel angle , and blood flow velocity at the abdominal aorta below the superior mesenteric artery ( sma ) . measurements were obtained from each subject at baseline and immediately after being placed in each device . the pasg was made by wrapping individual folded hospital sheets around each leg and the abdomen / pelvis . after the first wrap a bicycle tube was placed on each sheet and the sheet was further wrapped around the patient and tube . a second set of three sheets was then wrapped firmly around the first set being careful to allow the tube stem but no other part to protrude . the tubes were then inflated with a bicycle pump to 2.83.5 bar ( 4050 psi ) . the ultrasonographer has 20 years experience and is certified by the american registry for diagnostic medical sonography ( rockville , md , usa ) as both a registered diagnostic medical sonographer ( rdms ) and a registered vascular technician ( rvt ) . data were analyzed with one sample and paired t tests using two - tailed p values . mean blood flow was 1.99 l / min at baseline with a range of 0.393.05 l / min . mean flow decrease was 1.11 l / min or 56% [ 95% confidence interval ( ci ) : 0.641.57 , p = 0.0003 for the difference ] for the pasg and 0.65 l / min or 33% ( 95% ci : 0.031.26 , p = 0.04 ) for the nasg . the pasg decreased blood flow more than the nasg ( mean difference : 0.46 , 95% ci : 0.020.90 , p = 0.04 ) . our results are compatible with the medical literature regarding the regional hemodynamic effects of circumferential abdominal pelvic pressure using pasg [ 2 , 3 ] . commercial pasg are quite rigid and their air bladders are considerably larger than bicycle tubes . a previous study showed that when inflated to two thirds of their maximum , commercial pasg decrease blood flow by approximately one half , which is similar to the magnitude of decrease we demonstrated with our improvised pasg . fully inflated commercial pasg decrease distal aortic flow by about three quarters [ 2 , 3 ] . pelvic hemorrhage and particularly postpartum hemorrhage bleeding can be arterial from branches of the internal iliac , pelvic , ovarian , and middle sacral arteries . bleeding will stop when externally applied pressure exceeds the perfusion pressure of the injured vessel . low pressure venous bleeding theoretically requires only a few millibars , but this must be at the actual bleeding sites . this is impossible to apply externally and would in addition completely eliminate tissue perfusion , causing undesirable side effects . it provides indirect pressure on the pelvic organs that exceeds venous and capillary filling pressure . this directly decreases arterial bleeding and hence should give normal hemostatic mechanisms more opportunity to act . it is unfortunate that the term anti - shock became incorporated into the names of these devices because they have essentially no utility in reversing shock in general or in preventing it from injuries that are above the pelvis . however , emergency care and childbirth providers need a means of slowing or stopping pelvic hemorrhage . this is particularly true in low resource environments or when patients present late or to facilities that lack interventional radiology , immediate surgical capability , and safe blood . delays in gaining access to definitive care are even more serious if patients suffer from preexisting anemia or have little reserve . although we did not do any formal testing of device acceptability , we did ask all of the subjects if they wanted the trial terminated because the devices were too uncomfortable and none did . one subject had participated in a previous pasg study and spontaneously volunteered that all three devices felt about the same which is reassuring given the long clinical experience with commercial pasg hypovolemia ordinarily causes peripheral vasoconstriction which would lead to less blood flow at the distal aorta it would be unethical and impractical to perform an initial trial on ill and bleeding subjects . our within - subject experimental design specifically compensates for differing amounts of pelvic blood flow between subjects . although the amount of blood flow in the distal aorta might be different , the effect of mechanical pressure and hence the comparison between devices would be the same . although the improvised device can be made locally , the nasg is available in a manufactured version which may make it more suitable in places that can afford the initial investment . the pasg decreased blood flow by a larger margin than the nasg , but this does not prove that it is clinically either equivalent or superior . it should be noted that the optimal pressure to treat pelvic and postpartum bleeding is not known but that the pressure in each device can be changed by adding or removing air from the tubes or adjusting the tension on the straps of the nasg . currently available reports of nasg use in obstetric hemorrhage have not noted problems with compartment syndrome , but the number of subjects studied is small .

the relationship between stress challenges and adverse health outcomes , particularly for the development of affective disorders , is now well established . the highly conserved neuroimmune mechanisms through which responses to stressors are transcribed into effects on males and females have recently garnered much attention from researchers and clinicians alike . the use of animal models , from mice to guinea pigs to primates , has greatly increased our understanding of these mechanisms on the molecular , cellular , and behavioral levels , and research in humans has identified particular brain regions and connections of interest , as well as associations between stress - induced inflammation and psychiatric disorders . this review brings together findings from multiple species in order to better understand how the mechanisms of the neuroimmune response to stress contribute to stress - related psychopathologies , such as major depressive disorder , schizophrenia , and bipolar disorder . backgroundpelvic bleeding from trauma and postpartum hemorrhage is often difficult to treat successfully by emergency providers particularly in low resource environments , when hospital presentation is delayed or there is a lack of immediate surgical , anesthesia , and transfusion capabilities . pneumatic anti - shock garments ( pasg ) decrease pelvic blood flow and hemorrhage . a tightly fitted neoprene non - pneumatic anti - shock garment ( nasg ) has been shown to decrease blood loss and improve survival rates from postpartum hemorrhage.aimsthe objective of this study was to determine whether blood flow to the pelvis is decreased by use of the nasg or by an improvised pasg.methodsa pasg was made using three bicycle tubes , placing one tube on each leg and one on the lower abdomen / pelvis , wrapping firmly with sheets and inflating the tubes to approximately 3.5 bar ( 45 psi ) . a doppler ultrasound was used to measure distal aortic blood flow in 12 healthy adults at baseline and in both devices . data were analyzed with one sample and paired t tests.resultsmean flow was 1.99 l / min at baseline . mean flow decrease was 1.11 [ 95% confidence interval ( ci ) : 0.641.57 , p = 0.0003 for the difference ] for the pasg and 0.65 ( 95% ci : 0.031.26 , p = 0.04 ) for the nasg . the pasg decreased blood flow more than the nasg ( mean difference : 0.46 , 95% ci : 0.020.90 , p = 0.04).conclusionsboth devices decreased distal aortic blood flow , but the improvised pasg device decreased it by a larger margin .

cell culture performed as previously described . briefly , k562 cells ( atcc ) were cultured in rpmi 1640 ( gibco ) media and supplemented with 10% fbs ( hyclone ) , penicillin ( 10,000 i.u./ml ) , streptomycin ( 10,000 ug / ml ) , and l - glutamine ( 2 mm ) . cells were grown in log phase during all biological assays by returning the population to 500,000 cells / ml each day . k562 cells were maintained in a controlled humidified incubator at 37 c , with 5% co2 . briefly , to generate sufficient lentivirus to infect the genome - wide shrna library into k562 cells , we plated 293 t cells on 15 cm tissue culture plates . 293 t cells were transfected with third generation packaging plasmids and shrna encoding vectors . after 48 hrs and 72 hrs of incubation we filtered the pooled lentivirus through a 0.45 m pvdf filter ( millipore ) to remove any cellular debris . approximately 560 million k562 cells were infected with our next - generation genome - wide lentiviral shrna library to maintain roughly 1,000-fold coverage of the shrna library after selection . infected cells grew for 3 days before selecting the cells with puromycin ( 0.7 g / ml , sigma ) . after three days of selection , infection efficiency was monitored using flow cytometry ( bd accuri c6 ) . once the cells reached 90100% mcherry positive cells , they were spun out of selection and allowed to recover in normal rpmi 1640 media . at t0 , 500 million cells were pelleted by centrifugation ( 300 g for 5 min ) . cells were then split into two populations and maintained at logarithmic growth ( 500,000 cells / ml ) each day for 14 days . after 14 days of growth genomic dna was extracted for all 3 time points separately following qiagen s blood maxi kit protocol . a previously designed 4 sgrna / gene crispr - cas9 library was used targeting 5 ends of conserved exons with sgrnas varying in length between 19 and 25 base - pairs . the library was generated first by infecting k562 cells with a sffv - cas9-bfp vector to create a stably expressed cas9 cell line . we then infected the lentiviral genome - wide sgrna library into approximately 120 million cells following the same protocol as the genome - wide shrna library to maintain at least 1,000-fold representation in cells . infected cells were selected with puromycin ( 0.7 g / ml , sigma ) for 3 days . percentage of mcherry positive cells was measured by flow cytometry ( bd accuri c6 ) . selected cells were spun out of selection and into normal rpmi 1640 media . at t0 cells were then split into two populations and grown for 14 days , maintaining logarithmic growth ( 500,000 cells / ml ) each day . after 14 days of growth , cells were pelleted by centrifugation , and genomic dna was extracted for all three time samples separately following qiagen s blood maxi kit instructions . data from previous shrna screen for modifiers of ricin toxicity were obtained as pre- computed hairpin - level enrichments averaged from two replicates , pooled data was analyzed with castle , and compared to signed , log - transformed published results ( supplementary data 5 ) . count data for two replicates from a previous crispr / cas9 cutting screen for lps - induced tnf expression were obtained from the authors , analyzed with castle , and compared to signed , log - transformed published deseq results ( supplementary data 6 ) . data from previous crispri and crispra screens were obtained as pre - computed guide - level enrichment averaged from two replicates , analyzed with castle , and compared to published results ( supplementary data 7 ) . where available , positive and negative results from published low - throughput validations are also presented . trimmed sequences were aligned to the library using bowtie with zero mismatches tolerated . all alignment from multi - mapped reads were used . enrichment of individual hairpins was calculated as a median - normalized log - ratio of fraction of counts , as previously described . raw count files are available ( supplementary data 2 ) , and raw fastq files have been deposited at the sequence read archive ( accession srp072806 ) and bioproject ( accession prjna317269 ; http://www.ncbi.nlm.nih.gov/bioproject/prjna317269 ) . briefly , we built cas9 high throughput maximum likelihood estimator ( castle ) that uses an empirical bayesian framework to account for multiple sources of variability , including reagent efficacy and off - target effects ( for more complete description , see supplementary methods ) . for each gene , we have the phenotypes of multiple targeting reagents . from this , and the phenotypes of negative controls , we obtain an effect size estimate for each gene and an associated log - likelihood ratio . in the figures we present this as the castle score , which is twice the log - likelihood ratio , signed to match the effect size . all screens are analyzed with the same parameters , and no optimization was performed using the gold standard or related sets . these , along with a complete screen analysis pipeline , are available at https://bitbucket.org/dmorgens/castle . for comparison , riger was implemented with default settings on precomputed element enrichments with gene - e ( http://www.broadinstitute.org/cancer/software/gene-e/ ) . rsa was implemented with python scripts available from http://carrier.gnf.org / publications / rsa/. mageck was implemented with default settings with software available from https://sourceforge.net / projects / mageck/. hitselect was implemented with default settings with software available from https://sourceforge.net / projects / hitselect/. median and highest effect heuristics as well as the mw test were implemented with custom python scripts . go terms were generated using gorilla available at http://cbl-gorilla.cs.technion.ac.il/ by ranking genes from highest confidence negative growth phenotypes to lowest confidence genes to highest confidence positive growth phenotypes . for direct comparison to the 4 sgrna per gene crispr / cas9 library , the 25 hairpin per gene library rnai library was down - sampled to 4 shrnas per gene . hairpins were ranked according to their original computational design and the top four unique shrnas were used as well as all negative control shrnas . note that this is independent of the data set used here and represents the 4 shrna library that would have been designed . essential gene predictions were then repeated using this reduced shrna library as above ( supplementary fig . gene sets were defined by 10% fpr cutoff for cas9 , shrna , and combination screens . cas9 and shrna unique gene sets were defined as genes under the 10% fpr cutoff in the cas9 or shrna screen but neither the other nor the combination screen . an overlap set was defined as genes under the 10% fpr cutoff for both cas9 and shrna screens . public rna - seq data ( accession encff934ybo ) for the k562 cell line was obtained from encode experiment encsr000aem . genes were filtered for fpkm greater than one and successful mapping to genes present in our libraries , leaving expression data for ~7,000 genes . genes were then ranked from highest to lowest expressed and binned in increments of 500 . the number of genes in each bin were counted for each gene set and normalized by the total number of genes in each gene set present in the rna - seq data . was then graphed versus the mean log(fpkm ) value for each bin ( supplementary figure 11a , b ; supplementary data 10 ) . yeast essential genes annotated as viable or inviable as well as their human homologs were obtained from the saccharomyces genome database . the number of homologs in each gene set were counted for both inviable and viable annotations , and the fraction inviable is presented ( supplementary figure 11c , d ; supplementary data 11 ) . p - values are calculated using fisher s exact test . the total fraction of all homologs mapped from inviable yeast genes is also presented . cell culture performed as previously described . briefly , k562 cells ( atcc ) were cultured in rpmi 1640 ( gibco ) media and supplemented with 10% fbs ( hyclone ) , penicillin ( 10,000 i.u./ml ) , streptomycin ( 10,000 ug / ml ) , and l - glutamine ( 2 mm ) . cells were grown in log phase during all biological assays by returning the population to 500,000 cells / ml each day . k562 cells were maintained in a controlled humidified incubator at 37 c , with 5% co2 . briefly , to generate sufficient lentivirus to infect the genome - wide shrna library into k562 cells , we plated 293 t cells on 15 cm tissue culture plates . 293 t cells were transfected with third generation packaging plasmids and shrna encoding vectors . after 48 hrs and 72 hrs of incubation , lentivirus was harvested . we filtered the pooled lentivirus through a 0.45 m pvdf filter ( millipore ) to remove any cellular debris . approximately 560 million k562 cells were infected with our next - generation genome - wide lentiviral shrna library to maintain roughly 1,000-fold coverage of the shrna library after selection . infected cells grew for 3 days before selecting the cells with puromycin ( 0.7 g / ml , sigma ) . after three days of selection , infection efficiency was monitored using flow cytometry ( bd accuri c6 ) . once the cells reached 90100% mcherry positive cells , they were spun out of selection and allowed to recover in normal rpmi 1640 media . at t0 , 500 million cells were pelleted by centrifugation ( 300 g for 5 min ) . cells were then split into two populations and maintained at logarithmic growth ( 500,000 cells / ml ) each day for 14 days . after 14 days of growth genomic dna was extracted for all 3 time points separately following qiagen s blood maxi kit protocol . a previously designed 4 sgrna / gene crispr - cas9 library was used targeting 5 ends of conserved exons with sgrnas varying in length between 19 and 25 base - pairs . the library was generated first by infecting k562 cells with a sffv - cas9-bfp vector to create a stably expressed cas9 cell line . we then infected the lentiviral genome - wide sgrna library into approximately 120 million cells following the same protocol as the genome - wide shrna library to maintain at least 1,000-fold representation in cells . infected cells were selected with puromycin ( 0.7 g / ml , sigma ) for 3 days . percentage of mcherry positive cells was measured by flow cytometry ( bd accuri c6 ) . selected cells were spun out of selection and into normal rpmi 1640 media . at t0 cells were then split into two populations and grown for 14 days , maintaining logarithmic growth ( 500,000 cells / ml ) each day . after 14 days of growth , cells were pelleted by centrifugation , and genomic dna was extracted for all three time samples separately following qiagen s blood maxi kit instructions . data from previous shrna screen for modifiers of ricin toxicity were obtained as pre- computed hairpin - level enrichments averaged from two replicates , pooled data was analyzed with castle , and compared to signed , log - transformed published results ( supplementary data 5 ) . count data for two replicates from a previous crispr / cas9 cutting screen for lps - induced tnf expression were obtained from the authors , analyzed with castle , and compared to signed , log - transformed published deseq results ( supplementary data 6 ) . data from previous crispri and crispra screens were obtained as pre - computed guide - level enrichment averaged from two replicates , analyzed with castle , and compared to published results ( supplementary data 7 ) . where available , positive and negative results from published low - throughput validations are also presented . trimmed sequences were aligned to the library using bowtie with zero mismatches tolerated . all alignment from multi - mapped reads were used . enrichment of individual hairpins was calculated as a median - normalized log - ratio of fraction of counts , as previously described . raw count files are available ( supplementary data 2 ) , and raw fastq files have been deposited at the sequence read archive ( accession srp072806 ) and bioproject ( accession prjna317269 ; http://www.ncbi.nlm.nih.gov/bioproject/prjna317269 ) . briefly , we built cas9 high throughput maximum likelihood estimator ( castle ) that uses an empirical bayesian framework to account for multiple sources of variability , including reagent efficacy and off - target effects ( for more complete description , see supplementary methods ) . for each gene , we have the phenotypes of multiple targeting reagents . from this , and the phenotypes of negative controls , we obtain an effect size estimate for each gene and an associated log - likelihood ratio . in the figures we present this as the castle score , which is twice the log - likelihood ratio , signed to match the effect size . all screens are analyzed with the same parameters , and no optimization was performed using the gold standard or related sets . these , along with a complete screen analysis pipeline , are available at https://bitbucket.org/dmorgens/castle . for comparison , riger was implemented with default settings on precomputed element enrichments with gene - e ( http://www.broadinstitute.org/cancer/software/gene-e/ ) . rsa was implemented with python scripts available from http://carrier.gnf.org / publications / rsa/. mageck was implemented with default settings with software available from https://sourceforge.net / projects / mageck/. hitselect was implemented with default settings with software available from https://sourceforge.net / projects / hitselect/. median and highest effect heuristics as well as the mw test were implemented with custom python scripts . go terms were generated using gorilla available at http://cbl-gorilla.cs.technion.ac.il/ by ranking genes from highest confidence negative growth phenotypes to lowest confidence genes to highest confidence positive growth phenotypes . for direct comparison to the 4 sgrna per gene crispr / cas9 library , the 25 hairpin per gene library rnai library was down - sampled to 4 shrnas per gene . hairpins were ranked according to their original computational design and the top four unique shrnas were used as well as all negative control shrnas . note that this is independent of the data set used here and represents the 4 shrna library that would have been designed . essential gene predictions were then repeated using this reduced shrna library as above ( supplementary fig . gene sets were defined by 10% fpr cutoff for cas9 , shrna , and combination screens . cas9 and shrna unique gene sets were defined as genes under the 10% fpr cutoff in the cas9 or shrna screen but neither the other nor the combination screen . an overlap set was defined as genes under the 10% fpr cutoff for both cas9 and shrna screens . public rna - seq data ( accession encff934ybo ) for the k562 cell line was obtained from encode experiment encsr000aem . genes were filtered for fpkm greater than one and successful mapping to genes present in our libraries , leaving expression data for ~7,000 genes . genes were then ranked from highest to lowest expressed and binned in increments of 500 . the number of genes in each bin were counted for each gene set and normalized by the total number of genes in each gene set present in the rna - seq data . this fraction of essential genes for each gene set was then graphed versus the mean log(fpkm ) value for each bin ( supplementary figure 11a , b ; supplementary data 10 ) . yeast essential genes annotated as viable or inviable as well as their human homologs were obtained from the saccharomyces genome database . the number of homologs in each gene set were counted for both inviable and viable annotations , and the fraction inviable is presented ( supplementary figure 11c , d ; supplementary data 11 ) . p - values are calculated using fisher s exact test . the total fraction of all homologs mapped from inviable yeast genes is also presented . distribution of targeting and control elements . ( a ) distribution of negative controls for a single replicate of cas9 and shrna screens . ( b , c ) distribution of targeting elements is shown in meta - gene plots for the top 50 ( b ) enriched and ( c ) disenriched genes found in a single replicate of the cas9 and shrna screens as identified by castle . to normalize , the enrichment of each individual element was divided by the effect size estimate for the gene generated by castle . ( a d ) enrichment of targeting elements and estimated effect size is shown for the top four disenriched genes from cas9 data from a single replicate . ( a d ) enrichment of targeting elements and estimated effect size is shown for the top four disenriched genes from shrna data from a single replicate . the unknown relationship between gene dosage and measured phenotype as well as the unknown distribution of shrna and cas9 efficacies restricts the predicted effect size of reagents to a bounded region , marked as the blue shaded region , between 0 and the maximum effect i , marked by the dotted line . some fraction ( 1 ) of the reagents the phenotype observed is thus the true effect obscured by noise , which is estimated using the distribution of non - targeting controls . the likelihood of models for different values of i and are calculated and by marginalizing the most likely effect size is selected . a likelihood ratio is then calculated by comparing to a null model where i is zero . ( a ) results are shown for a previously published shrna screen for ricin sensitivity reanalyzed with castle and compared to published results based on a mw test . ( b ) previous crispr / cas9 deletion screen for lps - induced tnf expression in primary mouse bone - marrow derived dendritic cells , analyzed with castle and the published deseq results . ( c ) previous crispri screen for sensitivity to the fusion toxin ctx - dta , analyzed with castle versus the average of the top three sgrna effects . ( d ) previous crispra screen for sensitivity to the fusion toxin ctx - dta , analyzed with castle versus the average of the top three sgrna effects . ( a d ) roc curves indicate screen performance in identifying essential genes from changing composition between the plasmid library and two weeks growth . true positive rates and false positive rates are calculated using a previously established gold standard set of essential and nonessential genes . genes are ranked by likelihood to be essential using the indicated methods , including castle . highest effect heuristic was calculated by ranking the genes according to their most disenriched element . data is shown from single replicates of the ( a , c ) cas9 and ( b , d ) shrna screens for ( a , b ) replicate 1 and ( c , d ) replicate 2 . supplementary figure 7 . ( a ) roc curves from combination of different replicates of cas9 and shrna using castle . roc curves indicate screen performance in identifying essential genes from changing composition between the plasmid library and two weeks growth . true positive rates and false positive rates are calculated using a previously established gold standard set of essential and nonessential genes . a large positive castle score indicates a high confidence increase in growth rate , while a highly negative castle indicates a high confidence decrease in growth rate , i.e. gene essentiality . the graphs compare replicate measurements of likelihood ratio between plasmid and t14 of the combination score based on replicates 1 for cas9 and shrna and replicates 2 for cas9 and shrna . ( a ) roc curves indicate screen performance in identifying essential genes by comparing the library composition between the plasmid library and cells after two weeks growth . roc curves for cas9 ( red ) and shrna ( blue ) screens based on duplicate data combined using castle . alternatively , data from single replicates of both cas9 and shrna screens were combined using castle ( purple ) . ( b ) the number of essential genes at 10% false positive rate and their overlap based on the duplicate data from cas9 and shrna screens , as well as combination of a single replicate from both screens . ( c ) precision recall curve for cas9 , shrna , and combination data using castle . results from the 25 shrna library were downsampled by only including four hairpins per gene , selected by previous computational ranking . ( a ) roc curves indicate screen performance in identifying essential genes by comparing the library composition between the plasmid library and cells after two weeks growth . ( b ) the number of essential genes at 10% false positive rate and their overlap based on the duplicate data from cas9 and shrna screens , as well as combination of a single replicate from both screens . ( c ) comparison of castle scores derived from castle between single replicates of cas9 and shrna data . ( d ) adjusted p - values for select go terms for shrna and cas9 screens as well as for data from both screens combined with castle . supplementary figure 10 . a large positive castle score indicates a high confidence increase in growth rate , while a highly negative castle score indicates a high confidence decrease in growth rate , i.e. gene essentiality . the graphs compare replicate measurements of castle scores between plasmid and t14 for ( a ) cas9 and ( b ) shrna screens . castle scores in different time - frames for ( c ) cas9 and ( d ) shrna screens . genesets are defined for cas9 , shrna , and combination by a 10% fpr cutoff . genesets are defined for cas9-combo and shrna - combo by the genes present in cas9 or shrna set and not in the combination set . overlap set is defined as genes present in both the cas9 and shrna set ( see supplementary fig . ( a , b ) ~7,000 genes with detectable expression in k562 were binned by expression . the fraction of genes identified as essential in each bin is reported versus the average expression level of the bin . ( c , d ) fraction of genes that are homologs of essential yeast genes versus genes that are homologs of nonessential yeast genes . clinical presentations vary from nonspecific systemic symptoms ( e.g. , fever , myalgia , palpitation , chest pain , dyspnea ) to fulminant hemodynamic collapse and sudden death . infections ( viral , bacterial , fungal , protozoal , parasitic ) , toxins , immunological syndromes , and hypersensitivity can cause myocarditis . among these lewin et al . reported that seven explanted hearts from 288 heart transplants ( 2.4% ) had histological evidence of hypersensitivity myocarditis . unlike other forms of myocarditis , the prognosis of hypersensitivity myocarditis is excellent , usually improving after administration of the causative drug is ceased . , we report a case of hypersensitivity myocarditis , as confirmed by cardiac magnetic resonance imaging ( cmr ) and endomyocardial biopsy ( emb ) , which subsequently improved after cessation of the causative drug and administration of prednisolone . she had a history of hypertension , transient cerebral ischemic attack , and osteoarthritis in both knees . her blood pressure was 91/53 mmhg , pulse rate was 63 beats per minute , respiration rate was 20 breaths per minute , and body temperature was 37. upon physical examination , heart sounds were normal without murmur . multiple itchy erythematous lesions were present on the hands , buttocks , abdomen , and trunk ( fig . the results of laboratory examinations were as follows : white blood cell count , 9,140/mm ; eosinophil count , 1,380/mm ; hemoglobin , 10.3 g / dl ; platelet count , 123,000/mm ; c - reactive protein level , 1.7 mg / dl ; and erythrocyte sedimentation rate , 72 mm / sec . the levels of brain natriuretic peptide , creatine kinase - mb ( ck - mb ) , and troponin - i were 944 pg / ml , 56.73 ng / ml , and 38.85 ng / ml , respectively . initial electrocardiography showed st segment elevation 1 mm in leads ii , iii , avf , v5 , and v6 ( fig . emergency coronary angiography was performed under the assumption of acute st segment elevation myocardial infarction at the inferolateral wall , but coronary atherosclerosis was not evident . echocardiography showed the " sparkling " appearance of the left ventricular ( lv ) myocardium , concentric lv hypertrophy , and mild hypokinesia on the anterior , anterolateral , and posterior walls from the mid - lv to the apex with 50% lv ejection fraction . peripheral blood eosinophilia , skin rash , and a history of taking multiple medications suggested hypersensitivity myocarditis . 3 . management of heart failure using aldosterone and furosemide was initiated promptly . on the second day of hospital admission , however , dyspnea worsened , and newly developed bilateral pulmonary congestion was noted on chest radiography . therefore , cmr and emb were carried out to confirm the diagnosis of hypersensitivity myocarditis . contrast - enhanced cmr ( ce - cmr ) showed high signal intensities in circular and nodular patterns mainly in the epicardium and septum of the left ventricle ( fig . these patterns suggested infiltrative myocarditis , such as amyloidosis , sarcoidosis , or eosinophilic myocarditis . emb obtained from the septum of the right ventricle revealed lymphocytic and eosinophilic infiltration in the interstitium and perivascular area , findings that were consistent with a diagnosis of hypersensitivity myocarditis ( fig . 5 ) . levels of ck - mb and troponin - i were normalized on days 6 and 9 , respectively . the peripheral eosinophil count decreased from 1,380/mm to 620/mm on day 2 and was normalized ( 50/mm ) on day 4 . the skin rash disappeared almost completely after use of 5 mg prednisolone , and the patient was discharged from the hospital on day 12 . echocardiography at the 6-month follow - up showed normalized regional wall motion abnormalities and improved lv systolic function from 50% to 70% of the ejection fraction . at the 6-month follow - up , ce - cmr showed that previously high signal intensities in circular patterns in the epicardium of the left ventricle had disappeared and the oval - shaped area of high signal intensity in the lv septum had decreased in size ( fig . eosinophilic myocarditis can be classified according to cause and includes those types associated with systemic disease ( e.g. , hypereosinophilic syndrome , churg - strauss syndrome , malignancies ) , parasitic infections ( e.g. , toxocara canis ) , and drugs or vaccines ( hypersensitivity myocarditis ) . eosinophilic myocarditis associated with hypereosinophilic syndrome is usually underpinned by a history of peripheral eosinophilia ( > 1.5 10/l ) lasting > 6 months and is related to systemic involvement ( e.g. , central nervous system , gastrointestinal tract , heart , skin , or lung ) . in the present case , hypereosinophilic syndrome and parasitic infection were excluded because the patient did not have a history of prolonged eosinophilia , other organ involvement , or ingestion of raw meat . instead , she had a history of taking multiple medications , urticaria , and peripheral eosinophilia drug - related myocarditis can be classified into five types : 1 ) hypersensitivity myocarditis ; 2 ) toxic myocarditis ; 3 ) endocardial fibrosis ( e.g. , ergotamine tartrate , methysergide , phentermine , fenfluramine ) ; 4 ) drug - induced cardiomyopathy ( e.g. , anthracycline , chloroquine ) ; and 5 ) giant cell myocarditis . numerous medications , including clozapine , sulfonamide , penicillin antibiotics , methyldopa , and some antiseizure drugs , have been reported to be associated with hypersensitivity myocarditis . delayed hypersensitivity is the main pathogenesis and occurs from several days to months after administration of the causative agent . hypersensitivity myocarditis is not easy to recognize because the clinical features suggestive of drug hypersensitivity reactions ( e.g. , urticaria , angioedema , malaise , fever , eosinophilia ) do not occur uniformly . however , if tissue necrosis or deterioration of clinical status is observed , corticosteroid and cytotoxic agents can be used . emb is the gold standard tool in the diagnosis of myocarditis . according to the dallas criteria , eosinophils may be a minor component of postviral and giant cell myocarditis but are predominant in hypersensitivity and parasitic myocarditis as well as hypereosinophilic syndrome . lymphocytic and eosinophilic infiltrates with slight myocyte necrosis were observed in our case , which coincided with hypersensitivity myocarditis . myocarditis is often a focal process , so sampling error may occur and increase the false - negative rate . a previous study of endomyocardial samples obtained from the hearts of patients who died of myocarditis performed at the mayo clinic reported a false - negative rate of 37% for the right ventricle . other limitations of emb include the aggressiveness of the procedure as well as the delay until confirmation of the result . the lake louise criteria were found to have a sensitivity of 67% , specificity of 91% , accuracy of 78% , positive predictive value of 91% , and negative predictive value of 69% , when the criteria were compared with clinical or histopathological data . mahrholdt and sechtem reported recently that cmr - guided biopsy improved the sensitivity of emb . thus , the combined application of cmr and emb may yield diagnostic synergy and overcome some of the limitations of cmr or emb when applied individually . in our case , we performed cmr - guided emb on the right ventricular septum , in which we detected regional high signal intensity on late gadolinium enhancement . in conclusion , careful history taking ( especially with regard to drugs ) , thorough physical examination , and clinical suspicion can help in recognizing hypersensitivity myocarditis both accurately and promptly . cmr - guided emb is a valuable tool for the early and appropriate diagnosis for hypersensitivity myocarditis . our patient recovered completely after cessation of administration of the causative drug and short - term administration of prednisolone for rapidly worsening hypersensitivity myocarditis .

we compare the ability of shrna and crispr / cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line k562 . we find that the precision of the two libraries in detecting essential genes is similar and that combining data from both screens improves performance . notably , results from the two screens show little correlation , which can be partially explained by identification of distinct essential biological processes with each technology . myocarditis often occurs due to viral infections and postviral immune - mediated responses . hypersensitivity myocarditis is a rare form of myocarditis . numerous drugs can induce myocarditis , which is typically reversible after withdrawal of the causative agent . here , we report a case of hypersensitivity myocarditis that was probably triggered by amoxicillin and that resolved completely with heart failure management as well as discontinuation of the drug . a 68-year - old woman presented with acute chest pain mimicking acute coronary syndromes , but the coronary angiography was normal . a recent history of taking medications , skin rash , and peripheral eosinophilia suggested a diagnosis of hypersensitivity myocarditis , which was confirmed by cardiac magnetic resonance imaging and endomyocardial biopsy .

liver cancer is the second most common cause of cancer - related death worldwide , the fifth most common cancer among men and the ninth most common cancer among women.1 hepatocellular carcinoma ( hcc ) is responsible for ~90% of primary liver cancers.2 in the united states , hcc is the fastest rising cause of cancer - related death , and its incidence has nearly tripled since the 1980s.3,4 despite increased focus and research , the overall prognosis for hcc remains poor . curative modalities , such as liver transplant , resection and radiofrequency ablation , do exist when hcc is diagnosed early ; however , only ~15% of patients are eligible for such treatments ( table 1).3,5,6 the majority of patients are diagnosed with advanced - stage hcc , for which the median survival time is < 50% at 1 year and only 12% at 5 years.7,8 chemotherapy is rarely used for the treatment of hcc due to its minimal success rate of 1020% and high levels of toxicity.2 the only therapy currently approved by the food and drug administration ( fda ) for the treatment of advanced hcc is sorafenib , a tyrosine kinase inhibitor that has been shown to decrease cell proliferation and tumor angiogenesis.9,10 while sorafenib represents a major advancement in the treatment of hcc , its effects are modest , with the sharp trial demonstrating an increase in overall survival from 7.9 months to 10.7 months and an increase from 2.8 months to 5.5 months in the median time to radiological progression.10 it demonstrated even less robust benefits in the asia - pacific trial , with overall survival in the treatment group of 6.5 months and median time to radiological progression of 2.8 months.9 furthermore , after experiencing an initial response to treatment , most hcc patients develop a decrease in efficacy with sorafenib.11 many novel therapies are currently under investigation in clinical trials . in a recently published phase iii trial , regorafenib , an oral multikinase inhibitor , has demonstrated an increase in median overall survival ( 10.6 months vs. 7.8 months ) and an increase in median progression - free survival ( pfs ; 3.1 months vs. 1.5 months ) compared to placebo in patients with advanced hcc who have progressed on sorafenib.12 to date , however , there are no fda - approved second - line or salvage therapies for those patients who progress with or are intolerant to sorafenib.13 one of the most promising new therapies under investigation is tivantinib ( arq 197 ) , which has demonstrated positive results in early - phase clinical trials as a second - line agent for multiple solid tumors . tivantinib is an oral , highly selective , non - atp competitive inhibitor of c - met receptor tyrosine kinase.14 the met proto - oncogene was initially discovered in human osteosarcoma , and it encodes for cellular mesenchymal epithelial transition factor ( c - met).15 c - met and hepatocyte growth factor ( hgf ) , its only known ligand , are necessary for normal embryonic development and tissue repair ; however , their expression is normally very low and only found in cells of epithelial or mesenchymal origin.1618 when hgf binds to c - met , there is activation of multiple metabolic pathways , including the mitogen - activated protein kinase , protein kinase b , mammalian target of rapamycin , phophatidylinositol 3-kinase and focal adhesion kinase , which can lead to tumor growth , proliferation , cell invasion and distant metastasis ( figure 1).1921 upregulation of the c - met pathway is found in multiple cancers , including hcc , where it is present 2040% of the time and is associated with increased risk for metastases and poor prognosis.22,23 hgf is also over - expressed in hcc , which results in increased hepatic regeneration and suppression of hepatocyte apoptosis.24,25 similarly , livers with hcc have been shown to have high levels of mitogen - activated protein kinase , which can go on to activate the raf / mek / erk signaling pathway , leading to unopposed cellular proliferation , migration and survival.25,26 the inhibition of c - met by tivantinib disrupts the deregulated signaling cascade , promotes apoptosis and inhibits cellular growth in those cells that express c - met . munshi et al14 demonstrated the selectivity of tivantinib by showing that tivantinib inhibited only four of 229 human kinases with a ki for c - met of 355 nmol / l . nmol / l is less than that of other available c - met inhibitors , cellular assays demonstrated similar biochemical potency . tivantinib targets the inactive , unphosphorylated form of c - met , locking it in the inactive configuration and preventing downstream signaling.27 controversy does exist regarding the mechanism of action of tivantinib and whether c - met inhibition is the major anti - tumor effect of the drug . several studies have shown that tivantinib possesses cytotoxic activity , even in cells that lack c - met.2832 other proposed mechanisms include microtubule , glycogen synthase kinase 3 , cyclin b1 and proteasome inhibition . microtubule inhibition is the most widely theorized alternative mechanism of action , with three independent in vitro studies corroborating this hypothesis.2931 significant challenges to this alternative hypothesis have been raised . notably , in vivo studies have not documented evidence of neurotoxicity , which is known to be a major adverse effect of microtubule inhibitors.23 similarly , tivantinib has demonstrated only minimal , if any , response in patients who have tumors with low levels of met expression , while those tumors with high levels of met have had the most profound responses to the drug.33,34 from a pharmacokinetic standpoint , tivantinib has an oral bioavailability of > 20% , with a half life of 29 minutes . it is metabolized by the cyp2c19 and cyp3a4 pathways , with 19% elimination via the kidneys and 68% via the stool.35 possible drug interactions could occur with known cyp2c19 inhibitors , such as clopidogrel , esomeprazole , fluconazole , fluoxetine and omeprazole , or with known cyp2c19 inducers , such as carbamazepine , phenytoin and rifampin . maximum inhibition of c - met is obtained after just 24 hours of treatment , and this effect is sustained for 12 hours after the drug is withdrawn.36 three phase i trials have evaluated tivantinib monotherapy in samples without hcc and found tivantinib to be well tolerated with mild clinical benefit.3739 the first of these was conducted as an open - label , sequential 3 + 3 dose escalation design with an initial dose of 100 mg bid at a single center in the united kingdom.37 fifty - one patients with advanced solid organ tumors including prostate , melanoma , gastric , colorectal , sarcoma and breast were included in the trial . the primary objective was to evaluate safety and tolerability and determine the maximum tolerated dose ( mtd ) , establish a recommended phase ii dose ( rp2d ) and define dose - limited toxicities . tivantinib was deemed safe and well tolerated , with an mtd / rp2d of 360 mg bid . stable disease was observed in 14 patients , as defined by response evaluation criteria in solid tumors.40 similarly , a multicenter open - label single - arm dose escalation trial with an initial dose of 10 mg bid was conducted in the united states.38 seventy - nine patients with advanced solid organ tumors , including colorectal , renal , ovarian , pancreatic , sarcoma and thyroid among others , were enrolled . the pfs for all 79 patients was 77% at 6 weeks , 52% at 12 weeks and 34% at 21 weeks . the same concept was evaluated in japan in a multicenter dose - escalating open - label trial with a 3 + 3 design at an initial dose of 70 mg bid.39 forty - seven japanese patients with solid organ tumors were enrolled , although no hcc patients were included in the cohort . it is of interest that these patients were enrolled based on their cyp2c19 polymorphism status . it has been shown that 3080% of asians have a genetic polymorphism of cyp2c19 , making them very poor metabolizers of tivantinib . this same polymorphism , in contrast , is found in only 1219% of whites or blacks.41 tivantinib was well tolerated by both normal and poor metabolizers of cyp2c19 , although a dose adjustment down to 240 mg bid was recommended for poor metabolizers . the first study of tivantinib monotherapy in patients with hcc was a phase ib study conducted by santoro et al.42 three hundred and sixty milligrams of tivantinib was administered twice per day to 21 adult patients with child - pugh a and b cirrhosis and hcc in 28-day treatment cycles . all 21 patients eventually discontinued the treatment , 17 due to disease progression and four due to adverse events . only 16 patients were able to be evaluated for tumor response , and while none of them demonstrated any objective response to therapy , nine did show stable disease ( table 2 ) . the overall median time to disease progression was 3.3 months ( range 1.475.3 months ) among those who were able to be evaluated and 1.8 months ( range 1.65.3 months ) in the intention - to - treat analysis . pharmacokinetic analysis found significant tivantinib accumulation in the plasma of hcc patients compared to the accumulation in other solid tumors , but no correlation was found between tivantinib exposure and adverse events . several years later , a phase i trial using tivantinib monotherapy was performed in a japanese sample with advanced hcc.43 only 28 patients were included in the trial , and though the authors found no complete or partial responses , they did see stable disease in 20 of the 28 patients . multiple phase i studies have been performed using tivantinib as part of a combination therapy with sorafenib , sunitinib , erlotinib and gemcitabine.4447 a preclinical study showed that tivantinib and sorafenib had an additive effect in three hcc cell lines ( jhh-4 , plc / prf/5 and sk - hep-1 ) but did not find any degree of synergy between tivantinib and sunitinib in any of the 87 cancer cell lines tested.48 one of the clinical trials , a phase i trial , assessed the combination of tivantinib and sorafenib in 87 adults with advanced solid organ tumors.44 twenty patients had hcc with child - pugh a ( n=14 ) and b ( n=6 ) cirrhosis . an overall response rate of 11.5% and a 10% response in the hcc population were observed . among the hcc patients , there was one complete response , one partial response and 12 cases of stable disease . interestingly , both the complete response and the partial response were noted in patients who had a high concentration of tumor met , and overall three of four patients with high concentrations of tumor met achieved disease control . the median pfs was 3.5 months ( 95% confidence interval [ 95% ci ] : 311.1 ) , and the disease control rate was 70% . patients with hcc who had received prior treatment with vascular endothelial growth factor ( vegf ) therapy , sorafenib and/or sunitinib had a longer pfs than those who did not receive prior vegf therapy ( 15.9 months [ 95% ci : 1.715.9 ] vs. 3.5 months [ 95% ci : 37.4 ] ) . a pooled analysis was conducted by chai et al46 on all phase i studies with tivantinib in hcc and biliary tract cancer patients . overall , 53 patients were treated with tivantinib : 42 were diagnosed with hcc , ten with cholangiocarcinoma and one with gallbladder adenoma . twenty - three patients received tivantinib monotherapy , and 30 received a combination of tivantinib and either sorafenib ( n=20 ) , gemcitabine ( n=8 ) or erlotinib ( n=2 ) . the overall response rate was 6% , but the disease control rate was 62% , with one complete response , two partial responses and 30 cases of stable disease . based on positive results from phase i trials , a phase ii international multicenter double - blind randomized placebo - controlled trial was conducted.34 one hundred and seven adult patients with advanced - stage hcc who had previously progressed or had demonstrated intolerance to one prior systemic therapy ( sorafenib or sunitinib ) were randomized in a 2:1 ratio to receive tivantinib or placebo . the initial dose of tivantinib was 360 mg bid ; however , this was later decreased to 240 mg bid due to a higher than expected incidence of grade 3 or higher neutropenia . in total , 38 patients received tivantinib 360 mg bid , 33 patients received tivantinib 240 mg bid and 36 patients received placebo . the median time to progression ( ttp ) was significantly longer in the tivantinib group ( 1.6 months [ 95% ci : 1.42.8 ] ) compared to that in the placebo group ( 1.4 months [ 95% ci : 1.41.5 ] ) , with a hazard ratio ( hr ) of 0.64 , 90% ci 0.430.94 , p=0.04 . the median pfs in the tivantinib group ( 1.5 months [ 95% ci : 1.42.7 ] ) was longer than that in the placebo group ( 1.4 months [ 95% ci : 1.41.5 ] ) , with this trend approaching significance , hr 0.67 , 95% ci : 0.441.04 , p=0.06 . there was no significant difference in overall survival between the two groups . according to the statistical analysis plan , the study participants were also subdivided based on their tumor met status into high - expression ( met - high ) and low - expression ( met - low ) subgroups . there were 37 patients with met - high tumors , 22 of whom received tivantinib ( ten patients at 360 mg bid and 12 patients at 240 mg bid ) , and 40 patients with met - low tumors , 27 of whom received tivantinib ( 13 patients at 360 mg bid and 14 patients at 240 mg bid ) . patients who had met - high tumors and received tivantinib had a longer median ttp ( 2.7 months vs. 1.4 months [ hr 0.43 , 95% ci : 0.190.97 , p=0.03 ] ) , a longer median pfs ( 2.2 months vs. 1.4 months [ hr 0.45 , 95% ci : 0.210.95 , p=0.02 ] ) and a longer median overall survival ( 7.2 months vs. 3.8 months [ hr 0.38 , 95% ci : 0.180.81 , p=0.01 ] ) compared to placebo . there was no difference in those with met - low tumors who received tivantinib and those who received placebo . based on the significant benefits seen in the met - high group in the above - mentioned phase ii study , two phase iii trials have been initiated to further evaluate the benefit of tivantinib in patients with met - high hcc tumors . the metiv - hcc trial ( nct01755767 ) is a large international randomized double - blind , placebo - controlled trial comparing tivantinib 120 mg bid to placebo in patients with met - high hcc tumors who have failed or progressed with sorafenib.49 the primary end point is overall survival , and secondary end points are pfs and safety . a planned interim analysis was recently completed , and the study will continue to the final analysis as recommended by the data monitoring committee.50 the jet - hcc trial ( nct02029157 ) is a randomized , double - blind , placebo - controlled trial being conducted in japan to evaluate the effect of tivantinib at 120 mg bid on met - high hcc in patients previously treated with one prior course of sorafenib therapy . the primary end point of this study is pfs , and the secondary end point is overall survival . tivantinib dosing for hcc has continued to evolve as more studies have been performed in this patient population . the two initial phase i dosing trials did not include any patients with hcc , and both recommended a tivantinib dose of 360 mg bid.37,38 the three most common adverse events in both trials were fatigue , nausea and vomiting . dose - limiting toxicities observed by yap et al included one case of grade 3 fatigue at 200 mg bid , two cases of febrile neutropenia at 400 mg bid and one incidence of a combination of mucositis , palmar two cases of pancytopenia , vomiting and dehydration at 360 mg bid were seen in the rosen et al trial . the hematologic toxicity associated with tivantinib is expected , as hgf promotes hematopoiesis by stimulating production of cytokines by the stromal cells in the bone marrow.51,52 the santoro et al42 trial was the first to evaluate tivantinib dosing exclusively in hcc patients . based on the results of prior studies , a starting dose of 360 mg bid was selected . overall , 20 of the 21 patients developed a drug - related event , with the most frequent events being neutropenia ( 52% ) , anemia ( 48% ) , asthenia ( 48% ) and leukopenia ( 38% ) . eleven patients ( 52% ) developed adverse reactions that were grade 3 or grade 4 in severity , the most common of which were neutropenia ( 73% ) , anemia ( 45% ) and leukopenia ( 36% ) . in general , neutropenia was the main cause of dose reduction , treatment interruption and treatment discontinuation . these reactions included one case of grade 3 anemia , one case of grade 3 anemia and grade 4 neutropenia , one case of grade 4 leukopenia and grade 4 neutropenia , and one case of grade 4 neutropenia with grade 5 septic shock leading to death . due to interpatient variability , the authors did not find any correlations among adverse events with tivantinib dose , exposure or child - pugh status ; however , they did recommend that tivantinib dosing in hcc patients be reduced to 240 mg bid due to the high amount of hematologic toxicity . a pooled analysis from all phase i / ib and phase ii studies assessed the exposure response of tivantinib in 289 patients , 73 of whom had hcc.53 the pharmacokinetic analysis demonstrated that the clearance of tivantinib in hcc patients was reduced by 67% , which resulted in a threefold increase in exposure compared to other types of cancer . a significant relationship was also found between tivantinib exposure and incidence of grade 2/3 neutropenia . the authors suggested that a starting dose of 240 mg bid of tivantinib rather than 360 mg bid would result in a decrease in the incidence of grade 3 neutropenia from 28% to 16% in hcc patients . similar results were seen in a later phase ii trial by santoro et al,34 in which the treatment dose was reduced from 360 mg bid to 240 mg bid due to a high incidence of grade 3 neutropenia . overall , hematologic toxicities ( neutropenia , anemia and leukopenia ) occurred more frequently in the tivantinib group than in the placebo group , and the incidence of grade 3 neutropenia was higher in the tivantinib 360 mg bid group than either the tivantinib 240 mg bid or the placebo group . there was even a trend toward longer overall survival in the tivantinib 240 mg bid group ( 7.5 months , 95% ci 4.4not available ) compared to the 360 mg bid group ( 6.4 months , 95% ci 49 ) . both the currently active phase iii trials , metiv - hcc and jet - hcc , are evaluating tivantinib at 120 mg bid . the metiv - hcc trial initially started patients on 240 mg bid ; however , the dose was decreased to 120 mg bid in response to a higher than expected incidence of neutropenia.54 however , it should be noted that the metiv - hcc trial used a different formulation of tivantinib than the santoro et al phase ii trial.23 once the dose was decreased to 120 mg bid , a safety analysis showed that a dose of 120 mg bid of the new formulation resulted in an incidence of neutropenia similar to that seen at a dose of 240 mg in the phase ii trial.55 in addition , a pharmacokinetic analysis showed that the plasma exposure of the new formulation at 120 mg bid was comparable to that of the capsule formulation used in the santoro et al trial.55 the jet - hcc trial set its dosing at 120 mg bid based on dosing studies in japanese samples.43 patients with a cyp2c19 polymorphism may require even lower doses of tivantinib . yamamoto et al39 were the first to examine the effect of the cyp2c19 polymorphism in patients taking tivantinib . forty - seven patients were included in the study , 14 of whom were considered poor metabolizers . the study was unable to demonstrate a relationship between dose level and plasma exposure ; however , it was found that the mean exposure of tivantinib in poor metabolizers at 240 mg bid was equivalent to that of normal metabolizers at a dose of 360 mg bid . based on this finding , the authors recommended that those with the cyp2c19 polymorphism receive doses of 240 mg bid , while those with normal cyp2c19 enzymes receive 360 mg bid . a second trial was conducted by okusaka et al43 to specifically look at tivantinib dosing in those with the cyp2c19 polymorphism and hcc . twenty - eight patients were included in the study , seven of whom had the cyp2c19 polymorphism . all patients tolerated tivantinib at a dose of 120 mg bid , but once the dose was increased to 240 mg bid , five of nine normal metabolizers developed dose - limiting toxicities , either neutropenia or febrile neutropenia , which resolved with drug interruption and treatment with granulocyte colony - stimulating factor medications . because normal metabolizers were unable to tolerate a dose of 240 mg bid , those with the cyp2c19 polymorphism were never escalated to a dose higher than 120 mg bid . the authors concluded that japanese patients with hcc were unable to tolerate a dose of tivantinib > 120 mg bid , irrespective of cyp2c19 polymorphism status . this study served as the basis for the dosing regimen in the jet - hcc trial . the future of tivantinib , like that of many other drugs , rests on appropriate patient selection . the identification of biomarkers for tivantinib sensitivity is an active area of research with promising early results . it was published that patients with met - high tumors showed a significant survival benefit , with longer median ttp ( 2.7 months vs. 1.4 months ) , longer median pfs ( 2.2 months vs. 1.4 months ) and longer median overall survival ( 7.2 months vs. 3.8 months ) on tivantinib compared to placebo.34 no difference in efficacy was found between tivantinib and placebo in patients with met - low tumors . in a combination study of tivantinib with sorafenib , the combination therapy demonstrated a trend toward significance regarding increased disease control rate , defined as the combination of complete responses , partial responses and stable disease for at least 8 weeks.44 while only four cases of met - high hcc were observed , three of the four patients achieved disease control . based on these promising results , both of the active phase iii trials are evaluating tivantinib in hcc patients with met - high tumor status . a preclinical study evaluated whether hgf tumor concentration could be used as a marker for hcc , but no significant differences were found between hgf - high and hgf - low groups in clinicopathological factors or patient outcomes.56 a more recent study that specifically evaluated met , hgf and afp as circulating biomarkers ( by 75 percentile ) found that they were prognostic markers for overall survival in patients with hcc.57 circulating met was also a pharmacodynamic biomarker for tivantinib : patients on treatment with a 10% decrease in circulating met levels demonstrated increased overall survival compared to those with a < 10% decrease ( 13.3 months vs. 6.3 months ; hr : 0.46 [ 95% ci : 0.240.86 ] , p=0.01 ) . additionally , tumor met levels were correlated with response to tivantinib treatment , the only biomarker thus far to predict response to treatment . these results support the use of tivantinib exclusively in met - high tumor patients , although more definitive data will be forthcoming with the results of the metiv - hcc trial , which will analyze over 900 tumor samples and validate the use of select biomarkers in hcc . additional studies are needed to assess the use of tivantinib in those with more severe cirrhosis . the vast majority of trials have evaluated tivantinib in patients who are child - pugh class a , with only a few trials including patients who are child - pugh class b. although theoretically tivantinib could also be used for those who are child - pugh class c , this has not yet been studied . moreover , it may not be safe to attempt such a trial given the anticipated side effects . nevertheless , future development of more specific targeted biologic markers may enable treating physicians to administer agents such as tivantinib in a safer and more effective way . tivantinib , a potent inhibitor of c - met , appears to be a promising therapy for those with advanced - stage hcc that has progressed or is intolerant to sorafenib . tivantinib has demonstrated increased overall survival , pfs and ttp compared to placebo in patients with met - high tumors . serious hematologic side effects ( neutropenia , anemia and leukopenia ) are anticipated to be less prominent at lower dosing regimens ( 120 mg bid ) , at which dose good treatment efficacy is still maintained . two large phase iii trials , metiv - hcc and jet - hcc , are currently ongoing . to date , there is no fda - approved salvage or second - line therapy for patients with hcc who have failed or progressed with sorafenib . conidiation in aspergillus involves many common developmental themes including spatial and temporal control of gene expression , specialized differentiation of cells , and intra and intercellular communications . conidiation is regulated by an asexual developmental signaling pathway that directs expression control elements and other genes required for conidiophore assembly . as developmental mechanisms in a. nidulans have been intensively studied and results have provided important clues for understanding conidiation in other aspergilli [ 2 , 4 , 7 ] , what is known in a. nidulans ( ani ) will be described first followed by what has been found in a. fumigatus ( afu ) . in ani , a key and essential step for conidiophore development is the activation of brla encoding a c2h2 zinc finger transcription factor ( tf ) , which induces expression of other genes required for conidiation . loss - of - function brla mutants form structures that resemble conidiophore stalks ( thus named " bristle " ) , except that they grow indeterminately and fail to produce vesicles , metuale , phialides , and conidia , indicating that brla controls the initiation of conidiophore development ( fig . , brla overexpression in vegetative cells causes termination of polar growth coupled with the commencement of abnormal sporulation leading to the formation of viable spores from hyphae . no environmental signals , including nutrient limitations or various ( osmotic and oxidative ) stresses , can bypass the brla requirement for conidiation , indicating that activation of brla expression early in conidiophore development represents a key and essential control point for commencing conidiation . the anibrla ( but not afubrla ) gene is a compound gene consisting of two overlapping transcriptional units designated brlaa and brlab . important information on the complex regulation of the two overlapping brla transcription units is well described elsewhere . afubrla , the afu brla homologue , shows 68% identity and 77% similarity to the ani brla . mah and yu tested whether brla - dependent conidiation is conserved in these two aspergilli using genetics and found that afubrla deletion completely eliminated asexual development in afu , resulting in elongated aerial hyphae and increased hyphal mass in the colonies . these studies clearly demonstrated that afubrla is essential for conidiophore development in afu , and that the role of the core downstream tf brla in conidiation is conserved in these aspergilli ( fig . brla is a putative tf ( activator ) with two c2h2 zinc finger motifs at the c - terminus . disruption of either one of the fingers by mutating the key cysteine to serine results in complete loss of brla activity . chang and timberlake further demonstrated that brla expression in the budding yeast saccharomyces cerevisiae results in brla dependent activation of aspergillus genes . furthermore , they proposed the consensus brla binding sites ( brla response elements [ bres ] ; 5'-(c / a)(g / a)aggg(g / a)-3 ' ) . although direct binding of brla to bres remains to be verified in vitro , a number of developmental genes including abaa , weta , roda , and ya have multiple bres in their promoter regions . importantly , tao and yu reported that multiple bres are present in the promoter regions of afu brla , abaa , weta , roda and the velvet regulators , implying a potentially conserved regulatory circuit in the two aspergilli . the abaa gene , activated by brla during the middle stages of conidiation when metulae and phialides are being formed , functions in the differentiation of phialides . abacus ( abaa ) mutants have nearly normal conidiophores bearing abacus - like structures with swellings at intervals instead of chains of conidia . morphological studies have revealed that the metulae of abaa mutants produce supernumerary tiers of cells with properties of metulae , not phialides , indicating that abaa is essential for the differentiation and functionality of phialides as conidiogenous cells . abaa expression is dependent on brla activity , and bres are found in the abaa promoter region , suggesting that brla directly activates abaa . the abaa gene encodes a developmental regulator , which begins to be expressed when phialides form at 15~18 hr after developmental induction ( fig . 3 ) , and loss of abaa function results in abnormal expression of a number of developmentally regulated genes . abaa overexpression in vegetative hyphae causes growth cessation and enhances cellular vacuolization but not spore formation in ani . abaa overexpression activates the expression of weta and brla , and it is thought that abaa induces the expression of brla at certain times of development . however , the genetic interaction between abaa and brla seems more complex , as brla mrna levels increase in abaa null mutants [ 15 , 20 ] , which might involve vosa ( fig . 3 ) . the abaa protein contains an atts / tea dna - binding motif [ 21 , 22 ] and a leucine zipper for potential dimerization . expression studies in both ani and s. cerevisiae have demonstrated that abaa binds to the cis - regulatory elements upstream of the ya gene . results of a gel mobility shift assay indicate that abaa binds to the consensus sequence 5'-cattcy-3 ' ( abaa response element [ are ] where y is pyrimidine ) . in fact , multiple ares are found in the promoter regions of developmentally regulated genes , including brla , weta , ya , roda , and abaa . importantly , park et al . demonstrated that abaa binds to the chitin synthase gene promoter , chsc , suggesting that abaa also regulates chitin biosynthesis during conidiophore development by controlling the expression of certain chitin synthetases . wet - white ( weta ) mutants produce colorless conidia that completely autolyze within a few days , leading to the formation of liquid droplets ( wet ) on the tops of conidial heads . sewall et al . have shown that the weta gene is required for the synthesis of crucial cell wall components late in the development of the inner c4 layer ( fig . the ani weta gene encodes two transcription units , and weta transcription is predicted to be complex and subjected to developmental control . abaa , but not brla , activates weta expression , as abaa overexpression activates weta in the absence of wild type brla . furthermore , weta is auto - activated , as it is not expressed in weta temperature - sensitive mutants ( fig . the ani weta gene is predicted to encode a 60 kda protein rich in serine ( 14% ) , threonine ( 7% ) , and proline ( 10% ) . while no known dna binding domains are present in weta , it has been proposed to function as a regulator of spore - specific gene expression . this is based on observations that weta alone is sufficient to activate many sporulation - specific genes , and that the weta mutants fails to accumulate many sporulation - specific mrnas . furthermore , weta overexpression in vegetative cells inhibits hyphal growth , resulting in excessive hyphal branching and the activation of spore - specific genes . however , weta overexpression does not result in brla or abaa activation or lead to precocious conidiation . taken together , weta is proposed to activate a set of genes required for spore formation and maturation , which may function to complete the final two conidial wall layers and/or direct their assembly ( fig . , the brlaabaaweta cascade has been proposed to define a central regulatory pathway that controls temporal and spatial expression of conidiation - specific genes during conidiophore development and spore maturation ( fig . vosa is a multifunctional regulator that acts in concert with the central regulatory genes , couples trehalose biogenesis and conidia maturation , and exerts negative feedback regulation of brla , thereby completing conidiation in ani ( fig . 3 ) . afuabaa ( eal88194 , 60% identity , 74% similarity to aniabaa ) and afuweta ( eal89470 , 58% identity , 68% similarity ) were previously identified through a genome search . the afuabaa open reading frame ( orf ) is composed of 2,518 bp with three exons and two introns and is predicted to encode a 797 aa - length protein containing an atts / tea dna - binding domain . the afuweta orf comprises 1,701 bp with no introns and is predicted to encode a 566 aa - length protein with a conserved c - terminal domain . tao and yu examined the levels of the afuabaa and afuweta transcripts throughout the lifecycle and found that afuabaa and afuweta are highly expressed during asexual development . afuabaa mrna starts to accumulate at 6 hr post - developmental induction , reaches its highest level at 12 hr , and disappears at 48 hr . afuweta mrna begins to accumulate at 12 hr post - developmental induction , reaches its highest level at 48 hr post induction , and is present in conidia . deletion of afuabaa results in the formation of aberrant conidiophores exhibiting reiterated cylinder - like terminal cells without conidia , indicating that , as in ani , afuabaa is essential for differentiation and functionalization of afu phialides as conidiogenous cells . importantly , tao and yu found that the most distal cells of the afuabaa conidiophores are capable of apical hyphal growth , whereas the fractions of cylinder - like elongated phialides do not undergo vegetative growth . they also examined hyphal dry weights and found that the absence of afuabaa causes delayed hyphal mass loss compared to that in the wild type . when cell viability was examined by determining the percent alamar blue reduction , which represents living cell mitochondrial activity , the afuabaa mutant clearly exhibited prolonged cell viability compared to the wild type . tao and yu then examined the effects of afuabaa overexpression and found that it caused accelerated hyphal fragmentation , disintegration , a dramatic reduction of mycelial mass , and precocious cell death . the afuweta gene is subsequently activated by afuabaa in the middle to late phases of conidiation . furthermore , tem analyses of the afuweta mutant revealed that weta conidiophores exhibit an interconnected conidia phenotype , i.e. , incomplete conidial separation and maturation . as afuweta plays an essential role in completion of the conidial wall , tao and yu hypothesized that the absence of afuweta may affect spore viability and/or integrity , and found that the afuweta mutant exhibits dramatically reduced viability starting from day 10 , whereas wild type conidia maintain high viability until day 20 . moreover , tem studies revealed that , even at day 2 , about 20% of the afuweta conidia appear to lack cytoplasm and exhibit sheared conidial walls . tao and yu then examined the effects of afuweta on spore tolerance to various stressors and found that the afuweta conidia were much more sensitive to heat , and oxidative and osmotic stresses . as trehalose is essential for long - term spore viability and stress resistance [ 15 , 27 ] , tao and yu determined that the afuweta conidia do not contain any trehalose , whereas wild type conidia harbor about 3.4 pg of trehalose per conidium . these results indicate that afuweta plays an essential role in trehalose biogenesis in conidia , which probably affects their viability and stress tolerance . this was the first report that weta is essential for trehalose biogenesis in fungal conidia . additionally , given that the afuweta gene is activated during the late stage of conidiation , and that its mrna accumulates preferentially in mature conidia , tao and yu further determined whether the absence of afuweta had an effect on spore germination and early vegetative growth . they found that after a 8-hr incubation in liquid submerged culture , the afuweta mutant conidia formed only one unidirectional germ tube without branching , which resulted in loosened mycelial aggregates , whereas conidia of wild type and complemented strains produced bi- or multi - directional germ tubes that formed branches . moreover , after a 14-hr incubation , the afuweta mutant clearly showed a five - fold reduction in hyphal branching . these results suggest that afuweta is associated with proper germ tube formation and vegetative growth . this is consistent with the previous finding that aniweta overexpression in hyphae causes excessive branching in ani . tao and yu further dissected the genetic interactions among the central regulatory genes in afu . the absence of afubrla eliminates the expression of all conidiation - specific genes tested , indicating that afubrla functions upstream of afuabaa , afuweta , afuvosa , and afuroda . as afuabaa is necessary for activating afuweta , afuweta mrna accumulation is not detected in the afuabaa mutant . moreover , levels of afuvosa mrna decrease considerably in both afuabaa and afuweta strains , indicating the role of these genes in proper afuvosa expression . however , as afuvosa is activated before afuweta in the wild type , it is proposed that afuabaa primarily activates afuvosa with the assistance of afuweta . importantly , afubrla is highly upregulated in the afuabaa and afuweta mutants , particularly in conidia ( aberrant conidiophores in afuabaa ) and early ( 6 hr ) and late ( 24 hr ) phases of vegetative growth , suggesting that afuabaa and afuweta are necessary for proper negative regulation of afubrla upon completion of conidiation and during certain phases of vegetative growth . afuroda is a small , moderately secreted hydrophobic polypeptide that forms the outermost rodlet layer of conidia . conidia of afuroda mutants lack the external rodlet layer and are hydrophilic . as no afuroda mrna is detected in the afubrla mutant and afuroda levels are low in the afuabaa mutant , tao and yu speculated that afuroda is primarily activated by afubrla with the assistance of afuabaa . a recent genetic study identified the novel regulator vosa , which functions in maturation of conidia and completion of ani development . importantly , vosa deletion results in the lack of trehalose in both types of spores , resulting in a rapid disappearance ( evaporation ) of the cytoplasm , disintegration of cellular organelles , and loss of long - term spore viability . moreover , the vosa mutant spores exhibit dramatically reduced tolerance to heat and oxidative stress . as vosa mainly localizes in the nucleus of mature conidia and contains a potential transcriptional activation domain at the c terminus , it may be a tf that primarily controls the late process of sporulation , including trehalose biogenesis . the vosa protein expressed in metulae and phialides and later localized to the nucleus of the conidia plays two chief roles : 1 ) activation of genes involved in spore maturation ; 2 ) negative feedback regulation of brla and developmental specific genes ( fig . 3 ) . vosa studies clearly divide conidiophore formation and conidia maturation into two distinct genetic phases . vosa has been identified with three other ani proteins sharing high levels of similarity ; vea , velb ( ef540815 ) , and velc ( ef540816 ) . these , together with vosa , define the velvet regulators , and they are highly conserved in many filamentous and dimorphic fungi , sharing at least one highly conserved domain [ 15 , 30 ] . importantly , recent studies have shown that vea , velb , and vosa bridge light - responding development and secondary metabolism by forming trimeric complexes with the nuclear master regulator of secondary metabolism , laea , in ani [ 30 , 31 ] . the afuvosa gene shows an mrna accumulation pattern almost identical to that of anivosa . to begin to understand the roles of the afu velvet proteins , we generated a fu mutants lacking afuvea , afuvelb , afuvosa , and afuvelc , and found that , while the deletion of afuvosa caused an approximate 50% reduction in the spore trehalose content and viability , it did not result in uncontrolled activation of conidiation in afu ( park and yu , unpublished ) . our recent unpublished data suggest that the feedback regulation of conidiation in afu is primarily exerted by velb and vea , whereas trehalose biogenesis is conferred by both afuvosa and afuvelb ( additive roles ) ( fig . identification and characterization of six upstream genes ( flug , flba , flbb , flbc , flbd , and flbe ) required for proper expression of brla in ani have illuminated genetic regulatory cascades for activating conidiation . among these , flbb , flbc , flbd , and flbe are defined by mutants exhibiting the fluffy delayed conidiation phenotypes . flbb , flbc , and flbd are putative tfs containing a basic leucine zipper ( b - zip ) , two c2h2 zinc fingers , and a cmyb - dna binding domain , respectively . thus , it has been thought that flbb , flbb , and flbd likely function as dna binding proteins that may control the transcriptional activation of other developmental regulators , such as brla , in response to sporulation signals . detailed functional studies of these potential tfs have demonstrated that flbb / c / d activate brla expression [ 33 - 38 ] . moreover , b - zip tf flbb is necessary for the activation of flbd expression , while flbb and flbd cooperatively activate brla . they found that flbc mrna is present throughout the lifecycle at relatively high levels during vegetative growth and during early asexual and late sexual developmental phases . deletion of flbc causes a delay / reduction in sporulation , brla and vosa expression , and conidial germination . while flbc overexpression does not cause conidiophore development , it inhibits hyphal growth and activates expression of brla , abaa , and vosa , but not weta . further reported that flbc contains two c2h2 zinc - fingers at the c - terminus and a putative activation domain at the n - terminus . they also found that flbc localizes to the nuclei of both hyphae and developmental cells . localization and expression of flbc is not affected by the absence of flbb or flbe and vice versa . importantly , as flbc overexpression inhibits growth and activates abaa and vosa in the absence of brla and abaa , respectively , kwon et al . proposed that flbc plays a direct activating role in the expression of these genes . consistent with this idea , an in vitro dna binding assay revealed that flbc binds to the promoter regions of brla , abaa , and vosa , but not to that of weta . the aniflbe gene is predicted to encode a 201 aa - length polypeptide with two conserved yet uncharacterized domains , and it has been demonstrated that flbe and flbb are functionally interdependent , that they physically interact in vivo , and co - localize to the hyphal tip in ani . our recent studies revealed that both deletion and overexpression of flbe in ani results in developmental defects , enhanced autolysis , precocious cell death , and delayed expression of brla / vosa , suggesting that balanced flbe activity is crucial for proper growth and development . the n - terminal portion of flbe exhibits a transactivation ability in budding yeast , whereas the c - terminal half negatively affects this activity . site - directed mutagenesis of certain conserved n - terminal amino acids abolishes transactivation ability , overexpression - induced autolysis , and complements the null mutation . these results suggest that the conserved n - terminal domain might be crucial for the functionality of flbe . finally , flbd overexpression , but not that of flbb or flbc , restores conidiation in ani dflbe , generally supporting the current genetic model of developmental regulation . in a series of recent studies , we characterized the functions of flbe and flbb in afu [ 39 , 40 ] . the predicted afu flbe protein is composed of 222 aa in length . while flbe is transiently expressed during the early growth phase in ani , deletion of afuflbe causes reduced conidiation and delayed expression of brla and vosa in both species . moreover , afuflbe is necessary for salt - induced development of afu in a liquid submerged culture . as the ani flbe null mutation can be fully complemented by afuflbe , the developmental function of flbe appears to be conserved in aspergilli . conducted a series of molecular analyses and found that afuflbb produces two transcripts predicted to encode two b - zip polypeptides , afuflbbb ( 420 aa ) and afuflbba ( 390 aa ) . afuflbb deletion results in delayed and reduced conidiation , precocious cell death , the absence of conidiophore development in liquid submerged culture , altered expression of afubrla and afuabaa , and the lack ( or a reduction ) in gliotoxin production . importantly , they found that while introducing the wild type afuflbb allele fully complemented these defects , disruption of the atg start codon to either one of the afuflbb polypeptides led to partial complementation , indicating that both polypeptides are needed for wild type level sporulation and gliotoxin biogenesis . consistent with these observations , introducing ani flbb encoding one polypeptide ( 426 aa ) into the afuflbb null mutant partially restores asexual and chemical development . . also found that the presence of 0.6 m kcl in a liquid submerged culture suppresses the defects caused by the lack of one , but not both , of the afuflbb polypeptides , suggesting a genetic prerequisite for afuflbb in afu development . northern blot analyses revealed that both afuflbb and afuflbe are necessary for afuflbd expression , suggesting that flbd functions downstream of flbb and flbe in both aspergilli . further suggested that afubrla might be necessary for gliotoxin biosynthesis , which was based on the observations that the afubrla mutant lacks gliotoxin production , and that multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes . regarding the upstream region of flbb / c / d / e , mah and as discovered in ani , levels of the afuflug transcript are relatively constant throughout the lifecycle . the afuflug deletion mutant conidiated normally , similar to wild type on solid medium , indicating that activation of afu conidiation in the presence of air does not require afuflug . however , the afuflug deletion mutant did not produce conidiophores in liquid submerged culture , whereas afu wild type strains sporulated abundantly at approximately 24 hr . moreover , the afuflug deletion mutant showed reduced conidiation levels and delayed afubrla expression upon induction of synchronized asexual development . these results led mah and yu to conclude that while the presence of air bypasses the need for afuflug in conidiophore development , afuflug plays a particular role in afu conidiation and afubrla expression . these findings led to the hypothesis that afu has multiple pathways to activate afubrla expression ( fig . in ani , a key and essential step for conidiophore development is the activation of brla encoding a c2h2 zinc finger transcription factor ( tf ) , which induces expression of other genes required for conidiation . loss - of - function brla mutants form structures that resemble conidiophore stalks ( thus named " bristle " ) , except that they grow indeterminately and fail to produce vesicles , metuale , phialides , and conidia , indicating that brla controls the initiation of conidiophore development ( fig . , brla overexpression in vegetative cells causes termination of polar growth coupled with the commencement of abnormal sporulation leading to the formation of viable spores from hyphae . no environmental signals , including nutrient limitations or various ( osmotic and oxidative ) stresses , can bypass the brla requirement for conidiation , indicating that activation of brla expression early in conidiophore development represents a key and essential control point for commencing conidiation . the anibrla ( but not afubrla ) gene is a compound gene consisting of two overlapping transcriptional units designated brlaa and brlab . important information on the complex regulation of the two overlapping brla transcription units is well described elsewhere . afubrla , the afu brla homologue , shows 68% identity and 77% similarity to the ani brla . mah and yu tested whether brla - dependent conidiation is conserved in these two aspergilli using genetics and found that afubrla deletion completely eliminated asexual development in afu , resulting in elongated aerial hyphae and increased hyphal mass in the colonies . these studies clearly demonstrated that afubrla is essential for conidiophore development in afu , and that the role of the core downstream tf brla in conidiation is conserved in these aspergilli ( fig . brla is a putative tf ( activator ) with two c2h2 zinc finger motifs at the c - terminus . disruption of either one of the fingers by mutating the key cysteine to serine results in complete loss of brla activity . chang and timberlake further demonstrated that brla expression in the budding yeast saccharomyces cerevisiae results in brla dependent activation of aspergillus genes . furthermore , they proposed the consensus brla binding sites ( brla response elements [ bres ] ; 5'-(c / a)(g / a)aggg(g / a)-3 ' ) . although direct binding of brla to bres remains to be verified in vitro , a number of developmental genes including abaa , weta , roda , and ya have multiple bres in their promoter regions . importantly , tao and yu reported that multiple bres are present in the promoter regions of afu brla , abaa , weta , roda and the velvet regulators , implying a potentially conserved regulatory circuit in the two aspergilli . the abaa gene , activated by brla during the middle stages of conidiation when metulae and phialides are being formed , functions in the differentiation of phialides . abacus ( abaa ) mutants have nearly normal conidiophores bearing abacus - like structures with swellings at intervals instead of chains of conidia . morphological studies have revealed that the metulae of abaa mutants produce supernumerary tiers of cells with properties of metulae , not phialides , indicating that abaa is essential for the differentiation and functionality of phialides as conidiogenous cells . abaa expression is dependent on brla activity , and bres are found in the abaa promoter region , suggesting that brla directly activates abaa . the abaa gene encodes a developmental regulator , which begins to be expressed when phialides form at 15~18 hr after developmental induction ( fig . 3 ) , and loss of abaa function results in abnormal expression of a number of developmentally regulated genes . abaa overexpression in vegetative hyphae causes growth cessation and enhances cellular vacuolization but not spore formation in ani . abaa overexpression activates the expression of weta and brla , and it is thought that abaa induces the expression of brla at certain times of development . however , the genetic interaction between abaa and brla seems more complex , as brla mrna levels increase in abaa null mutants [ 15 , 20 ] , which might involve vosa ( fig . 3 ) . the abaa protein contains an atts / tea dna - binding motif [ 21 , 22 ] and a leucine zipper for potential dimerization . expression studies in both ani and s. cerevisiae have demonstrated that abaa binds to the cis - regulatory elements upstream of the ya gene . results of a gel mobility shift assay indicate that abaa binds to the consensus sequence 5'-cattcy-3 ' ( abaa response element [ are ] where y is pyrimidine ) . in fact , multiple ares are found in the promoter regions of developmentally regulated genes , including brla , weta , ya , roda , and abaa . importantly , park et al . demonstrated that abaa binds to the chitin synthase gene promoter , chsc , suggesting that abaa also regulates chitin biosynthesis during conidiophore development by controlling the expression of certain chitin synthetases . wet - white ( weta ) mutants produce colorless conidia that completely autolyze within a few days , leading to the formation of liquid droplets ( wet ) on the tops of conidial heads . sewall et al . have shown that the weta gene is required for the synthesis of crucial cell wall components late in the development of the inner c4 layer ( fig . the ani weta gene encodes two transcription units , and weta transcription is predicted to be complex and subjected to developmental control . abaa , but not brla , activates weta expression , as abaa overexpression activates weta in the absence of wild type brla . furthermore , weta is auto - activated , as it is not expressed in weta temperature - sensitive mutants ( fig . the ani weta gene is predicted to encode a 60 kda protein rich in serine ( 14% ) , threonine ( 7% ) , and proline ( 10% ) . while no known dna binding domains are present in weta , it has been proposed to function as a regulator of spore - specific gene expression . this is based on observations that weta alone is sufficient to activate many sporulation - specific genes , and that the weta mutants fails to accumulate many sporulation - specific mrnas . furthermore , weta overexpression in vegetative cells inhibits hyphal growth , resulting in excessive hyphal branching and the activation of spore - specific genes . however , weta overexpression does not result in brla or abaa activation or lead to precocious conidiation . taken together , weta is proposed to activate a set of genes required for spore formation and maturation , which may function to complete the final two conidial wall layers and/or direct their assembly ( fig . together , the brlaabaaweta cascade has been proposed to define a central regulatory pathway that controls temporal and spatial expression of conidiation - specific genes during conidiophore development and spore maturation ( fig . vosa is a multifunctional regulator that acts in concert with the central regulatory genes , couples trehalose biogenesis and conidia maturation , and exerts negative feedback regulation of brla , thereby completing conidiation in ani ( fig . afuabaa ( eal88194 , 60% identity , 74% similarity to aniabaa ) and afuweta ( eal89470 , 58% identity , 68% similarity ) were previously identified through a genome search . the afuabaa open reading frame ( orf ) is composed of 2,518 bp with three exons and two introns and is predicted to encode a 797 aa - length protein containing an atts / tea dna - binding domain . the afuweta orf comprises 1,701 bp with no introns and is predicted to encode a 566 aa - length protein with a conserved c - terminal domain . tao and yu examined the levels of the afuabaa and afuweta transcripts throughout the lifecycle and found that afuabaa and afuweta are highly expressed during asexual development . afuabaa mrna starts to accumulate at 6 hr post - developmental induction , reaches its highest level at 12 hr , and disappears at 48 hr . afuweta mrna begins to accumulate at 12 hr post - developmental induction , reaches its highest level at 48 hr post induction , and is present in conidia . deletion of afuabaa results in the formation of aberrant conidiophores exhibiting reiterated cylinder - like terminal cells without conidia , indicating that , as in ani , afuabaa is essential for differentiation and functionalization of afu phialides as conidiogenous cells . importantly , tao and yu found that the most distal cells of the afuabaa conidiophores are capable of apical hyphal growth , whereas the fractions of cylinder - like elongated phialides do not undergo vegetative growth . they also examined hyphal dry weights and found that the absence of afuabaa causes delayed hyphal mass loss compared to that in the wild type . when cell viability was examined by determining the percent alamar blue reduction , which represents living cell mitochondrial activity , the afuabaa mutant clearly exhibited prolonged cell viability compared to the wild type . tao and yu then examined the effects of afuabaa overexpression and found that it caused accelerated hyphal fragmentation , disintegration , a dramatic reduction of mycelial mass , and precocious cell death . these results indicate that afuabaa functions in afu autolysis and cell death . the afuweta gene is subsequently activated by afuabaa in the middle to late phases of conidiation . furthermore , tem analyses of the afuweta mutant revealed that weta conidiophores exhibit an interconnected conidia phenotype , i.e. , incomplete conidial separation and maturation . as afuweta plays an essential role in completion of the conidial wall , tao and yu hypothesized that the absence of afuweta may affect spore viability and/or integrity , and found that the afuweta mutant exhibits dramatically reduced viability starting from day 10 , whereas wild type conidia maintain high viability until day 20 . moreover , tem studies revealed that , even at day 2 , about 20% of the afuweta conidia appear to lack cytoplasm and exhibit sheared conidial walls . tao and yu then examined the effects of afuweta on spore tolerance to various stressors and found that the afuweta conidia were much more sensitive to heat , and oxidative and osmotic stresses . as trehalose is essential for long - term spore viability and stress resistance [ 15 , 27 ] , tao and yu determined that the afuweta conidia do not contain any trehalose , whereas wild type conidia harbor about 3.4 pg of trehalose per conidium . these results indicate that afuweta plays an essential role in trehalose biogenesis in conidia , which probably affects their viability and stress tolerance . this was the first report that weta is essential for trehalose biogenesis in fungal conidia . additionally , given that the afuweta gene is activated during the late stage of conidiation , and that its mrna accumulates preferentially in mature conidia , tao and yu further determined whether the absence of afuweta had an effect on spore germination and early vegetative growth . they found that after a 8-hr incubation in liquid submerged culture , the afuweta mutant conidia formed only one unidirectional germ tube without branching , which resulted in loosened mycelial aggregates , whereas conidia of wild type and complemented strains produced bi- or multi - directional germ tubes that formed branches . moreover , after a 14-hr incubation , the afuweta mutant clearly showed a five - fold reduction in hyphal branching . these results suggest that afuweta is associated with proper germ tube formation and vegetative growth . this is consistent with the previous finding that aniweta overexpression in hyphae causes excessive branching in ani . tao and yu further dissected the genetic interactions among the central regulatory genes in afu . the absence of afubrla eliminates the expression of all conidiation - specific genes tested , indicating that afubrla functions upstream of afuabaa , afuweta , afuvosa , and afuroda . as afuabaa is necessary for activating afuweta , afuweta mrna accumulation is not detected in the afuabaa mutant . moreover , levels of afuvosa mrna decrease considerably in both afuabaa and afuweta strains , indicating the role of these genes in proper afuvosa expression . however , as afuvosa is activated before afuweta in the wild type , it is proposed that afuabaa primarily activates afuvosa with the assistance of afuweta . importantly , afubrla is highly upregulated in the afuabaa and afuweta mutants , particularly in conidia ( aberrant conidiophores in afuabaa ) and early ( 6 hr ) and late ( 24 hr ) phases of vegetative growth , suggesting that afuabaa and afuweta are necessary for proper negative regulation of afubrla upon completion of conidiation and during certain phases of vegetative growth . afuroda is a small , moderately secreted hydrophobic polypeptide that forms the outermost rodlet layer of conidia . conidia of afuroda mutants lack the external rodlet layer and are hydrophilic . as no afuroda mrna is detected in the afubrla mutant and afuroda levels are low in the afuabaa mutant , tao and yu speculated that afuroda is primarily activated by afubrla with the assistance of afuabaa . a recent genetic study identified the novel regulator vosa , which functions in maturation of conidia and completion of ani development . importantly , vosa deletion results in the lack of trehalose in both types of spores , resulting in a rapid disappearance ( evaporation ) of the cytoplasm , disintegration of cellular organelles , and loss of long - term spore viability . moreover , the vosa mutant spores exhibit dramatically reduced tolerance to heat and oxidative stress . as vosa mainly localizes in the nucleus of mature conidia and contains a potential transcriptional activation domain at the c terminus , it may be a tf that primarily controls the late process of sporulation , including trehalose biogenesis . the vosa protein expressed in metulae and phialides and later localized to the nucleus of the conidia plays two chief roles : 1 ) activation of genes involved in spore maturation ; 2 ) negative feedback regulation of brla and developmental specific genes ( fig . 3 ) . vosa studies clearly divide conidiophore formation and conidia maturation into two distinct genetic phases . vosa has been identified with three other ani proteins sharing high levels of similarity ; vea , velb ( ef540815 ) , and velc ( ef540816 ) . these , together with vosa , define the velvet regulators , and they are highly conserved in many filamentous and dimorphic fungi , sharing at least one highly conserved domain [ 15 , 30 ] . importantly , recent studies have shown that vea , velb , and vosa bridge light - responding development and secondary metabolism by forming trimeric complexes with the nuclear master regulator of secondary metabolism , laea , in ani [ 30 , 31 ] . the afuvosa gene shows an mrna accumulation pattern almost identical to that of anivosa . to begin to understand the roles of the afu velvet proteins , we generated a fu mutants lacking afuvea , afuvelb , afuvosa , and afuvelc , and found that , while the deletion of afuvosa caused an approximate 50% reduction in the spore trehalose content and viability , it did not result in uncontrolled activation of conidiation in afu ( park and yu , unpublished ) . our recent unpublished data suggest that the feedback regulation of conidiation in afu is primarily exerted by velb and vea , whereas trehalose biogenesis is conferred by both afuvosa and afuvelb ( additive roles ) ( fig . identification and characterization of six upstream genes ( flug , flba , flbb , flbc , flbd , and flbe ) required for proper expression of brla in ani have illuminated genetic regulatory cascades for activating conidiation . among these , flbb , flbc , flbd , and flbe are defined by mutants exhibiting the fluffy delayed conidiation phenotypes . flbb , flbc , and flbd are putative tfs containing a basic leucine zipper ( b - zip ) , two c2h2 zinc fingers , and a cmyb - dna binding domain , respectively . thus , it has been thought that flbb , flbb , and flbd likely function as dna binding proteins that may control the transcriptional activation of other developmental regulators , such as brla , in response to sporulation signals . detailed functional studies of these potential tfs have demonstrated that flbb / c / d activate brla expression [ 33 - 38 ] . moreover , b - zip tf flbb is necessary for the activation of flbd expression , while flbb and flbd cooperatively activate brla . they found that flbc mrna is present throughout the lifecycle at relatively high levels during vegetative growth and during early asexual and late sexual developmental phases . deletion of flbc causes a delay / reduction in sporulation , brla and vosa expression , and conidial germination . while flbc overexpression does not cause conidiophore development , it inhibits hyphal growth and activates expression of brla , abaa , and vosa , but not weta . further reported that flbc contains two c2h2 zinc - fingers at the c - terminus and a putative activation domain at the n - terminus . they also found that flbc localizes to the nuclei of both hyphae and developmental cells . localization and expression of flbc is not affected by the absence of flbb or flbe and vice versa . importantly , as flbc overexpression inhibits growth and activates abaa and vosa in the absence of brla and abaa , respectively , kwon et al . proposed that flbc plays a direct activating role in the expression of these genes . consistent with this idea , an in vitro dna binding assay revealed that flbc binds to the promoter regions of brla , abaa , and vosa , but not to that of weta . the aniflbe gene is predicted to encode a 201 aa - length polypeptide with two conserved yet uncharacterized domains , and it has been demonstrated that flbe and flbb are functionally interdependent , that they physically interact in vivo , and co - localize to the hyphal tip in ani . our recent studies revealed that both deletion and overexpression of flbe in ani results in developmental defects , enhanced autolysis , precocious cell death , and delayed expression of brla / vosa , suggesting that balanced flbe activity is crucial for proper growth and development . the n - terminal portion of flbe exhibits a transactivation ability in budding yeast , whereas the c - terminal half negatively affects this activity . site - directed mutagenesis of certain conserved n - terminal amino acids abolishes transactivation ability , overexpression - induced autolysis , and complements the null mutation . these results suggest that the conserved n - terminal domain might be crucial for the functionality of flbe . finally , flbd overexpression , but not that of flbb or flbc , restores conidiation in ani dflbe , generally supporting the current genetic model of developmental regulation . in a series of recent studies , we characterized the functions of flbe and flbb in afu [ 39 , 40 ] . the predicted afu flbe protein is composed of 222 aa in length . while flbe is transiently expressed during the early growth phase in ani , deletion of afuflbe causes reduced conidiation and delayed expression of brla and vosa in both species . moreover , afuflbe is necessary for salt - induced development of afu in a liquid submerged culture . as the ani flbe null mutation can be fully complemented by afuflbe , the developmental function of flbe appears to be conserved in aspergilli . xiao et al . conducted a series of molecular analyses and found that afuflbb produces two transcripts predicted to encode two b - zip polypeptides , afuflbbb ( 420 aa ) and afuflbba ( 390 aa ) . afuflbb deletion results in delayed and reduced conidiation , precocious cell death , the absence of conidiophore development in liquid submerged culture , altered expression of afubrla and afuabaa , and the lack ( or a reduction ) in gliotoxin production . importantly , they found that while introducing the wild type afuflbb allele fully complemented these defects , disruption of the atg start codon to either one of the afuflbb polypeptides led to partial complementation , indicating that both polypeptides are needed for wild type level sporulation and gliotoxin biogenesis . consistent with these observations , introducing ani flbb encoding one polypeptide ( 426 aa ) into the afuflbb null mutant partially restores asexual and chemical development . . also found that the presence of 0.6 m kcl in a liquid submerged culture suppresses the defects caused by the lack of one , but not both , of the afuflbb polypeptides , suggesting a genetic prerequisite for afuflbb in afu development . northern blot analyses revealed that both afuflbb and afuflbe are necessary for afuflbd expression , suggesting that flbd functions downstream of flbb and flbe in both aspergilli . xiao et al . further suggested that afubrla might be necessary for gliotoxin biosynthesis , which was based on the observations that the afubrla mutant lacks gliotoxin production , and that multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes . regarding the upstream region of flbb / c / d / e , mah and as discovered in ani , levels of the afuflug transcript are relatively constant throughout the lifecycle . the afuflug deletion mutant conidiated normally , similar to wild type on solid medium , indicating that activation of afu conidiation in the presence of air does not require afuflug . however , the afuflug deletion mutant did not produce conidiophores in liquid submerged culture , whereas afu wild type strains sporulated abundantly at approximately 24 hr . moreover , the afuflug deletion mutant showed reduced conidiation levels and delayed afubrla expression upon induction of synchronized asexual development . these results led mah and yu to conclude that while the presence of air bypasses the need for afuflug in conidiophore development , afuflug plays a particular role in afu conidiation and afubrla expression . these findings led to the hypothesis that afu has multiple pathways to activate afubrla expression ( fig . our further studies suggest that afuflbd is essential for proper asexual development in afu ( xiao and yu , unpublished data ) . to examine potential genetic interactions among upstream developmental regulators in afu , xiao et al . conducted a series of expression studies and proposed that afu has an upstream regulatory cascade slightly different from the one proposed for ani ( fig . afuflbe expression is independent , and both are required for proper afuflbd expression , xiao et al . proposed that afuflbb and afuflbe function upstream of afuflbd and cooperatively activate afuflbd , which subsequently activates afubrla . while the transcriptional activation of afuflbb and afuflbe is independent , it may be that the afuflbb and afuflbe proteins interact and form a functional complex as in ani . furthermore , as afuflbc expression is independent of afuflbb and afuflbe , afuflbc is predicted to function in a separate pathway . because the afubrla mutant is defective in gliotoxin production and multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes , xiao et al . further functional and molecular studies of afuflbc and the identification of additional developmental regulators , including afusfga and afuwetb , are in progress in my laboratory . in this review , i have briefly summarized our current understanding of the upstream and downstream regulation of aspergillus conidiation focusing on key tfs . in summary , these two aspergilli have common essential downstream activators of conidiophore development ; brla , abaa , and weta . moreover , velvet regulator functions are generally conserved in these aspergilli . however , as clear differences in the upstream regulation of conidiation between the two species exist , much remains to be learned . further genetic and genomics studies aimed at identifying and characterizing the unique regulators and control networks in afu will illuminate the complex mechanisms of development in this opportunistic pathogenic fungus .

hepatocellular carcinoma ( hcc ) is the fastest rising cause of cancer - related death in the united states and carries a very poor prognosis , with a median survival time of < 50% at 1 year for advanced disease . to date , sorafenib is the only therapy approved by the food and drug administration for the treatment of advanced hcc . tivantinib ( arq-197 ) , a non - atp competitive inhibitor of cellular mesenchymal epithelial transcription factor ( c - met ) , has shown a survival benefit in patients with advanced hcc who have failed or are intolerant to sorafenib in phase i and ii trials . those patients who have tumors with high concentrations of met ( met - high ) appear to derive the greatest benefit from tivantinib therapy . currently , two large randomized double - blind placebo - controlled phase iii trials ( metiv - hcc [ nct01755767 ] and jet - hcc [ nct02029157 ] ) are evaluating tivantinib in patients with met - high advanced hcc , with the primary end points of overall survival and progression - free survival , respectively . this study reviews the evidence for the use of tivantinib in advanced hcc . specific topics addressed include the pharmacology , dosing , toxicity , and biomarkers associated with tivantinib use . members of the genus aspergillus are the most common fungi and all reproduce asexually by forming long chains of conidiospores ( or conidia ) . the impact of various aspergillus species on humans ranges from beneficial to harmful . for example , several species including aspergillus oryzae and aspergillus niger are used in industry for enzyme production and food processing . in contrast , aspergillus flavus produce the most potent naturally present carcinogen aflatoxins , which contaminate various plant- and animal - based foods . importantly , the opportunistic human pathogen aspergillus fumigatus has become the most prevalent airborne fungal pathogen in developed countries , causing invasive aspergillosis in immunocompromised patients with a high mortality rate . a. fumigatus produces a massive number of small hydrophobic conidia as the primary means of dispersal , survival , genome - protection , and infecting hosts . large - scale genome - wide expression studies can now be conducted due to completion of a. fumigatus genome sequencing . however , genomics becomes more powerful and informative when combined with genetics . we have been investigating the mechanisms underlying the regulation of asexual development ( conidiation ) and gliotoxin biosynthesis in a. fumigatus , primarily focusing on a characterization of key developmental regulators identified in the model fungus aspergillus nidulans . in this review , i will summarize our current understanding of how conidiation in two aspergilli is regulated .

there is evidence corroborating the relationship of vascular dysfunction during the first decade of life with risk factors such as familial hypercholesterolemia and hypertension . however , the question whether or not people exposed to cigarette smoke also develop these dysfunctions remains controversial . numerous studies have been conducted on adults exposed to passive smoking , particularly partners of smokers , and various aspects of cigarette smoke on these individuals have been evaluated[2 , 3 , 4 , 57 ] . however , only a few limited studies have addressed the issue of children exposed to passive smoke and the consequences on their blood pressure.[8 , 9 , 10 , 11 ] parental smoking affects children and neonates , and is associated with low birth weight , sudden infant death , asthma , bronchitis , pneumonia , otitis media , increased risk of contracting tuberculosis on exposure , crohn s disease , learning disorders , development retardation and dental caries . nevertheless , it is not yet clear whether children exposed to passive smoke develop vascular dysfunction or major changes in blood pressure . since the impact of cigarette smoke on children s blood pressure will provide yet another logical reason for parents to quit smoking or avoid smoking in the presence of their children , we have conducted this study to assess this relationship . according to reference 8 , standard deviation of blood pressure in exposed group was 4 mmhg and in not - exposed group was 3mmhg . therefore , with precision of 7mmhg ( = 0.01 and = 0.90 ) , sample size for our study was 8 in each grade and sex and total sample size in both sexes and five grades was calculated as 80 ( 825 = 80 ) in each group . 80 elementary school children exposed to cigarette smoke and 80 not exposed to smoke were studied in fall 2010 . range of age was7 to 12 years and average was 9.5 years in both groups . passive smoking was defined as breathing in a place where another person has been smoking over a period of at least 3 years with a daily consumption of 5 cigarettes or more[13 , 8 ] . after acquisition of necessary permissions from the department of education of the province and its triple regions , 8 elementary schools in kermanshah were selected through cluster sampling . in each school one class from each grade was randomly allocated . in each class , if we did not find enough cases of exposure in a grade , another school was substituted . finally , 80 cases of exposed and 80 cases of not - exposed children with same sex and age were enrolled from 12 elementary schools . subsequently , the principals and teachers were provided with enough information regarding the study , and questionnaires were submitted to parents to collect information regarding parental consent for participation in the study , as well as the health status of the children and their exposure to passive smoke . the questionnaires did not record the children s names and were only indentified with a code . we asked about parents and other families smoking habits who lived with the students and if the answer was positive continued questions about the location of smoking and how long and how many cigarettes do they smoke near their children ? once the questionnaires were collected , their data were matched with those in the children s health certificate . subsequently , the passive smokers of each class were identified and for each exposed child , one non - exposed counterpart of the same age and gender was randomly selected from the same class . the exclusion criteria included : chronic cardiac or renal diseases as expressed by parents or discovered in physical examination prior to measurement of blood pressure , any acute diseases , including respiratory and gastrointestinal infections and consumption of any medicine , and discrepancy between information recorded in parental questionnaires and those in the children s health certificate . during physical examination and measurement of blood pressure therefore , the examinations were performed in school laboratories which created an attractive ambience for the students and also entailed the smallest stress and greatest facilities . after explaining the situation to participants in a simplified language , the children were entertained with fruit snacks . the students did not have any activity for 15 minutes prior to blood pressure measurement and did not receive stimulant substances such as tea or coffee . the measurements were performed on the right arm in the level of the heart and supported on a desk while the child was in a sitting position and his / her feet were on the ground . appropriate cuff was defined as the width of cuff equal to 40% of arm circumference at the mid - arm . systolic and diastolic blood pressures were measured using a digital instrument ( e-7051-hem omron n3 intellisense , made in china ) and repeated under the same conditions by a resident of pediatrics in the morning shift . systolic blood pressure was defined as first korotkoff ( k1 ) and diastolic as fourth or fifth korotkoff ( k4 or k5 ) sound . values are expresses as meansd . a p - value < 0.05 was considered statistically significant . the values of blood pressure were compared for gender using one - way anova test , and compared for gender and school grade using 2-way anova test . in this study , we measured the blood pressure of 160 elementary school ( grades 1 to 5 ) children in kermanshah in fall 2010 . thirty - two students were selected from each grade ( consisting of 16 boys and 16 girls ) equally distributed over the case and control groups . the mean values of systolic and diastolic blood pressure of girls were 107.1610 mmhg and 63.098.7 mmhg , respectively . for boys , the mean values of systolic and diastolic blood pressures were 107.169.32 mmhg and 63.987.13 mmhg , respectively . in the exposure group , the mean values of systolic and diastolic blood pressures were 109.39.97 mmhg and 64.987.36 mmhg , respectively , while in the not - exposure group , the systolic and diastolic values were 105.478.98 mmhg and 62.157.01 mmhg , respectively . the mean systolic and diastolic blood pressures of the exposure group were higher than those of the non - exposure group , with a confidence interval of 0.95 and p values of 0.012 and 0.016 , respectively . girls exposed to cigarette smoke had a mean systolic blood pressure of 109.269.7 mmhg and a mean diastolic blood pressure of 64.6610 mmhg , whereas girls not exposed to smoke had a mean systolic blood pressure of 105.0610 mmhg and a mean diastolic blood pressure of 61.517.4 , indicating no significant difference between the two groups ( p=0.06 and 0.059 , respectively ) . boys exposed to cigarette smoke had a mean systolic blood pressure of 109.3410.3 mmhg and a mean diastolic blood pressure of 65.187.5 mmhg , whereas boys not exposed to smoke had a mean systolic blood pressure of 105.889.2 and a mean diastolic blood pressure of 62.796.61 , also indicating no significant difference between the two groups ( p=0.097 and 0.13 , respectively ) ( tables 1 ) . table 2 shows mean systolic and diastolic blood pressure in children exposed and not exposed to cigarette smoke according to their school grade . comparison of mean ( sd ) systolic and diastolic blood pressure in exposed and non - exposed children to cigarette smoke sd : standard deviation comparison of mean systolic and diastolic blood pressure in children exposed and not exposed to cigarette smoke according to their school grade cigarette smoke consists of two constituents : 15% of cigarette smoke is the main stream and 85% is the side stream distributed into the environment from the burning cigarette between puffs . while these two streams are similar in quality , their quantity differs as the amounts of carbonmonoxide , benzopyrene , ammonia and other carcinogenic substances are higher in the side stream than in the smoke inhaled by the smoker . passive smoking has been associated with endothelial damage in healthy children and young adults , suggesting early arterial damage . the impact of cigarette smoke is exerted on peripheral vasculature via the above mentioned substances to disrupt the function of vascular endothelial cells and jeopardize perfusion to vital organs , resulting in disorders of peripheral vasculature and increased risk of cardiovascular diseases . our findings indicate that systolic and diastolic blood pressures are higher in the exposure group compared to the non - exposure group ; however , after consideration of the gender variable , the difference between exposed and non - exposed girls and boys was not significant . previous studies conducted on the effects of cigarette smoke on children s blood pressure or studies on adults who were exposed to cigarette smoke in childhood have yielded different results . in the study of simonetti ( the mean sys bp in exposed children was + 1.2 mmhg higher than in not - exposed children , p<0.05 ) . in another study by feely and mahmood , acute changes of blood pressure before and after exposure to cigarette smoke were measured in 21 young adults and compared to 12 healthy controls . the findings indicated that exposure to cigarette smoke after 60 minutes only resulted in systolic hypertension in brachial artery and aorta in the male group . the diastolic blood pressure in aorta and brachial artery of the male and female groups did not alter considerably before and after exposure to cigarette smoke . in a turkish study by agirbasli et al family history of coronary artery disease and current smoking history of family members were not significantly associated with systolic and diastolic hypertension in their children ( hypertension was defined as having a systolic or diastolic blood pressure > 95% percentile for age and gender ) . geerts et al reported the exposure of pregnant mothers to cigarette smoke , whether as secondary exposure to smoke or smoking by the mother herself , to be associated with higher systolic blood pressure in their newborns . a study by hunk evaluated the impact of smoking parents on diabetes , hypertension and metabolic syndrome in their male adult children retrospectively . data regarding the smoking status of parents during the prenatal period and childhood were obtained with questionnaires . the findings indicated that after correction for age , gender , race , education and smoking status of the person , participants whose parents were both smokers had a 1.55 fold increase in their risk for adulthood hypertension . our study also indicates that systolic and diastolic blood pressures are higher in school children who are exposed to cigarette smoke . since signs of atherosclerosis begin to develop during the first decade of life , particularly in high - risk individuals such as those with hypertension , diabetes mellitus , and familial hypercholesterolemia , the exposure group is more likely to develop cardiovascular diseases in the future . after addition of the gender variables , both the girls and the boys groups did not indicate a significant difference between exposed and non - exposed individuals . however , in the girls group , the difference was close to the level of significance , and therefore larger sample sizes may yield significant differences . the greater impact of cigarette smoke on girls may be due to their longer presence at home and consequently greater exposure to smoke or it may reflect their intrinsic sensitivity to cigarette smoke . unlike the other factors of cardiovascular diseases , exposure to cigarette smoke is easily omissible ; therefore , the authors recommend that given the sensitivity of parents about their children s health , the results of this study and similar studies be used for encouraging parents to quit smoking or smoke in places isolated from their children . there are certain limitations in our study , the most important of which is the provision of inaccurate information by parents in questionnaires . nevertheless , we tried to circumvent this challenge by not mentioning the children s names on questionnaires and comparing the information with the data recorded in students health certificates ( which contain information regarding the smoking status of the parents ) to eliminate those questionnaires with discrepancy . another challenge was to control children s anxiety and activity , particularly those in the first and second grade , during blood pressure measurement . this study revealed that exposure to cigarette smoke can increase blood pressure in children with possible risk of cardiovascular diseases in future . the present analysis was performed as part of the diabetes and informatics ( dai ) study , a multicenter cohort study conducted by the dai study group ( diainf ) together with the italian association of clinical diabetologists ( amd ) and the italian national institute of health ( iss ) since september 1998 . the aim of the dai study was to evaluate the prevalence and incidence of cvd ( myocardial infarction , ischemic heart disease [ ihd ] , percutaneous coronary intervention angioplasty [ ptca ] , coronary artery bypass graft [ cabg ] , stroke , and peripheral amputations ) among type 2 diabetic patients regularly attending hospital - based diabetes clinics . in italy , a public network of 700 diabetes clinics provides care for up to 80% of patients with known diabetes , delivering diagnostic confirmation , therapy , prevention , and early diagnosis of acute and chronic complications through close patient follow - up by a team of specialists and the scheduling of regular checkups . most patients are referred to these care units by their general practitioner , and care is free of charge . a detailed description of the dai study methodology has been presented elsewhere ( 5 ) . for the incidence study , 157 clinics participated , with a total of 14,432 patients : 11,644 patients free of cvd at enrollment and 2,788 patients who entered the study with a known prior cvd event . the recurrence of cvd events , described herein , was analyzed in the 2,788 patients with a prior cvd event ( cohort a ) and in the 844 of the 11,644 patients without events at enrollment who developed their first cvd event during the observation period ( cohort b ) . the data used in this analysis were collected in four waves of follow - up between 2000 and 2003 . during the enrollment and follow - up visits , a standard questionnaire was used to collect , in addition to anthropometric data , information on lifestyle habits , drug therapy , laboratory measures ( specified below ) , clinical history , microvascular complications ( retinopathy , blindness , and foot ulcers ) , and cardiovascular complications . for patients who did not appear for the scheduled visits , information , including death , was obtained through telephone interviews with the patient , a relative , or the primary care physician . a1c was measured at each clinic , not in a centralized laboratory ; for this reason , glycemic control was estimated as the percent increment of the individual patient 's a1c above the upper limit of the normal range of the local laboratory . urinary albumin excretion ( uae ) was obtained in a timed overnight collection ; microalbuminuria was defined as uae of 30300 mg / l in at least three successive measurements in the absence of other known causes of proteinuria . hypertension was defined as systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg and/or antihypertensive treatment . retinopathy was assessed by a comprehensive dilated eye examination and by the acquisition of high - quality stereoscopic photographs assessed by an ophthalmologist . familial cvd was identified when the patient had a first - degree relative ( parent , sibling , or child ) who had had a major cardiovascular event at age < 55 years . alcohol consumption was calculated in equivalent milliliters of wine and transformed to grams per week ( 0 g / week , no consumption ; 1225 g / week , moderate consumption ; and 226 g / week , high consumption ) . study events were all major ihd events ( myocardial infarction , ptca , and cabg ) , minor ihd events ( angina and other forms of ihd ) , stroke , and limb amputations . patients were classified as having ihd if they had one of the following : 1 ) a history of hospital admission for either fatal or nonfatal myocardial infarction or an episode of angina ; 2 ) a 12-lead electrocardiogram with positive results for prior myocardial infarction or angina by the minnesota coding system ( criteria i 13 , iv 13 , v 12 , and vii 1 ) ; or 3 ) a history of cabg or ptca . all patients had had at least one electrocardiogram in the 12 months preceding enrollment to exclude prior myocardial infarction . stroke was defined according to world health organization criteria for confirmed and possible stroke ( i.e. , a clinical syndrome consisting of a rapidly developing neurological deficit persisting for > 24 h or leading to death , in the absence of other diseases that could explain the symptoms ) . a hospital discharge record or a specialist visit a cvd event was considered as recurrent if it occurred at least 28 days after the first event . a minor ihd event after another minor or a major ihd event was not counted as a bona fide recurrent event , i.e. , a new episode . similarly , a major ihd event after a minor ihd event was not considered as recurrent . such episodes were collectively indicated as events not classifiable as recurrent , in contrast with the truly recurrent cvd events . this choice was made on the grounds that , very often , angina and a subsequent myocardial infarction or revascularization are consequences of the same arterial lesion . the analysis was conducted separately in the two study cohorts ( a and b ) . the variables considered in the analyses were those collected at enrollment for cohort a and those collected at the follow - up visit at which the first cvd event was reported for cohort b. data for the continuous variables are expressed as means sd or median ( interquartile range ) for non - normal variables and as proportions for categorical variables . the incidence density of recurrent cvd events was standardized on the basis of the age distribution of the 1998 italian population . univariate and multivariate cox proportional hazards models were used to examine the risk factors for recurrent events . preliminary data analysis was performed with univariate cox models of all covariates : duration of diabetes , waist circumference , bmi , total cholesterol level , hdl cholesterol level , triglyceride level , a1c , blood pressure level , alcohol intake , smoke , familial cvd , microvascular complication , lipid - lowering treatment , antihypertensive treatment , and treatment of diabetes . the interaction between blood pressure level and antihypertensive treatment and between total cholesterol level and lipid - lowering therapy was evaluated . for cohort a , the overall model was also adjusted by the number of years from the first event to the enrollment to partially consider the patient 's history of cvd preceding the beginning of the study . all covariates with p 0.1 ( p 0.05 in cohort b , given the smaller sample size ) were entered into the final multivariate models . the data used in this analysis were collected in four waves of follow - up between 2000 and 2003 . during the enrollment and follow - up visits , a standard questionnaire was used to collect , in addition to anthropometric data , information on lifestyle habits , drug therapy , laboratory measures ( specified below ) , clinical history , microvascular complications ( retinopathy , blindness , and foot ulcers ) , and cardiovascular complications . for patients who did not appear for the scheduled visits , information , including death , was obtained through telephone interviews with the patient , a relative , or the primary care physician . a1c was measured at each clinic , not in a centralized laboratory ; for this reason , glycemic control was estimated as the percent increment of the individual patient 's a1c above the upper limit of the normal range of the local laboratory . urinary albumin excretion ( uae ) was obtained in a timed overnight collection ; microalbuminuria was defined as uae of 30300 mg / l in at least three successive measurements in the absence of other known causes of proteinuria . hypertension was defined as systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg and/or antihypertensive treatment . retinopathy was assessed by a comprehensive dilated eye examination and by the acquisition of high - quality stereoscopic photographs assessed by an ophthalmologist . familial cvd was identified when the patient had a first - degree relative ( parent , sibling , or child ) who had had a major cardiovascular event at age < 55 years . alcohol consumption was calculated in equivalent milliliters of wine and transformed to grams per week ( 0 g / week , no consumption ; 1225 g / week , moderate consumption ; and 226 g / week , high consumption ) . study events were all major ihd events ( myocardial infarction , ptca , and cabg ) , minor ihd events ( angina and other forms of ihd ) , stroke , and limb amputations . patients were classified as having ihd if they had one of the following : 1 ) a history of hospital admission for either fatal or nonfatal myocardial infarction or an episode of angina ; 2 ) a 12-lead electrocardiogram with positive results for prior myocardial infarction or angina by the minnesota coding system ( criteria i 13 , iv 13 , v 12 , and vii 1 ) ; or 3 ) a history of cabg or ptca . all patients had had at least one electrocardiogram in the 12 months preceding enrollment to exclude prior myocardial infarction . stroke was defined according to world health organization criteria for confirmed and possible stroke ( i.e. , a clinical syndrome consisting of a rapidly developing neurological deficit persisting for > 24 h or leading to death , in the absence of other diseases that could explain the symptoms ) . a hospital discharge record or a specialist visit a cvd event was considered as recurrent if it occurred at least 28 days after the first event . a minor ihd event after another minor or a major ihd event was not counted as a bona fide recurrent event , i.e. , a new episode . similarly , a major ihd event after a minor ihd event was not considered as recurrent . such episodes were collectively indicated as events not classifiable as recurrent , in contrast with the truly recurrent cvd events . this choice was made on the grounds that , very often , angina and a subsequent myocardial infarction or revascularization are consequences of the same arterial lesion . the analysis was conducted separately in the two study cohorts ( a and b ) . the variables considered in the analyses were those collected at enrollment for cohort a and those collected at the follow - up visit at which the first cvd event was reported for cohort b. data for the continuous variables are expressed as means sd or median ( interquartile range ) for non - normal variables and as proportions for categorical variables . the incidence density of recurrent cvd events was standardized on the basis of the age distribution of the 1998 italian population . univariate and multivariate cox proportional hazards models were used to examine the risk factors for recurrent events . preliminary data analysis was performed with univariate cox models of all covariates : duration of diabetes , waist circumference , bmi , total cholesterol level , hdl cholesterol level , triglyceride level , a1c , blood pressure level , alcohol intake , smoke , familial cvd , microvascular complication , lipid - lowering treatment , antihypertensive treatment , and treatment of diabetes . the interaction between blood pressure level and antihypertensive treatment and between total cholesterol level and lipid - lowering therapy was evaluated . for cohort a , the overall model was also adjusted by the number of years from the first event to the enrollment to partially consider the patient 's history of cvd preceding the beginning of the study . all covariates with p 0.1 ( p 0.05 in cohort b , given the smaller sample size ) were entered into the final multivariate models . during the 4-year follow - up , in cohort a , 414 of 2,788 patients ( who had entered the study with a known previous cvd event ) developed at least an event that fulfilled the stipulated criteria of a recurrent event . in cohort b , 54 of 844 patients ( who had had their first event during the study ) had a recurrent event . with regard to multiple events , 38 patients in cohort a and 4 in cohort b had two recurrent events , and only 1 patient in cohort a had three recurrent events . events occurring in 386 patients of cohort a and 46 patients of cohort b were not included in the analysis because they did not meet the required criteria for recurrent events ( almost all ihd events ) . as summarized in table 1 , the study population consisted of a high proportion of elderly patients with rather good glycemic control . the most common cvd risk factor was hypertension , which was found in almost all patients . in cohort a , patients with recurrent events were older , were more often male with a previous history of microvascular complications , and had more use of insulin and lipid - lowering medications . in cohort b , patients with recurrent events had higher total cholesterol and triglyceride levels , more often had a previous history of microvascular complications , myocardial infarction , or amputation , and had more use of insulin . of note , the age - standardized incidence rate of recurrent cvd was 40% lower in cohort b than in cohort a ( table 2 ) . the average time between enrollment and the occurrence of the recurrent event in cohort a was 1.6 1.04 years , whereas in cohort b the average time between the first and recurrent event was 1.5 0.8 years . the average time between the first and the recurrent event in cohort a was 1.6 1.04 years , whereas the average time between the first and the recurrent event was 8.1 6.9 years in cohort a and 1.5 0.8 years in cohort b. in cohort a , the standardized incidence of recurrent episodes was 89.2 per 1,000 person - years ( 95% ci 71.3107.2 ) among patients with a major ihd event , 11.4 ( 3.619.2 ) among those with a minor ihd event , 49.2 ( 35.562.9 ) among those with a prior stroke , and 79.8 ( 36.0123.5 ) among those with amputation or combined events . a multivariate cox model for cohorts a and b a , age , male sex , and use of insulin , alone or in combination with oral agents , were independent predictors of recurrence . however , having had a major ihd event as the first event was the strongest factor of all , with stroke and combined events also being powerful predictors , whereas time between first and recurrent events did not make a significant independent contribution . in cohort b , the risk factor pattern was similar to that of cohort a , with prior events carrying the highest risk . in this model , the only metabolic factor with an independent risk prediction was a serum triglyceride level 1.69 no interaction between blood pressure level and antihypertensive treatment or between total cholesterol level and lipid - lowering therapy was found in either cohort . given the dominant role of the first event as a predictor , the multivariate models were also run after excluding this variable . in cohort a , some difference emerged : receiving lipid - lowering therapy ( hazard ratio [ hr ] 1.32 [ 95% ci 1.071.63 ] ) , number of years from first event ( 1.57 , [ 1.212.03 ] for > 6 years ) , and living in southern italy ( 0.77 [ 0.620.96 ] ) became significant . in cohort finally , given its value from the clinician point of view ( although not considered in our analysis of recurrent events ) , we report the standardized incidence of a minor ihd event : 39.5 per 1,000 person - years ( 95% ci 30.348.7 ) in men and 57.1 ( 36.777.5 ) in women in cohort a and 21.3 ( 5.736.8 ) in men and 8.3 ( 2.414.3 ) in women in cohort b. in our population of diabetic patients receiving usual care , we found that every year , 6.1% of the patients with a prior cvd event developed a new major atherosclerotic complication . this percentage compares very well with those ( 5.96.0% per year ) of the cohorts of diabetic patients in secondary prevention analyzed in the proactive ( 4 ) study and the scandinavian simvastatin survival study ( 4s ) ( 7 ) . a higher rate of 7.6% per year was reported in the drugs and evidence - based medicine in the elderly ( debate ) study ( 8) , but that study was performed in subjects with a higher mean age ( 80 years ) . the incidence rate of recurrent events in cohort b was almost half that of cohort a. likely explanations of this difference are that among cohort b patients , there were more women , age was somewhat younger , familial cvd was less prevalent , and prior myocardial infarction was less frequent relative to prior stroke . moreover , and more importantly , these patients had a shorter and more recent cvd history and were observed for only 1.7 year on average . survivors of a cvd event that preceded the recurrent event by a longer span of time ; in fact , there was a remarkable difference in terms of number of years from first and recurrent event , which could be the explanation for this different outcome . . this result may be due to the greater predisposition to necrotic events of men than of postmenopausal women , who are more prone to nonmyocardial infarction ihd ( angina ) and , probably , heart failure ( 911 ) . in the framingham study , after the onset of angina , men had a twofold greater risk than women for both myocardial infarction and coronary death after adjustment for age and ihd risk factors ( 1 ) . we described this sex effect previously in the prevalence ( 5 ) and incidence analyses of the first ihd event ( 12 ) and stroke ( 13 ) in the dai population . in the present study , on the other hand , the incidence of minor ihd in cohort a was higher in women than in men . second , in the search for risk factors specific to recurrent major cvd , we found that age played an important role , with a 10-year difference translating into a 26% risk increment . the impact of age as a cvd risk factor in the general population is well known ( 1418 ) , but the current findings document the fact that age is an independent risk factor for relapsing major cvd in a population of diabetic patients not selected for age . the fact that use of insulin , alone or in combination with oral agents , was an independent risk factor for recurrence in cohort a should not be overlooked . this finding was also reported in the proactive cohort of type 2 patients with prior myocardial infarction who developed a second fatal or nonfatal myocardial infarction ( 4 ) and in the cardiovascular health study ( 19 ) , in which insulin was found to be associated with greater cvd mortality compared with oral agents . the association was true also when serum insulin concentrations were measured ( 20 ) . in the dai study , insulin treatment was an independent risk factor for ihd ( 12 ) and stroke ( 13 ) in patients free of cvd at baseline as well . on the other hand , the adverse prognosis associated with insulin use may be regarded as the result of an indication bias : more severe , longer - standing diabetes is preferentially treated with insulin and has a worse cardiovascular prognosis ( 21,22 ) . in fact , in clinical trials such as the diabetes control and complications trial in type 1 diabetic patients and the uk prospective diabetes study ( ukpds ) in type 2 diabetic patients ( 23,24 ) , insulin treatment had a clear - cut antiatherogenic effect . therefore , the possibility that prolonged use of insulin in these type 2 diabetic patients resulted in less cardioprotection than control of glycemia by other pharmacological means can not be ruled out . dyslipidemia , as indicated by a higher frequency of lipid - lowering treatment in cohort a and higher serum triglyceride levels in cohort b , emerged as an independent risk predictor . this finding resonates with the previous observation in the dai study ( 12 ) that high triglyceride levels are a significant predictor of the first ihd event in women without prior cvd . the dominant observation in the dai study seemed to be the strong impact of the type of the first cvd events on relapses an observation that emerged from both study cohorts . having had a major ihd event , alone or combined with another cvd event , was associated with the highest relative risk , possibly leaving little room for other modifiable factors . a limitations of this study is the lack of centralized laboratory measurements . also , given the fact that the observation period was relatively short , there may not have been time for other potentially significant factors to emerge . finally , information on other potential risk predictors such as lipoprotein levels , postprandial glucose excursions , or genetic markers was lacking . the strength of this study , in addition to the large size , is that it provides a detailed description of the burden of secondary cvd prevention in diabetic patients under real - life conditions . because in italy the percentage of patients who seek care at the hospital - based diabetes clinics is very high ( up to 80% of patients with known diabetes ) , our results can be confidently extrapolated to the entire type 2 diabetic population in the country . in summary , this nationwide observational study outlines the natural history of recurrent events in type 2 diabetic patients managed by usual care . first , diabetic patients , especially elderly men , whose cvd onset is myocardial infarction or revascularization ( or combined events ) warrant close follow - up and intensive management . third , diabetic patients with high triglycerides levels may be targeted by aggressive treatment in secondary cvd prevention .

objectivemany diseases form their basis during childhood . one example is the changes in vascular structure and function , leading to atherosclerosis . in this study , we have assessed the impact of exposure to cigarette smoke on blood pressure of elementary school children in kermanshah.methods80 elementary school children exposed to cigarette smoke and 80 not exposed to smoke were studied in fall 2010 . information regarding the smoking status of parents and the children s health were obtained through questionnaires completed by parents . after physical examination and exclusion of those children with acute and chronic diseases as well as those consuming medicine , we measured and compared blood pressure in the exposure and non - exposure groups . data were analyzed using the anova statistical test . values are expresses as meansd.findingsthe mean systolic and diastolic blood pressures of the exposure group were higher than those of the non - exposure group ( 109.39.97/64.927.36 vs105.478.98/62.57.01 , respectively ; ci : 0.95 , p<0.05 ) . meanwhile , difference between two groups according to sex was not statistically significant.conclusionour study indicates that systolic and diastolic blood pressures are higher in those elementary school children exposed to cigarette smoke compared to those who are not . objective the purpose of this study was to assess incidence of and risk factors for recurrent cardiovascular disease ( cvd ) in type 2 diabetes.research design and methods we estimated the incidence of recurrent cardiovascular events in type 2 diabetic patients , aged 4097 years , followed by a network of diabetes clinics . the analysis was conducted separately for 2,788 patients with cvd at enrollment ( cohort a ) and for 844 patients developing the first episode during the observation period ( cohort b).results during 4 years of follow - up , in cohort a the age - adjusted incidence of a recurrent event ( per 1,000 person - years ) was 72.7 ( 95% ci 58.387.1 ) in men and 32.5 ( 21.243.7 ) in women , whereas in cohort b it was 40.1 ( 17.462.9 ) in men and 22.4 ( 12.932.0 ) in women . after controls were included for potential predictors ( familial cvd , obesity , smoking , diabetes duration , glycemic control , microvascular complications , geographic area , and antihypertensive and lipid - lowering treatment ) , male sex , older age , and insulin use were significant independent risk predictors ( cohort a ) and serum triglyceride levels 1.69 mmol / l emerged as the only metabolic ( negative ) prognostic factor ( cohort b ) . in both cohorts , a prior cvd episode , especially myocardial infarction , was by far the strongest predictor of recurrent cvd.conclusionsapproximately 6% of unselected diabetic patients in secondary prevention develop recurrent major cvd every year . those with long - standing previous cvd show a higher incidence of recurrence . male sex , age , high triglyceride levels , and insulin use are additional predictors of recurrence .

electrophysiological brain signals are widely studied to get insights into the inner function of the brain . the electro - encephalogram ( eeg ) , as an example , has been analyzed for decades and is particularly popular because of its noninvasiveness , wide availability , relatively small cost and excellent temporal resolution which enables capturing the fast neural dynamics . because it is known that electrophysiological brain signals exhibit important spectral characteristics , frequency transforms are often applied . however , since in general brain signals do not possess the statistical property of stationarity , time - frequency transforms are of special interest . such methods are able to represent a given signal jointly in the time - frequency domain , called its time - frequency representation ( tfr ) . thereby the signal 's spectral components can be analyzed in relation to its temporal dynamics . in a wide sense an important feature of biological signals , and particularly brain signals , is their inter- and intraindividual variability . that is , under fixed experimental conditions , the obtained signals exhibit heterogeneousness not only between groups of subjects , but also between subjects within the same experimental group and even within the same subject between multiple experimental trials . existing signal analysis techniques either do not take this issue into account adequately or usually treat it by defining frequency bands of interest rather than single frequencies , and similarly time intervals instead of sharp instants . however , this strategy requires a priori knowledge about the variability to appropriately set the interval widths , and it is imprecise because it blindly includes all information contained in that time / frequency region . a good example is the time - frequency coherence analysis of two given input signals , for instance , by means of the cross short - time fourier transform , the cross wigner - ville distribution , or the wavelet coherence [ 1 , 3 ] . all of these methods relate both signals at fixed time / frequency ( or scale ) locations . thus , if signal a exhibits the same neural activation as signal b , but signal a 's pattern is shifted in frequency just a little , none of the abovementioned techniques will be able to find the strong similarity of a and b. although coherence estimation and other rigid strategies have been successfully applied for more than 30 years , this issue has inspired us to develop a general flexible method of pattern analysis and corresponding feature extraction in electrophysiological tfr data . by abstracting from the tfr images and working with the representation of a tfr pattern , tfr patterns reduce dimensionality by representing neural activity in a wide spectrotemporal region by comparably few quantities . pattern - based outlier detection has the potential to become a useful tool for data quality assurance . in ongoing studies we are employing the presented method , for instance , to estimate functional brain connectivity by means of a pattern - based approach . in the following , we present the developed neuroinspired interpretable model which is able to capture general time - frequency patterns . we use solely eeg data for demonstrations here , but our method is applicable to general electrophysiological signals or even to other signals showing similar behavior . algorithms for robustly fitting the novel model to time - frequency representations are presented . a strategy for finding an appropriate model order is given , and distance functions are defined which quantify ( dis-)similarity of two given models . these methods are tested in section 3 , where real as well as simulated data are used to demonstrate our technique 's functionality and robustness . while our technique is not restricted to specific time - frequency distributions , we employ the smoothed pseudo wigner - ville distribution in this work . this is a quadratic transform estimating signal power in the time - frequency domain , whereby all quantities in this work are real numbers . the transform generates quite smooth tfrs , which means that neighboring pixel values are correlated . although only positive values can be interpreted as signal power , the wigner - ville distribution introduces also negative values in general . we will refer to time - frequency representations as mappings y : t f which estimate signal power for each point in the time - frequency domain . of the numerous ways to quantify tfr patterns , we choose to fit a parametric surface to the data . because of the spatial correlation inherent in the data , traditional regression assumptions about independence of observations do not hold here . this absence of strong gradients in the tfr images also invalidates most image feature extraction techniques , which are often based on edges and texture . our model , however , is especially designed for spatially correlated data ; furthermore its parameters are interpretable . these quantities are useful features which embody important information about the underlying signal and thereby considerably reduce data dimensionality . using our method , we propose an extension of the well - known gaussian model for tfr analysis . the gaussian model for multivariate data x is defined by ( 1)y(g)(x)=c+a exp(12(x)t1(x ) ) with c being a constant additive offset , a the amplitude relative to the offset , the constant n - dimensional mean vector , and denoting a n n symmetric positive definite matrix . positive definiteness ensures that the argument to the exponential function is always negative ; additionally we know that e is bounded by zero and one for negative x. therefore , the exponential factor scales the final amplitude between 0 and a relative to the offset c. the term ( x)(x ) is also known as the squared mahalanobis distance of x with respect to and . because in our context this function represents arbitrary data in contrast to statistical distributions , will also be called the position vector , and is the spread matrix , its entries ij are denoted spread parameters . firstly , the model will be shaped like a peak for all possible parameter values by imposing the constraint that ( and thus also ) is symmetric positive definite . thereby , the model will never be able to completely degenerate . because the model is not flexible enough to fit small local variations of an expected pattern , the gaussian model is relatively insensitive to local data outliers and is also unsusceptible to overfitting . an additional aspect of robustness is that extreme peak deformations are directly reflected in extreme parameter values . thereby degenerated models can be easily detected or may even be prevented by imposing parameter constraints . the gaussian model is well - suited to extract bivariate peaks from brain signals ' tfr data , reflecting short intervals of neural excitement in a specific frequency range . an instance of the above described surface , in the bivariate case , is fully identified by its parameter vector ( 2)p=(c , a,1,2,11,12,22)t . the absolute peak height c + a , the peak position ( 1 , 2 ) , and the peak orientation can be derived from the parameter vector . further relevant quantities are the temporal peak onset , peak offset , and peak duration ( as the difference of the previous two ) . as already mentioned , the gaussian model 's robustness comes at the cost of inflexibility . while some local effects in tfr data can be appropriately explained by ( 1 ) , more general patterns of activation do not follow peak - like shapes , as will be shown later . thus a generalization of the gaussian model would be desirable , especially concerning the ability to represent patterns of activation rather than just independent events in the spectrotemporal domain . at the same time , a generalized method should maintain maximum robustness in order not to degenerate easily and to prevent overfitting . the so - called gaussian mixture modeling ( gmm ) is a straightforward extension , but this method still assumes ( multiple- ) peak - shaped data . in the following , the smooth natural gaussian extension ( snage ) model is presented as a flexible extension of the multivariate gaussian model . before giving a formal definition , we explain the idea in an intuitive way , guided by figure 1 . the n - variate gaussian model can be described by its ( n + 1)-dimensional peak point p = ( 1 , , n , a ) and its spread parameters controlling the exponential flattening relative to p along the independent variables ' dimensions . now the idea is to not use only one , but k peak points p , i = 1 k , interpolated by a smooth ( n + 1)-dimensional curve of peaks . a way to think of the surface in figure 1 modeled by snage is to shape it by sliding an n - variate gaussian model along the curve , its peak point being connected to the curve and thus varying in height ( a ) and position ( vector ) . thereby complex smoothly bent patterns of data with varying amplitude ( dependent variable ) can be captured . analogously to the gaussian model , an n n spread matrix determines the model 's shape , which is a surface for the tradeoff between robustness and flexibility can be controlled by choosing the number of peak points k. using many peak points will allow for good fits to complex patterns but will also increase the danger of overfitting . choosing a small k yields a robust model , but its ability to capture complex patterns will be limited . by setting k = 1 , snage reduces to the traditional gaussian model as a special case . regarding the standard gaussian model , each function value y(x ) is fully determined by the mahalanobis distance of the point x to the unique mean point . but since the snage model offers infinitely many mean points this issue will be addressed in the next section , where a formal definition is given . let the number of peak points be denoted by k , k 1 . let : [ 1 , k] denote a smooth n - dimensional curve of means , and let a : [ 1 , k] be a smooth one - dimensional curve of amplitudes along . let further the offset c , and let be a symmetric positive definite matrix . define ( 3)y~(snage):n[1,k]y~(snage)(x , u)c+a(u ) exp(12(x(u))t 1(x(u ) ) ) . note that y~(snage ) is a family of traditional gaussian models , parameterized by the curve parameter u. in order to construct a function which is independent of u , we define ( 4)u(x)argmaxu[1,k]|y~(snage)(x , u)c| . the snage model is then given by ( 5)y(snage):ny(snage)(x)y~(snage)(x , u(x ) ) . the function u*(x ) defines that traditional gaussian model which assigns to x the largest absolute amplitude relative to the offset c among all members of the gaussian family y~(snage ) . this is necessary to cope with positive as well as negative a(u ) . in tfr analysis , a(u ) can be restricted to only positive values ( see section 2.3.1 ) , in which case ( 5 ) in fact simplifies to ( 6)y(snage)+(x)maxu[1,k]y~(snage)(x , u ) . for y as well as y the max ( ) function curve , in the sense that ||(u1 ) (u2)|| is small , but |a(u1 ) a(u2)| is large . we assume that the diagonal entries of the spread matrix , that is , the spread along each dimension , are sufficient to control a tfr pattern 's width . therefore , we fix offdiagonal entries to zero for the sake of robustness . thereby , the mahalanobis distance in ( 3 ) reduces to the weighted euclidean distance . the two curves a(u ) and (u ) are yet to be defined in terms of discrete parameter values . for good parameter interpretability , we choose to form both curves by interpolating k points p , i = 1 k by b - splines of degree 3 , which yields the curve : ( 7)p:[1,k]n+1 , p(u)=(p1(u) pn(u)pn+1(u))=(1(u) , we obtain a sufficiently smooth model which inherits both the splines ' flexibility and the gaussian standard model 's robustness . our model further inherits the b - splines ' local control property ; that is , varying a p will affect the model only in the p 's vicinity . additionally , the degree of flexibility can be adapted to the data at hand by varying k from 1 ( single gaussian peak ) to arbitrary flexibility with k > 1 . an instance of the n - variate snage model with k points ( or of order k ) is fully represented by the parameter vector of length 1 + n + ( n + 1)k : ( 8)p=(c,11,22, ,nn , p1(1), while the offset c and the spread parameters are mainly responsible for the prediction of data values , the curve p(u ) interpolating the p is directly interpretable as it models the main path of peaks in the data . in the next section we show how to fit the model to data in a robust manner . given a time - frequency representation y(x ) , x t f , we aim to find a parameter vector p so that the respective model y fits the data best , in the sense that it minimizes a cost function . we use the sum of squared differences of the data and the modeled surface : ( 9)sse(p)=x=(t , f)ttt , ff(y(tfr)(x)y(snage)(x))2 . this quantity is also called the sum of squares due to error which is zero if the model perfectly fits the data . sse is a nonlinear function dependent on p. using squared differences pronounces outliers , but these are not expected to occur frequently in our smooth tfr data . in order to find a locally minimum solution of sse , a nonlinear least squares algorithm implemented in the matlab optimization toolbox , lsqnonlin , given an initial parameter vector and the cost function sse , this optimizer produces a sequence of models pi . the iteration hopefully converges to a p * with minimum cost , that is , best resemblance between model and data . we attempt to provide advantageous starting conditions for the optimizer by preprocessing the tfr and by obtaining an initial parameter vector p0 which is expected to be close to the optimum with respect to sse . moreover , an iterative refinement scheme is proposed to be able to robustly fit models of high order . tfr data are badly scaled , showing differences of several orders of magnitude in values of time , frequency , and signal power , which affects optimization performance . to address this problem , lsqnonlin offers a way to take into account typical values for each dimension for gradient estimation . also concerning this issue , any euclidean distance operating on tfr data in our algorithms is weighted appropriately . furthermore , smooth objective functions are desirable so that the low - order taylor approximations used during optimization resemble the cost function in a relatively large neighborhood around the current point . to this end , the tfr images are smoothed and subsampled , which has the additional benefit of faster cost function evaluations . finally , since negative values in the time - frequency domain are not interpretable , they are usually set to zero . since lsqnonlin performs local optimization , a start parameter vector should be chosen such that it already lies in the basin of the cost function 's ( unknown ) global minimum , that is , a vector whose cost is already low . to this end , the constant offset and the spread parameters are initialized to c = 0 , 11 = ( tmax tmin)/5 and 22 = ( fmax fmin)/5 if the underlying tfr 's time and frequency axes are bounded by tmin , tmax and fmin , fmax , respectively . one may choose any sensible values alike , but the spreads should not be initialized too small in order to obtain a generalizable model . yet , more thought has to be put in choosing the number and coordinates of the peak points p. in the following we propose a way to compute initial estimates of the time , and frequency coordinates of all p directly from the data . a reasonable approximation to the unknown optimal curve of peaks can be found by tracing a path through the tfr image y from left to right , which runs through areas of high pixel intensity . more precisely , the sum of all pixel intensities along this path should be as high as possible . this is an optimization problem in turn , yet its solution can be computed in quadratic time complexity ( provided that the path 's slope is bounded ) by a dynamic programming algorithm , see . because a global optimum is guaranteed to be found , this strategy is insensitive to local outliers and noise . to this end , a similar approach as described in is employed . following the notation therein , we define our energy function to be equal to the tfr values themselves , that is , e(i ) = i. a horizontal path s * ( called seam in ) which maximizes ( this is in contrast to , where minimum energy seams are computed ) this simple cost function is found by dynamic programming . see figure 3 for an example . additionally , the paths are constrained by imposing an upper bound k on their slopes . this value depends on the time - frequency resolution here and once more represents a compromise between robustness and flexibility . given the found path s * and the desired model order , k evenly spaced samples are subsequently drawn from a smooth approximating curve to obtain estimates of the first two coordinates of the p , i = 1 k. we choose to empirically set the ps ' last components , interpretable as amplitudes relative to the initial constant offset c = 0 , to max(y ) c = max(y ) . once a parametric representation of the data is available , its accuracy can be improved in a step - wise manner , as is presented in the following section . as already stated , the number of points p controls the model 's robustness which complements its ability to resemble complex patterns . therefore , the demand for a near - optimal initial parameter vector increases with the model order k. employing the optimal image path method described in the previous section yields a reasonable estimation , but sampling k equidistant points p , i = 1 k from the resulting curve is a simplification . in fact , it can be observed that the optimizer tends to concentrate the p in time - frequency regions of high signal variability . for low model order k , this shortcoming of our initial parameter estimation algorithm can be compensated easily by the optimization algorithm , but it may become a problem for increasingly flexible models . for this reason we propose an iterative scheme.find initial parameters p0 by means of an optimal path ( see section 2.3.2 ) for a first , robust model of low order k0 . let i0.fit the model to the data to obtain optimal parameters pi*.construct the optimal curve of peaks p(u ) by interpolation of the peak points ( see section 2.2.1).obtain the ki+1 = ki + 1 peak points for a refined model by uniformly sampling ( with respect to the spline 's sites uj ) the curve computed in the previous step.construct the parameter vector pi+1 of the refined model from pi by replacing the old ki peak points with the ki+1 new ones.if ki < kmax , let ii + 1 and continue with step 2 . find initial parameters p0 by means of an optimal path ( see section 2.3.2 ) for a first , robust model of low order k0 . fit the model to the data to obtain optimal parameters pi*. construct the optimal curve of peaks p(u ) by interpolation of the peak points ( see section 2.2.1 ) . obtain the ki+1 = ki + 1 peak points for a refined model by uniformly sampling ( with respect to the spline 's sites uj ) the curve computed in the previous step . construct the parameter vector pi+1 of the refined model from pi by replacing the old ki peak points with the ki+1 new ones . the curve found in steps 2 and 3 will exhibit smaller gradient magnitude , that is , traversal speed , in areas of high signal variability than in other regions . we aim at maintaining the curve - defining points ' optimum distribution found by the fitting algorithm and at enhancing the model 's flexibility mainly in these areas . it turns out that simply by uniformly sampling the fitted curve ( step 4 ) we obtain a new interpolated curve which retains these properties . an application of this algorithm is demonstrated in section 3.1 , where the resulting sequence of nested models is evaluated . in this section distance measures are necessary , for instance , to quantify how well the data exhibit an expected pattern . distance functions which are based solely on the curve of peaks p(u ) were found to be quite effective . other possibilities include parameter vector distances and pixel - wise differences of signal power of the models ' generated data . by comparison , curve - based distance functions have the advantage of being able to interrelate models of different orders . additionally , they are not influenced by the less informative parameters ( offset c and the entries of ) . a popular distance measure for parametric curves is the frchet distance . in the continuous case , the frchet distance of two parametric curves p1(u ) and p2(u ) is defined by ( 10)df_max(p1,p2)=inf, maxu d ( p1((u)),p2((u ) ) ) . here , (t ) and (t ) are monotone reparameterizations of the two curves , and d ( ) denotes ( weighted ) euclidean distance . in words , we search for those reparameterizations which make the curves the most similar with respect to maximum point - wise euclidean distance along the curves . this maximum for these reparameterizations is returned as the two curves ' continuous frchet distance . in practice , the discrete frchet distance is frequently applied , whose computation is based on dynamic programming once more . in the discrete case , an additional distance function df_sum(p1 , p2 ) can be obtained by replacing the max function with a sum over u. that way , df_sum represents an average distance , being less prone to outliers in the curves . we demonstrate the workflow to determine the appropriate model order k by fitting a tfr of real eeg data in this section . typically we determine the necessary model complexity by fitting data with good signal to noise ratio ( snr ) in order to prevent the overestimation of k. for example , one possibility to achieve sufficient data quality is to average several tfrs which are expected to show similar patterns . the averaged tfr of real eeg data shown in figure 5 will guide the following explanations . the depicted brain signals located in the lower frequency bands were recorded from the temporal brain region during a face recognition experiment . these data exhibit a pattern of activity which is too complex to be captured by a traditional gaussian model . the iterative scheme described in section 2.3.3 an optimal path ( see section 2.3.2 ) estimates the initial parameters for the first , least flexible model of order k0 . since the appropriate k is still unknown , a sufficiently large number kmax = 7 is chosen for the refinement . at each refinement stage the respective model is evaluated , and in the end the most suitable ( 11)k{k0, ,kmax } , k0kkmax is chosen as the model order for future fittings on lower - quality data . these are the cost function value ( sse , see section 2.3 ) , the coefficient of determination r and its adjusted version radj . although the use of quantities based on the coefficient of determination is discouraged for nonlinear models , they are applied here nonetheless for two reasons . they are found to perform well for our purposes , and the proposed alternatives ( aic and bic ) are not easily applicable here . this is because the assumption of normally distributed residuals often does not hold , which is supported by a highly significant shapiro - wilk test at = 5% yielding p < 10 for this experiment . the results show that for the data at hand a model of order k * = 4 is sufficient to capture the variability . having determined the maximum model complexity on high - quality data , such a model can now be fitted to the rest of the data . if the tfrs are not expected to vary substantially , like when fitting a model to signals from several nearby sensors , a previous fit may serve as the initial model . however , if , for instance , multiple data segments of the same sensor should be fitted , the tfrs ' patterns may vary strongly . in this case , initial parameters should be chosen depending on the data by using the method of optimal paths described in section 2.3.2 . since in this example k * = 4 is a quite moderate number , the iterative refinement may also be skipped . however , in general we would start with k0 = 3 or k0 = 4 and refine up to the determined k * , as proposed in section 2.3.3 . we want to assess our model 's robustness by simulating data and measuring how strongly the model is affected by additive gaussian noise . to this end , artificial data are created in the time domain , and their tfrs are computed to which our model will be fitted . we created a signal consisting of three consecutive oscillations , representing an alpha - theta - alpha eeg pattern at 10 hz/4 hz/10 hz respectively over a time span of 2.5 seconds . the simulated sample rate is 250 hz . a plot is shown in figure 9 . these data are quite challenging for our model because three distinct peaks emerge in the tfr which could be more appropriately modeled by a mixture of independent gaussian peaks . however , we want to demonstrate the flexibility of the snage model which should also be able to cope with patterns of this form . for the following experiments we chose to start the fitting with a model of order k = 5 to account for the pattern 's complexity and perform one refinement step . initial parameters are estimated by finding optimal paths , which means that no a - priori information about the known optimal model is passed to the fitting procedure other than the number of p to use . we define the optimal model by fitting the noise - free simulation in the same way . the distance measures from section 2.4 are used to determine how well the simulated pattern is found . in this experiment we added gaussian noise , which is appropriately filtered with respect to the sampling frequency , to the simulated data in the time domain . signals exhibiting signal to noise ratios of 15 db up to + 10 db were generated in steps of 2.5 db . at each snr , ten distinct noise realizations are created to obtain representative results . this independent noise in the time domain will produce correlated noise in the time - frequency domain due to smoothing . this experiment serves to assess how strongly our algorithm is affected by pattern variability , respectively , to investigate its robustness . small pattern distortions should ideally only slightly alter the optimal model , reflecting its robustness and avoidance of overfitting . we further want to find out to what degree our model is able to find the simulated pattern at all . we note here that adding noise increases the tfrs ' maximum amplitudes exponentially which strongly affects the comparability of different models . without normalization but this would merely reflect the decreasing data amplitudes and contain no information about the quality of fit . normalizing maximum data values are not an appropriate option either , because , for negative snrs , this would keep the noise constant while exponentially shrinking the pattern 's pixel intensities . even if the optimal model was perfectly recovered from the noisy simulation , high distances only if signal power is excluded from model distance estimation , the returned values are useful representatives of how well the pattern was found . the snage 's robustness to noise with respect to the pattern 's power is therefore not regarded here . this is done by setting the third dimension of the path of peaks p(u ) to zero during frchet distance computation . convergence to the optimal model seems to require high signal to noise ratios . at 7.5 db , the distance measures ' variances fall off , reflecting the point of reliable pattern extraction . apparently , the noise and interferences introduced in this experiment considerably impair the fitting process . in figure 11 , this issue is exemplarily investigated . at the positive snr of 5 db , where distance variances across the noise realizations are still high the pattern was in fact found , but only in a different way than was expected . nevertheless , this example shows that the impact different kinds of noise may have on the fitting process . to get a better feel for the average ability to fit the pattern under the influence of noise , see figure 12 . at each noise level , shown is a sequence of mean models which progressively look more similar to the true pattern . in fact , the average fitting capability concerning both the positioning of the peak points in the time - frequency domain and the estimation of surface values is better than expected after having studied figure 10 . apparently , although the mean distances are still decreasing at negative signal to noise ratios , they are already small enough for successful pattern extraction on average . an example is the subplot corresponding to snr = 0 db in figure 12 , which already clearly resembles the simulated pattern.this experiment shows that interferences between the desired signal and additive noise affect the fitting process quite strongly in the worst case . positive signal to noise ratios of at least 7.5 db are found to be necessary for reliable pattern extraction in this investigation . however , successful data modeling is also possible at lower snrs , as is seen in the average case . the snage model is especially suited for time - frequency representations of electrophysiological signals because of their ( expected ) nonnegativity , smoothness and their patterns following a path of peaks . however , our robust model is able to cope with data which do not exactly meet these requirements . in order to retain robustness , we imposed several restrictions on our model , like neglecting offdiagonal spread parameters and holding the spread matrix constant over the curve of peaks . an interesting question remains how the model 's flexibility and robustness would be affected if these constraints were dropped . little effort would be necessary to include the stated extensions . as is typical for nonlinear optimization problems , therefore , a - priori knowledge about the optimal model can be incorporated by starting the optimization with a model which was previously fitted to similar data . moreover , an algorithm based on an optimal path is developed to estimate initial model parameters directly from the data . however , this method is limited to positive peak polarity by trying to maximize the path 's average amplitude . additionally , the technique will not be able to find initial models which exhibit multiple contemporary components . in such a case , the nonlinear optimization algorithm , which was found to work well , must compensate . further strategies for the estimation of initial parameters would be desirable . in particular , the extraction of the curve interpolation points p from the optimal path possesses potential for improvement . an open question is how we should deal with the spatial correlation of both the dependent variable and the residuals in a statistical inferential context . further work is necessary to facilitate statistical testing , for instance , to assess the null hypothesis that an expected pattern is not contained in the data . concerning the presented measures of model distance , the frchet distances were found to be very useful to assess model similarity in our experiments involving simulated noise . their distinct advantage is their independence of model order and the disregard of the less interpretable parameters . on the other hand , spurious high distances this can be attributed to the fact that the simulated data exhibit three independent peaks , which is a violation of the snage 's assumption of a connected path of peaks . therefore , a combination of frchet values and a pixel - wise distance function based on the models ' generated data seems advantageous . when applied to noisy time signals , the snage model adapts too well to the corresponding smooth time - frequency representations . because the optimized cost function does not take into account information about the expected pattern , the model simply tries to capture the tfr data as accurately as possible . data preprocessing and tfr interference suppression are therefore extremely important . adding penalty terms to the cost function and/or providing explicit initial parameters are ways to point the optimizer in the right direction . however , even without specifying a - priori knowledge , the model was able to find the simulated pattern for low signal to noise ratios in the average case . the analysis of time - frequency representations of electrophysiological signals calls for flexible methods accounting for inter- and intraindividual data variability . we present the flexible , robust , and interpretable model snage , which extends the established gaussian model . its ability to extract 3d features from time - frequency representations of electrophysiological data is demonstrated . however , the model applies to general multivariate data which exhibit similar behavior . in this work , several techniques to improve the model fitting performance are described . we show how to estimate start parameters directly from the data . an iterative scheme to refine optimized models experiments with real as well as simulated data demonstrate the snage model 's robustness and flexibility . under the influence of severe noise , the developed technique is best suited for patterns which are too complex to be appropriately captured by a gaussian model , but still simple enough to facilitate robust fits . to summarize , due to its robustness and flexibility the snage model possesses the potential to become a beneficial tool for practical eeg / meg analysis , including functional brain connectivity analysis , outlier detection , time - frequency denoising , and feature extraction . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request . lumbar hernias are rare and good history and general physical examination can rule out most of the differential diagnosis.ct scan should be done as routine prior to planning surgical approach unless patient is in life threatening emergency situation.even though the data available for us regarding the management of lumber hernias are limited current literature suggests laparoscopic approach is the best method of treatment.open surgery should be reserved for patients who have very large defects or failed laparoscopic approach.the operative technique should be decided on the size of the hernia , location , contents , etiology , recurrence and availability of facilities and expertise of the hospital . lumbar hernias are rare and good history and general physical examination can rule out most of the differential diagnosis . ct scan should be done as routine prior to planning surgical approach unless patient is in life threatening emergency situation . even though the data available for us regarding the management of lumber hernias are limited current literature open surgery should be reserved for patients who have very large defects or failed laparoscopic approach . the operative technique should be decided on the size of the hernia , location , contents , etiology , recurrence and availability of facilities and expertise of the hospital .

electrophysiological signals such as the eeg , meg , or lfps have been extensively studied over the last decades , and elaborate signal processing algorithms have been developed for their analysis . many of these methods are based on time - frequency decomposition to account for the signals ' spectral properties while maintaining their temporal dynamics . however , the data typically exhibit intra- and interindividual variability . existing algorithms often do not take into account this variability , for instance by using fixed frequency bands . this shortcoming has inspired us to develop a new robust and flexible method for time - frequency analysis and signal feature extraction using the novel smooth natural gaussian extension ( snage ) model . the model is nonlinear , and its parameters are interpretable . we propose an algorithm to derive initial parameters based on dynamic programming for nonlinear fitting and describe an iterative refinement scheme to robustly fit high - order models . we further present distance functions to be able to compare different instances of our model . the method 's functionality and robustness are demonstrated using simulated as well as real data . the snage model is a general tool allowing for a wide range of applications in biomedical data analysis . lumbar hernias are uncommon and about 300 cases have been reported till date . they commonly occur due to trauma , surgery and infection . they are increasingly being reported after motor vehicle collision injuries . however , spontaneous lumbar hernias are rare and are reported infrequently . it is treated with different surgical approaches and methods . we report a case of primary spontaneous lumbar hernia which was repaired by transperitonial laparoscopic approach using vypro ( polypropylene / polyglactin ) mesh and covered with a peritoneal flap .

gastroesophageal reflux disease ( gerd ) is one of the most frequently occurring benign functional disorders in western industrial countries . the effectiveness of laparoscopic antireflux surgery for recalcitrant gerd has been clearly demonstrated in several series . these laparoscopic results , in combination with a shorter hospital stay and a more rapid return to normal activities , have promoted the emergence of minimally invasive antireflux surgery as the method of choice for the operative management of gerd . antireflux surgery has a higher failure rate in mo , which is in direct relation to high body mass index . the increased intraabdominal pressure and the morbid obesity - related comorbidities lead to a higher failure rate of the standard antireflux procedures in this group of patients . over the past 40 years , surgery has become the most effective long - term treatment for morbid obesity . the national institutes of health during their consensus development conference on gastrointestinal surgery for morbid obesity in 1991 recognized the role of bariatric surgery in the treatment of highly selected , well - informed , motivated patients who are acceptable operative risks and fail or are likely to fail a medical weight loss program . bariatric operations allow for substantial weight loss , extended weight maintenance , and control or reversal of obesity - related health problems . several series have now reported that lrygbp improves gerd symptoms , but few have included standardized quality of life tools . the objective of this study was to evaluate the efficacy of lrygbp as an antireflux procedure on gerd - related symptoms in morbidly obese patients by using a heart - burn - related quality of life score and other standardized outcomes tools . patients with recalcitrant gerd and a bmi greater than 35 were offered lrygbp or nissen fundoplication . patients who chose lrygbp an extensive preoperative evaluation , including history and physical examination , the usage of antacid medication and its efficacy , nutritional and psychiatric evaluation , and indicated specialty consultations , was performed before surgery . all the patients had an upper endoscopy or upper gastrointestinal imaging to document and evaluate their gerd severity and upper gi anatomy . laboratory evaluation included complete blood count , serum chemistries , and thyroid function testing ; 24-hour ph monitoring was done in select patients . patient preparation for surgery consisted of a detailed explanation in written and oral form of the developmental aspect of lrygbp and its benefits and risks , including short- and long - term complications , side effects , nutritional sequelae , and the possibility of conversion to the open procedure . prophylaxis against venous thrombosis and pulmonary embolus consisted of perioperative pneumatic compression devices and low - dose subcutaneous heparin . data were collected prospectively and verified retrospectively , then entered into a customized computer database . data sources included office charts , follow - up notes , hospital charts , and patient interview in the outpatient clinic and phone interviews . parameters included patient demographics , comorbidity , hospital stay , recovery , complications , weight loss , gerd symptoms , and the need for antacids . quality of life changes and patient satisfaction were evaluated using the heart burn related quality of life ( hrqol ) and sf-36 quality of life questionnaires . recovery was defined as the number of days after surgery when patients resumed common activities of daily living , such as driving , shopping , household activities , and employment . follow - up weights were obtained from the university of pittsburgh surgical weight loss clinic scale with a capacity of 400 kg . on occasion the patient was placed in a supine position with the surgeon on the right and the assistant on the left , and 2 monitors were above the patient 's shoulders . after creation of carbon dioxide pneumoperitoneum ( 15 mm hg ) using the open hasson technique , cannulas ( u.s . the operating table was placed in a steep reverse trendelenburg position . to expose the esophagus and stomach , a 5-mm liver retractor ( genzyme , tucker , ga ) was placed through the inferior right subcostal port , and the left lateral segment of the liver was elevated . gastric pouch creation was performed . to localize the esophagogastric junction and size the pouch , then , a window was created in the lesser omentum near the gastric wall at the lesser curvature . the endo gia stapler ( u.s . surgical ) , 60-mm length and 4.8-mm staples , was inserted and applied 3 or 4 times to staple and cut the gastric pouch with 3 rows of staples on each side . a smaller staple size ( 3.5 mm ) was later substituted to reduce staple line bleeds at the transected stomach . the greater omentum and transverse colon were passed to the upper abdomen to expose the ligament of treitz . to create the roux limb , the jejunum was transected with an endo gia ii stapler ( u.s surgical ) , 45-mm length and 3.5-mm staples , at approximately 50 cm from the ligament of treitz , where a comfortable length of mesentery exists . a smaller staple size ( 2.5 mm ) the jejunal mesentery was then divided with 2 applications of the endo gia ii stapler , using the vascular load ( 45-mm length , 2.0-mm staples ) . a 6-cm length of penrose drain a window was created in the meso - colon immediately anterior and lateral to the ligament of treitz to gain access to the lesser peritoneal sac . the roux limb was then passed in a retrocolic retrogastric fashion to lie next to the gastric pouch ( figure 2 ) . the gastrojejunostomy was then created using the endo gia ii stapler , and the gastrojejunostomy anastomosis was closed with interrupted 20 surgideck suture material ( u.s . the gastrojejunostomy and enterotomy site were endoscopically inspected and tested for leakage after insufflation and submerging them in irrigation fluid . the roux limb was then measured 150 to 180 cm distally , and a stapled side - to - side anastomosis was created with the proximal jejunal limb using 1 application of the endo gia stapler ii ( 60-mm length , 3.5-mm staples ) . the enterotomy sites were stapled closed , and the mesentery of the jejunojejunostomy was sutured closed ( figure 2 ) . the laparoscopic port placements and their use during the procedure . the completed gastric bypass : gastric pouch - jejunal anastamosis , the jejunojejunostomy , closure of the mesentery . pain management consisted of morphine patient - controlled analgesia ( pca ) intravenously as needed . an upper gastrointestinal series was performed on the morning of the first postoperative day using gastrografin followed by barium . a clear liquid diet was begun that day , and the patient was discharged from the hospital after demonstrating tolerance for the diet and return of bowel function , usually on the second postoperative day . patient follow - up was scheduled for every 2 months , with laboratory evaluation every 6 months , until weight loss stabilized ( usually 1 to 1.5 years after surgery ) , then twice per year . all the patients are routinely treated with a low dose of 150 mg / day ranitidine hydrochloride , to prevent anastomotic ulcers . the patient was placed in a supine position with the surgeon on the right and the assistant on the left , and 2 monitors were above the patient 's shoulders . after creation of carbon dioxide pneumoperitoneum ( 15 mm hg ) using the open hasson technique , cannulas ( u.s . the operating table was placed in a steep reverse trendelenburg position . to expose the esophagus and stomach , a 5-mm liver retractor ( genzyme , tucker , ga ) was placed through the inferior right subcostal port , and the left lateral segment of the liver was elevated . gastric pouch creation was performed . to localize the esophagogastric junction and size the pouch , then , a window was created in the lesser omentum near the gastric wall at the lesser curvature . the endo gia stapler ( u.s . surgical ) , 60-mm length and 4.8-mm staples , was inserted and applied 3 or 4 times to staple and cut the gastric pouch with 3 rows of staples on each side . a smaller staple size ( 3.5 mm ) was later substituted to reduce staple line bleeds at the transected stomach . the greater omentum and transverse colon were passed to the upper abdomen to expose the ligament of treitz . to create the roux limb , the jejunum was transected with an endo gia ii stapler ( u.s surgical ) , 45-mm length and 3.5-mm staples , at approximately 50 cm from the ligament of treitz , where a comfortable length of mesentery exists . a smaller staple size ( 2.5 mm ) the jejunal mesentery was then divided with 2 applications of the endo gia ii stapler , using the vascular load ( 45-mm length , 2.0-mm staples ) . a 6-cm length of penrose drain was sewn to the end of the roux limb using the endostitch ( u.s . a window was created in the meso - colon immediately anterior and lateral to the ligament of treitz to gain access to the lesser peritoneal sac . the roux limb was then passed in a retrocolic retrogastric fashion to lie next to the gastric pouch ( figure 2 ) . the gastrojejunostomy was then created using the endo gia ii stapler , and the gastrojejunostomy anastomosis was closed with interrupted 20 surgideck suture material ( u.s . the gastrojejunostomy and enterotomy site were endoscopically inspected and tested for leakage after insufflation and submerging them in irrigation fluid . the roux limb was then measured 150 to 180 cm distally , and a stapled side - to - side anastomosis was created with the proximal jejunal limb using 1 application of the endo gia stapler ii ( 60-mm length , 3.5-mm staples ) . the enterotomy sites were stapled closed , and the mesentery of the jejunojejunostomy was sutured closed ( figure 2 ) . the laparoscopic port placements and their use during the procedure . the completed gastric bypass : gastric pouch - jejunal anastamosis , the jejunojejunostomy , closure of the mesentery . pain management consisted of morphine patient - controlled analgesia ( pca ) intravenously as needed . an upper gastrointestinal series was performed on the morning of the first postoperative day using gastrografin followed by barium . a clear liquid diet was begun that day , and the patient was discharged from the hospital after demonstrating tolerance for the diet and return of bowel function , usually on the second postoperative day . patient follow - up was scheduled for every 2 months , with laboratory evaluation every 6 months , until weight loss stabilized ( usually 1 to 1.5 years after surgery ) , then twice per year . all the patients are routinely treated with a low dose of 150 mg / day ranitidine hydrochloride , to prevent anastomotic ulcers . from february 1999 to april 2001 , 57 patients with a median bmi of 43 underwent attempted lrygbp at the thoracic division of the university of pittsburgh medical center . patient demographics ( n=57 ) hiatal hernia or esophagitis , or both , were present in 48 patients ; barrett 's esophagus was documented in 2 . lrygb was possible in 52 patients ; 5 required open conversion , 3 due to multiple adhesions and 2 for poor exposure . complications included 1 leak , 1 pulmonary emboli , 2 reoperations for internal roux intestinal limb hernia , and 7 gastrojejunal strictures , which were treated with endoscopic dilatations , average of 1 postoperative dilatation ( range , 1 to 3 ) . no deaths occurred . at a mean follow - up of 18 months ( range , 3 to 30 ) , all patients report improvement or no symptoms of gerd and a mean weight loss of 40 kg ( range , 16 to 70 ) . previous and current antacid medication usage is summarized in table 2 . before and after surgery antacid medication usage quality of life scores ( sf-36 ) were above national norms for physical and mental components ( median=55 , mode=50 ) . the gerd - health related quality of life median score was < 1 ( scale , 0 to 45 , 0=asymptomatic , 45=worse ) ( table 3 ) . excessive body weight is a significant independent risk factor for a hiatal hernia and is significantly associated with esophagitis . fisher et al demonstrated a correlation between both weight and body mass index with gastroesophageal reflux . since the early 1990s , the status of laparoscopic antireflux surgery has moved from experimental to routine , with large experiences now reported by many centers . furthermore , the outcome in the majority of patients undergoing surgery is good , with relief of reflux symptoms in about 90% of the patients . laparoscopic surgery for the correction of gastroesophageal reflux has become common throughout the western world , partly because of better patient acceptance due to a perception that surgical procedures are now less invasive and also because of an apparently increasing incidence of reflux presenting as a clinical problem . our series of patients is the first description and evaluation of the lrygbp as an antireflux procedure . previously , it was reported that one of the major morbidities after ring vertical gastroplasty is severe acid reflux ; the roux - en - y gastric bypass suggests a solution to this complication although it has never been studied from the perspective of treating gastroesophageal reflux disease . all the patients in this study had symptomatic gerd , and they opted to be treated with lrygbp to address the initial gerd problem and the other comorbidities associated with their morbid obesity . they were all treated with low - dose ranitidine hydrochloride postoperatively to prevent anastomotic ulcers ; however , the dosage used is nontherapeutic for gerd symptoms . the use of antacid medications after the surgery was in less than 10% of the patients , and all of these were on a lower dosage than that prior to the surgery . all our patients were refractory to medical treatment before surgery , and 53% were on a high dosage and a conventional dosage of proton pump inhibitors . all the patients were interviewed , and the heartburn - related symptoms were recorded . according to our results , all were free of gerd symptoms after the operation , and it was not always correlated to their excess body weight lost . our result may suggest that the antireflux mechanism of this surgery is a combination of acid reflux reduction due to gastric stapling or gastroplasty and the reduction of increased intraabdominal pressure by reduction of excess body weight resulting in lower intraabdominal pressure . thoracic surgeons with laparoscopic expertise should consider lrygbp for treatment of gerd in the mo . acute epidural hematoma ( edh ) accounts for 2.7% to 11% of traumatic brain injuries ( tbis ) , and if the size is large , the disease leads to devastating results.1712 ) generally , a small edh does not cause neurological deficits , and it has a good prognosis . however , if an initially small edh suddenly expands , the patient 's condition may deteriorate quickly.2813 ) this may cause death or a serious neurological disorder in a patient , and thus , it always requires attention and quick action . so far , much of the literature has reported on the expansion of acute edh , and most types have been analyzed , including asymptomatic hematoma expansion found in the follow - up of computed tomography ( ct).5111719 ) the change of the hematoma in edh treatment - not only the change of radiologic imaging size but also the degree of expansion of the hematoma leading to operation - is important . thus , we investigated the early predictors and risk factors of hematoma expansion in patients who underwent delayed surgery when their neurological condition worsened due to the expansion of the hematoma during the conservative treatment of edh . this was a retrospective study of 151 patients who opted for conservative treatment during initial hospitalization among patients who presented with traumatic edh from january 2011 to january 2014 . of the 151 patients , 61 patients were excluded ; eight patients were excluded for surgery because of hematoma size increases without significant neurological symptoms or signs . four patients who underwent emergency surgery because of severe neurological deficits without follow - up ct were excluded . ten patients were excluded due to hemorrhagic diseases , such as hepatic cirrhosis , hematologic malignancy , and end - stage renal disease with hemodialysis . two patients with diffuse axonal injury and 19 patients with other severe accompanying tbis ( e.g. , subdural hematoma , intracerebral hemorrhage ) were excluded . eighteen patients with abbreviated injury scale ( ais)6 ) scores of more than 3 with traumatic multi - organ injury were excluded . accompanying head injuries included skull fracture , contusional hemorrhage of less than 5 ml , and traumatic subarachnoid hemorrhage of less than a fisher grade 3 . a total of 90 patients were enrolled in this study and divided into a delayed - surgery group ( dg ) and a non - surgical group ( ng ) . the dg was the group that received surgery due to changes in symptoms and hematoma growth in the course of the conservative treatment period . in other words , the dg was a group of patients , who were not indicated for operation initially , that underwent surgery later due to edh growth with neurological deterioration . members of this group had to have shown neurological change and hematoma increase in the follow - up ct and to have undergone surgery ( figure 1 ) . the follow - up ct of the ng was performed within 6 to 12 hours . the gender , age , initial glasgow coma scale ( gcs ) , presence of drinking at trauma attack , and medical history ( hypertension , diabetes , anticoagulant use ) of all patients were investigated . the laboratory studies - including platelet counts , prothrombin time ( pt ) , and partial thromboplastin time ( ptt)-were also investigated . in addition , the initial amounts and changes in edh amounts ( ml ) , changes in mean arterial pressure ( map , mm hg ) , accompanying head injury , and time interval between trauma and initial ct time ( hours ) were collected . the prognostic outcome of both groups was evaluated with the glasgow outcome scale . only in the dg , we also analyzed the relationship between the prognosis of patients and the gcs of both initial and later time points , where the same patients went through neurological aggravation . the abc/2 method was used to measure the amount of edh.9 ) the indications for edh surgery were defined as a volume of more than 30 ml , a thickness of more than 10 mm , or a mid - line shift of more than 15 mm . map change was defined as the difference between the first time map was measured in the emergency room and just before the follow - up ct . whether platelet counts were normal additionally , normal pt and ptt were determined based on 9.5 to 12.8 seconds and 27.9 to 37.8 seconds , respectively . each factor was examined with univariate analysis and multivariate analysis . the statistical package for the social sciences ( spss ) version 18.0 for windows ( spss inc . , chicago , il , usa ) was used as the analysis program . in the univariate analysis , an independent t - test was conducted for continuous variables , and a chi - square test was conducted for nominal variables . the average amounts of change in the hematomas of the dg and the ng were 33.0231.02 ml and 2.4156.272 ml , respectively ( p<0.001 ) ( figure 2 ) . independent variables of the dg and ng are shown in table 1 . in the univariate study , the presence of drinking , map change , and time interval between trauma and initial ct were statistically significant . in the ng , 20 patients with drinking ( 29.58% ) were reported to be higher than one patient ( 5.26% ) in the dg ( p=0.0345 ) . the map change of the ng showed a decrease of 0.428 mm hg , while the dg showed an increase of 9.684 mm hg ( p=0.031 ) . the time interval between trauma and initial ct time was 5.7076.963 hours for the ng and 1.8021.565 hours for the dg . initial brain ct was performed more quickly in the dg than the ng ( p=0.0001 ) . other factors - including age , anticoagulant use , medical history , and accompanying head injury - were not statistically significant . in the multivariate study , only the time interval between trauma and initial ct was statistically significant ( p=0.0135)(table 2 ) . according to the presence of surgery , the receiver operating characteristic ( roc ) curve for the time interval between trauma and initial ct time a specificity of 88.2% and sensitivity of 68.3% are shown in figure 3 ( area under the curve=0.854 ) . in comparison with the ng , the dg revealed a relatively poor outcome ; however , this difference between the two groups was not statistically significant ( p=0.0673 ) ( table 3 ) . on the other hand , the gcs checked at later time points , where the patients were neurologically aggravated , statistically significantly affected the prognosis ( p=0.020 ) . nonetheless , the initial gcs and the degree of change in gcs score were not statistically influential ( table 4 ) . this study examined the risk factors and predictors of expansion of edh for surgery from the clinical rather than radiologic perspective of initial edh , which is not indicated for surgery . anatomically , the surgical approach of edh is relatively easy and does not require proficiency . therefore , in a clinical situation , methods of surgical management alone can not be the problem . a problem in the clinical situation occurs when conservative treatment is determined to be sufficient for early acute edh , but it suddenly expands in size , leading to neurological disorders . in other words , due to the relatively small acute edh in the initial , clear state of consciousness , the patient can suddenly worsen . at that time , if surgery is required due to edh expansion on the ct , there is no rapid response in the process , and several problems can occur . several vectors associated with edh progression from existing reports have already been revealed . however , the majority involved asymptomatic radiologic change and did not focus on changes in the clinical status of patients that needed operations.5719 ) therefore , we evaluated risk factors for surgery , using only cases of expanded edh for which surgery was necessary . thus , the patients of this study were selected initially for conservative treatment , and the dg was defined as the group that received surgery due to changes in symptoms and hematoma growth in the follow - up ct during conservative treatment . in this study 's univariate analysis and multivariate analysis results , the most significant factor was the time interval between the trauma and the initial ct . we considered the main reason to be that ct was undergone during hematoma expansion.2417 ) based on the results of this study and a review of previous literature , the time interval between head trauma and ct was shorter . in that time , edh had a high probability of expansion . according to oertel et al.,15 ) the time between the initial injury and the first ct of the edh - enlarged group was 1.3 hours , while the time of the non - enlarged group was 2.3 hours . in the study by bezirciolu et al.,3 ) they recommended that ct be taken within 6 hours in the follow - up of edh . therefore , our research also considered time interval between trauma and initial ct time as a definite risk factor , and the roc curve was calculated . at the 2.08-hour cut - off value , specificity of 88.2% and sensitivity of 68.3% were confirmed . additionally , this result requires re - evaluation and reinforcement from large - scale studies , and we should note that it is more important to obtain a cut - off value of high sensitivity than that of specificity for the prediction of edh expansion and faster operation . ultimately , this study aimed to evaluate and analyze the variable factors that may serve as prognostic parameters in both groups of patients . the parameters with statistical significance were strongly considered for the risk factors of delayed operation in the aggravating of edh . of the factors discussed , the interval time between trauma onset and initial ct , which was statistically presented with time - dependent sensitivity and specificity , was found to have predictive potential in the diagnosis of disease progression . in addition to the time interval between trauma and initial ct , changes in map and the presence of drinking were also statistically significant . neurological change is a clinically important factor for predicting prognoses in tbis ; therefore , intracranial pressure changes during the expansion of the edh were also thought to be accompanied by map change , and map was set up as an independent variable . it was found that the average map change of the dg was increased by about 10 mm hg more than that of the ng . however , a 10 mm hg change of map is not clinically meaningful . the increase of map can be seen as a change in systolic map due to cushing 's reaction , pain , or external factors , and as variance due to the difference between observers . this means that map was measured before follow - up ct in the ng as a routine check - up , but in the dg , map was measured after neurological deterioration or severe headache . additionally , pain controllers , inotropes , or antihypertensive agents can be confounding factors for map . additionally , the authors excluded the patients who presented with ais values of 3 or over and injuries to other organs , as these factors can act as confounding variables . drinking alcohol was another aspect the authors considered an important , independent factor influencing the initial gcs and the severity of edh in the dg . drinking was reported in only one of 20 patients of the dg , which was thought to reflect a selection bias due to the small sample . this also means further study is required to reveal the correlation between drinking and the prognosis for edh expansion . this has been found to have meaning in the literature ; however , whether accompanied by brain injury , such as a fractured skull , or anticoagulants , no statistically significance correlation was found in the present study.710121618 ) however , this is also thought to be because it was conducted at a single institution . the relatively small sample size and different inclusion criteria compared with other studies could have caused conflicting results . additionally , in edh , factors of age are known to affect the dural adhesion to the skull.14 ) we subsequently carried out a statistical analysis on the effect of age ; however , it was not found to be statistically significant ( p=0.078 ) . we hypothesized that gender differences may also lead to variable states of dural adhesion and cause diverse types of trauma as well as affect the severity of the disease progression . however , these independent factors were concluded to be not statistically significant . in this study , the prognostic outcome of the patients in the dg were poor compared to those in the ng , but we were not able to prove this statistically . we understand that this may be due to the retrospective nature of the study and the small pool of patients . when focusing only on the patients in the dg , the mechanical damage made by the hematoma expansion itself was statistically more influential in determining the prognostic outcome than the degree of clinical changes in the patients . obviously , it is logical to assume that larger changes in gcs scores mean poorer prognostic outcomes for patients , and this was clinically witnessed in our patients . thus , the small number of patients and retrospective nature of the study are the greatest limitations of this study . , the patients who arrived at our emergency department during the process of hematoma expansion were included in our study . however , if the hematoma was already large enough to be considered an end - point of hematoma expansion , requiring an emergency operation , they were excluded from our study . in order to overcome this limitation , a well - organized time - matched study will be required in the future . in reality , the time interval between trauma and initial ct is a non - modifiable factor that can be applied in the clinical situation . the statistics have shown that the parameters - such as changes in map , presence of drinking , and time interval between trauma and initial ct - are statistically significant to support our hypothesis . nonetheless , they are unfortunately non - modifiable clinical factors , which can not be used in treatment guidelines . however , this particular study has proven that these factors have critical implications as predictive risk factors of progressive edh requiring surgery . in summary , our data revealed a sensitivity of 68.3% and a specificity of 88.2% at a 2.08-hour time interval between trauma and initial ct . this present finding indicated that if the time interval between trauma and initial ct was shorter in acute edh , the probability of the patient suffering subse quent neurological deterioration following a delayed operation increased . since the time interval between trauma and initial ct is a non - modifiable factor , if it is shorter , the neurosurgeons should be alerted to the possible neurological deterioration of the patients due to the growing hematoma and promptly respond with appropriate treatments .

background and objectives : gastroesophageal reflux disease ( gerd ) is commonly associated with morbid obesity ( mo ) . antireflux surgery has a higher failure rate in mo and addresses only one of the comorbidities present . this paper reviews the results of laparoscopic rouxen - y gastric bypass ( lrygbp ) performed for recalcitrant gerd in mo.methods:patients with recalcitrant gerd and a body mass index ( bmi)>35 undergoing lrygbp were included . lrygb included crural repair , creation of a small gastric pouch ( 30 ml ) , and intestinal bypass ( 150 to 180 cm ) . all patients were followed in clinic and by telephone.results:from february 1999 to april 2001 , 57 patients ( 51 f , 6 m ) with a mean age of 43 ( range , 22 to 67 ) and a median bmi of 43 underwent lrygbp . hiatal hernia or esophagitis , or both , were present in 48 , barrett 's in 2 . lrygbp was possible in 52 patients ; 5 required open conversion . the median hospital stay was 3 days . complications included 1 leak , 1 pulmonary emboli , 2 reoperations for internal roux limb hernia , and 7 gastrojejunal strictures . at a mean follow - up of 18 months ( range , 3 to 30 ) , all patients report improvement or no symptoms of gerd and a mean weight loss of 40 kg ( range , 16 to 70 ) . quality of life scores ( sf-36 ) were above national norms for physical and mental components ( median 55 , norms=50 ) . gerd - health related quality of life median score was < 1 ( scale , 0 to 45 , 0=asymptomatic , 45=worse).conclusion : lrygbp was effective for recalcitrant gerd in mo . lrygbp also led to weight loss and improvement in other comorbidites . surgeons with minimally invasive expertise should consider lrygbp for treatment of gerd in the morbidly obese . objectivea small epidural hematoma ( edh ) that has been diagnosed to be nonsurgical by initial brain computed tomography ( ct ) can increase in size and need surgical removal , resulting in a poor prognosis . however , there have been few studies , which focused delayed operated edh . therefore , we analyzed the clinical factors to determine the predicting factors of delayed operated edh.methodsbetween january 2011 and january 2014 , 90 patients , who were admitted due to edh , were enrolled in this study . none of the patients were indicated for operation initially . based on the presence of surgery , we classified the patients into a delayed - surgery group ( dg ) and a non - surgical group ( ng ) . additionally , we analyzed them according to the following : time interval between the trauma and the initial ct , gender , age , medical history , drinking , change of mean arterial pressure ( map ) , volume of edh and other traumatic brain lesion.resultsamong the 90 patients , the dg was 19 patients . compared with ng , the dg revealed increased map , less presence of drinking , and a short time interval ( dg vs. ng : + 9.684 mm hg vs. -0.428 mm hg , 5.26% vs. 29.58% , 1.802 hours vs. 5.707 hours , respectively , p<0.05 ) . analyzing the time interval with receiver operating characteristic , there was 88.2% sensitivity and 68.3% specificity at the 2.05-hour cut - off value ( area under the curve=0.854).conclusionaccording to our results , the time interval between the trauma and the initial ct along with blood pressure change are potential predicting factors in the cases of delayed operation of edh .

duodeno - duodenal intussusception , which involves the distal invagination of a segment of the duodenum , is a rare condition owing to the retroperitoneal fixation of the duodenum , which is sometime absent in cases of intestinal malrotation . the vast majority ( 8090% ) of cases of intestinal intussusception are secondary to pathological masses in the intestine , such as brunner s gland hamartomas , lipomas and adenomas . the presence of a duodenal membrane may cause retention of food resembling a pathologic mass , which may in turn facilitate intussusception . we describe the interesting case of a patient with intestinal malrotation who developed acute pancreatitis as a result of a duodenal intussusception involving the head of pancreas which was caused by a duodenal membrane . other symptoms include gastro - oesophageal reflux , weight loss , fatigue , anaemia due to iron deficiency ; pancreatitis due to obstruction of the outlet duct has also been described [ 36 ] . owing to the intermittent nature of the symptoms delay in diagnosis symptoms had started three days previously and consisted of intermittent peri - umbilical pain , nausea and vomiting . the initial physical examination indicated a healthy young man with mild epigastric tenderness . in infancy he had undergone surgery for a congenital heart malformation involving closure of atrial and ventricular septal defects . during childhood he had been treated with proton - pump inhibitors for symptoms of gastro - oesophageal reflux , and he had a history of intermittent post - prandial abdominal pain , but had never undergone endoscopic examination , or any abdominal surgery . presented with a normal height and weight for his age with a body mass index of 25 . routine blood analyses revealed elevated levels of s - amylase ( 408 u / l ; ref . subsequently the patient developed fever , ( core body temperature 38.6 c ) ; acute cholecystitis was suspected so he was treated with intravenous antibiotics . subsequent abdominal ultrasonography was inconclusive , although there were signs consistent with a tumour in the head of the pancreas . a ct - scan revealed a duodeno - duodenal intussusception involving the head of the pancreas and the common bile duct ( figs . 1 and 2 ) . these finding were confirmed at laparotomy and non - rotation of the duodenum was also noted ( fig . the intussusception was released , and a duodenotomy revealed a duodenal membrane with a narrow central opening ( fig . s - bilirubin and s - amylase levels were within the normal range at discharge . to our knowledge this is the first case report of pancreatitis resulting from duodenal invagination caused by a duodenal membrane . high peristaltic pressure proximal to the stenosis may have contributed to the development of the invagination , which may also have been facilitated by the mobile , non - rotated duodenum , which had a vertical straight course without retroperitoneal fixation . cases in which an intraluminal polypoid mass or tumour in the duodenum resulted in duodeno - jejunal intussusception have been reported . in a few cases the intussusception involved the head of the pancreas , resulting in acute pancreatitis . these patients were treated by surgical reduction of the intussusception and duodenal resection or polypectomy . membranous duodenal stenosis is often treated by duodenotomy and excision of the membrane followed by a duodenoplasty , or by duodeno - jejunal anastomosis , with good results . in our case the patient had persistent symptoms of gastro - oesophageal reflux , which should have triggered an upper gi endoscopy ; had this revealed the intestinal malformation the patient could have been treated in a timely fashion and which would have avoided this serious complication . the intussusception was not detected by ultrasonography but was revealed by a ct - scan . a previous study found that 27.1% of patients with intestinal malrotation had a history of congenital cardiovascular defects . this suggests that all children with intermittent upper gastrointestinal symptoms such as vomiting , abdominal pain and gastro - oesophageal reflux , particularly those with a history of congenital heart malformation , should undergo upper gi endoscopy and imaging of the gi - tract to exclude gi anomalies . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request .

asbestos and silica are well - known fibrogenic dusts . however , there is no comprehensive understanding of the molecular and cellular events that lead to fibrosis as a consequence of asbestos or silica inhalation . previous studies have shown that asbestos stimulates superoxide anion production in alveolar macrophages through the phospholipase c / protein kinase c pathway . in contrast , silica does not appear to activate this pathway nor stimulate superoxide anion production , but silica does stimulate cytokine release by some undetermined pathway . therefore , using human alveolar macrophages isolated from normal healthy volunteers , we evaluated the potential involvement of intracellular calcium and tyrosine kinases as potential signal transduction pathways . in the absence of serum , crystalline silica , and to a lesser extent amorphous silica , caused a rapid and dose - dependent elevation of intracellular calcium coming from the extracellular space . however , in the presence of serum , which is required for silica - stimulated cytokine release , neither form of silica caused noticeable elevation of intracellular calcium . silica , however , did increase the extent of tyrosine phosphorylation , most notably of proteins at approximately 46 and 50 kda , suggesting activation of a tyrosine kinase pathway . preincubation of alveolar macrophages for 24 hr with silica - primed human alveolar macrophages for enhanced interleukin-1 beta ( il-1 beta ) release stimulated by endotoxin ( lps ) that was dose dependent . the enhanced lps - stimulated release of il-1 beta correlated with enhanced mitogen - activated protein kinase activity . taken together , these results indicate that a tyrosine kinase pathway is activated during silica stimulation of human alveolar macrophages.imagesfigure 4 . afigure 4 . b highlightsintussusception is not always seen on ultrasonography but can revealed by a ct-scan.any child , and especially those with previous congenital disorders , must undergo upper gi- endoscopy at the presence of intermittent upper gastrointestinal symptoms as vomiting , abdominal pain and gastro - oesophageal reflux , to exclude upper gi anomaly .

it is characterized by autoantibodies that target against liver - specific and non - organ - specific antigens ( 1 ) . corticosteroids , especially prednisone or prednisolone ( pd ) , have been the key drugs of initial antiinflammatory and immuno - suppressive therapy of autoimmune hepatitis in children ( 2 ) . however , it is well known that long - term use of corticosteroid is associated with the risk of steroid - induced toxicities , and this requires newer immunosuppressive agents for the treatment of autoimmune hepatitis , especially in growing children . we report on the case of a 11-yr - old korean girl with type-1 autoimmune hepatitis who discontinued pd due to steroid - induced toxicities , but who remained in biochemical and clinical remission while undergoing deflazacort and ursodeoxycholic acid ( udca ) combination therapy . a 9-yr - old girl was admitted to eul - ji hospital because of jaundice and right upper quadrant pain . her height was 135 cm ( 75 - 90 percentile ) , and weight was 43 kg ( over 97 percentile ) . the sclera was icteric , and the liver edge descended 4 cm below the right costal margin . the results of laboratory studies were as follows : aspartate aminotransferanse ( ast ) 1,106 iu / l , alanine aminotransferase ( alt ) 1,888 iu / l , total bilirubin 25.7 mg / dl , direct bilirubin 18.2 mg / dl , total protein 6.9 g / dl , albumin 4.2 g / dl , serum alkaline phosphatase 1,160 iu / l , ggtp 71 iu / l , inr 2.4 , aptt > 120 sec , antinuclear antibody ( ana ) ( + ) speckled pattern , with 1:320 titer , anti - smooth muscle antibody ( ab ) ( - ) , anti - mitochondrial ( ama ab ) ( - ) , igg 1,397 mg / dl , anti - hav igm ( - ) , anti - hbc igm ( - ) , hbsag ( - ) , ebv - vca igg / m ( -/- ) , and ceruloplasmin 28 mg / dl . liver biopsy results showed piecemeal necrosis at the portal - lobular interface accompaning mild to moderate infiltration of lymphocytes , moderate centrilobular cholestasis , moderate lobular inflammation , and moderate fibrosis ( fig . 1 ) . according to the scoring system of international autoimmune hepatitis group ( 3 ) , she had definite autoimmune hepatitis ( score=16 ) . initially , on 40 mg of pd and 300 mg of udca daily , she gradually recovered both clinically and biochemically . pd was tapered gradually , and three months later , she was taking 15 mg of pd and 300 mg of udca daily . at eight months , her aminotransferase level had elevated to 400 iu / l , but reduced rapidly when pd was increased to 40 mg / day . at nine months , she was taking 15 mg / day of pd and 300 mg / day of udca , but she complained of hirsutism , striae , moon face , and buffalo hump , at ten months . the results of a second round of laboratory studies conducted at this time were ; ast 21 iu / l , alt 52 iu / l , total bilirubin 0.7 mg / dl , total protein 7.2 g / dl , albumin 4.7 g / dl , glucose 86 mg / dl , ana ( + ) speckled pattern , with 1:160 titer . accordingly , deflazacort ( calcort , handok pharm co. , ltd , eumseonggoon , chungcheongbuk - do , korea ) was administered at dose of 18 mg a day , instead of pd at dose of 18 mg a day , and udca was continued . her biochemical test remained within normal ranges on this regimen , and the pd - associated complications improved rapidly . fourteen months later , deflazacort was reduced at dose of 15 mg a day , and after 19 months of deflazacort treatment , a follow - up liver biopsy was performed . this showed no lobular activity , no portal activity , near - normal periportal inflammation , and fibrosis ( fig . . the girl has been in clinical and biochemical remission for two years now , without therapy . there are few diseases to be differentiated from autoimmune hepatitis , overlap syndrome , in which the features of autoimmune hepatitis is combined with those of primary biliary cirrhosis , or primary sclerosing cholangitis , and autoimmune cholangitis . in overlap with primary biliary cirrhosis , serologic test of ama is usually positive and histologic study shows findings of cholangitis . in overlap with primary sclerosing cholangitis , serologic test of ama is negative , and histologic evidence suggests various forms of cholangitis . in autoimmune cholangitis , serologic test of ama is negative , and histologic examination shows bile duct injury , suggestive of primary biliary cirrhosis . autoimmune cholangitis , also , is generally not steroid - responsive . in the present case , despite the cholestatic presentation , serum level of ggtp was not so high , histologic study showed no evidence of definite cholangitis , and response to steroid was excellent . according to the scoring system of international autoimmune hepatitis group , this case has definite autoimmune hepatitis ( pre - treatment score 16 , post - treatment score 18 ) . the long - term use of corticosteroid is known to be associated with the risk of steroid - induced toxicities . generally , there are several ways of reducing steroid - associated toxicities , i.e. , administering steroid on an alternate - day schedule , administering adjunctive agent to reduce the need for steroid , and finding a substitute agent . however , of these three options in case of autoimmune hepatitis , the risk of relapse is higher if steroids are administered on an alternate - day schedule ( 4 ) , and thus this method is not recommended . some authors add azathioprine to steroids if normalization of the liver function is not obtained by steroids alone or if too high a dose of steroids is required to maintain normal transaminase levels ( 4 ) . initial combination therapy with steroid and azathioprine , is considered by some authors as standard therapy of autoimmune hepatitis in children , as in adults ( 1 ) . however , there is concern about the oncogenecity associated with long - term azathioprine therapy ( 5 ) , and because of its hepatotoxicity , some authors do not recommend azathioprine as first line treatment , particularly in severely jaundiced patients ( 4 ) . thus , udca was prescribed as an adjuvant medication with pd or deflazacort . in a clinical study on japanese adults , udca improved aminotransferase levels and reduced inflammation in liver biopsy ( 6 ) ; however , a clinical study on caucasian adults showed no significant difference in the results of biochemical tests or in histologic indices between a udca group and a placebo group ( 7 ) . deflazacort is a synthetic steroid , an oxazoline derivative of pd , with potentially lower toxicities than prednisone or pd ( 8) , and has been widely used as an immunosuppressant in the field of pediatric nephrology ; nephrotic syndrome and renal transplantation ( 9 ) , and in duchenne dystrophy ( 10 ) . only one case of adult study has indicated that deflazacort might be a suitable substitute for pd in the treatment of autoimmune hepatitis ( 11 ) . in clinical trials in spain , deflazacort was administered to patients with type-1 autoimmnune hepatitis who were in remission on a stable dose of pd . only clinical and biochemical remission were maintained during the follow - up period without histologic examination . in the present case , deflazacort was substituted for pd as a therapeutic drug in a child who was in remission on pd and udca , but significant pd - associated toxicities occurred . after switching to deflazacort , these toxicities improved rapidly , and the patient remained in clinical and biochemical remission . after 19 months on deflazacort , a follow - up liver biopsy showed near - normalization of auto - immune hepatitis . this is the first known clinical report to show that deflazacort is a suitable substitute for pd in the treatment of autoimmune hepatitis . a recent study showed the measurement of organ- and non - organ - specific auto - antibodies and igg levels correlated with disease activity in autoimmune hepatitis ( 12 ) . the patient in the present case remained ana - positive with 1:160 titer , when on pd and udca , but became ana negative at about the time of the follow - up liver biopsy performed after 19 months on deflazacort and udca . this may provide additional evidence that deflazacort is a possible substitute for pd in the treatment of autoimmune hepatitis . this case poses the possibility that deflazacort can be a possible substitute for prednisone in type-1 autoimmune hepatitis in children , and demonstrates the need for further study . congenital mitral valve anomalies are very rare clinical entities involving one or more components of the mitral valve apparatus . the majority of these abnormalities are causing mitral valve insufficiency and commonly presenting in the early part of life . common clinical presentations include left ventricular outflow tract obstruction , valvular insufficiency and heart failure . we report a rare and unique case of congenital mitral valve anomaly associated with anomalous coronary artery in a middle age man who presented with stroke . a 55-year - old male , smoker and previously healthy , presented to our emergency department for abrupt onset of difficulty reading and right sided weakness , signs and symptoms suggestive of a new onset stroke . stat computed tomography ( ct ) and computed tomography angiogram ( cta ) of the brain revealed no abnormality . magnetic resonance imaging of the brain showed acute infarction of the left posterior insular cortex and left parietal subcortical area . he underwent a routine transthoracic echocardiogram ( tte ) as to rule out any possible cardiac source of embolism . tte revealed an abnormally thickened anterior mitral leaflet associated with mild eccentric mitral regurgitation ( mr ) . tee revealed some form of redundant tissue along the anterior mitral leaflet and the base of the anterior leaflet in the left ventricular outflow tract ( lvot ) . it is thin and supple , the appearance was neither consistent with a fibroelastoma , nor a tumor , an abscess , a true aneurysm of the sinus of valsalva nor perimembranous septum . the anterior mitral leaflet is itself thickened and prolapses , which might represent myxomatous disease . a mild eccentric posteriorly directed mr jet was noted , likely originating from the mild prolapse of the anterior leaflet . at the very distal end of the anterior leaflet , there is an echo bright , independently mobile soft tissue entity , 2 - 3 mm in size . the left circumflex coronary artery ( lcx ) appears to have both an anomalous origin ( very low in the left sinus of valsalva immediately above the annulus , in closer association to the mitral valve than usual ) . the ostium of the right coronary artery appears larger than the left coronary artery ( fig . 1 , 2 , and 3 , supplementary movie 1 , 2 , and 3 ) . coronary cta revealed ; anomalous lcx originating from the proximal right coronary artery off the right sinus of valsalva with a retro - aortic benign course . the mitral valve was described as ; thickened anterior mitral valve leaflet with a band like attachment to the membranous inter - ventricular septum , the portion of the band closer to the anterior leaflet forms a thin curvilinear band ( fig . 4 and 5 ) . this abnormal appearance could represent a congenital anomaly , myxomatous changes are still probable although may not explain the atypical attachment site . the patient had no fever , blood cultures were negative and all other laboratory investigations were within normal limits . the patient remained in hospital for one week and discharged later with a minor neurological deficit . in the absence of obstruction of the lvot , the patient is being followed up without surgical intervention . most commonly , mitral valve prolapse due to myxomatous degenerative disease and connective tissue diseases , congenital mitral stenosis , accessory valvular tissue , isolated cleft mitral valve , double orifice and ebstein - like malformation were also reported . supra - mitral ring is one of the components of shone 's complex . in addition , there are also anomalies of the tensor apparatus i.e. , archade or hammock valve , straddling mitral valve with abnormal attachment of mitral valve chordae to both ventricles and parachute mitral valve characterized by unifocal attachment to a single or fused papillary muscle.1,2,3 ) blood cyst of the sub - valvular apparatus of the mitral valve are commonly reported as postmortem findings in infants and they regress spontaneously by age of 6 months , few cases in adults resulting in embolisation and valvular dysfunction have been reported.4 ) accessory mitral valve tissue is a rare congenital cardiac anomaly , commonly associated with other congenital heart diseases , such as ventricular septal defects and transposition of the great arteries . prifti et al.5 ) have classified accessory mitral valve tissue as type i - fixed type ( a : nodular , b : membranous ) , type ii - mobile type ( a : pedunculated , b : leaflet - like ) . type iib is divided into 1 ) rudimentary chordae and 2 ) developed chordae . in this case , the appearance might fit into type iib - mobile type ( leaflet like ) . however , they further described attachment of the mitral accessory tissue into six different locations in the left ventricle through the accessory tissue 's chordae . in our case , the accessory tissue was attached to the interventricular septum directly , not through a chordae , confirmed by echocardiogram and ct scan . in the majority of cases of accessory mitral valve tissue , presentations were left ventricular outflow tract obstruction , significant valvular insufficiency , palpitation and heart failure . the lvot obstruction is the result of systolic ballooning into the lvot producing a mass effect and the continuing turbulence producing this effect , results in a fibrous tissue reaction with permanent deposits of fibrous tissue and progressive lvot obstruction.6,7,8 ) in this case , there was no lvot obstruction and the presentation was stroke . however , infective endocarditis was excluded in this case on the basis of negative blood culture and absence of other criteria suggesting endocarditis . we reviewed all echocardiographic features of reported mitral valve anomalies , and none have shown similar appearance to the anomalous mitral valve presented in this case . proximal course of coronary arteries.9 ) the transesophageal echocardiogram revealed an anomalous course of the lcx , later confirmed by cta . pathological diagnosis of this uncommon mitral valve anomaly is unavailable , which might be considered as a limitation . however , given the clinical presentation of stroke , there is an ongoing debate , as to consider this anomaly being a definite cardiac source of embolism . in addition , there was no lvot obstruction and the final decision was to treat the patient conservatively with regular follow - up , which was also the patient 's preference . this case highlights an under - recognized anomaly of the mitral valve associated with anomalous coronary arteries . we advise regular clinical and echocardiographic evaluation in this situation to identify any progression . in future , such anomaly might be considered as potential source of embolism and surgical removal might be considered as a reasonable indication .

prednisone or prednisolone are the mainstay drug treatments for autoimmune hepatitis in children . however , long - term use of corticosteroid is associated with the risk of steroid - induced toxicities , and this situation requires newer immuno - suppressive agents for the treatment of autoimmune hepatitis , especially in growing children . an 11-yr - old korean girl with type-1 autoimmune hepatitis discontinued prednisolone due to toxicities , i.e. , hirsutism , buffalo hump , and skin striae , and remained clinical and biochemical remission under replacement of deflazacort and ursodeoxycholic acid combination therapy . a follow - up liver biopsy after 19 months of deflazacort and ursodeoxycholic acid treatment showed histologic remission . a 55-year - old male presented with stroke . transesophageal echocardiogram and cardiac computed tomography revealed an unrecognized congenital malformation of the anterior mitral leaflet associated with anomalous left coronary circumflex artery , arising from the right coronary artery , diagnosed first by echocardiogram . this case represents a unique unforeseen mitral valve anomaly that might be considered as potential cardiac source of embolism . this finding broadens the spectrum of known mitral valve anomalies .

nontraumatic spontaneous intracranial hemorrhage ( ich ) causes significant morbidity and mortality throughout the world . ten to 20% of strokes are caused by ich in western countries.6 ) over 45 years of age , ich is common,5)16)21 ) but rare before then . therefore , the goal of our study was to perform a descriptive analysis of ich etiology and prognostic factors in patients aged 40 years . we selected 39 consecutive patients who were 40 years of age diagnosed between january 2001 and june 2012 . all cases of ichs were confirmed using computed tomography ( ct ) or magnetic resonance imaging ( mri ) . traumatic ich , insufficient medical information , previously diagnosed intracranial tumors , arteriovenous malformation ( avm ) , and primary subarachnoid hemorrhage were exclusion factors for this study . for each patient , we analyzed the following prognostic factors : smoking history ( the consumption of 5 cigarettes at least 2 days per week over a period of 12 months ) , alcohol use ( the ingestion of 100 g / d or more every day for at least 20 days per month during the last 2 months or acute alcoholic intoxication during the 24 hours prior to the attack ) , previously diagnosed underlying disease such as hypertension or diabetes mellitus , the use of antihypertensive or antidiabetic agents , high serum cholesterol ( fasting cholesterol values 200 mg / dl at admission ) or low serum cholesterol ( < 150 mg / dl ) , and bleeding tendency ( prolongation of prothrombin time > 14.3 seconds or activated partial thromboplastin time > 43.5 seconds ) . the hematoma volume was estimated via the analysis of ct scans using the abc/2 method.12 ) the hematoma site was denoted by lobe ( frontal , parietal , temporal or occipital ) , thalamus , basal ganglia , cerebellum , or brain stem . hypertensive ich was defined as hemorrhage into the brain parenchyma in the absence of causal trauma or a structural disease process on a macroscopic level , such as a tumor , aneurysm , vascular malformation , or arteriovenous fistula , in patients treated for hypertension ( or who were previously diagnosed with hypertension ) , as well as the absence of another cause of ich . avm , venous angioma and other vascular malformations were confirmed using ct angiography , conventional cerebral angiography , or magnetic resonance angiography . if there was no explanatory underlying disease or abnormal structural lesion in the image workup , the ich was defined as cryptogenic ich . patients with an ich volume greater than 30 ml in the basal ganglia or thalamus received surgical treatment , with the exception of 4 patients . one patient was a high surgical risk because of disseminating intravascular coagulopathy associated with acute myelocytic leukemia , and the others showed no definite neurological deterioration and tolerated the event with conservative management . for the patients with an ich volume of less than 30 ml , conservative management was indicated . however , for the patients with an ich volume of less than 30 ml , if progressive acute hydrocephalus due to intraventricular hemorrhage and neurological deterioration was present , external ventricular drainage was performed . in cerebellar ich , regardless of the hemorrhage volume , a decompressive craniectomy with hematoma removal was performed if the patient showed progressive neurological deterioration . a good outcome was defined as a gos score 4 , and a poor outcome was defined as a gos score < 4 . fisher 's exact test was applied to assess statistical significance , and null hypotheses of no difference were rejected if p values were less than 0.05 . the mean age was 33.21 6.42 years ( range 15 - 40 years ) , and there were 11 females and 28 males . the characteristics of the study subjects by outcome at discharge and 6 months after discharge are described in table 1 . we assessed outcomes by reviewing the gos of medical record at discharge and 6 months after discharge . based on the outcomes at discharge and 6 months after discharge , smoking history was significantly associated with prognosis . alcohol use , previously diagnosed hypertension and bleeding tendency were not statistically significantly associated with outcome . all of the patients with cholesterol levels less than 200 mg / dl had good outcomes at 6 months after discharge ; this finding was statistically significant ( p = 0.047 ) . patients with an initial systolic blood pressure ( sbp ) < 160 mmhg had better outcomes ( good outcomes in 65.4% of patients ) , while those patients with an sbp 160 mmhg had poorer prognoses ( poor outcomes in 69.2% of patients ) ; this result was statistically significant ( p = 0.044 ) . the level of diastolic blood pressure ( dbp ) upon admission showed similar results ( < 90 mmhg : 66.7% experienced good outcomes ; 90 mmhg : 57.1% experienced poor outcomes ) although , these results were not significant . the ich location , as well as the presence or absence of intraventricular hemorrhage ( ivh ) , had no significant effect on outcome ( p = 0.451 ) . there were also no statistically significant findings associated with ich direction ( left or right ) . in particular , patients with gcs 8 had a higher proportion of poor outcomes , compared to patients with gcs > 8 ; this finding was statistically significant ( p = 0.02 via fisher 's exact test ) . variations in the volume of the hematoma showed no significant difference ( p = 0.051 ) in gcs score . lobar hemorrhage was the most common location of ich in young adults , and there were no statistically significant findings associated with hematoma location ( table 3 ) . in our institution , 280 patients were diagnosed with ich during the investigation period ; of these patients , 39 ( 13.9 % ) were aged 40 years . in western countries , only 3 - 5% of ich patients were younger than 45 years.14)24 ) the distribution of ich reflects the characteristics of the population ( ethnic / racial groups and the criteria of a young adult ) although these studies were not standardized.4 ) in this study , 11 ( 28.2% ) of the patients diagnosed with ich had also been diagnosed with hypertension . in young adults , hypertension is a primary cause of ich , and this hypertension / ich ratio varies from 11 to 15%.2)3)23 ) as with other published literature , our study showed that a poor prognosis is more likely in patients with hypercholesterolemia . many studies describe serum cholesterol level and smoking history as risk factors for ich.2)18)20)22)23 ) in our study , the serum cholesterol level and smoking history were also prognostic factors . according to ruiz - sandoval and coworkers,18 ) avm contributes to 33% of ich cases , cavernous angioma contributed to 16% of cases , and hypertension contributed to 11% of cases ( all under the age of 40 years ) , similar to our study results . however , among the elderly , hypertension was the most common cause of ich , and the putamen was the most common location.11)20 ) our results indicate that the most common causes of ich were cryptogenic cases ( n = 16 , 41.03% ) and the most common structural etiology of ich was avm . we found that an initial sbp greater than 160 mmhg indicated a poor prognosis , and patients with a sbp less than 160 mmhg were more likely to have a good prognosis . some authors reported a low ( < 130 mmhg ) or high ( 200 mmhg ) initial sbp as a prognostic factor that was independently associated with mortality.9 ) smoking , alcohol , and bleeding tendency in younger patients were determined to be prognostic factors of ich.13)18 ) regarding the serum cholesterol , the patients with levels < 200 mg / dl ( 63.3% ) showed a good prognosis , and patients with levels 200 mg / dl ( 77.8% ) showed a poor prognosis . six months after discharge , this difference was statistically significant and suggests that serum cholesterol levels can be a prognostic factor , as seen in the literature.1 ) ruiz - sandoval and coworkers19 ) reported that hypertension and hypercholesterolemia are 2 main risk factors for ich . some authors reported that hypocholesterolemia can cause ich and that the interaction between high diastolic blood pressure and low cholesterol levels weakens the endothelium of the intracerebral arteries , resulting in a hemorrhagic stroke in the presence of hypertension.10 ) however , in that study , the association between low cholesterol levels and the prognosis was not clearly presented . a study to investigate the association between serum low - density lipoprotein cholesterol ( ldl - c ) and mortality in ich found that lower ldl - c levels were associated with higher mortality rates.17 ) in our study , 7 patients ( 58.3% ) with ldl - c levels < 150 mg / dl had good outcomes and statistically significant improvements 6 months after discharge . however , the sample size was limited in our study , age was limited to 40 years , and cholesterol was the only factor used to assess the outcome via lipid profile . a statistically significant difference in outcomes ( p = 0.034 by the t - test ) by the initial gcs score was observed . a score of 8 or less predicted a higher risk of death within 2 days ( odds ratio , or = 3.9).7 ) among patients with a pre - hospital neurological decline , the gcs score was lower by an average of 6 points , and the mortality rate was 75%.15 ) the usefulness of surgical treatment of ich is still under debate.8 ) in this study , there was no statistically significant difference in prognosis between patients who did or did not receive surgical treatment ( p = 0.109 ) . ich is rare in young adults aged 40 years ; thus , the existing large - scale studies are not sufficient for studying either etiology or prognosis in this group of patients . due to the small number of patients and limited study period , more accurate and reliable outcome results could be obtained be comparative analysis of across several institutions and groups in the regional community . in this study , smoking , serum cholesterol , sbp on admission , and the initial gcs were significant prognostic factors . avm was the most common structural etiology of non - traumatic spontaneous ich in the patients aged 40 years . eight points or more on the initial gcs , sbp < 160 mmhg , serum cholesterol < 200 mg / dl , and low initial sbp indicated a good prognosis . a history of smoking was associated with a good prognosis in this study , although , the initial gcs score influenced this result . our study had some limitations , including its retrospective nature , its involvement of a single institution , the small number of cases , and the relatively short follow - up periods . thus , longer follow - up periods in prospective , multi - institutional studies that include large numbers of patients are necessary to better understand the prognosis of ich in young adults . given that almost three quarters of older adults lack decision - making capacity when urgent choices about life - sustaining treatment need to be made , older adults have long been encouraged by nurses and other health care providers to express their wishes for health care while they are healthy enough to meaningfully participate in treatment decision - making . advance care planning has been recently promoted by the centers for disease control and prevention as a process contributing to overall public health through its focus on supporting the individuals ' health care choices and preventing unnecessary suffering . widespread social marketing of advance care planning has made many excellent online and print resources available to the public [ 24 ] . although advance care planning is now seen as an iterative process that includes the way in which people think about and communicate their values and preferences so that they may receive the health care they desire in the case of life - threatening illness , much of the extant research in this area has focused upon the completion of written advance care plans . using a population - based approach , this study addressed the research question how do community - dwelling adults aged 55 years and older plan for serious illness , either formally ( e.g. , by designating a substitute decision maker and preparing written advance care plans ) or informally ( e.g. , by discussing states of health considered to be unacceptable to continue living or desired medical care in the event of serious illness ) ? the overall objective of this study was to identify the associations between formal and informal planning for serious illness and key sociodemographic , health , and knowledge variables . based on a review of current literature on advance care planning , it was hypothesized that sociodemographic variables ( including age , gender , urban or rural location , education level , and income level ) , values ( importance of planning for future care ) , and self - reported knowledge of advance care planning would be statistically significant predictors of behaviors relating to planning for serious illness . it is estimated that between 18% and 36% of all american adults in the general public have completed an advance directive . rates of advance directive completion amongst distinct subpopulations , such as persons with terminal illnesses and older adults , have been reported to range from 15% to 84.9% [ 1 , 713 ] . completion of an advance directive is associated with a wide range of factors , including older age , greater disease burden , type and acuity of condition , white race , higher socioeconomic status , knowledge about advance directives or end - of - life treatment options , a positive attitude toward end - of - life discussions , a long - standing relationship with a primary care physician , and whether the patient 's primary care physician has an advance directive . while the clinical utility of advance directives has been rightfully questioned [ 1420 ] and the uptake by intended consumers less than hoped in spite of widely available resources , support for the overall process of people considering and sharing key values and preferences for health care in the face of serious illness remains strong . social marketing campaigns have increasingly focused public attention on promoting dialogue , rather than completing forms , as a means of supporting the preferences and autonomy of individuals who may not be able to communicate his or her wishes for care in the future . a number of studies have examined the extent to which discussions related to advance care planning are taking place , as well as the outcomes of these dialogues . between 75% and 91% of participants over age 65 in the canadian study of health and aging had considered who might make health decisions for them if they were unable to do this for themselves . the same study revealed that between 46% and 69% had discussed their preferences for end - of - life care with someone else . the benefits of having discussed preferences for future care were noted in a study of patients with advanced cancer by wright and colleagues and encompassed less aggressive medical care , including lower rates of ventilation ( adjusted or 0.26 ; 95% ci 0.080.83 ) , resuscitation ( adjusted or 0.16 95% ci 0.030.80 ) , icu admission ( adjusted or 0.35 ; 95% ci 0.140.90 ) , and earlier hospice admission ( adjusted or 3.37 95% ci 1.1210.13 ) , as well as better patient quality of life and improved bereavement adjustment . discussions early in the trajectory of cancer have also recently been demonstrated to be associated with less aggressive treatment and greater use of hospice care . despite these positive outcomes , many people remain skeptical about the benefits of advance care planning , fuelled in part by society 's denial of the inevitability of death [ 24 , 25 ] and death 's sequestration from mainstream society . the choice not to engage in advance care planning is complex and highly individual but may include any one or more of the following reasons : believing that one 's health is good and it is not necessary ; believing that advance care planning is only for the terminally ill , elderly , or infirm ; challenges discussing death ; not being ready ; lack of knowledge ; difficulty completing the form ; reluctance to broach the subject with the physician ; fear of being a burden ; incompatibility with cultural and spiritual traditions ; preference to delegate treatment decision - making to family or others ; lack of confidence that a written document would change the course of treatment received [ 15 , 23 , 2730 ] . this study was conducted in april , 2012 , within the province of saskatchewan , a province located in western canada with a population just over one million people . one third of the province 's population is considered rural , with the remainder divided between two urban centres . the sample of 238 community dwelling older adults over the age 55 years represents a subgroup of the entire sample of 827 individuals obtained for a larger project , who were stratified by age , region , and sex to be representative of the population of the province . the entire sample was randomly selected from a pool of volunteers who had agreed to participate in online commercial market surveys ( saskwatch research ) . participants provided consent for the data to be used in subsequent presentations and publications . the survey was comprised of structured , closed - ended items salient to planning for serious illness care . demographic data included age ; sex ; personal income ; education ; residence ( urban or rural ) . respondents were asked to identify the number and type of health conditions with which they lived from a list of common illnesses , as well as to respond to the following question : how important do you think it is to plan for medical care at the end - of - life ? ( not at all important , somewhat important , important , or very important ) . respondents were asked to indicate their level of knowledge about advance care planning and living wills on a three - point scale ( not at all familiar ; some basic understanding ; fairly or very good understanding ) . those who reported a written living will or advance directive were also asked to identify sources of help received with preparing this document ( consultation with lawyer ; consultation with lawyer and family ; consultation with family ; consultation with someone else ; prepared by myself ) . statistical analysis was completed using spss 19.0 . given that an age of 65 years has been recognized as the point at which many canadians retire from paid employment , respondents were divided into two groups : those 65 and younger and those older than 65 years . comparisons between the groups were completed using the kruskal - wallis test ( with multiple mann - whitney tests adjusted with bonferroni corrections for post hoc analysis ) and chi - square tests of proportion where appropriate . in order to determine the characteristics influencing outcome behaviors variables ( discussions of unacceptable states of health ; discussion of wishes for treatment , designation of a substitute decision maker ; preparation of a written directive ) , binary logistic regression was conducted . the strength of association was measured by the odds ratio ( or ) and 95% confidence interval ( ci ) . table 1 compares the demographic and health characteristics of the sample between respondents aged 5564 years ( younger group ) and those who were 65 years and older ( older group ) . respondents ranged in age from 55 to 88 years . the majority ( 82.1% ) of all respondents had education beyond completion of high school . a higher proportion of the older group reported incomes of less than $ 60,000 than the younger group , while the opposite was true for incomes above $ 60,000 . participants residing outside of primary urban centres ( populations > 250,000 ) comprised 58.6% of the total sample . a significantly higher ( p < 0.001 ) proportion of the older group reported four or more health conditions compared to the younger group , although the proportions were similar between the groups for those reporting one to three health conditions . table 2 compares the responses of the younger and older age groups in terms of values , knowledge , and behaviors related to planning for the end of life . the majority of respondents indicated they felt it was important or very important to plan for medical care in the event of serious illness ( 78.4% ) and to plan for one 's own funeral ( 62.2% ) . while 61.5% of respondents reported a good understanding of the term living will , substantially fewer indicated they had a good understanding of the term advance care plan . approximately half of all respondents had discussed states of health in which they would find it unacceptable to live with someone close to them in the past year . one - third of those aged 65 and older reported they had a written directive compared to 20.5% of adults below age 65 . respondents in the older group were significantly more likely ( p < 0.05 ) to have discussed wishes for treatment , designated a substitute decision maker , and to have prepared either an advance care plan or living will . respondents without a written advance care plan were asked to identify reasons for not preparing this document . the majority ( 60.0% ) indicated they had not considered this yet , while 21.2% suggested that their families would make decisions about future health care for them and 18.2% indicated that their families together with their physician would decide . for the 72 respondents who had prepared a written advance care plan , the most common source of assistance to prepare the document was a lawyer ( 50% ) . a third of respondents cited families as a source of assistance , while 22.2% indicated they had completed the plan by themselves . few ( 4.2% ) respondents had the assistance of a health care professional in writing the directive . table 3 displays the adjusted association between the outcome variables ( discussions about treatment wishes or unacceptable states of health ; designation of a substitute decision maker ; preparation of a written advance care plan ) and the hypothesized predictor variables . after adjustment for all the variables listed in table 3 , women were twice as likely as men ( o.r . = 2.05 , 95% ci 1.163.62 ) to have discussed treatment wishes but were similar to men in terms of reporting discussion regarding unacceptable conditions , designating a substitute decision maker , or completing a written advance care plan . those over 65 years of age were significantly more likely than those between the ages of 55 and 65 to have designated a substitute decision maker ( o.r . the older group was not more likely to have completed a written advance care plan , although this association approached significance ( p = 0.06 ) . education , income , and the number of self - reported health conditions were not associated with any of the four planning behaviors under consideration . = 0.47 , 95% ci 0.250.86 ) than those from urban centres to have completed a written advance care plan . those who believed it was important to plan for care were more than twice as likely to have designated a substitute decision maker ( o.r . = 2.32 , 95% ci 1.154.70 ) and more than four times as likely to have completed a written advance care plan ( o.r . respondents who reported a good understanding of advance care planning had a significantly greater likelihood of engaging in all planning behaviors : discussion of treatment wishes ( o.r . = 3.45 , 95% ci 1.876.34 ) ; discussion of states of health they would find unacceptable to live with ( o.r . = 5.62 , 95% ci 2.9210.92 ) ; designating a substitute decision maker ( o.r . = 2.49 , 95% ci 1.876.34 ) ; completing a written advance care plan ( o.r . the findings of this study demonstrate that neither formal nor informal planning for serious illness can yet be considered widespread amongst community - dwelling adults aged 55 and older . formal planning for serious illness , in the form of designating substitute decision makers and preparing advance care plans , was reported by fewer than half of the respondents in this study . these proportions are similar to those reported in other studies [ 1 , 713 ] . the proportion of respondents who engaged in informal planning for serious illness , such as conversations about treatment preferences and unacceptable health conditions , was only marginally higher , at 50% and 60% . in examining the demographic and health variables associated with planning behaviors , women were twice as likely as men to have discussed treatment wishes with someone close to them , but just as likely as men to have discussed unacceptable states of health , to have designated a substitute decision maker , or to have prepared a written advance care plan . similar findings were reported by garrett et al . , who reported women to be more likely to consider having a conversation about end - of - life wishes , but found no sex differences with respect to preparing an advance care plan . in the adjusted model , there were no associations between age and informal planning behaviors . older age proved significant only for the formal planning behavior of having designated a substitute decision maker , although the association between older age and having prepared an advance care plan approached significance ( p = 0.06 ) . given the formal nature of both these activities and the fact that half of the advance care plans were completed with the assistance of lawyers , it may be that older adults are more willing to consider planning for serious illness under the umbrella of estate planning than health . interestingly , the model found no associations between the number of health conditions reported by respondents and planning behaviors . it may be that an increasing number of health conditions are an expected sequela of the aging process and insufficient in and of themselves to trigger concern that planning for serious illness is warranted . the reason most frequently cited by respondents in this study for not preparing an advance care plan was that they those individuals may have been in the precontemplation phase , described in the transtheoretical model , and were not ready to engage in formal planning behaviors . the decision to participate in planning for serious illness activities would , according to this model , mean that the benefits must outweigh the the costs associated with the uncomfortable recognition and acceptance that one is personally vulnerable to poor health or death in the foreseeable future may have been such that respondents were not yet ready to engage in planning behaviors . if planning for serious illness is indeed a way to promote autonomy that is valued by older adults , it is worthwhile to further consider the factors that enhance readiness to participate in planning behaviors . a significant proportion ( 21.6% ) of respondents those who did not consider planning to be important were significantly less likely to have designated a substitute decision maker or have completed a written advance plan . while governments and health care providers continue to promulgate the benefits of planning for serious illness , it is important to bear in mind that a significant proportion of our patients may not share this value . nurses who seek to educate patients about strategies to plan for future medical care must first take the time to appraise whether , in fact , a given individual believes these activities to be of value . understanding the experiences that shape the individual 's perspective on advance care planning is critical to a truly patient - centred approach . knowledge about advance care planning was consistently and independently associated with all types of planning for serious illness behaviors ( discussions about treatment wishes or unacceptable states of health ; designation of a substitute decision maker ; preparation of a written advance care plan ) . the fact that respondents from rural areas , however , were only half as likely as those from urban areas to have an advance care plan suggests that access to information about planning for serious illness may , in fact , be a key driver for planning behavior . while the survey did not evaluate the access respondents had to education about advance care planning , access to health services is often limited in rural settings ( cihi ) . it is worthwhile to bear in mind , however , that the association between knowledge and behavior does not demonstrate causality , whereby more education about advance care planning leads directly to greater participation in planning . individuals who were already receptive to the importance of planning for serious illness may have availed themselves of opportunities to educate themselves about this process . teachable moments and providing support and education to those who demonstrate an interest and willingness to learn more about ways to best plan for future serious illness . tailoring interventions to the individual 's level of readiness is referred to as stage - matching within the transtheoretical model . while this survey was representative of the population of the province in terms of sex and region , the sample did not include the entire population of the province . stratification by socioeconomic status or additional variables was not possible , although level of education may reflect socioeconomic status to some extent . random selection occurred from the pool of those individuals who had agreed to participate in online surveys conducted by saskwatch research , and thus selection bias may have been present . education of the patients with respect to the process of advance care planning has been recognized as a public health issue in which nurses may make a significant contribution . as advocates for supporting the autonomy of older adults , however , nurses who engage in practice related to advance care planning must keep in mind that , for almost one quarter of respondents in this study , planning for serious illness was not considered important . that some individuals do not hold planning as a value is an important consideration when planning nursing interventions designed to educate and foster participation in advance care planning , highlighting the need to match interventions to levels of readiness .

objectivespontaneous intracerebral hemorrhage ( ich ) in young adults is rare . the purpose of this study was to investigate causes , sites and other factors affecting the prognosis of ich in young adults aged 40 years.methodswe reviewed 39 consecutive patients diagnosed with spontaneous ich between january 2001 and june 2012 . patients with primary subarachnoid hemorrhage , previously diagnosed brain tumor bleeding , or vascular malformation were excluded . we analyzed the differences in prognostic factors such as hemorrhage location and vascular structural etiology . the outcome was measured using the glasgow outcome scale ( gos ) , and a good outcome was defined as a score of 4 or more.resultswe retrospectively evaluated 39 patients ( mean age , 33 years ; sd = 6.4 , range 17 to 40 years ) . the most common structural etiology was arteriovenous malformation . a statistically significantly higher proportion of patients with good outcomes had a lower initial systolic blood pressure ( sbp 160 mmhg , p = 0.036 ) , a higher initial glasgow coma scale ( gcs ) ( 9 or more , p = 0.034 ) , lower cholesterol levels ( < 200 mg / dl , p = 0.036 ) , and smoking history ( at discharge , p = 0.008 ; 6 months after discharge , p = 0.019).conclusionin this study , cryptogenic ich was the leading cause of spontaneous ich . a gcs score of 9 or more on admission , a lower serum cholesterol level ( < 200 mg / dl ) , and a lower sbp ( < 160 mmhg ) predicted a good outcome . older adults have long been encouraged to maintain their autonomy by expressing their wishes for health care before they become too ill to meaningfully participate in decision making . this study explored the manner in which community - dwelling adults aged 55 and older plan for serious illness . an online survey was conducted within the province of saskatchewan , canada , with 283 adults ranging in age from 55 to 88 years . planning for future medical care was important for the majority ( 78.4% ) of respondents , although only 25.4% possessed a written advance care plan and 41.5% had designated a substitute decision maker . sixty percent of respondents reported conversations about their treatment wishes ; nearly half had discussed unacceptable states of health . associations between key predictor variables and planning behaviors ( discussions about treatment wishes or unacceptable states of health ; designation of a substitute decision maker ; preparation of a written advance care plan ) were assessed using binary logistic regression . after controlling for all predictor variables , self - reported knowledge about advance care planning was the key variable significantly associated with all four planning behaviors . the efforts of nurses to educate older adults regarding the process of advance care planning can play an important role in enhancing autonomy .

lung cancer is the leading cause of cancer death in most countries . although much effort has been made over the last few decades to improve the survival of lung cancer patients , overall five - year survival rates remain unsatisfactorily low ( 14% in the u.s.a . ) brain metastasis ( bm ) is a major cause of the low survival rate and poor quality of life of cancer patients . the prognosis for patients with bm who go untreated is extremely poor ( about one month following diagnosis ) ( 2 ) , whereas patients with non - small cell lung cancer ( nsclc ) who are treated with radiation therapy survive for about 8 months ( 3 ) . the incidence of bm in patients with locally advanced nsclc is 12 - 28% over the entire course of the disease . in most cases of nsclc , bm are diagnosed only after the development of symptoms , which is partly responsible for the poor prognosis of patients with nsclc . in patients with small cell lung cancer ( sclc ) , the incidence of detectable bm at the time of initial diagnosis is 10 - 14% ; the cumulative incidence at three years for patients that are in complete remission and that have few signs of disease is 59% ( 4 ) . for patients with nsclc , the incidence of bm at the time of initial diagnosis is about 6% according to a prospective study in which computerized axial tomography ( ct ) was used ( 5 ) . the prognoses of patients with symptomatic bm are substantially worse than those in which the metastases are asymptomatic ( 6 - 8 ) . clearly , improvements in systemic and local therapies can improve the long - term survival of cancer patients , which means that early and accurate diagnosis of bm has become crucial to improving the quality of life and poor survival rates of cancer patients . the use of imaging to detect extrathoracic metastasis , particularly bm , at the time of initial staging in asymptomatic patients is the subject of debate ( 8 - 10 ) . imaging is usually recommended in patients who form part of a population in which the overall incidence of bm is particularly high , such as in cases with adenocarcinomas ( 6 , 11 - 13 ) . of the different imaging methods , mri is more sensitive than ct and is the method of choice with which to screen for intracranial metastasis ( 12 , 14 - 17 ) . however , a major drawback to the routine use of brain mri is its high cost . therefore , we carried out the modified standard mri or limited mri procedure to detect bm at lower cost and without loss of sensitivity ( 18 ) . in our pilot study limited mri showed no difference with conventional mri for detecting bm ( sensitivity 97.67% , specificity 100% ) ( 18 ) . the cost of the modified mri was us$ 180 , which is substantially lower than the cost of conventional brain mri ( us$ 480 ) . based on these results , we conducted the present study to screen for bm at the time of initial staging and validated the clinical significance of the early detection of bm using limited brain mri . the results have implications for the quality of life and survival of patients with nsclc . between may , 2001 and april , 2002 , 183 patients were newly diagnosed with primary nsclc at chungnam national university hospital . all patients underwent the following initial staging procedures : clinical examination ; routine blood tests ; chest radiography ; chest ct ( including liver and adrenal glands ) ; whole - body bone scan ; and limited brain mri . the limited brain mri was modified from conventional mri by omitting t2-weighted axial , proton density axial , and contrast- enhanced t1-weighted images ( table 1 ) to decrease the cost from us$ 480 to us$ 180 without any decrease in sensitivity ( 18 ) . as a control group , we used 131 patients who were newly diagnosed with nsclc between may , 2000 and april , 2001 . control patients underwent the initial staging procedures described above , except that a brain mri was carried out only in cases that exhibited neurologic symptoms related to bm ( e.g. headaches , seizures , or unexplained weakness ) at the time of initial diagnosis . the incidence of bm in the initial staging of patients with primary nsclc and the incidence of metastases to other systemic sites were compared . we compared the clinically relevant characteristics of both groups ( age , sex , cell type , performance status , clinical stage , and incidence of brain metastasis ) . patients were followed up until the 30th of april , 2003 ( i.e. , a minimum period of one year ) and we analyzed the clinical significance of early detection of bm and the relationships among the various prognostic factors . differences between the two groups were evaluated for statistical significance using a chi - squared test . survival data were calculated using the kaplan - meier method and statistical differences were confirmed by means of the log - rank test . there were no statistically significant differences between the groups with respect to gender , age , performance status , tumor subtype , or clinical stage ( excluding brain metastasis ) ( table 2 ) . bm was detected in 38 of 183 ( 20.8% ) and 6 of 131 ( 4.6% ) patients in the study and control groups , respectively ( p<0.001 ) . twenty - three of 139 ( 16.5% ) males and 15 of 44 ( 34.1% ) females had bm . patients with non - squamous - type cells ( 32.6% ) had a higher frequency of bm than those with squamous - type cells ( 8.8% ) . during the follow - up period , subsequent bm developed in 3 of study group , and 9 of control group . in these patients , 1 of study group and 5 of control group had synchronous bm ( within 6 months after initial diagnosis ) ( table 3 ) . eighteen patients ( 47.4% ) had a single focus of metastasis within the brain , 17 patients ( 44.7% ) had multiple intracranial foci , and 3 patients ( 7.9% ) had a leptomeningeal pattern of metastasis ( table 4 ) . only 7 of 38 patients ( 18.4% ) in which bm was observed using limited mri also exhibited clinical symptoms related to bm ( table 4 ) . in 15 of 38 patients ( 39.5% ) in which bm was observed using limited mri , the brain was the only site of systemic metastasis , and these 15 patients were upstaged to stage iv based solely on the diagnosis of bm . this number included 11 of 68 patients ( 15.9% ) that were considered initially to be resectable surgically in the absence of bm . two stage i , three stage ii , six stage iiia , and four stage iiib patients were upstaged to stage iv ( table 5 ) . for the survival analysis , 10 of 183 patients in the study group were excluded due to insufficient follow - up data . the survival analysis was carried out on 173 patients . the overall median survival time and one - year survival rate of patients with bm were 43 weeks and 39.5% and 31 weeks and 16.7% for the study and control groups , respectively . the number of patients in the control group was too small to determine whether there were any statistically significant differences between the two groups . the one - year survival rate and median survival time of patients who had bm ( 46.7% and 49 weeks , respectively ) was greater only than patients who had multiple systemic metastases that included the brain ( 34.8% and 27 weeks , respectively ) . eighteen patients had between one and three metastatic nodules in the brain that could be accessed for stereotactic radiosurgery and 20 patients ( including three with leptomeningeal involvement ) had multiple ( > 3 nodules ) foci within the brain . the one - year survival rate and median survival time for the former and latter type of patient was 44.4% and 45 weeks and 35.0% and 24 weeks , respectively . of the 38 patients who had bm and were strongly recommended to receive radiation therapy to the brain with systemic chemotherapy , 14 were treated with whole brain irradiation only and 23 were treated with both radiation and chemotherapy . the one - year survival rate and median survival time of irradiation- treated patients was 21.4% and 21 weeks , respectively , and the one - year survival rate and median survival time of those treated with both radiation and chemotherapy was 52.2% and 49 weeks , respectively . the one - year survival rate and median survival time for symptomatic vs. asymptomatic patients was 42.9% and 45 weeks and 38.7% and 43 weeks , respectively ( table 6 ) . the one - year survival rate and median survival time overall for stage iv patients were 36.1% and 37 weeks , respectively . the median survival of 12 patients in whom bm was diagnosed during the follow - up treatment period was 8 weeks . even though locoregional control rates of 50% and higher have been achieved after neoadjuvant chemoradiotherapy and definitive resection ( multimodal therapy ) in patients with locally advanced nsclc , the brain is the most frequent site of initial treatment failure and is the site of approximately one third of all relapses that occur ( 11 , 13 , 19 , 20 ) . therefore , treatment strategies that reduce the risk of bm are needed to optimize the efficacy of multimodal therapy and to improve survival . between 25 and 30% of all patients with lung cancer develop bm at some stage , and this incidence is likely to increase if survival is prolonged by means of aggressive multi - modal treatment . the increasing frequency of treatment failure , the development of new imaging techniques and treatments , as well as the reports of prolonged survival of patients that have received aggressive treatment , suggest that approaches to treating patients who have bm should be reassessed . specifically , novel treatment paradigms should be developed to ensure that such patients receive optimal therapeutic intervention . to this end , prophylactic cranial irradiation in nsclc and sclc patients has been introduced and has led to several reports of positive outcomes , although a large - scale study is needed to confirm this ( 11 , 13 , 21 ) . an alternative means to the same end is the introduction of brain imaging when the initial workup is carried out . this approach would facilitate the early detection of bm and promote the timely application of appropriate treatments , which would improve the survival rates of cancer patients . however , such early imaging should be considered only if it is practicable and available at a reasonable cost . considering each of the aforementioned factors , we designed the present study to assess the clinical effectiveness of using limited brain imaging at the time of initial screening of lung cancer patients . the rate of bm in patients with locally advanced nsclc has been estimated previously to be between 17 and 32% ( median : 22% ) with a median time - to - brain recurrence of 7.5 - 9.3 months ( 8 , 11 , 13 ) . it has been shown previously that most recurrences of metastases within the central nervous system occur within 2 yr of the initial diagnosis ( 13 ) . furthermore , bm is a direct cause of death in between one third and one half of affected patients ( 12 ) . the incidence of bm in the initial screening of patients with primary lung cancer has been reported to be between 12 and 18% ( 22 , 23 ) . in the present study , we suspect that this slightly higher incidence was the result of using the limited brain mri procedure , which is more sensitive than brain ct ( 5 , 17 ) . there are several risk factors that are related to bm , including non - squamous histological characteristics ( particularly adenocarcinomas ) , bulky lymph nodes in the mediastinum , younger age , stage iiib , etc . our data supported the same trend and , in addition , suggested that the incidence of such risk factors is greater in females than in males . specifically , in the present study , the incidence of bm and non - squamous tumors in females was 30.6 and 26.2% , respectively , which was statistically higher than in males ( 15.5 and 8.8% , respectively ) . the incidence of bm in all patients with adenocarcinomas was 33.3% , whereas the incidence in females with adenocarcinomas was 50% . in clinical stage iv patients , the brain was the second most common site of metastasis ( 38/87 patients or 43.7% ) , following bone ( 39/87 , 44.8% ) , and was a more common site of metastasis than lung , liver , and the adrenal glands . although neurological symptoms have been reported to be associated with a poor prognosis ( 7 , 8 , 14 ) , the survival rates in the present study were unrelated to the presence of clinical symptoms . we found that patients who had a single focus of bm , and isolated bm , and who were treated with chemotherapy had better survival , as reported by others . the survival rate of patients who had bm was equivalent to those who did not have any clinical symptoms of bm ; this was due to early treatment using whole brain radiotherapy , stereotactic radiosurgery , and chemotherapy . reported that the use of chemotherapy to control not only bm but also extracranial lesions prolonged the survival of patients with bm due to nsclc ( 26 ) . in the present study , the survival rates of patients who had bm and who received both chemotherapy and radiotherapy were similar to those of patients in stage iiib ( data not shown ) , which would appear to support our hypothesis that early detection of bm and subsequent application of appropriate treatment may improve overall survival . in the present study , 15 patients ( 15/111 , 13.5% ) were upstaged from clinical stage i ( n=2 ) , ii ( n=3 ) , iiia ( n=6 ) , or iiib ( n=4 ) to stage iv , because of the presence of bm , including 11 patients ( 11/68 , 15.9% ) who had been considered initially to be suitable for surgical resection in the absence of evidence for the presence of bm . all of the aforementioned patients were treated with either radiotherapy alone ( n=7 ) or both radiotherapy and chemotherapy ( n=8 ) . the median survival time and one - year survival rate of the former and latter patients was 43 weeks and 28.6% and 72 weeks and 66.7% , respectively . that was greater than the survival rate of all patients with bm ( median survival time and one - year survival rate for radiotherapy only and radiotherapy plus chemotherapy for all patients with bm were 21 weeks and 21.4% , and 49 weeks and 52.2% , respectively ) ( table 6 ) . during a follow up period of at least one year , 12 patients ( 3 of study group and 5 of control group ) subsequently developed bm . all of these patients ( except one ) were treated with chemotherapy after the diagnosis of bm . initially , these patients responded to chemotherapy such that four exhibited partial responses , five exhibited a stable disease state , and three exhibited progression of the disease . the median time to relapse in treated patients ( n=11 ) was 38 weeks , which is similar to the time reported by ceresoli et al . these 11 patients were treated with radiation only without any further chemotherapy ; their median survival time after the diagnosis of bm was 8 weeks , which was shorter than that of patients in whom metastasis was detected during the initial screening and who were treated with radiation therapy alone ( 21 weeks ) . their overall median survival time and one - year survival rate ( after the diagnosis of lung cancer ) was 38 weeks and 27.3% , respectively , worse than the study group that had bm at the time of the initial screening ( 43 weeks and 39.5% ) and worse than patients who received whole - brain radiation and chemotherapy ( 49 weeks and 52.2% ) following the initial screening . nevertheless , these outcomes were slightly better than those of the control patients , who were diagnosed and treated for brain metastasis only upon the development of symptoms ( 31 weeks and 16.7% ) . in conclusion , limited brain mri appears to be a useful and cost - effective means by which to detect bm at the time of initial staging of lung cancer patients . such early detection of bm enabled patients to be treated immediately with radiotherapy and chemotherapy and led to improved rates of survival . consequently , we recommend that limited brain mri should be used as an initial screening procedure for the early detection of bm . this will allow lung cancer patients to receive the appropriate treatment in a timely manner , which should improve both the survival and quality of life of such patients . hyponatraemia is the most commonly encountered electrolyte abnormality in hospitalised patients particularly in the intensive care unit ( icu ) . in a post - surgical icu , the syndrome of inappropriate secretion of antidiuretic hormone ( siadh ) is a frequent cause for hyponatraemia . vaptans , a new group of vasopressin receptor antagonists , are increasingly being used for the treatment of euvolaemic , hypervolaemic forms of hyponatraemia . conivaptan is now gradually becoming the most popular drug among the vaptans for correction of this condition in the icu . this is administered as an initial intravenous ( iv ) bolus followed by an infusion , which may be continued for days . in this study , we aimed to assess the effectiveness of a single dose iv conivaptan for correction of hyponatraemia in post - operative icu patients . this was a prospective , randomised trial conducted in 40 patients from april 2013 to december 2014 . based on the key article , and due to the availability of limited number of cases satisfying the inclusion criteria , the study was restricted to 40 patients , with 20 in each group ( a and b ) . post - operative icu patients who had undergone major head and neck surgeries for malignancies with a serum sodium level of 130 meq / l and were symptomatic ( headache , nausea , vomiting , lethargy , confusion , disorientation ) were included in the study . patients were randomly allocated to one of the two groups ( a and b ) using computer generated sequence of random numbers . pre - operative nutritional status and hydration of the patients in both groups were assessed with body mass index ( bmi ) , haemoglobin , serum albumin and sodium levels , pre - operative heart rate ( hr ) and mean arterial pressures . perioperative normovolaemia was assessed by comparing pre - operative and post - operative urea levels in addition to intraoperative urine output per hour . the 24 h fluid balance of the patients 2 days and 1-day prior to commencement of study was also documented . when symptomatic , for correction of hyponatraemia , group a patients received iv conivaptan 20 mg over 30 min , whereas in group b infusion of 3% hypertonic saline was started as an infusion at the rate of 2030 ml / h , the exact dose being calculated using the formula , the rate of correction in serum sodium was aimed to be 68 meq / l in 24 h , 1214 meq / l in 72 h. no diuretics were concurrently administered and there was no fluid restriction . hypotension was defined as a reduction of > 30% of systolic bp from baseline value . serum sodium was checked at beginning of treatment , 12 , 24 , 48 and 72 h after initiation of treatment . the daily fluid balance was recorded at 24 , 48 and 72 h following initiation of treatment . the statistical software ibm spss version 20.0 was used for data analysis . for comparing american society of anesthesiolgists ( asa ) physical status , gender and thrombophlebitis , mann - whitney test was used to compare the pre - operative nutritional status and hydration , post - operative serum sodium values , fluid balance and volume of hypertonic saline used at different time periods . mean age , weight , bmi , distributions of sex and asa physical status among patients in group a and b were comparable [ tables 1 and 2 ] . pre - operative haemoglobin , serum albumin , sodium , hr and mean arterial pressures of both groups were also comparable [ table 2 ] . comparison of pre - operative and post - operative urea levels , intraoperative urine output as well as the post - operative daily fluid balance 2 days and 1-day prior to commencement of study did nt show any significant difference between the groups [ p < 0.05 , table 3 ] . comparison of demographics and asa physical status comparison of pre - operative nutritional status and hydration comparison of perioperative fluid volume status the serum sodium level before initiating treatment for hyponatraemia was also comparable between the groups . however , the subsequent sodium level measured at 12 , 24 and 48 h showed significantly high values in group a ( p < 0.05 ) . at 72 h the mean sodium value was high in group a but the difference was not statistically significant [ p = 0.159 , table 4 and figure 1 ] ] . comparison of serum sodium levels and daily fluid balance during study changes in sodium levels group a showed a significantly high fluid loss ( negative fluid balance ) on day 1 , 2 and 3 following initiation of treatment as compared to group b. in addition , it was also observed that on day 3 , the fluid balance in group b had become positive [ table 4 ] . the mean volume of hypertonic saline used in group b showed a steady decline from day 1 to 3 [ 430 92.3211.5 65 ml , figure 2 ] and only 13 patients required hypertonic saline on the third day . this could probably explain the reason for a positive fluid balance observed in group b on the third day . hypertonic saline requirement in group b though less number of patients in group a developed thrombophlebitis compared to group b ( 10 vs. 50% ) , the difference was not statistically significant ( p = 0.014 ) . dysregulation of arginine vasopressin ( avp ) frequently occurs in hospitalised patients resulting in hyponatraemia and is associated with increased morbidity and mortality . non - osmotic secretion of avp is the main pathophysiology in patients with euvolaemic , hypervolaemic hyponatraemia . acute onset hyponatraemia ( duration of < 48 h ) requires prompt correction , whereas chronic hyponatraemia should be corrected cautiously as there is a higher risk of development of central pontine myelinolysis following rapid correction . although earlier recommendations supported a correction of < 1012 mmol / l of sodium during the first 24 h of treatment and a target < 18mmol at the end of 48 hrs , the occurrence of osmotic demyelination even at these values has led to caution in the rates of correction . meq / l in 24 h , 1214 meq / l in 48 h and 1416 however , in symptomatic patients regardless of chronicity , a rapid rise of 46 meq / l is adequate . conivaptan is a non - selective vasopressin receptor antagonist available in an iv form and is approved by the food and drug administration to treat euvolaemic , hypervolaemic hyponatraemia . though conivaptan has high affinity for both v1a ( vascular ) and v2 ( renal ) receptors , affinity for v2 is tenfold higher and the aquaretic effect is pre - dominantly v2-associated . v1 receptor antagonism in vascular smooth muscle cells results in vasodilatation whereas antagonism of v2 receptors present in the renal collecting duct results in aquaresis . side effects of conivaptan include minimal to clinically significant fall in bp , postural hypotension and hypokalaemia . though in most patients rate of increase in sodium level is moderate , there are reports of rapid correction as fast as 14 meq / l within 4 h when conivaptan 4080 mg / day was used . even though no serious neurological sequelae had been reported because of rapid correction , possible development of osmotic demyelination syndrome should be kept in mind . other possible side effects include rebound hyponatraemia and renal damage due to significant hypovolaemia leading to hypotension and acute tubular necrosis . increased frequency of adverse cardiac events , atrial dysrhythmias and sepsis in the presence of congestive cardiac failure however , v2 receptor antagonists ( v2ra ) are now indicated selectively for the treatment of hyponatraemia caused by congestive cardiac failure . tolvaptan or other v2ra may be of benefit in selected patients with severe congestive symptoms . recent research indicates that conivaptan and tolvaptan are safe and not associated with increased morbidity such as renal failure and arrhythmias . conivaptan is usually started at a dose of 20 mg iv over 30 min , followed by an infusion of up to 20 mg over the next 24 h , given to a maximum of 4 days , closely monitoring serum sodium levels , bp and volume status . though conivaptan treatment is costlier , it involves a significantly lower volume of medication and possibly reduces icu stay . data from the study by galton et al . suggest that in non - hyponatraemic patients with severe traumatic brain injury , the use of single dose conivaptan is safe and may reduce intracranial pressure . a single bolus dose of 20 or 40 mg was found to be effective for the correction of acute hyponatraemia in neurologically injured patients and the effect of intermittent bolus dosing lasts up to 72 h. similar results were obtained in our study also though the study population was different . while giving an iv infusion , to avoid the risk of an accidental administration of a bolus dose , it is always preferable to administer the drug using an infusion pump . the advantages of using single dose conivaptan are that only a lesser amount of the drug is required , which will reduce the cost of treatment by avoidance of an infusion , which requires careful monitoring or an infusion pump for safe administration . the superiority of 3% hypertonic saline in raising the serum sodium concentration in hyponatraemia patients is well proven . it is less expensive but carries a risk of volume overload in oliguric or anuric patients . a drawback of hypertonic saline is that it has to be given as an infusion and the duration of drug administration may extend over many hours . in the present study , the pre - operative nutritional and hydration status of patients in both the groups were comparable and well within normal limits [ table 2 ] . perioperative normovolaemia was ensured as reflected by adequate urine output and comparable pre - operative and post - operative blood urea levels [ table 3 ] . the positive daily fluid balance observed 2 days prior to patients becoming symptomatic indicated the gradual onset of development of hyponatraemia [ table 3 ] . the main limitations of our study were a low sample size and the fact that urine osmolarity was not analysed . we did not statistically analyse the haemodynamic parameters during the period of the study , but there were no alterations in hr or bp during the 72 h study period . none of the patients developed hypokalaemia during our study , but another limitation was that this was again not statistically analysed . based on the results of our study we suggest that a single dose of conivaptan can be considered for the initial correction of hyponatraemia , with serial serum sodium level monitoring , rather than prescribing a bolus dose followed by an infusion . a single dose of conivaptan was found to be effective in increasing the serum sodium levels up to 72 h , with a significantly negative fluid balance , in post - operative icu patients following major head and neck surgeries .

the purpose of this prospective study was to determine whether using magnetic resonance imaging ( mri ) for early screening for brain metastases ( bm ) can improve quality of life , survival in patients with non - small cell lung cancer ( nsclc ) . the study group comprised 183 patients newly diagnosed with nsclc . all patients underwent limited brain mri and routine workups . the control group comprised 131 patients with nsclc who underwent limited brain mri only if they had neurologic symptoms . the incidence of bm was 20.8% ( 38/183 ) in the study group and 4.6% ( 6/131 ) in the control group . the rate of upstaging based on the mri data was 13.5% ( 15/111 ) overall and 15.9% ( 11/69 ) in patients that had been considered initially to be resectable surgically . there was no significant difference in survival outcome between the groups . patients who had bm alone had a greater overall survival time ( 49 weeks ) than those who had multiple systemic metastases ( 27 weeks ; p=0.0307 ) . in conclusions , limited brain mri appears to be a useful , cost - effective method to screen for bm at the time of initial staging . and it may facilitate timely treatment of patients with nsclc and improve their survival and quality of life . background and aims : conivaptan , a vasopressin receptor antagonist , is commonly used for the treatment of euvolaemic , hypervolaemic hyponatraemia . usually , an intravenous ( iv ) bolus followed by infusion is administered for many days . we decided to assess the effectiveness of single dose conivaptan for correction of hyponatraemia in post - operative patients.methods:this was a prospective , randomised trial conducted in 40 symptomatic post - operative intensive care unit ( icu ) patients with a serum sodium level of 130 meq / l . group a patients received iv conivaptan 20 mg over 30 min , whereas in group b infusion of 3% hypertonic saline was started as an infusion at the rate of 2030 ml / h . serum sodium levels were measured at 12 , 24 , 48 and 72 h and the daily fluid balance was measured for 3 days . the chi - square test , wilcoxon signed rank test and mann - whitney tests were used as applicable.results:the serum sodium levels before initiating treatment were comparable between groups . however , subsequent sodium levels at 12 , 24 and 48 h showed significantly high values in group a. though at 72 h the mean sodium value was high in group a , it was not statistically significant . group a showed a significantly high fluid loss on day 1 , 2 and 3 . the mean volume of hypertonic saline required in group b showed a steady decline from day 1 to 3 and only 13 patients required hypertonic saline on the 3rd day.conclusion:single dose conivaptan is effective in increasing serum sodium levels in post - operative icu patients up to 72 h associated with a significant negative fluid balance .

the introduction and subsequent sustained global spread in the human population of influenza a viruses with a novel hemagglutinin ( ha ) subtype leads to an influenza pandemic . the novel influenza pandemics that occurred during the last century resulted in considerable mortality and morbidity . genetic investigations revealed that these pandemic strains were partially or entirely derived from viruses of avian origin [ 14 ] and that most of them first appeared in southern china , a hypothetical influenza epicenter . the avian h5n1 influenza virus that infected poultry and humans in hong kong in 1997 caused the death of 6 of 18 persons that were confirmed infected with this virus [ 68 ] . this virus was proposed to be a naturally occurring avian virus produced by the reassortment of h5n1 and h9n2 or h6n1 viruses [ 6 , 7 ] . h9n2 influenza viruses have become panzootic during the last decade and have been isolated from different types of terrestrial poultry worldwide [ 911 ] . two distinct lineages of h9n2 viruses , represented by the prototype a / duck / hong kong / y280/97 ( h9n2 ) ( dk / hk / y280/97 ) and a / quail / hong kong / g1/97 ( h9n2 ) ( qa / hk / g1/97 ) viruses , have become established in terrestrial poultry : dk / hk / y280/97-like viruses are found predominantly in chickens whereas qa / hk / g1/97-like viruses are most often found in quail [ 10 , 12 ] . the qa / hk / g1/97-like viruses are thought to have been involved in the generation of the highly pathogenic h5n1 virus first isolated in 1997 . h9n2 viruses of each lineage have been isolated from humans [ 13 , 14 ] , and the dk / hk / y280/97-like lineage also has been isolated from pigs in southern china . during 1997 , an h6n1 influenza a virus , a / teal / hong kong / w312/97 was isolated from a green - winged teal . subsequent characterization of the virus showed that seven of its eight gene segments were closely related to those of the h5n1 influenza viruses isolated from humans in 1997 [ 68 , 17 ] . later studies showed that a / teal / hong kong / w312/97-like viruses continued to circulate in the hong kong live - bird markets and that all h6n1 viruses isolated up until 2000 belonged to this lineage . similarly , the host range of these h6n1 viruses remained stable , with most isolates originating from quail and minor " poultry species such as chukka , pheasant , and guinea fowl but rarely originating from chickens . beginning in 2001 , the host range appeared to be changing , with an increase in the number of h6n1 viruses isolated from chicken and silky chicken . to address whether this phenomenon was real and , if so , whether it was due to a change in the h6n1 viruses circulating in the markets , we investigated the antigenic , genetic , and biologic characteristics of various h6 influenza viruses isolated from the hong kong live - bird markets between 2001 and 2003 . this paper describes the results of these studies and demonstrates an abrupt increase in the genetic diversity of h6 viruses in these markets . the viruses isolated in the hong kong region and the abbreviations used in this study are listed in table 1 . viruses present in fecal and cloacal samples from various bird species were grown in 10- to 11-day - old embryonated chicken eggs . the h6 virus antigens were compared by using the hemagglutination inhibition ( hi ) test as previously described in . all sera were pretreated with the receptor - destroying enzyme from vibrio cholerae ( denka seiken , tokyo ) to abolish interference by nonspecific serum inhibitors . the viruses , which came directly from infected allantoic fluid , were diluted to doses equivalent to four ha units for use in the tests . viral rna was extracted from infected allantoic fluid by using rneasy kits ( qiagen , valencia , ca ) according to the manufacturer 's instructions . reverse transcription and pcr were performed under standard conditions by using primers specific for the various genes of influenza viruses . sequencing reactions were performed by the staff of the hartwell center for bioinformatics and biotechnology at st . template dna was sequenced by using drhodamine dye terminator cycle sequencing ready reaction kits with amplitaq dna polymerase fs ( perkin - elmer applied biosystems , inc . [ pe / abi ] , foster city , ca ) and synthetic oligonucleotides . samples were subjected to electrophoresis , detection , and analysis on pe / abi model 3700 dna sequencing instruments . dna sequences were compiled and edited by using the lasergene sequence analysis software package ( dnastar , madison , wi ) . multiple sequence alignments were made by using clustal w , and phylogenetic trees were generated by using the neighbor - joining algorithm in the treeview version 1.6.6 software package ( available at http://taxonomy.zoology.gla.ac.uk/rod/treeview.html ) . nucleotide and amino acid sequences of other influenza viruses were obtained from the influenza sequence database of the los alamos national laboratory . the ability of the h6 viruses to hemagglutinate chicken , horse , and guinea pig erythrocytes was determined as previously described in . briefly , 32 ha units of virus ( as determined by binding to chicken erythrocytes ) were used in a hemagglutination assay with 0.5% ( v / v ) horse or guinea pig red blood cells . chicken and guinea pig erythrocytes contain both 2 - 3- and 2 - 6-linked sialic acid whereas those from horse contain almost exclusively 2 - 3 linkages , thereby indirectly inferring receptor preferences of the agglutinating viruses . we used 5-to 6-week - old japanese quail and white leghorn chickens to determine the 50% infectious doses ( qid50 and cid50 ) of a selection of h6 viruses . infectious allantoic fluid 10-fold serially diluted ( 1 : 10 to 1 : 10 ) was intranasally administered to three quails ( 0.2 ml inoculum ) or chickens ( 0.5 ml inoculum ) of each group . birds were examined daily for disease signs , and cloacal and tracheal swabs were taken 3 and 5 days after inoculation . tracheal swabs were placed in 0.5 ml of sample medium ( 50% glycerol in phosphate - buffered saline ( pbs ) that contained 1000 u / ml penicillin , 200 g / ml streptomycin , 50 u / ml mycostatin , 100 u / ml polymyxin b , and 250 g gentamicin ) ; cloacal swabs were placed in 1 ml of sample medium . each of two embryonated chicken eggs were inoculated with 100 l of the sample medium containing tracheal or cloacal swabs and were incubated for 48 hours at 37c . ha assays using chicken red blood cells were performed to verify virus growth in the harvested allantoic fluid . briefly , uninfected birds were placed in direct contact with or in cages above and below the inoculated birds ( 10 pfu per bird ) 1 day after inoculation . intensive surveillance systems in the hong kong live - bird markets have been in place since 1997 . from 2001 through 2003 , these systems showed that 253 isolates of 42495 tested specimens were h6 influenza viruses ( 232 in 2001 , 12 in 2002 , and 9 in 2003 ) . we arbitrarily selected 18 h6 influenza viruses isolated from domestic poultry for further study ( table 1 ) . since 1997 , h5 viruses in hong kong have undergone antigenic and genetic change . to determine whether similar antigenic changes have occurred in h6 viruses in hong kong , we characterized the antigens of recent isolates by using hi tests . the h6 influenza viruses isolated in hong kong reacted with hyperimmune goat antisera raised against a / turkey / massachusetts/1/65 ( h6n2 ) ( ty / ma/1/65 ) , hyperimmune rabbit antisera raised against a / shearwater / australia/1/72 ( h6n5 ) ( sh / aus/1/72 ) , and postinfection chicken antisera against a / teal / hong kong / w312/97 ( h6n1 ) ( tl / hk / w312/97 ) , a / quail / hong kong / yu1654/00 ( h6n1 ) ( qa / hk / yu1654/00 ) , a / quail / hong kong / yu39/01 ( h6n1 ) ( qa / hk / yu39/01 ) , and a / duck / shantou/5540/01 ( h6n2 ) ( dk / st/5540/01 ) ( table 2 ) . the antisera against ty / ma/1/65 and sh / aus/1/72 were broadly reactive against all recent h6 viruses , as were antisera to qa / hk / yu39/01 and dk / st/5540/01 . all 2002 and 2003 isolates were antigenically indistinguishable from other viruses isolated during the same year whereas some heterogeneity existed among the 2001 isolates . although there were detectable differences in reactivity patterns to antiserum against qa / hk / yu39/01 between viruses of subsequent years , the h6 viruses of 2001 , 2002 , and 2003 represented a relatively antigenically homogeneous group of viruses . they had , however , changed in comparison to tl / hk / w312/97 , particularly in reactivity to antisera against ty / ma/1/65 and sh / aus/1/72 . the full - length ha sequences of the 18 h6 influenza viruses were determined to analyze their phylogenetic relationships ( figure 1 ) . the ha gene of contemporary h6 influenza viruses isolated from terrestrial poultry had an open reading frame of 1704 bp that encodes a precursor protein of 567 amino acids . the insertion of a conserved aspartic acid between positions 144 and 145 ( h3 numbering ) of the precursor polypeptides distinguishes the contemporary terrestrial poultry viruses from aquatic bird viruses . all h6 isolates possessed the typical nonpathogenic sequence pqietr / g at the ha cleavage site [ 2628 ] . the homology between the ha nucleotide sequences of the contemporary isolates and that of tl / hk / w312/97 ranged from 96% to 98% . the two strains isolated from chicken in 2001 ( ck / hk / sf3/01 and ck / hk / sf4/01 ) were typical of other h6 viruses within the terrestrial host clade . in addition to the ha genes , the na genes of all 18 h6 isolates were sequenced . the n1na genes from 14 virus isolates from domestic poultry , including the two isolates from chicken , belonged to the cluster represented by tl / hk / w312/97 . this cluster was distinguishable from that containing a / goose / guandong/1/96-like ( h5n1 ) n1na genes . the remaining two n1na genes , which were from two of the 2001 isolates ( qa / hk / fb611/01 and qa / hk / fb801/01 ) , were slightly different from those of the tl / hk / w312/97-like n1na cluster and instead were more similar to that of a / quail / hong kong/1721 - 20/99 . alignment analysis revealed a 19-amino acid deletion from the stalk region of the n1nas of all contemporary terrestrial isolates . unexpectedly , two of the contemporary h6 viruses isolated from domestic poultry contained n2 genes . the n2na gene of sc / hk / ap46/01 was similar to that of qa / hk / g1/97-like ( h9n2 ) viruses ( 98% identity to a / hk/1074/99 ) ; the high degree of similarity suggests that sc / hk / ap46/01 was a reassortant derived from cocirculating h6n1 and h9n2 viruses . the n2na gene of gf / hk / ssp99/02 did not belong to the qa / hk / g1/97-like ( h9n2 ) lineage but instead to a lineage represented by recent duck h9n2 isolates ; this finding suggests reassortment between h6n1 viruses and aquatic bird viruses . in 1998 and 1999 , the h6n1 viruses isolated from the hong kong markets were genetically stable and were of the same genetic lineage . to determine whether the stability of h6 viruses has continued since 1999 , we analyzed partial sequences for each of the remaining genes from the isolated h6 viruses . phylogenetic analysis of the pa polymerase ( pa ) gene revealed that , after 2001 , reassortment occurred not only with na genes but also with internal genes . pa genes from all nine viruses isolated in 2001 as well as those of qa / hk / yu404/02 , and qa / hk / yu421/02 were similar to tl / hk / w312/97-like viruses . the pa genes of ph / hk / ssp44/02 , gf / hk / ssp99/02 , and all of the 2003 isolates showed high homology to those of h5n1 viruses isolated from domestic poultry in 2000 and 2001 . also belonging to this lineage were the h5n1 viruses isolated from humans in hong kong in 2003 [ 29 , 30 ] . the np gene segments of ph / hk / ssp44/02 and all of the 2003 isolates were distinct from that of the tl / hk / w312/97-like lineage , as were the ns gene segments of ph / hk / ssp44/02 , gf / hk / ssp99/02 and all of the 2003 isolates . these np and ns gene segments were most similar to those of dk / hk / y280/97-like viruses ( h9n2 ) . the np and ns gene segments of a / pheasant / hk / fy294/00 ( h6n1 ) also clustered with those of dk / hk / y280/97-like viruses ; this clustering indicated that the reassortment events between h6 and h9 viruses had happened as early as 2000 . although there were minor differences , the remaining gene segments ( pb2 , pb1 , and m ) of the h6 viruses isolated from domestic poultry belonged to the tl / hk / w312/97-like lineage ( table 3 ) . the nucleotide sequences for 18 h6 strains presented in this paper have been submitted to dna data bank of japan ( ddbj ) under the accession numbers ab586744 to ab586887 . the receptor specificity of the ha is considered a likely determinant of host range for influenza viruses . additionally , studies have shown that h9n2 viruses circulating in the hong kong live - bird markets have human virus - like receptor specificity . to explore the receptor binding specificity of tl / hk / w312/97 and contemporary h6 isolates , the ability of these viruses to agglutinate erythrocytes from chicken , horse , and guinea pig was measured . tl / hk / w312/97 and recent h6 viruses from aquatic birds [ dk / st/5540/01 ( h6n2 ) and he / hk / lc10/03 ( h6n8 ) ] were uniform in their ability to agglutinate erythrocytes from all sources to similar levels ( table 4 ) . conversely , the recent h6 isolates from domestic poultry were able to agglutinate erythrocytes of chicken and guinea pig origin but not those from horse . the binding of the contemporary h6 isolates was similar to that of qa / hk / g1/97 , a virus with human virus - like receptor specificity . these results demonstrate that the receptor binding properties of the recent h6 isolates from poultry have evolved from those of tl / hk / w312/97 . an increase in the number of h6 viruses isolated from chickens in the hong kong live - bird markets raised the possibility that the host range of these viruses was expanding and that they were adapting to chickens . to test this hypothesis , we determined for chicken and quail the 50% infectious doses of selected h6 viruses isolated from different host species . tl / hk / w312/97 and the domestic poultry h6 isolates from 2001 , 2002 , and 2003 were able to infect quail and chickens ( table 5 ) , although in each case the id50 was lower for quail than for chicken . none of the tested strains showed a markedly increased infectivity for chicken ; the lack of an increase suggests that the contemporary h6 viruses , including those isolated from chickens , were not more adapted to chickens than was tl / hk / w312/97 . the ability of a virus to replicate within a host does not always correspond to its ability to be transmitted between individuals of that host . the ability of the domestic poultry h6 viruses to grow in chickens upon experimental infection conflicts with the low rates of isolation from this host in the markets . to determine the role of transmission in this apparent contradiction , we assessed the ability of the h6 viruses to be transmitted from chicken to chicken and from quail to chicken . although the h6 viruses we selected replicated in the inoculated animals , no virus was isolated from the animals in direct contact with infected birds ( table 6 ) ; this result shows that transmission of the h6 viruses to chickens is poor . in contrast , qa / hk / csw106/01 , ck / hk / sf4/01 , and ph / hk / ssp44/02 were efficiently transmitted from infected to contact quail ( data not shown ) . these infection and transmission results suggest that the h6 viruses circulating in southeastern china have not adapted to chicken populations and are unlikely to do so without further genetic change . the outbreak of h5n1 influenza in 1997 prompted researchers to hypothesize that chickens have the capacity to act as an intermediate host for influenza virus in humans . in light of this possibility , the observation that the host range of h6 viruses within the hong kong live - bird markets might be increasing to include chickens caused concern . h5n1 and h9n2 viruses that had been circulating in poultry in hong kong have caused human infection [ 7 , 8 , 13 , 14 , 17 ] . h6n1 viruses have been cocirculating with these h5n1 and h9n2 viruses in hong kong but have not been isolated from humans , and these viruses appear to have remained genetically stable with regard to antigenic drift and reassortment . the results of our study show that , although the 2003 h6 viruses are not more adapted to chicken than are earlier isolates , h6 viruses in hong kong are evolving and reassorting with other viruses . antigenic and genetic analyses of the h6 influenza viruses isolated from domestic poultry in southeastern china from 2001 through 2003 provide convincing evidence that the h6 influenza viruses are reassorting with h9n2 , h5n1 , and other viruses in the region . previous studies have suggested that h9n2 viruses have a two - way transmission between terrestrial and aquatic birds . this two - way transmission resulted in the generation of multiple genotypes of h9n2 viruses containing internal genes of aquatic avian origin . it is unclear whether the same type of transmission is occurring with h6n1 viruses or whether they are acquiring aquatic bird genes from h9n2 or similar viruses . this pattern parallels the re - emergence of h5n1 influenza viruses in hong kong in 2001 during which multiple new genotypes were identified . although all h6 ha genes appear to be of the tl / hk / w312/97 lineage , the result of hi assays indicate that despite a high level of genetic similarity , there has been a gradual decline in the reactivity of the recent isolates to serum raised against isolates collected in 2001 . the ability of the tl / hk / w312/97-like h6n1 or h6n2 viruses to replicate and transmit in quail but not in chickens suggested that the removal of quail from the hong kong markets ( accomplished in february 2002 ) would substantially affect the isolation rates of h6 viruses . in 2001 , 232 h6 viruses were isolated in the market but only 12 during 2002 and 9 in 2003 were isolated . this dramatic reduction in the isolation rate does indeed suggest that quail were playing an important role in maintaining h6 viruses within the markets . the comparatively small number of birds such as chukkas and pheasants in the markets corresponds with the small number of h6 viruses isolated since the removal of quail . the lack of efficient transmission of the h6 viruses to chicken under experimental conditions , however , conflicts with the isolation of these viruses from chicken in the markets . many market samples are fecal samples taken from cages beneath a given species of bird . although the isolates are likely from the species housed in the above cage , there is the possibility of environmental contamination . it is likely that the birds in the markets are under additional stresses exerted by temperature and other environmental factors in addition to the presence of other pathogens . such stresses which are impossible to mimic in laboratory settings may increase the susceptibility of the birds to influenza infection . regardless , our results showing the unlikely role of chicken as efficient hosts of h6 viruses in the hong kong market system suggest that h6 viruses are now maintained by species such as chukkas and pheasants but not by chickens . correspondingly , it should be expected that their incidence will remain low unless further adaptation to the chicken host is achieved . in addition to the reduction in isolation rates , the increase in the genetic diversity within the h6 viruses in the markets also corresponds with the removal of quail . exactly what role quail may have had in maintaining a stable tl / hk / w312/97-like lineage is uncertain . reassortants were present as early as 2000 , but tl / hk / w312/97-like viruses were the dominant genotype . it could be speculated that the tl / hk / w312/97-like viruses are more adapted for replication and transmission in quail than some of the reassortant viruses . hence , while quail remained in the market tl / hk / w312/97-like viruses predominated . the association of qa / hk / g1/97 ( h9n2)-like viruses , that share 6 gene segments in common with tl / hk / w312/97 , with quail would support this possibility . it is of course also possible that it is coincidence that links the removal of quail and the emergence of multiple genotypes . studies on h9n2 and h5n1 isolates in the region have also revealed an increasing degree of genetic diversity within these viruses . a more sinister scenario for the increase in h6 diversity is that there was an across - the - board enhancement in the amount of genetic reassortment occurring in the domestic poultry viruses throughout southeast asia . although we were unable to detect an increase in the chicken infectivity of recent h6 viruses , some biologic differences to tl / hk / w312/97 similar to what has been described for h9n2 viruses , the contemporary h6 viruses have erythrocyte binding properties more reminiscent of human viruses . human viruses , in contrast to avian viruses , are unable to bind to horse erythrocytes which contain primarily 2 - 3-linked sialic acids . unlike the contemporary h6 viruses , tl / hk / w312/97 has an avian virus - like binding pattern and retains the ability to bind to horse erythrocytes . interestingly , the contemporary h6 viruses do not contain any of the amino acid changes in the ha that matrosovich and colleagues associated with the human virus - like binding properties of the h9n2 viruses . similarly , no correlation between predicted carbohydrate moieties and binding preferences of the h6 viruses was seen . it is interesting that both h9 and h6 viruses have evolved towards a human - like virus binding preference . the fact that it has occurred in distinct subtypes of influenza virus would strongly support a selective advantage accompaning the change . exactly what environmental or host factor is driving the selective pressure is unclear . the hypothesis that the host range of h6 viruses underwent expansion in the hong kong markets appears to be incorrect in view of our findings . h6 viruses do have the capacity to be maintained in chicken populations as demonstrated by the emergence of multiple genotypes of h6n2 virus in chicken in the united states [ 3537 ] . what further molecular changes are required for establishment of the hong kong h6 viruses in chickens , if at all possible , is unknown . although the removal of quail has reduced the h6 virus burden in the hong kong bird markets , there has been an abrupt and corresponding increase in the genetic diversity of these viruses . it is unclear what effect this increase has on the continued presence and importance of these viruses for animal and human health . using best evidence to inform healthcare decisions is widely recognized as a key competency for all healthcare professionals . academic institutions are implementing evidence - based healthcare ( ebhc ) as part of learning in the curriculum of healthcare professionals . these curricula usually cover critical enquiry and formulating clear questions when faced with a scenario of uncertainty , finding best research evidence applicable to the problem , critically appraising the evidence for validity , clinical relevance and applicability , interpreting and applying the findings in the clinical setting and evaluating the performance . clinically integrated teaching and learning , with a focus on learning linked to real - world problems in the clinical setting and learning by doing , as modelled by sackett are considered to be the more effective approaches for improving ebhc knowledge , skills and attitudes . successful teaching and learning depends on factors related to the learner , the educator / lecturer , and on having a supportive environment with teaching and learning opportunities . educators play a critical role in the delivery and facilitation of ebhc teaching and learning , in encouraging critical enquiry , in fostering reflective practices and in being role models for the practice of ebhc in the clinical setting . within the clinical setting , clinician lecturers / educators who have received ebhc training are more likely to teach the application of ebhc . a study in new zealand among clinical teachers found that those who received training in ebhc are more likely to teach its application in the clinical setting and are more comfortable to engage students on topics and issues related to ebhc , and this was more often seen among general practitioners than specialists . among nurse educators , melnyk et al . found significant relationships between educators knowledge of ebhc and their beliefs about the value of ebhc , the ability to practice ebhc and the relationship between teaching ebhc and advancing the profession and their comfort in teaching ebhc . the create framework , which provides an international consensus statement on ebhc assessment tools , defines self - efficacy as the individual 's judgments regarding their ability to perform a certain activity and notes that educators confidence in their ability may increase their likelihood to engage in practicing the various ebhc steps . internationally , training initiatives thus focus on training trainers in ebhc to enhance their capacity to integrate ebhc teaching and learning in the clinical setting . also on the increase are initiatives to build confidence in teaching and learning principles and theories . in south africa , the colleges of medicine of south africa includes critical appraisal skills in curricula for medical specialist training while the medical and dental professions board of the health professions council of south africa states in its regulations for registration of students , undergraduate curricula and professional examinations in medicine and dentistry that : the emphasis in teaching should be on fundamental principles and methods that promote understanding and problem - solving skills and not only on the purely factual knowledge which , in any event , becomes outdated . they should be taught at all times to be critical of old and new knowledge and to evaluate data , statistics , thinking and methods objectively . the emphasis in teaching should be on fundamental principles and methods that promote understanding and problem - solving skills and not only on the purely factual knowledge which , in any event , becomes outdated . they should be taught at all times to be critical of old and new knowledge and to evaluate data , statistics , thinking and methods objectively . the health professions council of south africa used the canmeds framework to define the desired graduate attributes of a newly qualified healthcare professional . this now serves as a guide to the essential abilities of a newly qualified health professional to optimize patient outcomes and defines the attributes of the graduate according to seven interdependent roles : medical expert , scholar ( which includes ebhc ) , professional , communicator , collaborator , manager and health advocate . at stellenbosch university an ongoing project aims to develop and implement undergraduate ebhc teaching and learning to medical undergraduates in an integrated manner . to inform curriculum development , an assessment of the medical curriculum was conducted , including a document review of the 2011 curriculum , a survey of recent graduates and interviews with faculty . this found that ebhc is covered to varying degrees with teaching in specific modules , which it was not explicitly integrated in a stepwise fashion and did not progress from foundational knowledge to the acquisition of skills and practical competencies throughout the curriculum . recent graduates felt that they lacked ebhc skills and proposed that ebhc teaching and learning be integrated into clinical rotations , making use of relevant examples in different disciplines . as educators play a key role in facilitating the teaching and learning of ebhc , especially within the clinical setting , the study reported here assessed educators confidence in practicing , and their attitude , to ebhc , as well as their confidence in teaching ebhc and the barriers they had experienced , or perceived , to practicing and teaching ebhc . the study population included all faculty members involved in teaching on the undergraduate medical curriculum across the 10 departments at the stellenbosch university faculty of medicine and health sciences . all those employed by the university , and those on joint appointments , whose role included teaching of undergraduate medical students were invited to participate by e - mail , and provided with a link to the online questionnaire . the structured questionnaire sought information regarding demographics and training received , experience in teaching and learning , and previous exposure to ebhc ( training received , research conducted , etc . ) . we used validated tools for assessing confidence in practicing , and attitude to , ebhc . confidence in practicing ebhc was assessed using the evidence - based practice confidence scale and questions were included to assess attitude . visual analogue scales measured attitudes to , and confidence in , teaching ebhc and open - ended questions explored barriers to practicing and teaching ebhc . the afrikaans version was back translated by a person independent of the research team , and the original english and the back - translated version compared to ensure that the meaning of the questions was not lost . stata 12 ( statacorp lp , college station , texas , usa ) was used for quantitative data analysis . categorical data were summarized using proportions . for questions on knowledge we assessed consistency and found high cronbach 's alphas ( 95% ) and therefore combined all 16 items into one knowledge score . the highest score was allocated to the best level of knowledge , giving a knowledge score with a maximum 80 . for questions on attitude to practicing ebhc we reversed the scores of the negatively phrased items , for example i rarely formulate questions about patients and then added all the items together to get an overall score between 10 and 50 ( cronbach 's alpha 57% ) . for confidence in practicing ebhc we grouped the scores into five categories aligned with the five steps in practicing ebhc namely ask clear questions , search for research evidence , appraise and interpret the evidence , apply the evidence and audit practices . scores for self - perceived confidence in teaching ebhc were also combined into an overall score ( maximum 55 ) . bivariate analysis of associations between factors such as demographics , education and exposure to ebhc , and outcomes such as attitude and confidence to practice and teach ebhc were assessed using correlation analysis , analysis of variance testing , pearson 's analysis and t tests as appropriate . responses to open - ended questions on barriers , and proposed strategies to overcome these , to practicing and teaching ebhc were coded and analysis and interpretation were done by the investigators , using thematic content analysis to identify key emerging themes . we linked these to the conceptual framework for integrated teaching and learning of ebhc , developed following semistructured interviews with 24 ebhc programme coordinators from around the world , which revolves around the engagement between the learner and the educator within the institutional context . the study proposal was approved by the stellenbosch university health research ethics committee ( s12/10/262(c ) ) . to enhance response rate , we had a lottery for respondents , with a sponsored conference registration , for a conference of their choice , to the value of r5000 as the prize . participants who wanted to enter the lottery had to provide their cell phone numbers , which were only be used to notify the winner . forty two ( 19% ) of 227 faculty members involved in teaching undergraduate medical students responded . they worked across various departments , were mainly senior lecturers and offered teaching to all years of medical students ( table 1 ) . about 75% had attended training on research methodology ( mainly epidemiology , research proposal writing , biostatistics , systematic reviews and qualitative research methods ) , and to a lesser extent training on knowledge translation and change management . most participants had conducted primary research , with only a few conducting systematic reviews . more than half ( 57% ) indicated that they had done some training in ebhc in the past 5 years by attending short courses , workshops / seminars , online courses , journal clubs or by reading articles on ebhc . these activities mainly addressed enabling competencies such as epidemiology , biostatistics , research methodology and the basic principles of ebhc . most ( 85% ) had attended teaching and learning training events that had content such as teaching and learning strategies , assessment , curriculum planning , teaching ebhc , supervision and promoting active learning . participants described ebhc as : supporting clinical decision - making by combining best available evidence with own experience , patient preference and local factors and healthcare practices ( of any nature e.g. prevention , diagnosis , treatment , prognosis etc . ) that are informed by evidence as far as possible ; and recognising where there is inadequate / insufficient evidence to inform these practices . it implies ongoing changes to healthcare practice when new evidence becomes available . supporting clinical decision - making by combining best available evidence with own experience , patient preference and local factors and healthcare practices ( of any nature e.g. prevention , diagnosis , treatment , prognosis etc . ) that are informed by evidence as far as possible ; and recognising where there is inadequate / insufficient evidence to inform these practices . profile of survey responders ebhc , evidence - based healthcare ; iqr , interquartile range . some emphasised use of research evidence and did not include reference to combining this with clinical experience and patient preferences . participants self - reported understanding of ebhc - related terms is described in table 2 . using the overall knowledge score for the 16 items , maximum score 80 , the median the knowledge scores were not significantly associated with training , highest qualification , years since qualifying , position , academic department or age . self - reported understanding of evidence - based healthcare - related terms often used in research articles figure 1 graphically depicts responses to the questions on attitude to practicing ebhc . the items were framed both positively , for example ebhc is useful on a daily basis and negatively , for example i rarely formulate questions about patients. most felt that ebhc is a realistic option in their practice and that lifelong learning is important . however , more than 50% felt that literature searches are too time - consuming to undertake in the clinic and that questions can be answered faster by referring to a textbook or a consultant . the overall mean score was 38 ( sd 5 ) ( maximum score 50 ) . data were normally distributed , so we compared the independent variables to attitude score using t tests and analysis of variance . training in ebhc and research methods , qualification , time since qualification , faculty position , age , conducting systematic reviews and years of teaching were not significantly associated with attitude . lecturers confidence in practicing ebhc is summarized in table 3 , highlighting levels of confidence that are high overall but with lower levels for interpreting statistics . they raised various barriers , relating to the individual or the context , to practicing ebhc . by far the most common were lack of time , clinical workload , limited access to internet and resources , knowledge and skills.no simple solution to increase time - clinicians responsibilities in the hospital , including patient and student load will only be addressed if more posts are made available . no simple solution to increase time - clinicians responsibilities in the hospital , including patient and student load will only be addressed if more posts are made available . educators self - perceived confidence in practicing evidence - based healthcare ( n = 42 ) they made suggestions on how this could be addressed , calling for more staff and dedicated time for research and for faculty development . they proposed capacity development opportunities to especially enhance their capacity to interpret and understand biostatistics , searching skills , how to read papers and on time management . support group to assist each other . to create an enabling environment , they suggested widely available and reliable internet access and wifi , access to relevant literature and having evidence informed clinical guidelines available at the point of care . furthermore , they suggested using auditing and feedback to enhance practices . this was done through lectures , small group tutorials , teaching at the bedside , online learning and including ebhc concepts in assessments . the content covered in these sessions focused on the enabling competencies ( epidemiology , biostatistics ) , basic ebhc principles and searching skills . the overall mean score for self - perceived confidence in teaching ebhc was 40 ( sd 11 ) of a maximum of 55 ( n = 42 ) . most ( 38 ) of the 42 respondents indicated that they were confident to help medical students find relevant articles in medline ( or other bibliographic databases ) , and to guide critical review of articles ( 60% or higher on the visual analogue scale ) . fewer ( 30 ) indicated that they were confident to assist students phrase a clear question following a clinical encounter with a patient , and to guide students in considering the application of the results of their critique of articles to the patient 's care . twenty seven selected 60% or higher on the visual analogue scale for confidence in evaluating students ebhc knowledge . 2 . confidence in teaching evidence - based healthcare ( ebhc ) per department . using total score ( maximum = 55 ) . educators raised a number of perceived barriers to teaching ebhc , which relate to the students , the context and the educators . they felt that the curriculum is full , that students are subjected to information overload , that there is no scaffolding of ebhc learning over the course of the degree and that ebhc is not integrated in practice . lack of internet access , especially at point - of - care , was commonly listed . with respect to students the size of student groups , immaturity and lack of interest of students were seen as barriers to ebhc learning while competing priorities and lack of time ( am involved with too many other activities ) , as well as limited ebhc knowledge and skills ( i need to upgrade and maintain my knowledge of ebhc ) and the tendency to stick to habits influence how educators facilitate ebhc learning . suggestions for addressing these challenges included improving departmental internet access , exploring wifi access for the whole faculty , and easy point - of - care access to databases and resources . respondents stressed the importance of increasing awareness of the value and utility of ebhc ( demonstrate to them the value of ebhc ) and thus the need for ebhc teaching . they also emphasized building capacity to both practice and facilitate learning of ebhc , and expressed the need for more time to devote to teaching students ( get somebody to take over my postgraduate activities . furthermore , they called for promotion of critical thinking among students , incorporating the teaching and learning of ebhc from the start of the curriculum , and making ebhc applicable and relevant to students. respondents felt that there needs to be dedicated time to apply ebhc principles especially within the clinical setting , and that it should be integrated in assessments . to support each other , educators requested help from others who are particularly proficient , for example a working group supporting educators , and highlighted the need for evaluation and ongoing refinement of teaching approaches and material . the items were framed both positively , for example ebhc is useful on a daily basis and negatively , for example i rarely formulate questions about patients. most felt that ebhc is a realistic option in their practice and that lifelong learning is important . however , more than 50% felt that literature searches are too time - consuming to undertake in the clinic and that questions can be answered faster by referring to a textbook or a consultant . the overall mean score was 38 ( sd 5 ) ( maximum score 50 ) . data were normally distributed , so we compared the independent variables to attitude score using t tests and analysis of variance . training in ebhc and research methods , qualification , time since qualification , faculty position , age , conducting systematic reviews and years of teaching were not significantly associated with attitude . lecturers confidence in practicing ebhc is summarized in table 3 , highlighting levels of confidence that are high overall but with lower levels for interpreting statistics . they raised various barriers , relating to the individual or the context , to practicing ebhc . by far the most common were lack of time , clinical workload , limited access to internet and resources , knowledge and skills.no simple solution to increase time - clinicians responsibilities in the hospital , including patient and student load will only be addressed if more posts are made available . no simple solution to increase time - clinicians responsibilities in the hospital , including patient and student load will only be addressed if more posts are made available . educators self - perceived confidence in practicing evidence - based healthcare ( n = 42 ) they made suggestions on how this could be addressed , calling for more staff and dedicated time for research and for faculty development . they proposed capacity development opportunities to especially enhance their capacity to interpret and understand biostatistics , searching skills , how to read papers and on time management . support group to assist each other . to create an enabling environment , they suggested widely available and reliable internet access and wifi , access to relevant literature and having evidence informed clinical guidelines available at the point of care . this was done through lectures , small group tutorials , teaching at the bedside , online learning and including ebhc concepts in assessments . the content covered in these sessions focused on the enabling competencies ( epidemiology , biostatistics ) , basic ebhc principles and searching skills . the overall mean score for self - perceived confidence in teaching ebhc was 40 ( sd 11 ) of a maximum of 55 ( n = 42 ) . most ( 38 ) of the 42 respondents indicated that they were confident to help medical students find relevant articles in medline ( or other bibliographic databases ) , and to guide critical review of articles ( 60% or higher on the visual analogue scale ) . fewer ( 30 ) indicated that they were confident to assist students phrase a clear question following a clinical encounter with a patient , and to guide students in considering the application of the results of their critique of articles to the patient 's care . twenty seven selected 60% or higher on the visual analogue scale for confidence in evaluating students ebhc knowledge . 2 . confidence in teaching evidence - based healthcare ( ebhc ) per department . educators raised a number of perceived barriers to teaching ebhc , which relate to the students , the context and the educators . they felt that the curriculum is full , that students are subjected to information overload , that there is no scaffolding of ebhc learning over the course of the degree and that ebhc is not integrated in practice . lack of internet access , especially at point - of - care , was commonly listed . with respect to students the size of student groups , immaturity and lack of interest of students were seen as barriers to ebhc learning while competing priorities and lack of time ( am involved with too many other activities ) , as well as limited ebhc knowledge and skills ( i need to upgrade and maintain my knowledge of ebhc ) and the tendency to stick to habits influence how educators facilitate ebhc learning . suggestions for addressing these challenges included improving departmental internet access , exploring wifi access for the whole faculty , and easy point - of - care access to databases and resources . respondents stressed the importance of increasing awareness of the value and utility of ebhc ( demonstrate to them the value of ebhc ) and thus the need for ebhc teaching . they also emphasized building capacity to both practice and facilitate learning of ebhc , and expressed the need for more time to devote to teaching students ( get somebody to take over my postgraduate activities . furthermore , they called for promotion of critical thinking among students , incorporating the teaching and learning of ebhc from the start of the curriculum , and making ebhc applicable and relevant to students. respondents felt that there needs to be dedicated time to apply ebhc principles especially within the clinical setting , and that it should be integrated in assessments . to support each other , educators requested help from others who are particularly proficient , for example a working group supporting educators , and highlighted the need for evaluation and ongoing refinement of teaching approaches and material . this is one of only a few studies conducted in south africa to assess one or more of the following : undergraduate educators confidence in practicing and teaching ebhc , attitudes to ebhc and to practicing and teaching ebhc . this study links to ongoing work at stellenbosch university supporting the implementation of graduate attributes , specifically linked to developing and implementing clinically integrated ebhc teaching and learning for medical undergraduates , a process within which educators play a critical role . despite respondents having a high self - reported level of knowledge and understanding of ebhc concepts attitudes towards ebhc varied ( fig . those who were teaching ebhc focused the curricula content on enabling competencies of ebhc , basic ebhc principles and searching skills , rather than on reading , interpreting and considering the application of different types of articles . as educators are faced with various competing priorities the need was expressed for dedicated faculty development and a community of practice to provide support in the implementation of ebhc teaching and learning . similar studies among south african psychiatrists and general practitioners with a special interest in mental health , general practitioner and specialist educators in new zealand and nurse educators in the united states found that those who had attended ebhc courses were more likely to teach ebhc . barriers to teaching ebhc , resonating with our survey , were centred around lack of time , lack of support , lack of evidence in some clinical areas and the need for more training in teaching ebhc . findings of this survey also resonate with a curriculum assessment , which found that there is no scaffolding of ebhc learning over the course of the degree and that ebhc is not integrated in practice . a supportive enabling institutional , and health sector , environment is important for advancing ebhc learning . educators need to be confident and competent to facilitate the learning and , to this end , require opportunities to enhance their capacity in ebhc and in how to facilitate learning . furthermore , through working together , building on each other 's strengths , sharing best practices and lessons learnt , in a supportive community of practice can build the critical mass of educators needed to facilitate learning across the various disciplines . our survey had limited the power to assess associations between confidence to practice and teach ebhc and variables such as attitude and training . the low response rate might also mean that those who took part are not representative of the target population . the nonresponders may have different levels of confidence to practice and teach ebhc , their attitude may vary and they could be experiencing different challenges . to assess this we conducted brief follow - up survey of nonresponders , to which 14 educators responded . they described ebhc in a similar way to responders and listed lack of time , length of the survey , limited involvement with undergraduate teaching and that ebhc is irrelevant to their practice as reasons for not participating . educators play a critical role in facilitating learning not just in the classroom , but also in practice . this survey , despite low response rate , shows that even for those with high levels of self - reported knowledge and understanding of ebhc ; adequate support , training and development and an enabling environment are important for educators to be the role models future healthcare professionals need .

until 2001 , h6n1 influenza viruses in the hong kong bird markets were represented by a single stable a / teal / hong kong / w312/97-like lineage . beginning in 2001 , despite a reduction in overall prevalence , an increase was observed in the number of h6 viruses isolated from chickens and other hosts . to assess any changes in h6 viruses , we characterized 18 h6 viruses isolated in the hong kong bird markets from 2001 to 2003 . experimental data showed that the 2003 h6 viruses had similar infectivity for chickens as did a / teal / hk / w312/97 , and they were unable to transmit . although all hemagglutinin genes were closely related to a / teal / hk / w312/97 , 7 isolates were reassortant viruses containing similar gene segments of co - circulating h9n2 or h5n1 viruses . the receptor specificity was different from that of a / teal / hong kong / w312/97 . interestingly , similar observations have been documented in h9n2 viruses in hong kong . this evolution strongly suggests that some change in the ecology of influenza in the region selected for these changes . taken together , these findings suggest that the h6 influenza viruses isolated in the hong kong markets are not well adapted to chickens and that the likely continued source of these viruses are other minor poultry species in which they are undergoing genetic and biologic evolution . abstractaim : medical student educators play critical roles in evidence - based healthcare ( ebhc ) teaching and learning and as role models practicing ebhc . this study assessed their confidence to practice and teach ebhc , their attitude to ebhc and barriers to practicing and teaching ebhc.methods:we conducted a cross - sectional online survey of educators of undergraduate medical students at a south african academic institution . stata 12 was used for quantitative data analysis . responses to open - ended questions were coded , and further interpretation done using thematic content analysis.results:forty two ( 19% ) educators from various departments responded to the invitation sent to everyone formally involved in teaching undergraduate medical students . they had high levels of knowledge and understanding of ebhc . many had received training in teaching and learning approaches , although ebhc training received was mainly on enabling competencies . limitations to practicing ebhc included lack of time , clinical workload , limited access to internet and resources , knowledge and skills . one quarter of the respondents indicated that they teach ebhc . perceived barriers to teaching ebhc reported related to students ( e.g. lack of interest ) , context ( e.g. access to databases ) and educators ( e.g. competing priorities ) . respondents suggestions for support included reliable internet access , easy point - of - care access to databases and resources , increasing awareness of ebhc , building capacity to practice and facilitate learning of ebhc and a supportive community of practice.conclusion:educators play a critical role in facilitating ebhc learning not just in the classroom , but also in practice . without adequate support , training and development , they are ill equipped to be the role models future healthcare professionals need .

the regulated expression of different families of retrotransposons and dna transposons has been described in different tissues and species . however , a detailed analysis of the transcripts or their temporal and spatial expression patterns has been reported in only a few cases . during drosophila embryogenesis , spatial differential expression of the retrotransposon 412 has been described , specifically in the gonadal mesoderm . in mouse , retrotransposons make a high contribution to the pool of maternal mrnas in early embryos , and the expression of these elements is developmentally regulated . in xenopus , the 1a11 retrotransposon - like element is specifically expressed in the mesoderm and its expression is regulated by fgf . likewise , the expression of the retrotransposon family xretpos is restricted to ventro - posterior regions during development . we extended these observations by showing that a dna transposable element of the tc1/mariner family is differentially expressed during x. tropicalis development . transcripts for some tc1-like elements have been detected in other est databases , but no detailed analyses has been performed . our studies showed that the tc1-like element tc12_xt mrna ( named according to the repeatmasker nomenclature ) is specifically expressed at the gastrula stage in the spemann s organizer . this region is required for the proper dorso - ventral and anterior - posterior patterning of the embryo and the neuroectoderm , the tissue that will give rise to the nervous system ( fig . then as development progresses , tc12_xt mrna is found restricted to neural tissue regions . both tc12_xt sense and antisense strands present similar expression patterns . the length of the mrnas suggests that these transcripts are not included in other genes . pirnas are specifically derived from tc12_xt , and analysis of the expression of selected tc12_xt - derived pirnas suggests that these elements control its temporal expression . ( a ) the sequence analysis of the 116 copies of the tc12_xt transposable element in the x. tropicalis genome showed the typical structure of tc1-like elements . it is flanked by two inverted repeats ( ir ) containing two direct repeats ( white triangles ) for the binding of the transposase . the transposase orf ( green ) contains a putative dna binding domain ( blue ) and the catalytic triad ( red , aspartic - aspartic - glutamic residues ( dde ) ) . ( b ) schematic representation of the expression of tc12_xt during x. tropicalis early development . the expression of tc12_xt is shown in blue for early stages of x. tropicalis development . no expression is detected before the beggining of the zygotic transcription ( st.6 is shown as an example , lateral view ) . expression is clearly detected from gastrula stage ( st.10 ) in the spemann s organizer and then in anterior and neural tissues . figures were downloaded from http://www.xenbase.org/anatomy/static/nf/nf-all.jsp and modified according to the expression pattern obtained by in situ hybridization . in the x. tropicalis genome , 72% of transposable elements correspond to dna transposons with seven families of tc1-like elements characterized . our analyses for these elements , albeit not detailed , have shown that several of these families are transcribed during x. tropicalis development . interestingly , we could not find tc12_xt in x. laevis by using rt - pcr or in situ hybridization . whether this is explained because this element invaded the x. tropicalis genome after both species diverged , or to a high divergence in the sequence in the x. laevis genome is not known . the availability of the x. laevis genome could be very useful to study these alternatives . however , in x. laevis , two tc1-like elements have been described in the genome , txr and txz . our studies have demonstrated that txr and txz are also expressed during x. laevis development . importantly , the expression pattern of these elements is very similar to tc12_xt in x. tropicalis and both strands are also expressed . at present , we do not know how the expression of these elements is controlled and whether one or several loci are being transcribed . the presence of regulatory elements controlling gene expression in the sequences of these tc1-like elements has not been studied . however , in contrast to the expression of tc1 elements in c. elegans , which occurs by fortuitous read - through transcription , most tc12_xt rna is not included in other protein - coding transcripts , for which read - through transcription is unlikely to explain the bulk of tc12_xt rnas . another alternative is the presence of clusters of several copies of transposable elements under the control of a single promoter . the transcription of clusters of transposable elements to generate pirnas has been described in drosophila . it is possible that these clusters can also be regulated during development to produce specific expression patterns . another explanation is that transcription is ubiquitous , and that tc12_xt rnas are degraded in ventral and posterior regions by a pirna dependent - mechanism . in any case , the specific temporal and spatial expression of all these elements strongly suggests that they could play a role during nervous system development . in the x. tropicalis genome , 111 out of 116 tc12_xt copies contain frame - shifts and mutations rendering putative transposase - inactive copies of the element . consistently , the analysis of 20 tc12_xt cdnas from gastrula stage embryos indicates that none of them codes for an active transposase . these results suggest that tc12_xt could play a role as a non - coding rna . in mouse , the transcription of the retrotransposon sine b2 is necessary for gene activation in the growth hormone locus . in this case , the expression of this retrotransposon regulates the formation of a euchromatin / heterochromatin boundary , resulting in the expression of genes in proximal regions . it is still unknown whether this is the case for tc12_xt expression , but we can propose that some of the tc12_xt loci may be regulating chromatin architecture , allowing the expression of genes involved in the formation of the nervous system . considering that pirnas derived from tc12_xt were detected , another possible function is to generate pirnas . the expression of both strands is consistent with the ping - pong model of amplification of pirnas . if this is the case , it is worth noting that the tc12_xt rna is stable enough to be detected by rt - pct , rna gel blot and in situ hybridization , suggesting that the pirnas produced are most likely not enough to degrade all of the tc12_xt rna during early development . in addition , the expression of sense and antisense strands is specifically detected in neural tissues , and therefore , the amplification of pirnas could occur in these tissues . alternatively , pirnas derived from tc12_xt may not be involved in the degradation of tc12_xt itself . rather than that , they may regulate endogenous genes that contain sites complementary to pirnas . although our analysis for two tc12_xt - derived pirnas showed that all the pirna sequences in the genome are included only in tc12_xt sequences , the possibility that other tc12_xt - derived pirnas map to genes can not be ruled out . for example , pirnas derived from transposable elements regulate the expression of endogenous genes and allow the clearance of maternal mrnas during early drosophila development . finally , recently it has been published that pirnas derived from non - repetitive regions have a role in spine morphogenesis in the central nervous system in mice . although all the tc12_xt cdna copies we analyzed do not contain a functional transposase open reading frame , 5 out of 116 tc12_xt complete sequences in the genome could putatively code for an open reading frame containing the catalytic triad and perhaps an active transposase ( fig . we do nt know if these five copies contain all the other residues required for transposition , such us the binding to dna domains . however , the presence of active copies in the genome and its possible expression can not be ruled out . interestingly , work from the gage laboratory showed that endogenous line-1 retrotransposition can occur during mouse development . in addition , line-1 retrotransposition in the vicinity of neural genes can alter the expression of these genes in neural precursor cells . furthermore , the same group showed that line-1 retrotranspositions can also occur in neural progenitor cells isolated from human fetal brain , suggesting that de novo line-1 retrotransposition events may occur in the human brain . these events produce mosaicism in the neurons due to different genomic modifications on different neurons in the same individual . this has been suggested as a novel mechanism involved in the generation of the astonishing neuronal diversity required for nervous system formation . therefore , the specific expression of tc12_xt ( and txr and txz ) in dorsal and neural tissues allows speculation about a similar role in xenopus . as active elements for transposition , these transposons could be involved in the generation of heterogeneity during xenopus nervous system development . in this section we will briefly discuss possible experimental approaches to evaluate transposition of tc1 elements during nervous system development and determine its possible contribution to produce neuronal diversity . one of the first questions is to determine whether expression of an active dna transposase occurs during early development . after cloning the possible candidates , transposase activity must first be determined by in vitro assays . for this , a plasmid reporter for excision events needs to be prepared . based upon the white - peach allele studied in drosophila the egfp open reading frame contains an insertion of a random sequence ( same length as the transposase ) in between of the inverted repeats the putative transposase recognizes . therefore , when co - expressed with the transposase mrna in an exogenous system ( e.g. , cell culture ) , successful translation into an active transposase would render egfp+ cells . the reporter construct contains an egfp open reading frame with an insertion of a random sequence ( same length as the original transposase ) . when transposase mrna is present ( either exogenous , when working in vitro , or endogenous , when in vivo ) and an active transposase is being translated , transposition will occur , rendering egfp+ cells . the random sequence insertion sites can then be characterized using deep sequencing of reverse pcr amplicons . experiments to demonstrate in vivo transposition could be performed . for this purpose , transgenic xenopus embryos containing a tissue - specific promoter would allow us to follow transposition in the central nervous system . the presence of an endogenously active transposase would render egfp+ cells that we could observe in later developmental stages , such as stage 50 tadpoles . furthermore , it would be possible to characterize transposon insertion sites in neural tissues of individual tadpoles using deep sequencing of reverse pcr amplicons ( see fig . 2 ) . the comparison of insertion sites in neural tissues with non - neural tissues could be an indicator of in vivo transposition events . all of these approaches can be useful to determine whether transposition events occur during xenopus development . although host cells contain mechanisms to avoid the expression of transposable elements , they could also be using these elements for cellular functions . a proper balance must exist to control these positive and negative effects of transposable elements . our work has shown that the specific expression of tc1 elements in neural tissues and suggests that transposable elements may play a role during the formation of the nervous system in vertebrates . we obtained formalin - fixed , paraffin - embedded tissue sections from 30 leiomyosarcoma patients at the university of texas md anderson cancer center ( mdacc ; houston , texas , usa ) . these included samples from the retroperitoneum ( n = 12 ) , gastrointestinal tract ( n = 9 ) , uterus ( n = 6 ) , and skin ( n = 3 ) . to validate the results obtained with the mdacc samples , another cohort of 45 formalin - fixed , paraffin - embedded tissues from patients with leiomyosarcoma were retrieved from tianjin medical university cancer institute & hospital ( tmucih ) for further study . the patients ' clinicopathologic characteristics , including sex , age , clinical stage , degree of cell differentiation , tumor location , tumor size , the presence or absence of hemorrhage / cyst , and histopathologic subtype , were also collected . the tissues had been acquired from 18 male and 27 female patients with a mean age of 55.8 years . histopathologic subtypes were assigned based on the 2002 world health organization classification of tumors of soft tissue and bone , . overall survival time was defined from the date of diagnosis or the first treatment to the date of death or last follow - up . overall survival ranged from 1 to 74 months , and the median survival was 19 months . the study protocol was approved by the institutional review boards of both hospitals , and consent was obtained from each patient . briefly , tissue sections ( 4 m ) were dewaxed with xylene , rehydrated with ethanol , soaked in citrate buffer solution ( 10 mmol / l , ph=6 ) , and boiled in a pressure cooker for 2.5 min to repair the antigen . the sections were then blocked for 20 min with normal serum ( vector laboratories , burlingame , ca ) and incubated overnight at 4c with rabbit polyclonal anti - prune2 antibody ( abcam company , abcam , cambridge , uk ; diluted 1:100 ) . secondary antibody was applied for 60 min ( one drop of biotinylated anti - rabbit with three drops of normal rabbit serum ) . dab ( dako corporation , carpinteria , ca ) staining was applied for 510 min and monitored by microscopy . two pathologists blinded to clinical information evaluated and scored prune2 staining based on staining extent and intensity . ten random high - power fields , each containing approximately 100 cells , were observed under the microscope ( 40 ) . first , staining extent was scored according to the proportion of positive tumor cells : 0% ( score 0 ) , 10% ( score 1 ) , 11% to 25% ( score 2 ) , 26% to 50% ( score 3 ) , 51% to 75% ( score 4 ) , and > 75% ( score 5 ) . second , staining intensity was scored based on the color observed : no color ( score 0 ) , yellow ( score 1 ) , tan ( score 2 ) , and brown ( score 3 ) . final scores were calculated by adding the extent and intensity scores , and the results were used to divide patients into two groups : low expression group , for final scores of 04 , and high expression group , for final scores > 4 . kaplan - meier survival analysis ( log - rank test ) and multivariate cox regression analysis were used to examine the relationship between overall survival and prune2 protein expression . we obtained formalin - fixed , paraffin - embedded tissue sections from 30 leiomyosarcoma patients at the university of texas md anderson cancer center ( mdacc ; houston , texas , usa ) . these included samples from the retroperitoneum ( n = 12 ) , gastrointestinal tract ( n = 9 ) , uterus ( n = 6 ) , and skin ( n = 3 ) . to validate the results obtained with the mdacc samples , another cohort of 45 formalin - fixed , paraffin - embedded tissues from patients with leiomyosarcoma were retrieved from tianjin medical university cancer institute & hospital ( tmucih ) for further study . the patients ' clinicopathologic characteristics , including sex , age , clinical stage , degree of cell differentiation , tumor location , tumor size , the presence or absence of hemorrhage / cyst , and histopathologic subtype , were also collected . the tissues had been acquired from 18 male and 27 female patients with a mean age of 55.8 years . histopathologic subtypes were assigned based on the 2002 world health organization classification of tumors of soft tissue and bone , . overall survival time was defined from the date of diagnosis or the first treatment to the date of death or last follow - up . overall survival ranged from 1 to 74 months , and the median survival was 19 months . the study protocol was approved by the institutional review boards of both hospitals , and consent was obtained from each patient . , tissue sections ( 4 m ) were dewaxed with xylene , rehydrated with ethanol , soaked in citrate buffer solution ( 10 mmol / l , ph=6 ) , and boiled in a pressure cooker for 2.5 min to repair the antigen . the sections were then blocked for 20 min with normal serum ( vector laboratories , burlingame , ca ) and incubated overnight at 4c with rabbit polyclonal anti - prune2 antibody ( abcam company , abcam , cambridge , uk ; diluted 1:100 ) . secondary antibody was applied for 60 min ( one drop of biotinylated anti - rabbit with three drops of normal rabbit serum ) . dab ( dako corporation , carpinteria , ca ) staining was applied for 510 min and monitored by microscopy . two pathologists blinded to clinical information evaluated and scored prune2 staining based on staining extent and intensity . ten random high - power fields , each containing approximately 100 cells , were observed under the microscope ( 40 ) . first , staining extent was scored according to the proportion of positive tumor cells : 0% ( score 0 ) , 10% ( score 1 ) , 11% to 25% ( score 2 ) , 26% to 50% ( score 3 ) , 51% to 75% ( score 4 ) , and > second , staining intensity was scored based on the color observed : no color ( score 0 ) , yellow ( score 1 ) , tan ( score 2 ) , and brown ( score 3 ) . final scores were calculated by adding the extent and intensity scores , and the results were used to divide patients into two groups : low expression group , for final scores of 04 , and high expression group , for final scores > 4 . kaplan - meier survival analysis ( log - rank test ) and multivariate cox regression analysis were used to examine the relationship between overall survival and prune2 protein expression . in the 30 leiomyosarcoma tissues from mdacc , prune2 protein staining was predominantly in the cytoplasm of cells ( figure 1 ) . low expression was discovered in 63.3% ( 19/30 ) of the samples ( figure 1a ) , while high expression was observed in 36.7% ( 11/30 ) ( figure 1b ) . to investigate the prognostic role of prune2 , we analyzed the association between prune2 protein expression and overall survival . univariate analysis showed that prune2 protein expression had no significant association with overall survival ( = 0.246 , p = 0.62 ) ( figure 2a ) . a , low expression of prune2 protein ; b , high expression of prune2 protein . to validate these results and further evaluate the prognostic role of prune2 in leiomyosarcoma , we used a larger cohort of leiomyosarcoma tissue samples from tmucih . in this set of 45 samples , prune2 was also predominantly detected in the cytoplasm ; and 62.2% ( 28/45 ) had low expression , while 37.8% ( 17/45 ) had high expression . we also analyzed the association between prune2 protein expression and clinicopathologic factors , and found that prune2 protein expression was significantly associated with tumor size ( p = 0.03 ) and the absence of hemorrhage / cyst ( p = 0.014 ) ( table 1 ) . in addition , univariate analysis showed that there was a significant association between prune2 protein expression and overall survival ( = 5.399 , p = 0.02 ) ( figure 2b ) , with longer survival noted among patients with high levels of the protein . furthermore , multivariate analysis revealed that high prune2 protein expression was an independent , favorable prognostic factor for overall survival ( hr = 0.373 , p = 0.025 ) . a , prune2 protein expression is not significantly associated with overall survival in patients from md anderson cancer center ; b , prune2 protein expression is significantly associated with overall survival in patients from tianjin medical university cancer institute & hospital . in the 30 leiomyosarcoma tissues from mdacc , prune2 protein staining was predominantly in the cytoplasm of cells ( figure 1 ) . low expression was discovered in 63.3% ( 19/30 ) of the samples ( figure 1a ) , while high expression was observed in 36.7% ( 11/30 ) ( figure 1b ) . to investigate the prognostic role of prune2 , we analyzed the association between prune2 protein expression and overall survival . univariate analysis showed that prune2 protein expression had no significant association with overall survival ( = 0.246 , p = 0.62 ) ( figure 2a ) . a , low expression of prune2 protein ; b , high expression of prune2 protein . to validate these results and further evaluate the prognostic role of prune2 in leiomyosarcoma , we used a larger cohort of leiomyosarcoma tissue samples from tmucih . in this set of 45 samples , prune2 was also predominantly detected in the cytoplasm ; and 62.2% ( 28/45 ) had low expression , while 37.8% ( 17/45 ) had high expression . we also analyzed the association between prune2 protein expression and clinicopathologic factors , and found that prune2 protein expression was significantly associated with tumor size ( p = 0.03 ) and the absence of hemorrhage / cyst ( p = 0.014 ) ( table 1 ) . in addition , univariate analysis showed that there was a significant association between prune2 protein expression and overall survival ( = 5.399 , p = 0.02 ) ( figure 2b ) , with longer survival noted among patients with high levels of the protein . furthermore , multivariate analysis revealed that high prune2 protein expression was an independent , favorable prognostic factor for overall survival ( hr = 0.373 , p = 0.025 ) . a , prune2 protein expression is not significantly associated with overall survival in patients from md anderson cancer center ; b , prune2 protein expression is significantly associated with overall survival in patients from tianjin medical university cancer institute & hospital . the most important contribution of this study is that we found a significant association between prune2 protein expression and overall survival , and that prune2 protein expression was an independent prognostic factor for leiomyosarcoma . more specifically , leiomyosarcoma patients with higher prune2 protein expression exhibited a better survival trend , suggesting that increased levels of the protein might be a favorable prognostic factor for leiomyosarcoma . similarly , in neuroblastoma and prostate cancer , prune2 protein is highly expressed and also plays a prognostic role , . on the other hand , we found no statistically significant association between prune2 mrna expression and survival time in tissue samples from patients at mdacc in our previous study . here , kaplan - meier survival analysis revealed no significant relationship between overall survival time and prune2 protein expression in samples from mdacc , which is consistent with our previous result . nevertheless , in the larger cohort of samples from tmucih , prune2 protein expression showed a significant association with overall survival and was an independent prognostic factor . possible causes are the differences in race among the patients from each center and in the number of cases . we also found that prune2 protein expression was significantly associated with tumor size and hemorrhage / cyst . specifically , prune2 protein expression was higher in the smaller tumor size group ( < 10 cm ) . because prune2 plays an important role in regulating cell differentiation and apoptosis , this result suggests that patients with larger tumors may have worse survival . leiomyosarcoma is a rare soft tissue tumor that can occur anywhere in the human body . several factors have been reported to predict poor outcomes in patients with leiomyosarcoma , including advanced age , vascular invasion , dna aneuploidy , and c - myc expression , , . in particular , c - myc expression was reported to be a marker for poor prognosis in leiomyosarcoma . in this study , we have identified that prune2 protein expression level can serve as a prognostic factor for leiomyosarcoma . in conclusion , we report the association between prune2 protein expression and prognosis in human leiomyosarcoma . survival time was longer in tmucih leiomyosarcoma patients with higher expression of prune2 protein . this suggests that prune2 may be involved in the process of leiomyosarcoma development and can be regarded as a biomarker for favorable prognosis . further study is necessary to better understand the role that racial factors play for the relationship between prune2 protein expression and prognosis in patients with leiomyosarcoma , and discover new therapeutic strategies against aggressive leiomyosarcoma .

transposable elements ( retrotransposons and dna transposons ) comprise a large proportion of animal genomes , for example 20% in d. melanogaster , 36% in x. tropicalis and 45% in humans . after invading a new genome , the transposable element increases its copy number and subsequently accumulates mutations . these may eventually result in inactive copies . until recent days transposons have been considered junk dna and no clear function have been assigned for this important amount of information on genomes . prune2 plays an important role in regulating tumor cell differentiation , proliferation , and invasiveness in neuroblastoma . our previous study revealed that prune2/obscn two - gene relative expression classifer accurately differentiated leiomyosarcoma from gastrointestinal stromal tumor . however , the association between prune2 expression and prognosis in leiomyosarcoma is poorly understood . in this study , we evaluated the prognostic role of prune2 in leiomyosarcoma . prune2 expression was detected using immunohistochemistry in 30 formalin - fixed , paraffin - embedded leiomyosarcoma tissues from md anderson cancer center , and high expression was detected in 36.7% ( 11/30 ) of the samples . to validate these results , immunohistochemistry was performed on another cohort of 45 formalin - fixed , paraffin - embedded leiomyosarcoma tissues from tianjin medical university cancer institute & hospital , and high prune2 protein expression was detected in 37.8% ( 17/45 ) of the samples . moreover , elevated prune2 expression was significantly associated with tumor size ( p = 0.03 ) and hemorrhage / cyst ( p = 0.014 ) , and was an independent favorable prognostic factor for overall survival in leiomyosarcoma patients from tianjin medical university cancer institute & hospital ( p < 0.05 ) . these data suggest that increased prune2 protein expression may serve as a favorable prognostic marker in human leiomyosarcoma .

child abuse is defined as any physical or emotional injury which causes harm or substantive risk of harm to the child 's health or welfare . it includes sexual abuse , neglect , or being physically dependent upon an addictive drug at birth . types of child abuse , include physical abuse , emotional abuse and neglect , healthcare neglect ( medical and dental ) , physical neglect , sexual abuse , failure to thrive , safety neglect , intentional poisoning , and munchausen syndrome by proxy ( fabricated or induced illness by parent ) . factors contributing to abuse include stress[35 ] ( e.g. life crises such as unemployment or homelessness ) , lack of a support network , substance / alcohol abuse , learned behavior ( many abusers were previously victims ) , and other forms of family violence in the home such as spousal or elderly abuse.[69 ] craniofacial , head , face , and neck injuries happen in more than half of the cases of child abuse . although the oral cavity is a frequent point of sexual abuse in children , obvious oral injuries or infections are rare . the american academy of pediatric dentistry defined dental neglect as the willful failure of a parent or guardian to seek and follow through with treatment necessary to ensure a level of oral health essential for adequate function and freedom from pain and infection . indeed , many dentists who regularly treat children assert that management of dental neglect is part of daily practice . however , based on the literature , dentists feel unprepared to play a child protection role and are unsure what to do if they suspect that a child has been maltreated.[1215 ] the aim of this study was to provide data on prevalence and factors of orofacial lesions relating child abuse in iran to lend evidence to support preventing child abuse . the overall approach was a case - note review of children with child abuse history recording by personnel of social services . the social emergency services are legally eligible to separate the victims of child abuse from their families as well as interview the children and their families in order to solve their problems . research ethical approval was sought from the central social service organization as well as ethics committee of isfahan university of medical sciences . this study was conducted in isfahan , iran . for study inclusion , participating children had to have identifiable records of abuse during 2007 - 2011 . inclusion criteria for children dictated that they had been exposed to at least one child abuse event during this period . the data collected retrospectively for each child , from their existing records , were as follows : type of abuse : physical , sexualpatient characteristics : age , gender type of abuse : physical , sexual patient characteristics : age , gender descriptive statistics were used to describe child - related sociodemographic and clinical data . type of child abuse , gender , age , and the type of abuser was described using appropriate measures of spread . exploratory analyses including bivariate analyses and correlation were used to analyze the relationship of different variables such as child gender , age , abuse experience , abuse type as well as abuser type . all statistical tests were repeated using these data and found the same relationships between variables as identified for the original data set . for study inclusion , participating children had to have identifiable records of abuse during 2007 - 2011 . inclusion criteria for children dictated that they had been exposed to at least one child abuse event during this period . the data collected retrospectively for each child , from their existing records , were as follows : type of abuse : physical , sexualpatient characteristics : age , gender type of abuse : physical , sexual patient characteristics : age , gender type of child abuse , gender , age , and the type of abuser was described using appropriate measures of spread . exploratory analyses including bivariate analyses and correlation were used to analyze the relationship of different variables such as child gender , age , abuse experience , abuse type as well as abuser type . all statistical tests were repeated using these data and found the same relationships between variables as identified for the original data set . data were obtained for 301 children from social services records . there was an equal gender distribution amongst children gender . the mean age of children when abuse had been occurred was 8 years ( sd = 1.68 ) which was categorized to seven age groups [ table 1 ] , and there were approximately an equal number of boys and girls [ table 2 ] . children had a high physical experience ( 66.1% ) ; of these children , at least 69% sustained trauma to the face and mouth . emotional abuse was 77.1% , neglect was 64.1% , and lower experience of sexual abuse which was 4.1% . age of children having child abuse records categorized to seven age groups the gender distribution of children having child abuse records during 2007 - 2011 exploratory bivariate analyses revealed a significant relationship between the frequency of abuse with gender and age , p = 0.008 and 0.015 , respectively . having problem such as being a mental retired and hyperactive child shows significant relationship with gender , p = 0.03 and 0.02 , respectively , which was in the favor of males . there was a strong relationship between gender and abuser which shows girls have been affected by stepfathers , p = 0.001 . there was also strong relationship between age of child and age of abuser , p = 0.001 . however , there were no significant differences regarding age of abuser and gender of the child . the mean age of children when abuse had been occurred was 8 years ( sd = 1.68 ) which was categorized to seven age groups [ table 1 ] , and there were approximately an equal number of boys and girls [ table 2 ] . children had a high physical experience ( 66.1% ) ; of these children , at least 69% sustained trauma to the face and mouth . emotional abuse was 77.1% , neglect was 64.1% , and lower experience of sexual abuse which was 4.1% . age of children having child abuse records categorized to seven age groups the gender distribution of children having child abuse records during 2007 - 2011 exploratory bivariate analyses revealed a significant relationship between the frequency of abuse with gender and age , p = 0.008 and 0.015 , respectively . having problem such as being a mental retired and hyperactive child shows significant relationship with gender , p = 0.03 and 0.02 , respectively , which was in the favor of males . there was a strong relationship between gender and abuser which shows girls have been affected by stepfathers , p = 0.001 . there was also strong relationship between age of child and age of abuser , p = 0.001 . however , there were no significant differences regarding age of abuser and gender of the child . the main purpose of this study was to obtain preliminary data to provide data on child abuse - related orofacial lesions in order to lend evidence to prevent child abuse . this was considered important as there is a paucity of current data on the incidence of child abuse in all over the world and there is recognized need to evaluate the evidence to support dentists in different aspects of service provision . results of previous studies show that trauma to the head and associated areas occurs in approximately 50% of the cases of physically abused children and soft tissue injuries most frequently bruises are the most common injury to head and face . these findings make it obvious that dentists are in a position to detect child abuse . the british study by skinner and castle ( 1967 ) documented the injuries to 78 abused children requiring medical attention ; of these children , at least 34 ( 43.5% ) sustained trauma to the face and mouth . this may be an underestimate since some of the bruises were reported without noting the region . the majority of the injuries were bruises , but they also included lacerations , bites , and abrasions which support the results of our study . however , in our study , 60% of children had trauma to the face and head which was a higher prevalence . national figures in usa indicate that as many as 1 million children are abused and/or neglected annually and of these about 1000 die each year . if we assume that half of these cases involve trauma to the head , as is indicated in the literature , our profession is definitely in a position to detect and assist substantial numbers of victim - abused children . in this way , we can help to refer them to social security agencies and prevent further continuing trauma to the children by bringing help to these troubled families . the majority of victim - abused cases in our study were lower than ten years old . stress and trauma of them permanently impacted any aspects of their future life , especially in girls . the cost of orofacial trauma makes heavy burden on social security agencies and their families . the main purpose of this study was to obtain preliminary data to provide data on child abuse - related orofacial lesions in order to lend evidence to prevent child abuse . this was considered important as there is a paucity of current data on the incidence of child abuse in all over the world and there is recognized need to evaluate the evidence to support dentists in different aspects of service provision . results of previous studies show that trauma to the head and associated areas occurs in approximately 50% of the cases of physically abused children and soft tissue injuries most frequently bruises are the most common injury to head and face . these findings make it obvious that dentists are in a position to detect child abuse . the british study by skinner and castle ( 1967 ) documented the injuries to 78 abused children requiring medical attention ; of these children , at least 34 ( 43.5% ) sustained trauma to the face and mouth . this may be an underestimate since some of the bruises were reported without noting the region . the majority of the injuries were bruises , but they also included lacerations , bites , and abrasions which support the results of our study . however , in our study , 60% of children had trauma to the face and head which was a higher prevalence . national figures in usa indicate that as many as 1 million children are abused and/or neglected annually and of these about 1000 die each year . if we assume that half of these cases involve trauma to the head , as is indicated in the literature , our profession is definitely in a position to detect and assist substantial numbers of victim - abused children . in this way , we can help to refer them to social security agencies and prevent further continuing trauma to the children by bringing help to these troubled families . the majority of victim - abused cases in our study were lower than ten years old . stress and trauma of them permanently impacted any aspects of their future life , especially in girls . the cost of orofacial trauma makes heavy burden on social security agencies and their families . this is the first study in iran to provide data for the evaluation of orofacial lesions relating child abuse . preliminary data suggest that there are strong evidence regarding the incidence of child abuse relating orofacial lesions which dentists should be aware of them . future trials may draw based on these useful baseline data to help their study design . oncocytes are observed in lesions of several organs such as the thyroid , kidney , pancreas , ovary , liver , and salivary gland . in the salivary glands , oncocytes are known to arise in warthin tumor , oncocytoma , oncocytic carcinoma , and oncocytosis . oncocytosis is a rare non - neoplastic lesion that is classified as diffuse oncocytosis and multifocal adenomatous oncocytic hyperplasia ( maoh ) ; it comprises approximately 0.1% of salivary gland lesions [ 3 , 4 ] . the correct cytological diagnosis of oncocytosis can be difficult because oncocytes are seen in a variety of other salivary gland lesions , and it is usually diagnosed by histological examination [ 5 , 6 , 7 , 8 ] . here she consulted an otolaryngology clinic because of discomfort at the left side of her neck . at the clinic , a mass was noted in the left side of her neck and she was referred to our hospital . a 1.5-cm mass with no lymph node swelling was identified using magnetic resonance imaging . from the above clinical findings , warthin tumor or a malignant parotid tumor was suspected . five slides were prepared for cytological evaluation , including 3 fixed smears for papanicolaou staining , 1 air - dried smear for may - grnwald giemsa ( mgg ) staining , and 1 smear examined by liquid - based cytology ( lbc ) based on the lbc technique . papanicolaou staining of direct smears revealed a loose sheet - like cluster formed of round to polygonal cells with granular cytoplasm against a hemorrhagic background . the cells had round to oval , centrally located nuclei with granular chromatin and without distinct nucleoli . in the lbc preparation , the cytoplasm showed microvacuoles in contrast to the direct smear ( fig . there were only a few lymphocytes , and basophils were not detected in any of the smears . the cut surface of the resected lesion revealed a milky - white lobular mass with an unclear border ( fig . microscopically , the lesion was formed of many variable - sized nodules that comprised oncocyte - like cells with small round nuclei and eosinophilic granular cytoplasm . a definite capsule was not seen around the nodules and the surrounding adipose tissue and acinus . the oncocyte - like cells were diffusely positive for cytokeratin antibodies and strongly positive for mitochondrial antibodies ( fig . basophils were not detected in the lesion , using immunohistological staining based on anti - cd117 ( c - kit ) antibodies . typical findings for low - grade cancer of the salivary glands were not observed . from the above findings it has been reported that maoh develops in women in their 60s and is localized unilaterally in the parotid glands . histologically , maoh is thought to arise from the ductal epithelium and the remnants of the original salivary gland . the lesion was observed unilaterally in the left parotid gland in a 71-year - old woman . the remnants of the existing salivary ducts were observed by p63 and ck34e12 staining , and a strong response to mitochondrial antibodies was found in the cytoplasm . from the above findings described the cytological findings of maoh in a case report : ( 1 ) a low n / c ratio ; ( 2 ) central round nuclei ; ( 3 ) anisonucleosis ; ( 4 ) prominent nucleoli ( in part of the cells ) , and ( 5 ) abundant eosinophilic cytoplasm . in the present case , however , we could not make a diagnosis of maoh cytologically because of the high n / c ratio and because the nucleoli were unclear . cytologically , the major lesions in which oncocytes are observed include warthin tumor , oncocytoma , and oncocytic carcinoma . typical cytological findings of warthin tumor reveal oncocytes and lymphocytes with an inflammatory or necrotic background . have reported that mast cells are present with oncocytes in fine - needle aspiration preparations . kobayashi et al . have investigated the frequency of mast cells in cytological samples , as compared with immunocytochemical identification using human mast cell tryptase antibodies in warthin tumor , and have indicated that mast cells are frequent in the epithelial cell component . in contrast , there are no reports to suggest any association with mast cells of other lesions . we also searched for mast cells by cytological mgg staining and immunohistochemical staining using the cd117 ( c - kit ) antibody . . the identification of basophils seems to be a diagnostic checkpoint when differentiation from warthin tumor is necessary . however , the typical cytological findings such as a lymphocyte and/or basophil appearance were not observed in any of the cases , which may thus be mistakenly diagnosed as oncocytoma or maoh . cytological features of oncocytoma comprise large , round to polygonal cells with abundant granular cytoplasm , centrally located nuclei , prominent nucleoli , binucleation , sheet - like clustering , and an often necrotic background . in oncocytic carcinoma , although the neoplastic cells are pleomorphic , with nuclear atypia and hyperchromatism , other findings overlap with oncocytoma [ 7 , 8 , 13 ] . thus , it can be difficult to differentiate between maoh , warthin tumor , and oncocytoma . in addition , a case of oncocytoma arising from maoh has been reported . however , the distinction between maoh and oncocytoma is possible by capsular interpretation . therefore , it should be noted that neoplastic identification can not be achieved from partial samples microscopically . furthermore , oncocytic metaplasia has recently been shown in other salivary gland lesions ( e.g. , pleomorphic adenoma , myoepithelioma , and mucoepithelial carcinoma ) , except for maoh , warthin tumor , and oncocytic tumor . therefore , it is necessary to carefully carry out the assessment of oncocytes . when oncocytes are observed , it is necessary to perform a surgical resection . the authors have no potential conflicts of interest with respect to the authorship and/or publication of this article .

background : family violence , including child abuse , neglect , and domestic violence , is a public health problem . the aim of this study was to provide data on prevalence and factors of orofacial lesions relating child abuse in iran to lend evidence to support preventing child abuse.materials and methods : the overall approach was a case - note review of children having child abuse note , recording by personnel of social services . research ethical approval was sought from the central social service organization . this study was conducted in isfahan , iran ( 2011).result : the mean age of children , when abuse had been occurred was 8 years ( sd = 1.68 ) , and there were approximately an equal number of boys and girls . children had a high physical experience ( 66.1%).of these children , at least 60% sustained trauma to the face and mouth . emotional abuse was 77.1% , neglect was 64.1% , and lower experience of sexual abuse which was 4.1% . there was a strong relationship between gender and abuser which shows girls have been affected by stepfathers ( p = 0.001).conclusion : preliminary data suggest that there are strong evidence regarding the incidence of child abuse relating orofacial lesions which dentists should be aware of them . future trials may draw on these useful baseline data to help their study design . multifocal adenomatous oncocytic hyperplasia ( maoh ) is a non - neoplastic lesion that is classified as oncocytosis . maoh is a rare entity of the parotid gland and accounts for approximately 0.1% of salivary gland lesions . here , we report a case of maoh of the parotid gland . the patient was a 71-year - old woman who presented with discomfort at the left side of her neck . fine - needle aspiration cytology of the parotid gland revealed a loose sheet - like cluster of round to polygonal cells with granular cytoplasm against a hemorrhagic background . the cells had round to oval , centrally located nuclei with granular chromatin and without distinct nucleoli . histologically , the lesion was formed of many variable - sized nodules , comprising oncocyte - like cells with small round nuclei and eosinophilic granular cytoplasm that was positive for mitochondrial antibodies . the diagnosis of maoh is difficult to make by cytology alone , because the findings overlap with those of other oncocytic lesions . in particular , the cytological findings of maoh have not been sufficiently reported to date . a correlation of cytology and histology was expected .

a 59 year - old female with a family history of mcd presented to our clinic with progressive loss of vision over a 40-year period . there were multiple irregular grayish - white , dense , poorly delineated spots in the stroma . because her parents died at an early age , her family history was unclear . of note , her only son complained of foggy vision and was 37 years old . his slit lamp examination revealed a similar , but less severe appearing corneal exam compared to that of his mother ( fig . one year post - operatively , that patient had a best - corrected visual acuity of 20 / 100 od and a clear cornea graft . informed consent for both the clinical examinations and dna analyses was obtained from the patient and her son in accordance with the declaration of helsinki . the study was approved by the institutional review board at the catholic university of korea , st . hematoxylin and eosin ( he ) , alcian blue , periodic acid - schiff ( pas ) , colloidal iron , and masson 's trichrome stains were performed on the specimen using standard techniques . for transmission electron microscopy , the corneal specimen was immersed immediately in a fixative solution containing 3% glutaraldehyde with 0.2 m sodium cacodylate at a ph of 7.4 . after overnight fixation , the fixative solution was removed and replaced with a phosphate buffer , followed by 1% osmium tetroxide buffered with sodium cacodylate . after one hour , the osmium was replaced with increasing concentrations of ethanol through propylene oxide , and the tissue was embedded in the epoxy . the embedded tissue was then sectioned with an ultra - microtome into 1 m - thick sections , and stained with toluidine blue . the area to be observed was placed under a light microscope , ultra - sectioned from 60 to 100 nm in thickness , double stained with uranyl acetate and lead citrate , and examined via transmission electron microscope ( jem 1010 ; jeol , tokyo , japan ) . the unique exon involving the coding region , exon 3 , was amplified via polymerase chain reaction ( pcr ) with the previously described primers . the cycling program began with an initial denaturing step of 5 minutes at 95 followed by 33 cycles of 94 for 30 seconds , 53 to 57 for 30 seconds , and 72 for 45 seconds with a final extension step at 72 for 10 minutes . the pcr products were purified and sequenced directly on both strands using an automatic dna sequencer ( abiprism 377xl ; applied biosystems , foster city , ca , usa ) . the nucleotide sequences were compared with the published cdna sequence of chst6 ( nm_021615 ) . he staining revealed faintly basophilic deposits between the stromal lamellae , and within keratocytes and endothelial cells . these deposits were positive to alcian blue , pas , and colloidal iron stain , but negative to masson 's trichrome stain ( fig . electron microscopy revealed keratocytes distended by membrane - bound intracytoplasmic vacuoles containing electron - dense fibrillogranular material . one missense mutation was identified in a homozygous state ( p.arg205trp [ c.613c > t ] ) , which had not been previously reported . samples from the patient 's son also showed the same missense mutation ( p.arg205trp [ c.613c > t ] ) ( fig . hematoxylin and eosin ( he ) , alcian blue , periodic acid - schiff ( pas ) , colloidal iron , and masson 's trichrome stains were performed on the specimen using standard techniques . for transmission electron microscopy , the corneal specimen was immersed immediately in a fixative solution containing 3% glutaraldehyde with 0.2 m sodium cacodylate at a ph of 7.4 . after overnight fixation , the fixative solution was removed and replaced with a phosphate buffer , followed by 1% osmium tetroxide buffered with sodium cacodylate . after one hour , the osmium was replaced with increasing concentrations of ethanol through propylene oxide , and the tissue was embedded in the epoxy . the embedded tissue was then sectioned with an ultra - microtome into 1 m - thick sections , and stained with toluidine blue . the area to be observed was placed under a light microscope , ultra - sectioned from 60 to 100 nm in thickness , double stained with uranyl acetate and lead citrate , and examined via transmission electron microscope ( jem 1010 ; jeol , tokyo , japan ) . the unique exon involving the coding region , exon 3 , was amplified via polymerase chain reaction ( pcr ) with the previously described primers . the cycling program began with an initial denaturing step of 5 minutes at 95 followed by 33 cycles of 94 for 30 seconds , 53 to 57 for 30 seconds , and 72 for 45 seconds with a final extension step at 72 for 10 minutes . the pcr products were purified and sequenced directly on both strands using an automatic dna sequencer ( abiprism 377xl ; applied biosystems , foster city , ca , usa ) . the nucleotide sequences were compared with the published cdna sequence of chst6 ( nm_021615 ) . he staining revealed faintly basophilic deposits between the stromal lamellae , and within keratocytes and endothelial cells . these deposits were positive to alcian blue , pas , and colloidal iron stain , but negative to masson 's trichrome stain ( fig . electron microscopy revealed keratocytes distended by membrane - bound intracytoplasmic vacuoles containing electron - dense fibrillogranular material . one missense mutation was identified in a homozygous state ( p.arg205trp [ c.613c > t ] ) , which had not been previously reported . samples from the patient 's son also showed the same missense mutation ( p.arg205trp [ c.613c > t ] ) ( fig . in this study , we describe histopathological findings and a novel mutation in the chst6 gene ( p.arg205trp [ c.613c > t ] ) in a case of mcd . light microscopy revealed abnormal deposits of glycosaminoglycans in bowman 's histiocytes and keratocytes , as well as between the stromal lamellae , descemet 's membrane , and endothelium . electron microscopy revealed that these deposits corresponded to electron - lucent fibrillogranular material visible within membrane - bound intracytoplasmic vacuoles . such abnormalities have been identified as sequelae of an error in glycosaminoglycans metabolism within the cornea . in particular , errors in proteoglycan keratan sulfate ( ks ) metabolism the product of the chst6 gene , corneal n - acetylglucosamine-6-sulfotransferase ( c - glcnac6st ) , has been demonstrated to catalyze the sulfation of glcnac in ks . in normal corneal tissue chst6 can transfer a sulfuric acid group from the 3'-adenosine 5'-phosphate acid to ks via competition with an endogenous or exogenous substrate . the variations in the coding region of chst6 may reduce enzyme activity or cause it to be lost , resulting in a low sulfated form or non - sulfated form of ks . due to the loss of its soluble properties , non - sulfated ks can not be completely metabolized , inducing the deposition of sediment in the corneal stroma . the mutation identified in this korean patient is different from those reported previously in asians ( japanese and chinese ) or whites ( american , british , and icelandic ) . although the p.arg205gln mutation has been reported in a southern indian patient , the p.arg205trp mutation has not been reported previously . the immunophenotype of mcd was not performed in our study since we could not test ks serum levels . regardless of the type of macular corneal dystrophy , the patient showed indistinguishable clinical characteristics linked to the same responsible gene . in summary , we identified a novel homozygous missense mutation in chst6 in a korean patient with macular corneal dystrophy . this novel gene mutation expands the mutation spectrum of the chst6 gene and contributes to the study of molecular pathogenesis of corneal dystrophy . further studies on these mutations would be helpful in further understanding the molecular mechanisms underlying macular corneal dystrophy . various methods including chemical , electro - chemical , and physical deposition techniques have been employed to synthesize 1d zno nanostructures [ 3 - 5 ] . on the other hand , the wet - chemical methods [ 6 - 8 ] have been used for producing varied zno one - dimensional ( 1d ) nanostructures , such as nanotube , nanopencil , nanoneedle , and nanoscrew . recent experiments have shown that the zno 1d nanostructures have excellent field emission properties , far better than other semiconductors . an important advantage of using aligned nanorods , nanowires , nanobelts , and nanotubes for field emission is their high aspect ratio . the field enhancement factor is a key parameter which is determined by turn - on field , threshold field , and work function . also , the value of relates to the structure , shape , size , alignment , crystalline , aspect ratio , etc . . many effects , such as morphological effects , surface states , and densities of nanorods have been studied . the result showed that the zno nanoneedle arrays exhibit excellent properties due to their small emitter radius and high nanorod density remarkably reduces the local field at the emitters owing to the screening effect . wang et al . investigated the density effects on the field emission of zno nanorods and pointed out that the mezzo density of zno nanorods had the best field emission properties . but there is no report about the aspect ratio effects on the field emission properties of zno nanorod arrays . in this work , zno nanrod arrays with different aspect ratios and densities are synthesized by controlling the reaction times and concentrations of solution . the field emission properties of zno nanorod arrays with different aspect ratios and densities have been investigated for showing the enhancement factor was enhanced with increasing the aspect ratio of zno nanorods and the enhancement factor was decreased with reducing the density of nanorods . an optimum density and aspect ratio of zno nanorod arrays ( sample c ) the zno nanorod arrays were prepared on a silicon wafer ( 4 5 cm ) through a multi - step hydrothermal process . firstly zno nanocrystals colloid ( 4 10 m ) is spin - coated 15 times on the silicon wafer at the speed of 3000 r / s to form a thick film of zno nanocrystals and zno nanocrystlas film annealed at 400 c for 2 h under atmosphere . then the silicon wafer is immersed into the aqueous solution ( 250 ml ) of 0.04 m zinc nitrate hexahydrate / hexamethylenetetramine at 75 c . after keeping it for 10 h in this solution , the surface of silicon wafer is coated for forming a layer of white film , which is washed by deionized water three times , and dried in air at room temperature . a piece of the silicon wafer ( 1 4 cm ) is cut as sample a. the remnant wafer ( 4 4 cm ) is reinserted into the aqueous solution ( 250 ml ) of 0.04 m zinc nitrate hexahydrate / hexamethylenetetramine at 75 c for 10 h and the sample b is obtained by cutting a piece from the above mentioned silicon wafer ( 1 4 cm ) . the nanorods are able to form bundles if the sample b is immersed into 0.04 m aqueous solution of zinc nitrate hexahydrate / hexamethylenetetramine at 75 c for 10 h. the concentration of reaction solution is reduced to 0.03 m and a remnant wafer ( 3 4 cm ) is kept into the aqueous solution ( 250 ml ) of 0.03 m of zinc nitrate hexahydrate / hexamethylenetetramine at 75 c for 10 h and the sample c ( 1 4 cm ) is obtained from the above method the arrays of zno nanorods are characterized and analyzed by field emission scanning electron microscopy ( fesem ) and x - ray diffraction ( xrd ) . the sem images are obtained with a jeol jsm 6700f field emission scanning electron microscope . the xrd patterns are recorded with a japan rigaku d / max-2500 rotation anode x - ray diffractometer equipped with graphite - monochromatized cu k radiation ( = 1.54178 ) , employing a scanning rate of 0.05sin the 2 range from 20 to 60. the field emission properties of zno nanorod arrays are measured using a two - parallel - plate configuration in a homemade vacuum chamber at a base pressure of ~1.0 10 pa at room temperature . the sample is attached to one of the stainless - steel plates which is cathode with the other plate as anode . a direct current voltage sweeping from 0 to 5000 v was applied to the sample at a step of 50 v. the emission current is monitored using a keithley 6485 picoammeter . 1 . the diffraction peaks are identified to match the hexagonal zno crystalline with wurtzite structure and preferentially aligned in the c - axis direction . as shown in fig . 2 , for the samples a , b , c , d , and e , the zno nanorod arrays aligned on the silicon wafers . the average radiuses of samples a , b , c , d , and e are 90 2 nm , 125 5 nm , 136 5 nm , 150 5 nm , and 168 5 nm , respectively . the average lengths of samples a , b , c , d , and e are 2.5 0.05 m , 4.2 0.05 m , 5.3 0.05 m , 6.3 0.1 m , and 7.4 0.1 m , respectively . from sample a to sample b the average radiuses and lengths of zno nanorods obviously increased about to 35 nm and 1.7 m when the reaction times are increased . from sample b to sample e , the average radiuses and lengths of zno nanorods equably increased by ~15 nm and ~1 m . the radiuses and lengths of zno are able to be accurately controlled by adjusting the concentration of reactants and reaction times . the average spacing of nanorods of samples a , b , c , d , and e are 195 10 nm , 183 10 nm , 167 10 nm , 143 10 nm , and 126 10 nm , respectively . the average radiuses , lengths , and spacing of the nanorods are listed in table 1 . the aspect ratios of the samples a , b , c , d , and e are 28 , 34 , 39 , 42 , and 44 , respectively . xrd patterns of those zno nanorod arrays the sem images of samples a , b , c , d , and e. the inset images are the section view of samples the morphological characteristic and field emission property of zno nanorod arrays . ( r : the average radius;l : the length of nanorod; : the field enhance factor;s : the spacing of nanorods ) the current density electric field ( j e ) curves and the corresponding fowler - nordheim ( f - n ) plots of zno nanorod arrays are illustrated in fig . the important field emission parameters extracted from these current density electric field ( j we define the turn - on field ( eto ) and the threshold field ( eth ) as the applied electric fields required to produce a current of 10 a / cmand 1 ma / cm , respectively . the etoof samples a , b , c , d , and e are 6.33 v/m , 5.16 v/m , 3.83 v/m , 4.16 v/m and 4.65 v/m , respectively . the ethof sample a , b , c , d and e are 8.58 v/m , 7.43 v/m , 5.65 v/m , 6.57 v/m , and 6.97 v/m , respectively . the sample c has the lowest turn - on field ( 3.83 v/m ) and threshold field ( 5.65 v/m ) , which indicates that the sample c has the best field emission properties in these samples . the field emission properties of zno nanorod arrays : ( a ) j e plots . ( b ) the corresponding f - n plots to further analyze the field emission properties of the zno nanorod arrays , the class fowler - nordheim ( f - n ) law , which was induced on the basis of the electron emission properties from a semi - infinite flat metallic surface , was used to describe the relationship between the j and the local field nearby the emitter . is usually related to the average applied field e as follows : where d is the inter - electrode spacing , v is the applied voltage , and is the enhance factor . the f - n law is expressed as where , , and is the work function , which is estimated as 5.2 ev for zno . the enhance factor can be determined by fitting the slope value and taking a reasonable value . for those zno nanorod arrays , the f - n plots ( fig . 3b ) show a rough linear relationship , implying that a quantum - tunneling mechanism is responsible for the emission . the obtained field enhancement factors from the f - n plots are summarized in table 1 . the field enhancement factors of samples a , b , c , d , and e are 1103 , 1772 , 2612 , 2382 , and 1760 , respectively . the apparent reasons might be due to the screening effect [ 20 - 25 ] . an empirical model can be used to explain this phenomenon.(1)(2 ) l and r are the length and the radius of zno nanorods . 0 is the intrinsic field enhancement factor for a single emitter which is determined by the aspect ratio . is the field enhancement factor of the emitter array , which can be determined by the aspect ratio and the interspacing of nanorods ( density effect ) . s is the interspacing of nanorods . when the aspect ratio increases gradually , 0 will keep up with it . when the interspacing s decreasing , there exists a negative effect on the increment of . when s l , eq . the alterable parameter h is chosen 26 to fit the data of samples a and b. then using the formula ( 3 ) , the stimulant relationship of and l / r can be shown in fig . 4b which shows the sample c ( = 2885 ) has the highest field enhancement factor . , is not only related to aspect ratio but also dependent on s. for the low - density , the interspacing is large , and the screening effect is weak resulting in the field enhancement factor increasing with the increase of aspect ratio . for the small interspacing , the screening effect is able to be domain factor leading to decrease in the field enhancement factor . these two opposite effects take place simultaneously ; the enhancement factor shows a maximum value at balance point ( aspect ratio : 38.9 , interspacing : 178 nm ) , which is consistent with the sample c ( aspect ratio : 39 , interspacing : 167 10 nm ) , for producing an optimization of field emission properties . the arrays of zno nanorod with different aspect ratios and densities are constructed using a multi - step hydrothermal process by controlling the reaction times and concentrations . the results show that the aspect ratio and the density of nanorod arrays play key roles in the field emission . the field enhancement factorenhances with increasing the aspect ratio of the nanorod . for the small interspacing ( s ) , the screening effect may become the domain factor which will decrease the field enhancement factor. when the interspacing ( s ) is larger than 167 nm , the enhancement factorincreases with aspect ratio , linearly , while the screening effect can be negligible . but when thesis smaller than 167 nm , the screening effect becomes the domain factor . there exists a balance point ( aspect ratio : 38.9 , interspacing : 178 nm ) , in which the optimization field emission can be obtained . this work was supported by the national nature science foundation of china ( 20531060 , 20473102 , and 20571078 ) and the national basic research 973 program of china ( grant no .

to report a novel mutation within the chst6 gene , as well as describe light and electron microscopic features of a case of macular corneal dystrophy . a 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty . further studies including light and electron microscopy , as well as dna analysis were performed . light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells , as well as extracellularly within the stroma . all samples stained positively with alcian blue , colloidal iron , and periodic acid - schiff . electron microscopy showed keratocytes distended by membrane - bound intracytoplasmic vacuoles containing electron - dense fibrillogranular material . these vacuoles were present in the endothelial cells and between stromal lamellae . some of the vacuoles contained dense osmophilic whorls . a novel homozygous mutation ( c.613 c > t [ p.arg205trp ] ) was identified within the whole coding region of chst6 . a novel chst6 mutation was detected in a korean macular corneal dystrophy patient . zno nanorod arrays are prepared on a silicon wafer through a multi - step hydrothermal process . the aspect ratios and densities of the zno nanorod arrays are controlled by adjusting the reaction times and concentrations of solution . the investigation of field emission properties of zno nanorod arrays revealed a strong dependency on the aspect ratio and their density . the aspect ratio and spacing of zno nanorod arrays are 39 and 167 nm ( sample c ) , respectively , to exhibit the best field emission properties . the turn - on field and threshold field of the nanorod arrays are 3.83 v/m and 5.65 v/m , respectively . importantly , the sample c shows a highest enhancement of factor , which is 2612 . the result shows that an optimum density and aspect ratio of zno nanorod arrays have high efficiency of field emission .

the adverse consequences of the acquired immune deficiency syndrome ( aids ) or t cell immunodeficiency provide evidence of the vital role of cytotoxic t - lymphocytes ( ctl ) in the immune response . indeed , ctl are well - known elements of the immune response to virus - infected , tumour and allogeneic cells [ 13 ] . more recently , the role of ctl was expanded significantly when the ability to mediate direct killing of microbial pathogens was identified . research defining the precise mechanisms underlying ctl killing of microbes , however , is still in its infancy . remarkably , granulysin ( in contrast to granzymes ) has emerged as a fundamental mediator of microbial killing . the mode of action of granulysin appears to be through the disruption of membrane permeability . it follows that granulysin has been found to insert into the microbial membrane through ionic interactions between the positively charged amino acid residues and negatively charged phospholipids . insertion of granulysin in turn disrupts membrane permeability resulting in the influx of fluid into the cytoplasm and death by osmotic lysis . other mechanisms identified in the killing of tumor cells may also play a role , including ca influx and k efflux , and activation of a sphingomyelinase associated with the cell membrane to generate ceramide . ctl killing of extracellular pathogens involves direct microbial recognition by the ctl ( figure 1(a ) ) . in contrast to tumour and virus - infected cells , recognition of extracellular pathogens occurs through an apparent mhc - independent mechanism ( as microbes have not been found to express mhc ) . successful recognition induces the release of granulysin which may directly bind and kill the microbe . in the case of intracellular microbes these interactions , like recognition of tumor and virus - infected cells , are often mhc - restricted and antigen - specific . binding triggers the release of granulysin , which must enter the infected host cell for microbial killing to occur ( figure 1(b ) ) . this is thought to be mediated through perforin - generated pores in the host cell membrane . these pores facilitate the influx of extracellular ca which triggers the target cell to endocytose the damaged region of the membrane and internalize nearby granulysin [ 79 ] . alternatively , ctl may mediate lysis of the infected host cells releasing microbes which may then be recognized and killed by nearby ctl ( figure 1(c ) ) . to acquire microbicidal activity , this priming event can occur in response to cytokines such as t cell growth factors , stimulation by a mitogen , or antigen - specific responses . the receptors and signalling pathways involved in priming may be the same , but also might be quite distinct from the receptors and signalling that lead to immediate killing . following the priming event , the microbicidal ctl is then ready to receive the signal that triggers them to kill the pathogen . t cells from mice immunized with p. aeruginosa polysaccharide were stimulated with macrophages from nonimmune mice with or without the addition of heat - killed bacteria . macrophages had to be present during the priming , but surprisingly , heat - killed bacteria did not . this suggested that the bactericidal activity of these t cells was dependent on their interaction with macrophages but did not require presentation of p. aeruginosa antigens . supernatants collected from immune t cells exposed to macrophages and p. aeruginosa were found to kill p. aeruginosa in addition to staphylococcus aureus and escherichia coli suggesting that these t cells were producing a soluble bactericidal product . ctl have also been found to mediate killing of mycobacterium tuberculosis through the release of bactericidal products [ 1113 ] . m. tuberculosis growth was reduced as much as 74% in the presence of cd4 ctl and 84% in the presence of cd8 ctl . when cd8 ctl were pretreated with strontium chloride ( which depletes granule contents ) , it caused a clear reduction in the ability to kill m. tuberculosis that correlated with a marked decrease in granulysin content . in vitro , purified granulysin was able to kill extracellular m. tuberculosis in a dose - dependent fashion . however , killing of intracellular m. tuberculosis required the addition of perforin , which was not directly bactericidal , but was able to lyse m. tuberculosis - infected macrophages . together this data suggests that perforin facilitates entry of granulysin into m. tuberculosis - infected cells where granulysin can access and kill the intracellular pathogen . in vivo studies have provided additional support for the conclusion that perforin is required for cd8-mediated clearance of m. tuberculosis . in these studies , irradiated mice infected with m. tuberculosis were found to have a significantly greater bacterial load after receiving adoptively transferred perforin - deficient cd8 t cells compared to wild - type cd8 t cells . studies conducted with listeria innocua support a perforin - independent mechanism in which granulysin is actively taken up by the infected cell . both healthy and l. innocua - infected dendritic cells ( dc ) were found to take up recombinant granulysin in a temperature - sensitive manner , indicative of active internalization . furthermore , cholesterol depletion abrogated granulysin uptake and killing of infected dc , suggesting lipid raft involvement . this was further supported by data showing colocalization of granulysin and cholera toxin ( used as a marker of lipid rafts ) during and shortly following uptake . ninety minutes after uptake , granulysin was found to colocalize with phagosomes containing l. innocua dna . indeed , granulysin was found to kill both extracellular l. innocua and l. innocua - infected dc in a dose - dependent manner . while granulysin may function independently , another study found that perforin treatment ( whether simultaneous or sequential to granulysin treatment ) augmented granulysin - dependent killing of intracellular l. innocua which was not due to the formation of stable pores in the dc membrane . rather , perforin treatment was found to stimulate a transient change in the plasma membrane permeability ( assessed by ca influx ) that enhanced fusion of granulysin - containing endosomes with phagosomes containing l. innocua . the highest level of expression of granulysin occurred in patients afflicted with the localized tuberculoid form , rather than the disseminated lepromatous form of the disease . this correlation suggests that granulysin release by ctl may limit the spread and severity of m. leprae infection . cd4 t cell lines derived from tuberculoid leprosy lesions were found to lyse m. leprae infected macrophages and kill intracellular mycobacteria . strontium treatment abrogated both the cytolytic and bactericidal activity of these cd4 t cells , which suggested that killing was mediated through the granule - exocytosis pathway . blocking fas using anti - fas antibody also partially inhibited the cytolytic activity of two of four cd4 t cell lines suggesting that the fas - fasl pathway may also play a minor role in the lysis of m. leprae - infected cells . it follows that the extent of fas - fasl mediated lysis may depend on the nature of the ctl or the infected target cell . t cells stimulated with phytohemagglutinin ( pha ) , a mitogenic lectin , as well as various oxidative mitogens , antigens , and alloantigens were found to kill schistosomula . killing was found to correlate with increased binding to schistosomula suggesting a contact - dependent mechanism . in support of this , supernatants from pha - stimulated t cells were ineffective at killing , although the concentration of parasiticidal products in the supernatants may not have been high enough to mediate an appreciable response . moreover , it remains unclear how pha induces ctl parasiticidal activity . removal of pha prior to incubation of t cells with schistosomula was found to impair killing suggesting that pha may have an ulterior function in addition to stimulating t cell activation and proliferation . ctl killing of entameoba histolytica closely resembled that of schistosoma mansoni in many respects . first , nonspecific activation of t cells with pha was found to significantly enhance killing of e. histolytica trophozoites compared to unstimulated t cells . pha - stimulated t cells killed as many as 92% of trophozoites whereas amoebic viability in the presence of unstimulated t cells remained relatively unchanged . third , and surprisingly , killing required the continuous presence of pha , again alluding to direct participation . however , bridging the t cells and entameoba can not be the only mechanism by which pha participates in killing because at least 18 hours of preincubation was required to elicit killing . similarly , in studies with s. mansoni , pretreatment of schistosomula with pha was not sufficient to induce parasiticidal activity of unstimulated t cells . thus , pha appears to act both by activating the t cells as well as possibly functioning as a bridge for binding . specific activation of t cells isolated from patients with an amoebic liver abscess with an e. histolytica lysate also induced killing . in contrast to prior studies with pha [ 20 , 21 ] , stimulation with antigens from e. histolytica was found to augment binding intensity of t cells to the trophozoites rather than the binding frequency . thus , the more important determinants in killing amoeba may be the binding characteristics of the ctl rather than the number of ctl bound . anti - tnf- antibody blocked killing of immune t cells from mice immunized against e. histolytica , although large doses of tnf- did not directly kill the amoeba . these results demonstrate the complexity of the system and the possible role of additional mediators . t cells from mice immunized with p30 ( a t. gondii membrane protein ) and subsequently restimulated in vitro with p30 were found to kill extracellular t. gondii . unlike studies conducted with s. mansoni and e. histolytica [ 20 , 21 ] , killing of t. gondii appeared to be antigen specific . p30-antigen - specific splenocytes killed significantly more t. gondii than splenocytes stimulated with the mitogenic lectin , concanavalin a ( cona ) , despite greater t cell proliferation in response to cona . although antigen stimulation resulting in microbicidal priming was likely to have been mhc - restricted , killing was direct . since there is no evidence for expression of mhc or mhc - like molecules by t. gondii , this suggests that direct cytotoxicity was mhc - independent . mhc - unrestricted t cell killing of extracellular t. gondii has also been reported by others . however , the mechanism by which antigen - specificity is accomplished in the absence of mhc is not yet defined . ctl responses against intracellular t. gondii , on the other hand , have been found to proceed through an mhc - restricted mechanism . splenocytes from mice immunized against t. gondii mediated killing of both infected macrophages and intracellular t. gondii [ 26 , 27 ] . concanamycin , an inhibitor of the vacuolar atpase that is required to maintain perforin in lytic granules , significantly impaired killing , implicating perforin in the response against t. gondii . perforin - deficient mice chronically infected with t. gondii were found to have a higher rate of mortality and a greater number of brain cysts compared to wild - type mice , although other studies came to disparate conclusions . moreover , the ability of cd8 t cells to kill intracellular t. gondii is controversial . in one study , cd8 t cells failed to kill t. gondii - infected b cells as assessed by quantitative pcr of the sag-1 gene . t cells were found to kill blood - stage p. falciparum through a contact - dependent mechanism [ 3133 ] . killing appeared to be stage - dependent with extracellular merozoites being the suggested targets [ 31 , 32 ] . in vitro studies revealed a correlation between the parasiticidal activity of the t cells and expression of granulysin . together , this data indicates that parasiticidal ctl mediate killing of p. falciparum via the release of granulysin . several studies have reported the ability of ctl to kill the opportunistic fungus candida albicans . il-2 stimulation of murine splenocytes was found to induce fungicidal activity against c. albicans as well as several other candida species . anti - candida activity required at least 3 days of priming with il-2 and peaked at 7 days . the induction of fungicidal activity correlated with enhanced killing of an nk cell - resistant tumor cell line . supernatants taken from il-2 stimulated splenocytes had no effect on the growth of c. albicans suggesting a contact - dependent fungicidal mechanism . moreover , another study found that the cd8 t cells , and not nk1.1 cells ( nk cells ) , mediated fungicidal activity against c. albicans in response to il-2 . the ctl response against the yeast - like pathogen cryptococcus neoformans has been described extensively . in one of the earlier studies , lymphoid cells were isolated after mice had been immunized with heat - killed c. neoformans in complete freund 's adjuvant . the anticryptococcal activity of these cells was measured by assessing colony forming units , and delayed - type hypersensitivity was assessed by footpad swelling following administration of c. neoformans antigen . only lymphoid cells from immunized mice furthermore , the magnitude of the anticryptococcal activity correlated with the intensity of the delayed - type hypersensitivity response . t cells within the peripheral lymphoid compartment were found to be the mediators of the anticryptococcal activity . in another study , t cells were observed to interact directly with c. neoformans in vitro suggesting a contact - dependent mechanism of killing ( figure 2 ) . however , between 11 and 35% of t cells were found to bind to c. neoformans suggesting recognition by a receptor other than the t cell receptor . other studies reported similar killing with human peripheral blood mononuclear cells ( pbmc ) stimulated with dead c. neoformans or cultured for 7 days with il-2 and gm - csf ( but not with tnf , ifn- , or vitamin d3 ) . in agreement with prior studies [ 36 , 37 ] , t cells ( as well as nk cells ) isolated from the pbmc were found to directly bind to , and possess fungicidal activity against c. neoformans after priming with il-2 . another study found that freshly isolated t cells could mediate killing of c. neoformans without the need for il-2 stimulation . however , t cells cultured without il-2 soon lost their ability to kill , suggesting that il-2 is required for t cells to maintain their fungicidal activity . treatment of t cells with the proteases trypsin and bromelain , which have been found to cleave several receptors on t cells , also impaired killing of c. neoformans suggesting that receptor - ligand interactions were involved in anticryptococcal activity . investigators have asked whether ctl kill cryptococcus or exert their anticryptococcal activity by inhibiting the growth of the organism . studies using the viability dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) showed that c. neoformans failed to demonstrate metabolic activity after incubation with t cells . c. neoformans also becomes permeable and labels with propidium iodide after incubation with ctl providing further evidence for killing ( unpublished observations , anowara islam ) . furthermore , ctl can reduce the burden of organisms below the starting innocula [ 41 , 44 ] , and consistent with previous studies that suggested a contact - dependent mechanism of killing , separation of t cells and c. neoformans with a porous membrane during incubation was found to abrogate anticryptococcal activity . early studies investigating the mechanism of ctl anticryptococcal activity examined possible receptors and effector mechanisms . antibody - mediated blockade of various t cell surface molecules such as lfa-1 and cd3 did not significantly inhibit killing . together this data suggests that ctl killing of c. neoformans may proceed through a novel receptor that is yet to be identified . these early studies also examined the role of reactive hydroxyl radicals , which have been suggested to play a role in eosinophil - mediated killing . additionally , cyclooxygenase inhibitors and prostaglandin e2 , which have been found to inhibit nk cell cytotoxicity [ 47 , 48 ] and nf-b activation , were assessed . among the 3 hydroxyl radical scavengers used similarly , only 1 ( salicylic acid ) of the 3 cycloxygenase inhibitors abrogated killing , while prostaglandin e2 failed to have any appreciable effect . one study , however , described an effector mechanism by which il-15 stimulated cd8 ctl kill c. neoformans . in vitro studies have previously shown that il-15 from c. neoformans - stimulated monocytes induced t cells to become anticryptococcal . this led to studies showing that the anticryptococcal activity of il-15-stimulated cd8 t cells correlated with the level of granulysin expression . furthermore , both strontium treatment and sirna knockdown of granulysin abrogated anticryptococcal activity , directly implicating granulysin in the ctl response against c. neoformans . perforin was not required , as neither concanamycin a nor egta treatment impaired anticryptococcal activity . these results are in contrast to nk cells , which depend on perforin , but not granulysin for killing . these results were extended using cd8 t cells purified from pbmc that had been stimulated with c. neoformans mitogen ( cnm ) , a protein mitogen within the cryptococcal cell wall and membrane [ 52 , 53 ] . priming of anticryptococcal activity and granulysin expression by stimulation with cnm was dependent on cd4 t cells . stimulation of pbmc with cnm in the presence of anti - il-15 abrogated anticryptococcal activity and granulysin expression suggesting that the dependence on cd4 t cells was mediated through il-15 . however , in the absence of accessory cells , cd4 t cells were not sufficient to induce cd8 t cell anticryptococcal activity and granulysin expression . together this data suggests that the proliferating cd4 t cells provide a retrograde stimulus to accessory cells that results in production of il-15 , which then primes cd8 t cell for granulysin expression and anticryptococcal activity ( figure 3 ) . indeed , both cd4 and cd8 t cells in vivo have been found to be indispensable for an effective response to c. neoformans [ 5458 ] . aside from their role in licensing accessory cells to prime cd8 ctl anticryptococcal activity , cd4 t cells have also been demonstrated to directly kill c. neoformans . upon stimulation with il-2 , cd4 t cells increased the expression of granulysin ( but not perforin ) which correlated with increased fungicidal activity . both strontium treatment and granulysin sirna knockdown abrogated cd4 t cell killing of c. neoformans . perforin involvement was excluded as neither concanamycin a nor perforin knockdown could interfere with anticryptococcal activity of cd4 t cells stimulated with both il-2 and anti - cd3 antibody ( which stimulated both perforin and granulysin expression ) . together this data suggests that cd4ctl ( like cd8 ) directly kill c. neoformans through a granulysin - mediated mechanism . the high prevalence of serious cryptococcal infection in hiv - infected patients [ 60 , 61 ] may be at least partially due to a defect in this fungicidal activity . indeed , cd4 t cells from these patients were found to exhibit defective killing of c. neoformans . neither il-2 nor il-2 plus anti - cd3 stimulation could induce granulysin expression in these patients ' cells . these results suggest that profound dysregulation of perforin and granulysin expression may account for the failure of cd4 t cells from hiv - infected patients to kill c. neoformans . the signalling pathways regulating granulysin expression in cd8 and cd4 ctl are relatively unknown . in preparation for killing of fungi , expression of granulysin correlated with short - term ( 060 min ) and long - term ( 15 days ) phosphorylation of extracellular signal - regulated kinases ( erk)1/2 , p38 mitogen - activated protein ( map ) kinase , and p54 c - jun n - terminal kinases ( jnk ) as well as the transcription factor signal transducers and activators of transcription ( stat)5 . meanwhile , no phosphorylation of the human oncogene akt ( signifying phosphoinositide 3-kinase ( pi3k ) activation ) could be detected following continuous il-2 stimulation . akt phosphorylation could only be detected when cd4 t cells were stimulated with il-2 for 35 days , rested ( 24 hours ) and restimulated with il-2 for 5 min , indicating that pi3k undergoes a transient activation after initial stimulation with il-2 . continuous stimulation of cd4 t cells with il-2 was also found to increase expression of the il-2r and il-2r subunits of the il-2 receptor ( by day 3 ) followed by expression of granulysin ( on day 5 ) . pharmacological inhibition of janus kinase ( jak)3/stat5 or pi3k abrogated both granulysin expression and anticryptococcal activity of il-2 stimulated cd4 t cells , which correlated with decreased expression of the il-2r subunits . together this data suggests that granulysin expression requires acquisition of one or more of the il-2r subunits . indeed , blockade of il-2r expression using anti - il-2r antibodies or sirna knockdown abrogated granulysin expression in il-2 stimulated cd4 t cells . thus , il-2 ( and perhaps other t cell growth factors ) signals t cells to increase expression of il-2r , which is then available for signalling that is necessary for granulysin expression . previous studies have shown that cd4 t cells from hiv - infected patients exhibit dysregulated expression of perforin and granulysin . comparison of il-2 signalling in cd4 t cells from healthy donors and patients infected with hiv revealed the underlying cause to be defective stat5 and pi3k signalling . il-2 stimulation of cd4 t cells from hiv - infected patients failed to induce phosphorylation of stat5 and failed to increase expression of il-2r. furthermore , in contrast to cd4 t cells from healthy donors , il-2 failed to induce phosphorylation of akt . together these results demonstrate a critical role for jak / stat and pi3k signalling in granulysin - mediated killing of c. neoformans by cd4 t cells . / stat signalling was also found to be required for granulysin expression in cd8 t cells in response to il-15 and il-21 . cd8 t cells stimulated with either il-15 or il-21 were found to increase granulysin expression , which correlated with increased phosphorylation of stat3 and stat5 . il-15 was found to induce phosphorylation of both stat3 and stat5 , while il-21 only induced phosphorylation of stat3 . pharmacological inhibition and sirna knockdown of jak / stat signalling was found to abrogate granulysin expression by cd8 t cells in response to il-15 and il-21 . together this data indicates that jak / stat signalling regulates granulysin expression by cd8 t cells in response to il-15 and il-21 . consistent with previous studies conducted with cd4 t cells [ 59 , 62 ] , hiv - infected cd8 t cells exhibited reduced phosphorylation of stat3 , stat5 , and granulysin expression in response to il-15 and il-21 as compared to mock - infected cells . it is clear that both cd4 and cd8ctl mediate direct killing of a wide range of bacterial , parasitic , and fungal pathogens . several studies reported a correlation between the frequency [ 20 , 21 ] or intensity of binding and killing . moreover , microscopy [ 37 , 40 , 41 ] revealed direct ctl - microbe interactions . finally , t cells separated from c. neoformans by a porous membrane were unable to mediate killing . contact may induce release of cytotoxic products by the ctl or provide a microenvironment or concentration at which the cytotoxic product can be efficacious . the latter may explain why supernatants from activated t cells were often unable to mediate killing [ 20 , 34 , 40 , 41 ] . second , killing is mediated primarily through the granule - exocytosis pathway . in many experimental systems , pretreatment of ctl with strontium ( which depletes granule contents ) was found to abrogate killing [ 13 , 17 , 44 , 59 ] . furthermore , granulysin , a granule constituent , has been implicated in the killing of several microbes [ 13 , 1517 , 33 , 44 , 59 ] . third , killing ( at least of extracellular microbes ) is neither antigen - specific nor mhc - dependent . nonspecific stimulation with t cell growth factors [ 34 , 35 , 39 , 40 , 44 , 59 ] or mitogenic lectins [ 20 , 21 ] was often sufficient to prime the effector cells for killing . while granulysin appears to be highly involved in killing , the role of perforin and other effector molecules is not as clear . in the case of extracellular pathogens , perforin has not been found to play a role in killing [ 54 , 59 ] , although it plays a role in direct killing by nk cells [ 51 , 64 , 65 ] . by contrast , killing of intracellular microbes requires perforin [ 13 , 27 ] , although some investigations have suggested a perforin - independent mechanism . to be sure , this topic demands the attention of future studies . jak / stat and pi3k signalling have been found to be essential in ctl killing of c. neoformans . defective jak / stat and pi3k signalling in ctl from hiv - infected patients [ 62 , 63 ] may explain why these patients experience such a high incidence of severe cryptococcal infection . during the process of evolution , ctl have developed the ability to specifically recognize altered self through complex tcr - mhc interactions . as a consequence , they have become the epitome of specific cell - mediated immunity . it is fascinating to now discover that during this process , ctl have , at the same time , preserved one of the most rudimentary immune functions of all , namely , the ability to directly recognize a microbe , bind , and without the help of antigen presenting cells or other effector cells kill the invading pathogen with competence and precision . skin tissue integrity is a crucial factor to maintain the homeostasis generated through physical barriers , separating the organism from the environment . every disruption of dermal integrity triggers a complicated cascade of events , including rapid blood clot formation , inflammatory response , and wound healing , leading to the restoration of the integrity and formation of new tissue . however , repaired structures , known as scars , are nonfunctioning , tight , and tense masses of fibrotic tissue that maintain 7080% of normal strength , with even less flexibility . inflammatory responses are necessary for wound healing , preventing multiple infection and contamination and stimulating the proliferation , revascularization , and remodeling of the extracellular matrix [ 2 , 3 ] . nevertheless , wound healing might become uncontrolled and , combined with the inflammatory response , results in massive fibrotic tissue formation called fibrosis . in this review , we will focus on the molecular mechanisms underlying skin fibrosis as a post - wound - healing pathological disorder and the impact of bone marrow - derived cells and inflammation on the formation of scars . fibrosis is a pathological process that occurs in many different organs ( organ specific fibrosis ) , such as skin , kidney , heart , lung , and liver , which might also take the form of systemic sclerosis ( ssc ) , a global , progressive , and autoimmune disorder , characterized by an extremely poor prognosis and high mortality [ 5 , 6 ] . according to the united states government , every year , in the usa although the etiology and triggering cascade might differ , fibrosis is characterized by the increased production and deposition of extracellular matrix ( ecm ) components , including collagen type i , fibronectin , hyaluronan , and elastin , and the accumulation of activated , sma - positive , and collagen - secreting fibroblasts , called myofibroblasts [ 4 , 8 , 9 ] . many authors refer to fibrosis in the context of uncontrolled or exceeded wound healing , as an effect of long - term inflammation or mechanical irritation . this process can be divided into four overlapping phases : coagulation , inflammation , proliferation , and remodeling . skin injury results , inter alia , in the disruption of endothelial and epithelial cells integrity . damaged cells release inflammatory mediators that trigger the coagulation cascade , platelet recruitment , and blood clot formation . degranulated and activated platelets present in the blood clot release multiple chemokines and growth factors ( tgf-1 , pdgf ) , which recruit inflammatory cells . platelets also participate in the chemotaxis and recruitment of fibroblasts and endothelial cells [ 1113 ] . the first two phases are often treated as one phase , representing the inflammatory stage . the blood clot comprises cross - linked fibrin and extracellular matrix proteins , such as fibronectin , vitronectin , and thrombospondin . this structure serves as a physical barrier that closes the blood vessel , a reservoir of growth factors , and a matrix on which regenerated tissue is formed [ 12 , 14 ] . the next phase , proliferation , results from hypoxic conditions and reactive nitrogen species ( rns ) production from macrophages [ 15 , 16 ] . during this phase , angiogenesis occurs , forming new capillaries and facilitating the delivery of nutrients to the wound . in addition to nutrients , collagen - secreting myofibroblasts are recruited to the wound microenvironment . as previously described , myofibroblasts are activated , sma - positive , and collagen - secreting fibroblasts that deposit new ecm components , primarily fibronectin and collagen type i , to replace the clot - formed matrix , often forming a scar [ 10 , 18 ] . in physiological wound healing , remodeling is the final phase . during this phase , myofibroblasts and some vascular cells moreover , the synthesis of ecm components is reduced but not fully terminated , and remodeling is primarily regulated through different matrix metalloproteinases ( mmps ) and their inhibitors ( tissue inhibitor of metalloproteinases , timps ) . after the degradation of the overexpressed ecm components , scarring is reduced and an equilibrium between synthesis and catabolism is reached [ 13 , 20 ] . in fibrosis , . the increased deposition of collagen type i and fibronectin stiffens and damages the surrounding tissue . in addition , connective tissue cells replace the original cells , creating a scar that in some cases might take an extremely severe form [ 13 , 21 , 22 ] . the wound healing process is shown in figure 1 . activated fibroblasts / myofibroblasts accumulate in the fibrotic tissue environment by three different , simultaneous mechanisms . first , these cells are derived from preexisting fibroblasts in the affected tissue through activation due to specific , profibrotic , and proproliferative mediators released from infiltrating inflammatory cells , such as t cells [ 2325 ] . second , myofibroblasts are recruited through bone marrow - derived fibroblast resembling cells , such as fibrocytes , cd45 and cd34 positive cells . fibrocytes transmigrate to the fibrotic environment , and in a tgf-1-controlled process , these cells undergo transdifferentiation into myofibroblasts . finally , fibroblasts / myofibroblasts accumulate through the transition from endothelial or epithelial cells to mesenchymal fibroblast - like cells [ 5 , 11 ] . during the endothelial and/or epithelial to mesenchymal transition ( endmt and emt , resp . ) , cells lose their origin markers , polarity , and cell - cell connections and gain promigratory phenotypes and mesenchymal markers [ 2830 ] . both endmt and emt are physiological processes that occur during embryonic organogenesis and wound healing . epithelial and endothelial cells establish close cell - cell contacts with a certain cell polarity , forming a solid barrier that maintains homeostasis . this barrier is formed through desmosomes and tight and adherent junctions [ 28 , 31 ] . in contrast , mesenchymal cells are spindle - shaped solitary cells , possessing migratory and ecm remodeling abilities . these cells produce and secrete ecm components , such as collagen type i and fibronectin [ 13 , 32 ] . during tissue development or regeneration therefore , after undergoing endmt / emt , these cells gain the migratory abilities of fibroblasts , facilitating the recruitment of these cells to certain locations . cells do not typically undergo full transitions , often terminating in intermediate phenotypes between endothelial or epithelial and mesenchymal , and maintaining some cell - cell contacts to perform group migration rather than single cell migration [ 28 , 33 ] . the endothelial to mesenchymal transition was first observed and described as the leaking and proliferation of endothelial cells during the development of chick and rat endocardial cushions ( cardiac mesenchyme ) . endmt is closely associated with dermal , renal , cardiac , pulmonary , intestinal , and cystic fibrosis through the establishment of fibroblasts and myofibroblasts [ 35 , 36 ] . emt is reversible , and fibroblasts might regain epithelial phenotypes ( mesenchymal to epithelial transition , met ) , whereas endmt reversibility is not well understood . the reversal of endmt ( mesenchymal to endothelial transition ) has been recently observed in cardiac fibroblasts that rapidly adopt an endothelial - cell - like phenotype after acute ischemic cardiac injury . however , more additional evidence suggests that endmt is irreversible , and transformed cells can not regain endothelial phenotypes , even after the removal of endmt inducing factors . therefore , endmt , which is not terminated at a certain time , could lead to the accumulation of collagen type i secreted from myofibroblasts and the irreversible transformation into fibrotic tissue . both endmt and emt are regulated through the zinc finger transcription factor snail family ( snail1 , snail2 , and snail3 ) . after activation , on the molecular level , snail1 stabilizes the quantity of twist1 transcription factor , and in cooperation , both of these proteins upregulate zeb1 gene expression [ 40 , 41 ] . as a repressor , snail proteins downregulate the expression of genes encoding junction proteins , such as claudin , occludin , e - cadherin ( in epithelial cells ) , ve - cadherin , and pecam1 ( in endothelial cells ) . it is not clear whether snail upregulates the genes encoding mesenchymal markers , as observed in the upregulation of myosin va in some highly metastatic cancer cell lines , such as human lung carcinoma cell lines ( a549 , pg , and calu6 ) , human colon cancer cell lines ( lovo and sw480 ) , human breast cancer cell lines ( bicr - h1 and mcf7 ) , and prostate cancer cell lines with the same genetic background ( pg3m-1e8 and pg3m-2b4 ) , or represses epithelial / endothelial genes and therefore indirectly upregulates mesenchymal markers . nevertheless , mesenchymal cell proteins , such as vimentin , fibronectin , collagen type i , sma , sm22 ( transgelin ) , n - cadherin , calponin , and fsp-1 ( fibroblast specific protein 1 ) , are expressed during and after the transition [ 4346 ] . the microrna profile also changes during mesenchymal transition , revealing the significant upregulation of mir-125 , let-7c , let7 g , mir21 , mir30b , and mir195 and downregulation of mir122a , mir127 , mir196 , and mir375 . a previous study reported that the accumulation of snail in colorectal cancer cells and in mice utricle sensory epithelia cells , after blocking the degradation of this protein through the glycogen synthase kinase-3 ( gsk-3 ) , via lithium chloride treatment or the overexpression of snail , might trigger the transition into mesenchymal - like cells [ 43 , 48 , 49 ] . however , this transition is typically induced through a variety of proinflammatory cytokines and growth factors secreted from leukocytes , which act synergistically . the most important proinflammatory / profibrotic molecules are transforming growth factors -1 and -2 ( tgf-1 and tgf-2 ) , tumor necrosis factor- ( tnf- ) , interleukins il-1 , il-6 , il-8 , and il-11 , and fibroblast growth factor-2 ( fgf-2 ) [ 38 , 39 , 5054 ] . it has been suggested that tgf- receptor is essential for mesenchymal transition signal transduction , and the overexpression of snail might be an insufficient factor . the inhibition of tgf- receptor accompanied by simultaneous upregulation of snail does not lead to endmt in mouse embryonic stem cell - derived endothelial cells ( mesecs ) . however , the upregulation of the transcription factor snail directly upregulates profibrotic and proinflammatory cytokines , such as il-8 . the secretion of tgf- into the fibrotic microenvironment during inflammation is the most important snail inducer . however , studies on skin cancer formation have shown that smad2 inhibits endmt , whereas smad3 acts as an activator . the binding of tgf- to tgf- receptor type ii ( trii ) triggers heterodimerization through the activation of the tgf- receptor type i kinase ( tri ) , which activates activin - like kinase 5 ( alk5 ) and transduces a signal through the smad2/3 complex with smad4 , which activates the expression of snail . the inhibition of either alk5 or alk2 results in the inhibition of endmt [ 29 , 57 ] . tgf- also activates snail in a non - smad pathway , involving wnt and noch , via the sequestration of gsk-3 and akt2 , through the transcriptional repression of the mir-200 superfamily and the activation of the inflammatory transcription factor nfb [ 30 , 46 , 50 , 58 ] . as previously discussed , chronic inflammation is one of the main factors triggering fibrosis , particularly endmt - based fibrosis , as endmt is an irreversible process . constant inflammation leads to the production of a variety of proinflammatory cytokines and growth factors secreted from different leukocytes present in the fibrotic microenvironment . however , fibrosis formation is a multidimensional and multistage process that not only involves endmt . leukocyte recruitment triggers many different mechanisms and pathways that might lead to disordered wound healing , myofibroblasts and collagen type 1 accumulation , scarring , and fibrosis . the recruitment of these cells is initiated immediately after activated platelets degranulate and release tgf-1 and pdgf . tnf- , il-1 , and il-8 released from endothelial cells also stimulate neutrophil recruitment , leading to selectin - mediated rolling adhesion towards the chemoattractant gradient . in the next phase , tight adhesion to endothelial cells occurs via integrin 2 , followed by transmigration through the endothelial tissue . when necessary , neutrophils cross the ecm barrier along fibroblasts and transmigrate through epithelial cells to enter the wound . neutrophils begin phagocytosing invading bacteria and damaged necrotic cells to clear the wound , preparing it for the regeneration of homeostasis through scar formation . however , fetal wounds heal without scar formation , and fetal neutrophils are physiologically distinct from adult neutrophils , as these cells are less adept than adult cells , producing less cytokines and presenting lower contributions to the inflammatory response [ 60 , 61 ] . neutrophil serine protease , elastase , is secreted into the microenvironment , increasing il-8 expression in the surrounding cells . il-8 not only is responsible for leukocyte recruitment but also might trigger endmt and increase the survival and proliferation of endothelial - derived fibroblasts / myofibroblasts , leading to fibrosis [ 6 , 63 ] . additionally , elastase is also believed to cleave the il-8 receptor cxcr1 , interfering with neutrophil functions and antibacterial abilities , thereby prolonging inflammation , which in turn increases additional fibrosis - based changes . prolonged inflammation might also occur through the elastase - mediated degradation of complement , releasing the strong neutrophil chemoattractant , c5a . moreover , neutrophil derived oxidative burst , leading to the formation of hocl from h2o2 catalyzed through myeloperoxidase , induces injury to epithelial cells , thereby implicating the switch to fibrotic tissue deposition [ 66 , 67 ] . two different populations of neutrophils have been observed to enter the wound in mice after the induction of acute inflammation : one population has a proinflammatory function , and the second population is responsible for anti - inflammatory responses . these cells differ in size , granularity , and the expression of cd11b and ly6 g . respectively , the anti - inflammatory neutrophil response is strongly associated with the secretion of the anti - inflammatory cytokine il-10 . moreover , a certain population of mature neutrophils , characterized as cd11c / cd62l / cd11b / cd16 , have been reported to suppress t cell proliferation via the expression of the integrin mac-1 ( m2 ) . the impact of neutrophils on fibrosis has been observed in pulmonary fibrotic disorders , as these cells transmigrate to pulmonary fluids ( such as bronchoalveolar lavage fluid ) and recruit other leukocytes [ 71 , 72 ] . however , only a few clinical studies have successfully established anti - inflammatory strategies in patients with pulmonary fibrosis . inhibition of neutrophil derived elastase as strategy of downregulation of self - destructive process of neutrophil derived protease activity as well as elastase derived il-8 expression is currently being elucidated and brings more questions than answers . clinically useful concepts have only just started to evolve and bring promising , but not yet convincing , answers [ 59 , 73 ] . macrophages appear as the second type of bone marrow - derived cells invading the wound site , and three to five days after injury , they become the dominant leukocyte type . similar to neutrophils , different populations of macrophages have been reported , depending on the activation path through different chemokines and growth factors , as shown in figure 2 . classical macrophage activation , or m1 , is obtained , in particular , through the combination of interferon gamma ( ifn- ) and tumor necrosis factor- ( tnf- ) signaling pathways . alternative activation , or m2 , is far more complex , leading to the formation of regulatory and wound - healing macrophages . regulatory macrophages release anti - inflammatory cytokines il-10 and tgf- , which downregulate inflammation , and also lead to the endothelial to mesenchymal transition and increase the fibroblast number at the wound site . its abilities might shift , depending on other cytokines availability and cell type [ 76 , 77 ] . it was shown that tgf- administered to animals with infection or inflammation reduces severity of disease and production of proinflammatory il-1 and tnf . a second group of m2 macrophages , wound - healing macrophages , are derived through il-4 induction . these cells secrete cc chemokine ligands , including ccl2 , ccl17 , ccl18 , and ccl22 . wound - healing macrophages are extremely profibrotic , as these cells produce high levels of fibronectin and through ccl18 activation promote collagen production from fibroblasts / myofibroblasts . moreover , arginase activation in m2 macrophages , stimulated through il-4 , leads to the conversion of arginine to ornithine , a precursor of collagen [ 74 , 80 ] . blocking il-4 with specific antibodies significantly decreases wound - healing , macrophage accumulation , and fibrosis formation . recent studies showed that overexpression of mmp9 in macrophage might attenuate bleomycin induced pulmonary fibrosis . respectively , production of mmp13 by kuppfer cells was shown to be sufficient in preventing pig serum - induced rat liver fibrosis . these data suggest that high level of mmps might play key role in fibrosis reversibility . macrophages are the main sources of mmps that facilitate ecm degradation during remodeling phase in wound healing process ; they also phagocytose apoptotic myofibroblasts and cellular debris preventing advance in the fibrotic process . however , some authors suggest strong profibrotic role of macrophage derived mmp13 , as they observed that liver fibrosis was suppressed , along with fibrotic markers and inflammatory mediator expression , in mmp13-deficient mice during cholestasis - induced liver fibrosis . surpassingly , prolonging inflammation and recruitment of activated macrophages might be involved in fibrosis reversing process , as accumulating evidence strongly correlates macrophages and the macrophages derived mmps ( mmp1 , mmp2 , mmp8 , mmp9 , and mmp13 ) with this process . after arrival to the wound site , these cells produce lymphokines , which in turn activate other inflammatory cells , such as macrophages . among all lymphocyte subpopulations , th1 and th2 are most relevant for tissue fibrosis . th1 and th2 lymphocytes contribute different responses to wounded tissue . th1 acts as an antifibrotic , releasing il-10 , and th2 acts as a profibrotic . studies using mouse models have shown that the polarized th2 response leads to massive collagen deposition and increased fibrosis formation . however , the th1 response activates the genes responsible for apoptosis and acute - phase reactions [ 86 , 87 ] . among all cytokines released from th2 , the two most important and most profibrotic cytokines are il-4 and il-13 . both il-4 and il-13 share functional similarities , as these molecules transduce signal via the il-4r / stat6 pathway [ 8890 ] . as previously described , il-4 activates m2 wound - healing macrophages , resulting in collagen production and deposition . moreover , il-4 stimulates in the dose - dependent manner collagen synthesis in fibroblasts and is two times more effective than tgf- . the scleroderma mouse model ( tight - skin mutant mouse tsk/+ ) presented treatment with an anti - il-4 antibody resulted in collagen downregulation and provided less fibrosis - based pathological changes . the th2-mediated secretion of il-4 and il-13 enhanced fibrocyte differentiation from cd14-positive precursors , thereby leading to increased fibroblast recruitment and potential fibrosis . the impact of th2-derived il-5 on fibrosis is strongly associated with the recruitment and activation of eosinophils . activated eosinophils secrete inflammatory factors , such as il-13 and tgf-1 , into wounded tissue , resulting in fibrosis development , as shown for dermal fibrosis in a mouse model of skin allograft rejection . both il-13 and tgf-1 might induce collagen secretion from fibroblasts present in the wound ; however , tgf-1 is strongly associated with endmt- and emt - based fibrosis [ 95 , 96 ] . as discussed above , th1 and th2 lymphocytes affect differently fibrotic tissue . recently , it was shown that patients with cystic fibrosis and p. aeruginosa infection present an age - dependent dysregulation of lymphocyte t response that shifts towards th2 lymphocyte , resulting in enhanced fibrotic tissue deposition . as previously described , these cells are both cd45- and cd34-positive cells that transdifferentiate into myofibroblasts . the name fibrocyte represents a combination of shared features of these cells : fibroblast and monocyte . circulating fibrocytes subsequently , tgf-1 triggers the transdifferentiation of these cells into sma - positive myofibroblasts that express collagen type i , fibronectin , and vimentin and increase the amount and deposition of ecm components [ 98 , 99 ] . normally , circulating fibrocytes comprise less than 1% of all leukocyte populations , but during fibrotic changes derived from inflammation , the amount of these cells systematically increases . fibrocytes can be distinguished in at least 4-day - old skin wounds , and the quantity of these cells raises with time and increasing wound age . for the differentiation of fibrocyte precursors , the induction of fibrosis through fibrocytes is primarily based on the deposition of ecm components , as discussed above . nevertheless , we can not omit a variety of proinflammatory cytokines secreted from fibrocytes into the wound , namely , tnf- , il-6 , il-8 , il-10 , and macrophage inflammatory protein 1/ ( mip 1/ ) . moreover , it has been demonstrated that the fibrocytes in burned patients secrete tgf-1 , which activates myofibroblasts from existing fibroblasts or triggers differentiation toward fibroblasts - like cells from surrounding endothelial or epithelial tissues through endmt or emt . however , due to dynamic nature of fibrocytes and constantly changing phenotype and functions of these cells , during their migration , some serious inconsistency appeared on the exact definition and identification of fibrocytes . these discrepancies are related to different methodology used to investigate fibrocytes involvement in variety of fibrotic disorders on variable stages . it has been suggested that one must categorize fibrocytes as functionally different depending on the isolation condition . what is more , it is still unclear whether fibrocytes contribute only to worsening or improving tissue repair , as they possibly represent the wrong cells in the wrong time . fibrosis is a complicated and composed process , leading to severe pathological disorders . the scarce formation of fibrotic tissue , comprising excess amounts of collagen type i , fibronectin , and other ecm components , deregulates normal tissue functions . inflammation and inflammation - associated bone marrow leukocyte recruitment in wounded tissue trigger a cascade of events , leading to wound enclosure , scar formation , and , in case of prolonged inflammation , massive fibrosis . the impact of leukocytes on fibrosis formation might be generally divided into direct and indirect effects as shown in figure 3 . the direct impact is strongly associated with the production and excess deposition of ecm components . this effect is primarily observed with fibrocytes and alternatively activated , wound - healing macrophages . the indirect impact is far more complicated , as this effect is multistaged and associated with the activation and recruitment ( including cells transdifferentiation and emt / endmt fibroblasts formation ) of collagen - secreting cells , such as macrophages and myofibroblasts , and increased myofibroblast survivability through the downregulation of proapoptotic signals and increased inflammatory response times . the most common , and likely , best - known indirect impact is correlated with tgf- family proteins , as these molecules trigger both the endothelial and epithelial to mesenchymal transition and activate myofibroblasts from fibroblasts and fibrocytes , thereby increasing the production of ecm components . however , although this mechanism is well known , no antifibrotic therapy , based on tgf- deactivation , has been implicated without disruption of the physiological function of this molecule . several drugs for the downregulation of tgf- transcription or signal transduction have been examined in the last stages of clinical trials . nevertheless , the impact of il-6 , il-8 , il-4 , il-13 , or tnf- can not be neglected . th2-derived il-4 and il-13 are primarily responsible for macrophage collagen deposition and fibrocyte generation , whereas tnf- , il-6 , and il-8 are strongly associated with emt and endmt and myofibroblast survival and stimulation of collagen production . thus , leukocyte interactions with wounded tissue cells and other leukocytes are extremely complicated and complex , bringing more questions than answers .

cytotoxic t - lymphocytes ( ctl ) are famous for their ability to kill tumor , allogeneic and virus - infected cells . however , an emerging literature has now demonstrated that ctl also possess the ability to directly recognize and kill bacteria , parasites , and fungi . here , we review past and recent findings demonstrating the direct microbicidal activity of both cd4 + and cd8 + ctl against various microbial pathogens . further , this review will outline what is known regarding the mechanisms of direct killing and their underlying signalling pathways . skin tissue scar formation and fibrosis are often characterized by the increased production and deposition of extracellular matrix components , accompanied by the accumulation of a vast number of myofibroblasts . scaring is strongly associated with inflammation and wound healing to regain tissue integrity in response to skin tissue injury . however , increased and uncontrolled inflammation , repetitive injury , and individual predisposition might lead to fibrosis , a severe disorder resulting in the formation of dense and stiff tissue that loses the physical properties and physiological functions of normal tissue . fibrosis is an extremely complicated and multistage process in which bone marrow - derived leukocytes act as both pro- and antifibrotic agents , and therefore , few , if any , effective therapies are available for the most severe and lethal forms of fibrosis . herein , we discuss the current knowledge on the multidimensional impact of leukocytes on the induction of fibrosis , focusing on skin fibrosis .

acinetobacter baumannii is an opportunistic pathogen that is frequently involved in outbreaks of infection occurring mostly in intensive care units.1 members of genus acinetobacter is gram negative , nonmotile nonspore forming encapsulated coccobacilli belonging to family neisseriaceae.2 it is an opportunistic pathogen found to be associated with wide spectrum of infection including nosocomial pneumonia , meningitis , endocarditis , skin and soft tissue infections , urinary tract infection , conjunctivitis , burn wound infection and bacteremia posing risk for high mortality.34 acinetobacter pneumonia generally occurs in patients with diminished host defenses ( e.g. alcoholism , tobacco use , diabetes mellitus , and renal failure , underlying pulmonary disease).57 outbreak of acinetobacter infections is linked to contaminated respiratory equipments , intravascular access devices , bedding materials and transmission via hands of hospital personal.8 it typically colonizes skin and indwelling plastic devices of the hospitalized patients.9 mdr strains of acinetobacter isolates are a growing problem and have been widely reported.10 most a. baumannnii are now resistant to ampicillin , carbenicillin , cefotaxime , and chloramphenicol , with some centers reporting up to 91% of nosocomial acinetobacter resistant to resistance to tobramycin and amikacin is increasing . fluoroquinolones , colistin , imipenem , and meropenem may retain activity against nosocomial acinetobacter.11 ertapenem , the newest of the carbapenems , has little intrinsic activity against acinetobacter and should not be used.12 however , the rapid development of significant quinolone resistance in france , aminoglycoside resistance in germany , and carbapenem resistance in selected regions worldwide raises an important therapeutic problem.1314 a total 4180 specimens like blood sample , pus , csf , and other body fluids were subjected to simplified phenotypic identification scheme [ table 1 ] . presumptive identification of acinetobacter was made by inoculation on macconkey agar medium incubated at 37c . all nonlactose fermenting members subjected to gram staining , oxidase , catalase , and hanging drop preparation . acinetobactor are gram - negative bacilli or coccobacilli , oxidase negative , nonmotile , catalase positive . speciation was done on the basis of glucose oxidation , gelatine liquefaction , haemolysis , growth at 35 and 42c . bauer disk diffusion method with imipenem , meropenem , piperacillin - tazobactum , netilmicin , amikacin , ceftazidime , gentamicin , ofloxacin , chloramphenicol disks . the screening test for detection of inducible beta lactamases ( ibl ) , extended spectrum beta lactamases ( esbl ) , and mbl was done by the disk approximation method and double disk synergy respectively . during the period of study from january 2010 to december 2011 a total of 4180 samples were examined from of different age group admitted in various medical wards , surgical wards and icu . nonfermenter isolates account for 12% and acinetobacter isolates account for 4.5% of total organism isolated during the study period . most of these patients had respiratory problems like copd , bronchial asthma , respiratory failure . infection in neonates was common in preterm babies . in 87.5% sample growth was monomicrobial . in 12.5% sample growth was polymicrobial . escherichia coli was the most common associated organism with acinetobacter in the case of uti . staphylococcus aureus was the associated organism in the case of wound infection , cellulitis , and abcess . acinetobacter was isolated most commonly from surgical ward , medical ward , burn unit , and two isolates were isolated from humidifier ventilator and two isolates from the ot table . the present study shows more strains belonging to acinetobacter baumanii complex 114 ( 74.02% ) of total acinetobacter isolates [ table 2 ] . other species includes a.lowfii 22 isolates ( 14.2% ) , a. haemolyticus 12 ( 7.79% ) isolates , a junni 06 ( 3.8% ) isolates . different spices of acinetobacter isolated from clinical samples one of the striking features of genus acinetobacter is the ability to develop antibiotic resistant extremely rapid in response to challenge with new antibiotics . in the present study , strains were resistant to imipenem ( 5.2% ) , meropenem ( 9.75% ) , piperacilin - tazobactum ( 18.2% ) , netilmicin ( 16.24% ) , amikacin ( 14.29% ) , ceftazidime ( 74.1% ) , gentamicin ( 70.13% ) , ofloxacin ( ( 42.21% ) , chloramphenicol ( 92.20% ) [ table 3 ] . the difference in susceptibility pattern was due to environmental factors and different pattern of antimicrobial usage . acinetobacter species has emerged as an important pathogen causing life - threatening infections both in community and hospital . rapid emergence of multidrug - resistant acinetobacter has further made the situation critical.15 acinetobacter is found ubiquitously in nature , soil and also in skin as commensal . prior use of broad spectrum antibiotics , cross infection by hand of hospital staff , ventilator machine are all potential risk factors for development of multidrug - resistant acinetobacter infection in hospital.16 in one study at jipmer hospital respiratory infections due to acinetobacter in mechanically ventilated patients in icu were 44.7% . was responsible for 35% of ventilator associated pneumonia ( vap ) , making it the most conspicuous and dominant pathogen among all other bacteria encountered in that study.17 in our study , a total of 4180 samples were studied , out of which 504 ( 12.05% , n=4180 ) nonfermenters were isolated [ table 4 ] . acinetobacter species accounted for ( 30.55% , n=504 ) of total nonfermenter and 4.5% of total positive culture . most of the isolated acinetobacter species were sensitive to imipenem , meropenem . however , 10% of them were ibl producers and 8% were esbl producers . in conclusion , multidrug - resistant a. baumannii was responsible for majority of the acinetobacter infections at our hospital . injudicious use of antibiotics , mechanical ventilation , cross infection was found to be potential risk factors for development of acinetobacter infection . during routine microbiological work nonfermenter gnb other than pseudomonas aeruginosa but the rate of isolation of acinetobacter in various studies indicates its role as nosocomial pathogen and also as an agent of community acquired infection . traditional typing methods like phenotyping and antibiogram typing have advantage over genotyping as they are readily available and cost effective . bone grafting is common in neurosurgery for spinal fusion and in orthopedic reconstruction surgery for revision hip replacement , repairing skeletal defects after a trauma , and for support when removing a tumor . three types of bone graft are used : autograft ( autologous graft ) , allograft , and alloplastic graft . an alloplastic graft is a synthetic substitute , usually created from ceramics ( calcium phosphate , calcium sulfate , or bioactive glass ) , polymer ( derivative from acrylate ) , or growth factors ( the family of bone morphogenetic proteins ) . intraoperative complications might occur and affect subsequent tissue healing , motor function recovery , and the surgical outcome . approximately 500,000 surgical site infections caused by bacteria and viruses occur every year in the united states . postoperative transmission of an infection can be devastating to the graft and the patient : it may lead to serious illness and even death . patients with a preexisting illness are at a higher risk for an infection than are those who are otherwise healthy . delayed union and nonunion fracture are two of the most common complications after orthopedic surgery , and both require additional reconstruction using bone grafts . moreover , after bone surgery , patients with an immunocompromised disease , like diabetes mellitus ( dm ) , have a greater risk of infection . two other increasing chronic infectious diseases in asia , tuberculosis ( tb ) and acquired immunodeficiency syndrome ( aids ) , may compromise the immunity of patients and relate to the incidence of bone infection . treating deep infection alternatively , a skin graft or flap might be required to improve the outcome after infection . for most patients , removing the graft might be the only way to fix the problem , because the fresh tissue may be the source or a nidus for infection recurrence . nevertheless , there is no prior study that examines the risk factors of infection after bone - grafting surgery . we examined the risk factors of infection in patients that underwent bone grafting in taiwan . data were collected from the taiwan national health insurance research database ( nhird ) ( http://nhird.nhri.org.tw/en/background.html ) and analyzed for all patients who were admitted to hospitals from 1997 through 2012 for primary bone - grafting treatment . the incidence of recipient graft site infection in patients who underwent bone grafting , and the risk factors associated with postoperative infection , were evaluated . this is the first large national database study of the factors associated with bone - grafting surgery . approximately 98% of taiwan 's population ( ca . 23 million ) participate in the national health insurance program ( http://www.nhi.gov.tw/english/index.aspx?menu=13&menu_id=486&wd_id=486 ) . the taiwan nhird , which uses the international classification of disease , ninth revision , clinical modification ( icd-9-cm ) diagnosis and procedure codes , covers all inpatient medical claims data for enrolled residents . the established categories of expenditure were provided in inpatient expenditure by admission ( dd files ) ( http://nhird.nhri.org.tw/date_01.html ) . the personal information of all patients was encrypted with a double scrambling protocol for research purposes to protect patient privacy . this study was approved by the institutional review board of jianan psychiatric center , and the protocol was evaluated by the national health research institutes ( nhri ) , which consented to this planned analysis of the nhird ( agreement number : nhird-103161 ) . the participant demographic characteristics , including age , gender , hospital stay , and low - income households , were identified at the index date . the risk factors included in this study were those that have been found to be associated with or possibly related to the development of bone infection . a diagnosis of bone infection was defined on the basis of deep infection after surgery ( excluding superficial infection ) as prescribed by the national health insurance system ( icd 9 codes 996.4 , 996.66 , 996.67 , 996.69 , and 730.10730.19 ; table 1 ) . data regarding deep infection - related surgeries ( icd 9 codes 77.177.9 , 79.3 , and 86.22 ) were collected . they were identified from data of the nhird based mainly on icd-9-cm diagnostic codes ( table 1 ) . the diagnosis in this study were judged and determined by related specialists and physicians according to the standard criteria . the minimum living expense standard was defined as 60% of the average monthly disposable income for each region . low - income households were defined as those with a monthly average per - member gross income of less than the monthly minimum living expense standard in the household 's residence region . the icd-9-cm procedure code used for alloplasts and allografts were the same ( 79.3 ) ; thus , grafts were grouped into three categories : alloplast / allograft , autograft , and combined ( autograft plus alloplast / allograft ) . the criterion for a nonunion fracture was an unhealed broken bone 9-month postfracture that had shown no healing progression between the affected ends of the bone for 3 months after any specific treatment . a fracture that failed to heal within the normal time for that fracture type and location was defined as a delayed union fracture . according to the chapter ii , laws of medical care act , ministry of health and welfare in taiwan ( http://law.moj.gov.tw/eng/lawclass/lawcontent.aspx?pcode=l0020021 ) , medical care institutions with wards for the inpatient care of patients shall be referred to as hospitals ; those with only out - patient facilities shall be referred to as clinics . clinics referred may set up no more than 9 beds for observation . in taiwan , local clinics are set up with different specificities . therefore , the data regarding to patients with bone infection can be collected from those local clinics specialized in orthopedics , general trauma , rehabilitation , infectious disease , etc . descriptive statistics were analyzed for all variables using frequency , percentage , mean , and standard deviation ( sd ) . the effects of outcome variables were measured using the adjusted odds ratio ( aor ) with a 95% confidence interval ( ci ) . approximately 98% of taiwan 's population ( ca . 23 million ) participate in the national health insurance program ( http://www.nhi.gov.tw/english/index.aspx?menu=13&menu_id=486&wd_id=486 ) . the taiwan nhird , which uses the international classification of disease , ninth revision , clinical modification ( icd-9-cm ) diagnosis and procedure codes , covers all inpatient medical claims data for enrolled residents . the established categories of expenditure were provided in inpatient expenditure by admission ( dd files ) ( http://nhird.nhri.org.tw/date_01.html ) . the personal information of all patients was encrypted with a double scrambling protocol for research purposes to protect patient privacy . this study was approved by the institutional review board of jianan psychiatric center , and the protocol was evaluated by the national health research institutes ( nhri ) , which consented to this planned analysis of the nhird ( agreement number : nhird-103161 ) . the participant demographic characteristics , including age , gender , hospital stay , and low - income households , were identified at the index date . the risk factors included in this study were those that have been found to be associated with or possibly related to the development of bone infection . a diagnosis of bone infection was defined on the basis of deep infection after surgery ( excluding superficial infection ) as prescribed by the national health insurance system ( icd 9 codes 996.4 , 996.66 , 996.67 , 996.69 , and 730.10730.19 ; table 1 ) . data regarding deep infection - related surgeries ( icd 9 codes 77.177.9 , 79.3 , and 86.22 ) were collected . they were identified from data of the nhird based mainly on icd-9-cm diagnostic codes ( table 1 ) . the diagnosis in this study were judged and determined by related specialists and physicians according to the standard criteria . the minimum living expense standard was defined as 60% of the average monthly disposable income for each region . low - income households were defined as those with a monthly average per - member gross income of less than the monthly minimum living expense standard in the household 's residence region . the icd-9-cm procedure code used for alloplasts and allografts were the same ( 79.3 ) ; thus , grafts were grouped into three categories : alloplast / allograft , autograft , and combined ( autograft plus alloplast / allograft ) . the criterion for a nonunion fracture was an unhealed broken bone 9-month postfracture that had shown no healing progression between the affected ends of the bone for 3 months after any specific treatment . a fracture that failed to heal within the normal time for that fracture type and location was defined as a delayed union fracture . according to the chapter ii , laws of medical care act , ministry of health and welfare in taiwan ( http://law.moj.gov.tw/eng/lawclass/lawcontent.aspx?pcode=l0020021 ) , medical care institutions with wards for the inpatient care of patients shall be referred to as hospitals ; those with only out - patient facilities shall be referred to as clinics . clinics referred may set up no more than 9 beds for observation . in taiwan , therefore , the data regarding to patients with bone infection can be collected from those local clinics specialized in orthopedics , general trauma , rehabilitation , infectious disease , etc . descriptive statistics were analyzed for all variables using frequency , percentage , mean , and standard deviation ( sd ) . only variables that were significantly associated with bone infection ( p < 0.05 ) were entered simultaneously into multivariate logistic regression models . the effects of outcome variables were measured using the adjusted odds ratio ( aor ) with a 95% confidence interval ( ci ) . we enrolled 1,303,347 cases ( mean age : 46.57 22.16 years old ; mean length of hospital stay : 8.04 7.73 days ; male : 58.4% ; low income : 1.3% ) ( table 2 ) . comorbid diseases included dm ( 83,416 ) , tb ( 1,353 ) , and aids ( 19 ) . the infection rate of bone - grafting cases with dm , tb , and aids was 4.6 , 6.6 , and 31.6% , respectively . more patients went to district hospitals ( 485,171 ) and regional hospitals ( 453,723 ) for surgery ( table 2 ) . all variables but previous bone graft surgery were significantly correlated with surgical site infection after bone grafting ( table 3 ) . characteristics of patients who underwent a bone graft bivariate associations between variables and bone infection univariate and multivariate analyses both showed that the likelihood of postoperative infection was significantly positively dependent upon the patient - related factors of age , gender , income , and comorbid diseases ( table 4 ) . patients more easily developed surgical site infections if they were older , were male , had a lower income , and had comorbid diseases . patients with comorbid aids had the highest odds of infection ( aor = 6.945 , 95% ci = 2.40520.054 , p < 0.001 ) . univariate and multivariate analyses also showed that the likelihood of postoperative infection was significantly dependent upon the surgery - related factors of hospital stay length and fracture , graft , and hospital type . patients with a history of nonunion have relatively high odds of developing a surgical site infection ( aor = 7.005 , 95% ci = 6.7747.245 , p < 0.001 ) ; our data also indicated that patients who underwent an autograft were the least likely to develop a postoperative infection . the aor of patients who had their bone grafts done in medical centers was 0.928 compared with those who had them done in local clinics ( 95% ci = 0.9040.953 , p < 0.001 ) . the aor of patients who had their bone grafts done in regional hospitals was 0.764 compared with those who had them done in local clinics ( 95% ci = 0.7430.785 , p < 0.001 ) . the aor of patients who had their bone grafts done in district hospitals was 0.544 compared with those who had them done in local clinics ( 95% ci = 0.3360.881 , p = 0.013 ) . the relative risk factor of surgical site infection was the highest for bone grafts done in local clinics , followed by those done in medical centers , regional hospitals , and district hospitals . to our surprise , the odds of infection in the medical centers were the second highest . in this study , we evaluated various risk factors of surgical site infection in patients who undergo bone grafting . among the 1,303,347 cases that had undergone a bone - grafting operation , 39,816 cases developed a postoperative infection . the infection rate was 3.05% , lower than the 5.4% allograft infection rate in 128 patients reported by delloye et al . our infection rate was also lower than the 7.8% rate in 140 patients after cranioplasty . our infection rate might be lower because our patients were recruited from the general population and because we did not restrict enrollment based on the surgery site or the graft materials used . surprisingly , except for local clinics , the smallest units where the aor was highest , the second highest one was in the medical centers , the largest units , followed by the regional hospitals and district hospitals . the commonsense and intuitive order should have been local , district , and regional hospitals , and then medical centers , that is , the smallest rate of infection should have been at the largest unit . this paradoxical finding might be explained by a national study of nosocomial infection , which found that the infection rate was highest in medical centers ( 0.42% ) followed by regional hospitals ( 0.34% ) . it may also explain why staying 1 extra day in a hospitals increased by 1.054 times the odds of developing a surgical site infection . patients given an autograft were less likely to develop an infection than those who were given a combined graft or an alloplast / allograft . similar results were reported in a study on anterior cruciate ligament reconstruction : bone - tendon - bone autografts were associated with a lower risk of postoperative infection than were allografts and combined grafts . the principles involved in successful bone grafts include osteoconduction ( guiding the reparative growth of the natural bone ) , osteoinduction ( encouraging undifferentiated cells to become active osteoblasts ) , and osteogenesis ( living bone cells in the graft material contribute to bone remodeling ) . however , an autograft might lead to complications such as pain , infection , scarring , and blood loss , as well as a variable quantity of bone to harvest from each patient . the alternative is an allograft , but that lacks osteoactive capacity and risks carrying infectious agents or leading to an immune rejection . other approaches have used alloplasts , which are various types of organic and synthetic substitutes for bone , and they have focused on improving the efficacy of bone grafts or other scaffolds by incorporating bone progenitor cells and growth factors to stimulate cell proliferation . based on our study , in addition to using an autograft alone , the combined use of an alloplast with an autograft might help reduce infection . we collected data without exclusion of prior infection history . although the bone grafting was performed when no sign of infection or the infection was totally under control in surgical sites , an exclusion of these patients with prior infection would underestimate the infection rate . the debridement of wound infection would not be considered to use bone grafts . however , in rare cases , when repetitively deep infection occurred , a vascular bone graft might be applied from the donor site of patients for bone grafting . in that case , this type of bone graft belonged to autograft that was the least likely to develop a postoperative infection compared to the other two groups in our study . nevertheless , preexisting complications can increase the risk of infection ; for example , a nonunion increases it 7.005 times and a delayed union increases it 2.781 times after bone grafting . careful attention is required when doing a bone graft , particularly in patients with these histories . our study indicated that patients 1 year older were 1.009 times more likely to develop a postoperative infection than patients 1 year younger . moreover , patients with comorbid chronic or infectious diseases were more likely to develop an infection . oxidative stress in the tissue of patients with dm reduces the phagocytic and antibacterial activity of neutrophils and macrophages and provides an intracellular niche for the pathogen to replicate . it should not , therefore , be surprising that patients with comorbid aids are 6.945 times more likely to become infected after bone - grafting surgery . notably , patients with comorbid tb , another infectious disease with a rapidly rising incidence worldwide , are 1.780 times more likely to become infected than those without tb . cd4 t cells are protective against mycobacterium tuberculosis : cd4 t cell depletion is responsible for the increased susceptibility to develop active m tuberculosis in hiv - infected patients . although the role of cd8 t cells in tb is less clear than the role of cd4 t cells , they are generally considered to contribute to optimal immunity and protection . these results suggested that compromised immunity because of aging and comorbid disease are determining risk factors of bone - graft infection . how other noninfectious comorbid diseases were involved in bone - graft infection required for further investigation . we also found that low - income earners were 1.634 times more likely compared with higher income earners to develop an infection . consistent with this risk factor for other bone surgery , economic stress might physically and psychologically make patients less aware of the state of their individual health . the national health insurance bureau has established a uniform system to control the quality of medical services , and the quality of the coded data in the present study was reliable . compared with self - reported surveys , the methodology used in this study reduced the patients recall bias and thereby increased its accuracy . nevertheless , the nhird provides only insurance - related data ; information such as education level and marital status are not provided . second , the limited data acquired made it difficult to explain the different infection rate between males and females . identified that several risk factors were associated with infection after total knee arthroplasty surgery , including previous history of open reduction internal fixation , remnants of prior internal fixation material , and male gender . this is probably why the mortality rate was higher for male patients orthopedic and trauma admissions . compliance with the doctor 's prescribed medication regimen may be different between men and women . in conclusion , we surveyed the epidemiology of surgical site infection in patients who had undergone bone grafting in taiwan between 1997 and 2012 . they consists of the patient - related factors like age , income , gender , and chronic diseases , and the surgery - related factors like the length of hospital stay , disease complications , type of graft used , and type of hospital in which the surgery was done . as the frequency of using different types of bone grafts for orthopedic reconstructive surgery increase , our findings should provide an understanding of the risk factors associated with infection and should be a valuable reference for orthopedic surgeons .

background : antibiotic - resistant acinetobacter nosocomial infection is a leading problem . it acts as an opportunistic pathogen to cause a wide spectrum of infection including nosocomial pneumonia , meningitis , endocarditis , skin and soft tissue infections , urinary tract infection , conjunctivitis , burn wound infection and bacteremia . multidrug - resistant acinetobacter infection creates a great problem in hospital setting.materials and methods : the clinical specimens obtained from icu and different surgical and medical wards were investigated using standard microbiological techniques to know the distribution of and their resistant profile . antimicrobial resistance was studied using the modified kirby bauer disk diffusion technique following the clsi protocol.results:major infections found in different medical wards , surgical wards and icu were due to acinetobacter baumannii ( 74.02% ) , a. lowfii ( 14.2% ) , a. haemolyticus ( 7.79% ) , a. junii ( 3.8% ) among acinetobacter spices . acinetobacter showed increased resistant against majority of commercially available drugs imipenem ( 5.2% ) , meropenem ( 9.75% ) , piperacillin - tazobactum ( 18.2% ) , netilmicin ( 16.24% ) , amikacin ( 14.29% ) , ceftazidime ( 74.1% ) , gentamicin ( 70.13% ) , ofloxacin ( 42.21%).conclusion : a . baumannii was found to be associated with uti , rti , septicemia , bacteremia , and meningitis and wound infection . a. baumannii displayed higher resistance to more number of antibiotics than other nosocomial pathogens from icu . abstractbone grafting is a commonly used orthopedic surgical procedure that will provide bone formation in bone defects or regions of defective bone healing . a major complication following bone grafting is a postoperative recipient graft site infection that is associated with substantial mortality and increased use of medical resources . the purpose of the study was to identify the risk factors associated with infection after bone - grafting surgery.data from 1,303,347 patients listed in the taiwan national health insurance research database ( nhird ) and admitted to hospitals from 1997 through 2012 who underwent primary bone grafting ( mean age : 46.57 years old ; mean length of hospital stay : 8.04 days ) were analyzed . the incidence of infection by age , hospital stay , gender , income , chronic disease ( tuberculosis [ tb ] ; diabetes mellitus [ dm ] ; acquired immunodeficiency syndrome [ aids ] ) , fracture complications ( nonunion ; delayed union fracture ) , types of graft and hospital was evaluated.three percent of the patients developed a postoperative recipient graft site infection . multivariable analysis revealed that patients were more likely to develop a post bone - grafting surgery infection if they were older , had a longer hospital stay , were male , had a lower income , or had comorbid tb , dm , or aids . patients were more likely to develop an infection if they had a nonunion , an alloplast graft , or treated in a local clinic.our findings should provide a clinically relevant reference for surgeons who perform bone grafting . patients should be informed of the potential risks .

globally , mother - to - child transmission of hiv-1 results in approximately 370 000 infant infections each year . hiv-1 early infant diagnosis ( eid ) programs aims to detect hiv infection as early as possible in hiv - exposed infant and to link them to care and treatment . eid of hiv confers substantial benefits to infants and families , both for hiv - infected and uninfected infants , as well as to programs providing prevention of parent - to - child transmission ( pptct ) services , including hiv counselling and testing , family centric approach , provision of care , support and lifelong treatment for hiv positive women , provision of arv and cotrimoxazole prophylaxis and early infant diagnosis services for hiv - exposed children , guidance on feeding and nutrition practices , and immunization services . maharashtra is one of the high prevalent states in the country with hiv prevalence of 0.4% among antenatal women and 0.42% in adults and ante natal case prevalence 0.40% . early infant diagnosis was launched in the country in april 2010 , which entails follow - up of babies born to hiv positive women for hiv dna polymerase chain reaction ( pcr ) test and subsequently final hiv test at the age of 18 months of age . a major priority for pptct services is ensuring pregnant women pursue their care and treatment , including testing of babies at regular intervals for eid . the common barriers reported in qualitative studies regarding low uptake of pptct - eid services for mother and baby both include confidentiality , stigma , availability of testing facility , financial issues , behavior of health staff , fear of baby 's hiv test result , cultural barriers , long waiting time . in addition , health workers are unable to monitor pregnant women because of inadequate data management system across the service continuum . who estimates that only 615% of hiv - exposed infants under 1 year of age accessed eid programs in 20082009 , highlighting that the implementation of effective eid programs has been challenging in resource - limited settings and demands careful attention . although there are many reasons for the lack of continuity of care , there is a need to improve the adherence and retention in care and this may be improved by maintaining better communication between program officers and field staff . mobile phone technology has the potential to serve as a strategic intervention medium to improve patient management . owing to the widespread use and low cost of this technology , it pervades all age groups and many cultures and socioeconomic backgrounds . but there is also a need for interactive program management by reacting promptly and effectively to deviations from program plans . this may include operational decisions for short - term management of patient retention at individual care centers , tactical decisions such as reconfiguring the messaging based on performance , and strategic decisions such as data collection or preservation . hence , an integrated mobile phone text messaging system was designed , developed , and implemented to follow the babies under eid in maharashtra ( excluding mumbai ) in june 2012 . this study was conducted to assess the utility of web - based mobile technology monitoring tool , for ensuring linkages , and tracking of hiv - exposed child until 18 months of age . maharashtra state aids control society developed a web - based system that provides support to eid program . a broad set of usability requirements was identified during brainstorming design sessions between the researchers , program officers , and developer . the program officers in maharashtra state aids control society , with strong program backgrounds and substantial experience in healthcare and understanding of the field problems , outlined the requirements that they anticipated the system should offer . these requirements were then documented and elaborated in more detail as new ideas were exchanged within the design team ( two program officers and one computer experts ) and final design principles of the system were derived . the development team and the program officers assessed the responses from field staff in the current system , which were stored in a microsoft structured query language server , to identify patterns that could be used to define relevant answers . the output was a list of possible automatic sms text message replies through symbian supported mobile . eid follow - up system was designed as a web - based tool using java swing framework which generates automated sms and e - mails for reminding the field level staff for follow - up of hiv - exposed babies 7 days prior to the follow - up date . the information was collected through field tested excel - based hiv positive pregnant women line - list format incorporating the major key indicators of pptct program . this line list is generated at the hiv testing facilities where the pregnant woman is detected hiv positive . format is then shared through e - mail with hiv treatment facilities to update the treatment details of the pregnant women . the line list is further updated on periodic basis whenever the pregnant women is accessing the pptct services at hiv facilities . the individual records in the line list are monitored and crossverified by district and state level officials during the supervisory visits to these centers on periodic basis . the data collected through line list from different districts is then compiled by the state . for the software , we used the variables like name of mother and child , date of birth of baby , mobile number of counsellor and district supervisor , email of district official , and name of the hiv testing facility . database of hiv positive mother and her exposed child with date of birth was updated on monthly basis . deliberation with various stakeholders on development of tracking system led to use of mobile technology in follow - up mechanism . the software developed sends required information ( name of mother / child , facility name , and follow - up date ) to the counsellor of the respective facility and district supervisor as mobile text message alerts on daily basis and simultaneously , system generated e - mail is send to district officials for monitoring and following the baby for eid . we used brainstorming techniques to solicit usability requirements , focus group meetings to discuss and define system architecture , and prototyping to test in real environments and to improve the system ( fig . eid follow up system : development process and work flow . in this kind of system , there are telecommunication costs involved . it is imperative to be able to make favorable bulk mobile text messaging contracts on the part of the healthcare provider , but also to be efficient in messaging so as to reduce the traffic . on a pilot basis , we have purchased sim card at state for sending mobile text message from the same number to counsellors and district supervisors . the connection between the server and the client mobile is made using a web service . a set of text messages was developed based on discussion involving the design team and the program officers . suggestions for message formulations were reviewed and amended during team meetings , resulting in a final set of three messages , which were approved by all involved . in both sets , messages for testing of baby for eid were divided into four categories : 6-week follow - up reminders , 6-month follow - up reminders , 12-month follow - up , and 18-month follow - up reminders . the messages did not include general information about the health status of the patient because such status information may be difficult to communicate in an unambiguous way . as the mobile networks in maharashtra are often unreliable , especially in rural areas , we designed a mechanism of e - mail to district aids prevention and control unit ( dapcu ) and dapcu will follow - up at field level . this way , we were able to , in most cases , get daily updates , although not necessarily at a predefined time every day . to accommodate the requirements , architecture the main elements of this architecture include the following : the sms server , as shown in fig . 3 , interacts with feild staff that uses their mobile phone set and the sms messaging service to send messages to the system . at the lowest level , the sms server interfaces with a global system for mobile ( gsm ) modem that sends field staff sms text messages through an sms service provider ( mobile operator ) . once the data are imported in software from excel sheet , the sms server performs checks on its content and responds to the field staff accordingly ( automatic reply feature ) . safeguarding the privacy , confidentiality , and security of any public health information is an important undertaking . the system only collects data necessary for the server to process and send text messages . the system comprises an application layer for the capture and delivery of patient information , including date of next appointment and next date of testing , to a remote server by means of a gsm modem . six sets of system requirements that need to be addressed for success are data collection , telecommunication costs , privacy and data security , text message content , connectivity , and system scalability . a text messaging system was designed and implemented in 578 hiv testing facilities and district supervisors in 33 districts in maharashtra . these sites feed data into a central data repository , which was used for analysis of operations and decision support . based on the follow - up schedule , the system automatically generates appointment reminders as mobile text message to counsellors and as emails to dapcu officers . healthcare professionals , technical experts , and an interdisciplinary group of researchers were involved in the design , development , and implementation processes to enhance the utility and functionality of the system . it is imperative to be able to make favorable bulk mobile text messaging contracts on the part of the healthcare provider , but also to be efficient in messaging so as to reduce the traffic . on a pilot basis , we have purchased sim card at state for sending mobile text message from the same number to counsellors and district supervisors . the connection between the server and the client mobile is made using a web service . a set of text messages was developed based on discussion involving the design team and the program officers . suggestions for message formulations were reviewed and amended during team meetings , resulting in a final set of three messages , which were approved by all involved . in both sets , messages for testing of baby for eid were divided into four categories : 6-week follow - up reminders , 6-month follow - up reminders , 12-month follow - up , and 18-month follow - up reminders . the messages did not include general information about the health status of the patient because such status information may be difficult to communicate in an unambiguous way . as the mobile networks in maharashtra are often unreliable , especially in rural areas , we designed a mechanism of e - mail to district aids prevention and control unit ( dapcu ) and dapcu will follow - up at field level . this way , we were able to , in most cases , get daily updates , although not necessarily at a predefined time every day . to accommodate the requirements , architecture the main elements of this architecture include the following : the sms server , as shown in fig . 3 , interacts with feild staff that uses their mobile phone set and the sms messaging service to send messages to the system . at the lowest level , the sms server interfaces with a global system for mobile ( gsm ) modem that sends field staff sms text messages through an sms service provider ( mobile operator ) . once the data are imported in software from excel sheet , the sms server performs checks on its content and responds to the field staff accordingly ( automatic reply feature ) . safeguarding the privacy , confidentiality , and security of any public health information is an important undertaking . the system only collects data necessary for the server to process and send text messages . the system comprises an application layer for the capture and delivery of patient information , including date of next appointment and next date of testing , to a remote server by means of a gsm modem . six sets of system requirements that need to be addressed for success are data collection , telecommunication costs , privacy and data security , text message content , connectivity , and system scalability . a text messaging system was designed and implemented in 578 hiv testing facilities and district supervisors in 33 districts in maharashtra . these sites feed data into a central data repository , which was used for analysis of operations and decision support . based on the follow - up schedule , the system automatically generates appointment reminders as mobile text message to counsellors and as emails to dapcu officers . healthcare professionals , technical experts , and an interdisciplinary group of researchers were involved in the design , development , and implementation processes to enhance the utility and functionality of the system . before the introduction of eid follow - up system , only 55.9% ( 637/1139 ) of the hiv - exposed babies born were tested at 6 weeks for dna - pcr during april 2011march 2012 . however , after its introduction , 68.4% ( 1117/1631 ) of them were tested during april 2012march 2013 . correspondingly , the 18 months confirmatory hiv testing in eligible babies increased from 45.6% ( 934/2044 ) to 54.7% ( 1118/2044 ) . this study summarizes the processes of design , development , and implementation of a remote management system using mobile technologies to help field staff and supervisors to track mother and children for hiv testing . our study endeavors to design , develop , and implement a tool to empower health staff in tracking hiv - exposed children . mobile phones are available and are practical , low - cost tools that may facilitate and support health staff in follow - up of babies . our study showed that mobile phone text messaging provides an effective communication mechanism between healthcare providers and supervisors , provided it is implemented well . it has been shown that regular follow - up is possible through mobile technology intervention . design of a system for follow - up of specific diseases like hiv / aids , based on mobile phone text message and e - mail technology , includes careful attention to not only the theoretical underpinning of the strategic intervention and associated content , but also the requirements for optimal design , including how each part of the system has to be placed together . crucial parts of a successful text messaging intervention include how messages are tailored , how participants may be directed to different content based on their situation , and the intensity of the communication . the system must also include cost - effective mechanisms so as to minimize costs of both technologies involved ( including costs for gsm network communication and gsm modems with its data plan ) and of sending messages to patients . trust , usefulness , and usability are crucial issues for both health staff and professional users , and it is important that features installed also work properly and meet privacy standards . therefore , it is better to move slowly in making the system more advanced and , at the same time , making sure to have users on board . the basic input information required for system to generate the follow - up is being manually compiled through excel - based format on monthly basis leading to delay in updating the base data in the system . this study demonstrated that it is possible to design , develop , and implement an integrated remote patient management system using the mobile phone 's text message feature to communicate with field staff regarding alerts on follow - up of children . the study shows that it is necessary to address the following six different requirements : data collection , telecommunication costs , privacy and data security , the content of text messages , connectivity , and system scalability . state may plan for an evaluation of the system , including a satisfaction survey of the health professionals and field staff that used it . additionally , plan to perform a cost - benefit analysis and to implement the system permanently with advanced technology using android - based smart phone . we thank all the ictc , dapcu , art and sacs staff of maharashtra state aids control society for their sincerity and hard work in facilitating this study . regulation of gene expression is fundamental for the coordinate synthesis , assembly and localization of the macromolecular structures of cells . this is achieved by a multi - step program that is highly interconnected and regulated at diverse levels ( fig . it starts in the nucleus , where transcription factors bind to specific dna sequences proximal to the genes they regulate and recruit rna polymerases for rna synthesis . as soon as rna precursors are formed , they get covered by a host of proteins forming ribonucleoprotein complexes [ 3 , 4 ] . messenger rna - binding proteins ( mrbps ) associate with nascent mrna precursors and mediate diverse rna processing reactions including 5-end capping , splicing , editing , 3-end cleavage and polyadenylation [ 24 ] . the transcripts are subsequently exported through nuclear pores to the cytoplasm where they may undergo localization to subcellular regions by complexes consisting of motor proteins and rbps or by the signal recognition particle . the transcripts assemble with translation factors and ribosomes for protein synthesis , which is controlled by global or transcript - specific mechanisms . the fate and location of proteins can be further controlled through modification of specific amino acids , cleavage by site - specific proteases and degradation through the proteasome . see text for details . besides many classical biochemical and genetic studies that revealed factors involved in and regulating these diverse steps in the gene expression program , the recent development of genome - wide analysis tools like dna microarrays allowed fundamental new insights into the systems architecture of gene regulatory programs . for instance , dna microarrays have been extensively used to study transcriptional programs by comparing steady - state rna levels between diverse cell types and stages , and by the mapping of binding sites for dna - associated proteins through chromatin immunoprecipitation ( so - called chip - chip assays [ 9 , 10 ] ) . integration of these data allowed the description of complex transcriptional regulatory networks , involving large sets of genes that control coherent global responses in physiological and developmental programs [ 1113 ] . in contrast , less is known about the systems architecture that underlies the post - transcriptional steps in the gene expression program ( although many rna regulatory processes also occur co - transcriptionally , we further classify them as post - transcriptional for simplicity ) . considering the large number of mrna molecules in the cell ranging from 15 000 to 150 000 mrna molecules in yeast and mammals , respectively it is rational to assume that the location , activity , and fates of these rnas is not left to chance but is highly coordinated and regulated by an elaborate system . such a post - transcriptional regulatory system may be controlled by the hundreds of rbps and non - coding rnas ( e.g. micrornas ) that are encoded in eukaryotic genomes , possibly defining specific fates of each rna by the combinatorial binding of distinct groups of rbps [ 1416 ] . here , we summarize recent work that applied genomic tools to decipher the principles and logic of post - transcriptional regulatory systems . we focus on studies considering the localization , translation and decay of mrnas in eukaryotes . nodes of these programs , which involves the characterization of rbps and the systematic identification of their rna targets ( fig . ( a ) determining the translation status of each mrna for the mapping of translational programs . cell - extracts are fractionated through a sucrose - density gradient and the absorbance at 254 nm is monitored . free rna and ribosomal subunits , monosomes ( 80s ) and polysomes , and analyzed with dna microarrays . the relative position of a message in this profile is an indicator for its translational activity . ( b ) systematic identification of rnas associated with specific rna - binding proteins . in this ribonomics approach , rbps are immunoprecipitated or affinity - purified via a tag from cellular extracts . rnas associated with rbps are isolated , cdna copies are fluorescently labeled and hybridized to dna microarrays . the cy5/cy3 fluorescence ratio for each locus reflects its enrichment by affinity for the cognate protein . ( a ) determining the translation status of each mrna for the mapping of translational programs . cell - extracts are fractionated through a sucrose - density gradient and the absorbance at 254 nm is monitored . free rna and ribosomal subunits , monosomes ( 80s ) and polysomes , and analyzed with dna microarrays . the relative position of a message in this profile is an indicator for its translational activity . ribonomics approach , rbps are immunoprecipitated or affinity - purified via a tag from cellular extracts . rnas associated with rbps are isolated , cdna copies are fluorescently labeled and hybridized to dna microarrays . the cy5/cy3 fluorescence ratio for each locus reflects its enrichment by affinity for the cognate protein . rna localization generally refers to the transport or enrichment of subsets of mrnas to specific subcellular regions . rna localization can be achieved passively by local protection from degradation or through the trapping / anchoring at specific cellular locations . moreover , asymmetric distribution of rna can also be established by the active transport of rnas via rbp - motor protein complexes [ 5 , 17 ] . here , we discuss studies that systematically mapped rna distribution to subcellular structures or organelles , and then refer to investigations aimed at globally identifying localized mrnas mediated through active mrna transport by rbps . in a pioneering study by pat brown and colleagues , cytosolic ribosomes by equilibrium density centrifugation in a sucrose gradient , and the distribution of transcripts in the fractions were quantified by comparative dna microarray analysis . as expected , transcripts known to encode secreted or membrane - associated proteins were enriched in the membrane - bound fraction , whereas those known to encode cytoplasmic or nuclear proteins were preferentially enriched in the fractions containing mrnas associated with cytoplasmic ribosomes . however , transcripts for more than 300 genes in the yeast saccharomyces cerevisiae were found in the membrane - fraction coding for previously unrecognized membrane or secreted proteins . rather unexpected , among these was also the message for ash1 coding for a well - known transcriptional repressor , suggesting alternative signals for membrane association . similarly , application of this method to map mrna distributions in the plant arabidopsis thaliana allowed the classification of 300 previously unknown transcripts as secreted or membrane - associated proteins . a recent extension of this approach to eleven different human cell lines provided a detailed catalog containing more than 5000 previously uncharacterized membrane - associated and 6000 cytoplasmic / nuclear proteins at high confidence levels . strikingly , this analysis predicts that 44% of all human genes encode membrane - associated or secreted proteins exceeding previous estimates ranging from 15% to 30% . in addition , the comparison of this catalog to data obtained from hundreds of dna microarray profiles from tumors and normal tissues allowed the identification of candidate genes that are highly overexpressed in tumors and , hence , could be particularly good candidates for diagnostic tests or molecular therapies . claude jacq s lab applied a subcellular fractionation approach to determine transcripts associated with free and mitochondrion - associated ribosomes in the yeast s. cerevisiae . besides the mrna for atp14 , which was previously known to localize in the vicinity of mitochondria , nuclear transcripts for diverse mitochondrial proteins were enriched in the mitochondrial fraction . interestingly , two characteristics correlated with this mrna localization : the phylogenetic origin and the length of the genes . mrnas enriched in the mitochondrial fraction were preferentially longer ( as deduced from average length of the encoded proteins ) and originate from genes with bacterial homologues , whereas mrnas in free cytosolic polysomes were shorter and of eukaryotic origin [ 22 , 23 ] . possibly , such coordinate localization of groups of mrnas could allow oriented access for controlling their fates . for instance , a low - density array study revealed that 22 out of 649 analyzed transcripts were enriched in the cytoskeleton fraction relative to the cytosolic fraction most of these encoding ribosomal proteins or structural proteins that interact with the cytoskeleton [ 24 , 25 ] . in polarized cells like neurons , mrna localization has major physiological implications . in dendrites , rna transport and subsequent local protein synthesis is thought to influence experience - based synaptic plasticity and long - term memory formation ; in axons , local translation modifies axon guidance and synapse formation [ 26 , 27 ] . however , to date there are only a handful of well - characterized examples of localized neuronal mrnas , among them the messages coding for microtubule - associated protein 2 ( map2 ) , the -subunit of a calmodulin - dependent protein kinase ( camkii ) , brain - derived neurotrophic factor ( bdnf ) , and activity - regulated cytoskeletal - related ( arc ) [ 5 , 26 ] . therefore , several genomics - based approaches have been undertaken to identify novel localized transcripts . for example , matsumoto et al . fractionated brain tissue and isolated rna from the heavy portion of polysomes and synaptosomes to provide a list of potentially dendritic mrnas that undergo localized translation . interestingly , the induction of neural activity by an electroconvulsive shock triggered a redistribution of the population of dendritic transcriptome , which may trigger changes in the translatability of this transcriptome , suggesting complex mechanisms of local translation in response to synaptic inputs ( fig . the hundreds of potentially localized mrna in neurons now await confirmation by in situ hybridization and exploration as to whether and how these may be regulated through activating stimuli , such as neurotransmitter release . to date , more than 100 mrnas are known to undergo active mrna transport in diverse organisms [ 17 , 29 ] . in neurons , mrnas are transported over long distances in a microtubule - dependent manner in the form of large granules consisting of rna - binding proteins , ribosomes and translation factors [ 27 , 30 ] . several rbps associated with neuronal rna transport have been identified , such as zipcode - binding proteins ( zbp1,2 ; named after their ability to bind to a conserved 54-nucleotide element in the 3-utr of the -actin mrna known as the zipcode ) , staufen , hnrnpa2 , cytoplasmic polyadenylation protein ( cpeb ) and members of the familial mental retardation proteins ( fmrps ) . at least for one of these rbps , fmrp , a systematic gene array - based screen was undertaken to identify the mrnas that are transported and possibly regulated by this protein . using a ribonomics approach , which involved the immunoprecipitation or affinity isolation of rbps followed by the identification of bound rnas with dna microarrays ( fig . 2b ) , the authors immunopurified the protein from mouse brain tissues and found 4% of all mrnas ( 435 messages ) associated with fmrp . in addition , they compared the mrna profiles of polyribosomes between normal human cells and cells derived from fragile x syndrome patients identifying over 200 messages with altered association and hence , these are potentially subject to translational regulation ( fig . notably , nearly 70% of the homologous messages found in both studies had a g - quartet structure , which was demonstrated as an in vitro fmrp target . these data provided a good starting point for further investigations on the most critical targets involved in fragile x pathophysiology and , possibly , on other related cognitive diseases . probably the best - studied example for actin - dependent rna transport concerns ash1 mrna localization to the bud tip of yeast cells during cell division . mrna is bound by she2p , an rbp tethered to the myosin motor protein myo4p via the adaptor protein she3p . this rna - protein complex travels along actin cables to the emerging bud for local protein synthesis . to identify other localized mrnas , affinity purification of components of the she complex followed by the analysis of bound mrna with microarrays was combined with a robust reporter system for in vivo visualization as a secondary screen [ 33 , 34 ] . this analysis revealed 23 additional transcripts that are localized to the bud - tip and encode a wide variety of proteins , several involved in stress responses and cell wall maintenance . these results reveal an unanticipated widespread use of rna transport in budding yeast possibly providing the daughter cells with a favorable start - up package. in conclusion , the few studies that investigated spatial distribution of mrnas in the cell on a global level challenge the long - standing assumption of a rather unorganized pool of mrnas that randomly diffuse in the cytoplasm to be eventually translated . possibly , many mrnas may be spatially organized even in non - polarized cells for local translation or decay in processing ( p ) bodies . further applications of both subcellular fraction techniques and ribonomics approaches will certainly reveal a more comprehensive picture of the spatial arrangement of rnas in cells . translational regulation has essential roles in development , oncogenesis and synaptic plasticity [ 3537 ] . it concerns the differential recruitment of mrna species to the ribosome for protein synthesis , which results in a lack of correlation between the relative amounts of mrna and the amount of the encoded protein . in an innovative study , the relative contribution of transcriptional and translational regulation in yeast was measured using large - scale absolute protein expression measurement called apex ( absolute protein expression index ) , which relies upon observed peptide counts from mass spectrometry . most ( 73% ) of the variance in protein abundance can be explained by mrna abundance , which is lognormal distributed around an average of 5600 proteins per mrna molecule . this indicates that the abundance of most proteins is set per mrna molecule ; however , one third of the mrnas must be regulated at additional levels including translation and/or protein turnover . in mammalian cells , the fraction of differentially expressed messages may be considerably higher ranging from 60% to 80% , indicating that gene expression of most messages is heavily controlled at diverse levels . translation can be divided into three steps : initiation , elongation and termination . during translation initiation , the primary target for translational control , translation initiation factors ( eifs ) recruit the mrna to the small ribosomal subunit ( 40s subunit ) . thereby , eif4e binds to the cap structure at the 5-end of the mrna and interacts with eif4 g , which binds to the poly(a)-binding protein ( pabp ) . the initiation complex then scans the mrna in 5 to 3 direction until the initiation codon is reached where the large ribosomal subunit ( 60s ) joins the complex leading to the formation of active ribosomes . notably , ribosomes can also be recruited cap - independently to some viral and cellular mrnas by direct binding of the small ribosomal subunit to internal rna structures , termed ires . the assembled ribosomes traverse the coding region with help of elongation factors ( eefs ) and synthesize the encoded polypeptide with multiple ribosomes covering them rna to form polysomes . at the termination codon , peptide chain - releasing factors ( erfs ) two basic modes of translation regulation have been described . during global regulation , translation of most mrnas for instance , phosphorylation of eif2 reduces the amount of active initiation complexes and hence leads to a rapid reduction of translation of most messages . the availability of eif4e is controlled by 4e - binding proteins ( 4e - bp ) that displace eif4 g from eif4e , and thus inhibit association of the small ribosomal subunit with them rna[6 , 41 ] . the second mode of translational regulation concerns mrna - specific control , where translation of defined groups of mrnas is modulated without affecting general protein biosynthesis . this can be carried out by specific rna - binding proteins , which often bind to sequence or structural elements in untranslated regions ( utrs ) of protein - coding transcripts and , hence , repress translation via interactions with eifs [ 6 , 42 ] . a prime example for such regulation represents cytoplasmic aconitase , an enzyme that regulates iron - dependent translation initiation through binding to a stem - loop structure in the 5-utr of messages involved in iron metabolism ( e.g. , ferritin mrna coding for an iron regulatory protein ) . specific control can also be exerted by micrornas ( mirnas ) small rnas of 22 nucleotides in length that have recently been shown to repress translation via base pairing to sequences located in 3-utrs of target mrnas . interestingly , it has recently become apparent that mirna- and rbp - mediated translational regulation may collaborate or compete on specific mrna substrates , suggesting interconnections between these different modes of translational regulation . genome - wide analysis of translational regulation . a reliable measure for translation of cellular mrna is the degree of its association with ribosomes . since the rate of initiation usually limits translation , most translational responses will alter the ribosome density on a given mrna . actively translated mrnas are typically bound by several ribosomes ( polysomes ) and can be separated from the small ( 40s ) and the large ( 60s ) ribosomal subunits and the 80s monosomes by sucrose gradient centrifugation ( fig . in classical experiments , total rna was isolated from fractions of the polysomal gradient and assayed for the mrna of interest by northern blot analysis . several laboratories have further extended this technique using dna microarray technology to perform genome - wide analysis of mrnas in polysomes in yeast , drosophila and mammals . the laboratories of pat brown and daniel herschlag performed a high resolution translation state analysis in rapidly growing yeast cells , providing profiles for mrna - ribosome association for thousands of genes . based on these data , they calculated the ribosome occupancy ( fraction of a specific mrna associated with ribosomes ) , the ribosome density and the translation rate for each expressed mrna . the average occupancy was calculated at 71% , indicating that most mrnas are likely engaged in active translation . however , about 100 mrnas showed only weak association with ribosomes and may therefore be considered as potential candidates for translation on demand. the average ribosome density was found to be 156 nucleotides per ribosome , which is about one fifth of the maximal packing density , supporting the premise that translation initiation is the rate - limiting step for protein synthesis . surprisingly , the orf length appears to be a major factor determining the ribosome density , which is expressed through an inverse correlation between the orf length and the ribosome density in diverse species [ 4749 ] . since poly(a ) tail length affects translational efficiencies and mrna stability , two recent studies systematically addressed its length in yeast [ 48 , 50 ] . in a procedure called polyadenylation state array analysis ( pasta ) , mrnas were captured with poly(u ) sepharose columns and differentially eluted by increasing temperature . rnas fractions were analyzed with dna microarrays to identify groups of mrnas with similar poly(a ) tail lengths . in the yeast s. cerevisiae , mrna coding for functionally or cytotopically related mrnas long poly(a ) tails were found among mrna coding for cytoplasmic ribosomal proteins , whereas short tails were enriched for dna / ty elements and among mrnas coding for nucleolar proteins involved in ribosome synthesis , and proteins with cell cycle - related functions . the comparison of the data with other genome - wide analysis revealed that poly(a ) tail length positively correlates with ribosome density and to some extend with mrna abundance , and it negatively correlates with orf and utr length . the poly(a ) tail length , and hence ribosome occupancy of messages correlate with the degree of association with poly(a)-binding protein ( pab1p ) global support for the concept that long poly(a ) tails stimulate translation via pab1p and eif4 g . therefore , it appears that translation rates are not directly coupled to mrna decay control , although poly(a ) shortening is a prerequisite for mrna decay . possibly , processes acting on oligo(a)-tailed intermediates may limit the decay rates of large number of yeast mrnas . a congruent study performed in fission yeast s. pombe monitored translational status , poly(a ) tail length , mrna abundance , mrna decay rates and rna polymerase ii association under identical conditions . functional groupings of mrna in respect to their translational efficiencies , length and abundance were identified with shorter and abundant mrnas having longer poly(a ) tails . notably , orf length correlated best with ribosome density and mrna abundance with ribosome occupancy . in conclusion , both studies revealed similar principles that may organize translation and therefore , these may be evolutionarily conserved . further studies in other organisms will reveal whether these principles are universally conserved and possibly affected in disease . several studies were aimed at the systematic identification of translationally regulated messages after subjecting cells to stress and other environmental stimuli . low - resolution profile analysis , where the mrna contents of high sucrose gradient fractions ( polysomes ) were compared with fractions from the low sucrose gradient ( the pool of non - translated mrnas ) ( fig . , changes in the levels of total rna were measured to study the relation between transcription / decay and translationally regulated messages . in yeast , global effects on translation were first studied for the rapid transfer of cells from a fermentable to a non - fermentable carbon source mimicking glucose starvation , followed by heat - shock response and rapamycin treatment , amino acid starvation , butanol addition ( an end product of amino acid breakdown ) , and application of hydrogen peroxide to induce oxidative stress . first , it should be noted that these relatively harsh treatments induced global translation inhibition that goes along with a decrease in cell growth . although this global translation inhibition is triggered by similar signaling pathways like phosphorylation of eif2 , the various forms of stress affected quite different sets of mrnas . amino acid and glucose starvation differentially regulated the translation of up to 20% of all mrnas , whereas more mild treatments , such as butanol and peroxides , affected less than 4% of transcripts . treatment of cells with heat / rapamycin or nutrient removal ( amino acid and glucose starvation ) co - activated similar translational and transcriptional programs . here , regulation at the translational level often reflects a magnification of the transcriptional activity an effect that has been termed potentiation . in contrast , the addition of butanol or peroxide provoked no potentiation , but instead changed the abundance and translation rates of different sets of mrnas . this could , at least in part , be explained by the recruitment of stored mrna for translation on demand. nevertheless , in all cases , the specific sets of mrnas that undergo treatment - specific regulation appear to share functional themes that can be attributed to logic response of the cell s altered physiological circumstances . for instance , mrnas coding for proteins related to sugar metabolism and transport , such as hexose transporters , remain associated with polysomes during glucose starvation ; rapamycin treatment , which blocks the target of rapamycin ( tor ) pathway controlling cell growth , led to a decrease of nearly all yeast mrnas coding for cytoplasmic ribosomal proteins , whereas mrnas for proteins acting in the nitrogen discrimination pathway were increased ; amino acid starvation strongly coregulated or potentiated transcripts encoding permeases , proteases and proteins involved in degradation pathways , which may reflect an early amino acid scavenging response to starvation . another interesting aspect of these studies is that the concentration of an applied compound may significantly matter for the outcome . . showed that low concentrations of peroxide ( 0.2 mm h2o2 ) induced the translation of mrnas coding for antioxidants , cellular transporters and proteins involved in diverse intermediary metabolism and may reflect the need for metabolic reconfiguration . a tenfold higher concentration of peroxide ( 2 mm ) resulted in the up - regulation of genes involved in ribosome biogenesis and ribosomal rna processing , possibly reflecting the need to repair factors for efficient protein synthesis . on the other hand , many translationally repressed mrnas showed increased steady - state ( total rna ) levels . again it was postulated that this group of messages may represent an mrna store that could become rapidly activated following relief of the stress condition . mild treatments with pharmacological compounds activate dose - dependent non - linear effects via distinct regulatory programs . if so , this may become of great medical relevance as diverse drugs are known to act differentially depending on their dose . finally , there are recurring and intriguing observations that mrnas coding for cytoplasmic ribosomes generally appear to undergo outstanding and strong coregulation . amino acid and glucose starvation coordinately repress these transcript s abundances and ribosome association very rapidly , whereas the addition of butanol or oxidative stress even lead to the opposite effect translational activation that possibly reflects the requirement of cells to replace ribosomal proteins and rrna that became damaged by free radicals or other toxic products . therefore , besides tight transcriptional control of these messages , they also undergo decent post - transcriptional control at diverse levels and hence , may represent the most tightly controlled genes in eukaryotic cells . first studies to investigate global aspects of translational regulation in mammalian cells focused on ires - dependent translation in poliovirus - infected cells , and the reaction of mitogenically activated fibroblasts , providing early proof - of - principle for the methodology introduced above that involves polysomal fractionation followed by dna microarray analysis of rna contents ( fig . 2a ) . a further landmark study by holland and colleagues analyzed polysomal profiles of murine cell lines after blocking oncogenic ras and akt signaling apparently , these pathways regulate the recruitment of specific mrnas to ribosomes to a far greater extent than de novo synthesis of mrnas by transcription and thus , ras and akt signaling pathways seem to have a more pronounced effect on translational versus transcriptional regulation . the authors postulated that the immediate and direct inductive oncogenic effect of these signaling pathways could be largely achieved through translational activation . the differences seen in rna abundances during chronic signaling alterations may be secondary to translational effects caused by mrnas encoding transcription factors . similar studies on different cancer types may lead to the identification of potential markers and possibly reveal novel drug targets [ 37 , 61 ] . moreover , a recent study identified specific subsets of mrnas regulated by eif4e overexpression , which is known to lead to tumor transformation . the authors postulated that down - regulation of eif4e and its downstream targets may represent a potential therapeutic option for the development of novel anti - cancer drug . as seen for ras / akt activation , it is intriguing that changes at translation can even outperform changes at the steady - state mrna level . this has also been noticed in a study analyzing radiation - induced changes in gene expression of human brain tumor cells or normal astrocytes . ten times more genes ( 15% ) were altered at the level of translation compared to the number of genes regulated at the level of transcription ( 1.5% of 7800 analyzed human genes ) . only a few transcripts were commonly affected at both the transcriptional and translational levels , suggesting that the radiation - induced changes in transcription and translation are not coordinated . those transcripts that were affected at translation fell into functional groups such as cell cycle , dna replication and anti - apoptotic functions . this indicates that dna damage affects post - transcriptional gene regulation of previously synthesized mrnas , possibly enabling cells to repair dna instead of being transcriptionally active . functional relations among messages were also recognized in a recent study performing translational profiling of mouse pancreatic -cells in response to an acute increase in glucose concentration . more than 300 transcripts ( 2% of the analyzed genes ) changed their association with polysomes more than 1.5-fold ; most of them encoding proteins acting in metabolism or transcription . notably , this set of messages is related to the group of genes translationally altered during glucose starvation in the yeast s. cerevisiae . therefore , in mammals and yeast , it appears that mrnas for functionally related messages may be coordinately regulated at the translational level . it is possible that a comparative analysis in different species may allow evolutionarily conserved translational regulatory programs to be deciphered , which are at the moment still rather speculative . whereas concomitant changes in rna abundance and translational rates were rarely detected during radiation response , a recent study identifying mrnas that remain associated with polysomes during hypoxia in transformed prostate cancer cells ( a condition that tumors prevent through the induction of angiogenesis ) found both homodirectionally / potentiated mrnas and distinctively regulated messages . after prolonged exposure of pc-3 cells to low oxygen levels , global translation was reduced by half ; however , 104 mrnas , representing about 0.5% of all analyzed features , became more associated with hypoxic polysomes compared to normoxic ones . among these , 71 mrnas were similarly increased in hypoxic polysomes compared with total rna levels representing homodirectional changes ; 33 mrnas were translationally enriched , some of them potentiated ( 11 of those coding for ribosomal proteins ) . in summary , the common principles of translational regulation that emerge from genome - scale studies in diverse eukaryotes suggest a complex but coordinate system of regulation . it must be triggered by a variety of factors that go well beyond the described pathways that influence global translation . in future , there will certainly be an increasing number of studies to decipher translational regulatory programs in cancer , neurogenesis and development . intriguingly , despite the impact of translational regulation during development , only one recent study systematically investigated translational programs during early embryogenesis in the fruit fly drosophila melanogaster . the mapping of translational programs in diverse species will likely reveal key regulatory networks and how these are affected in disease . steady - state mrna levels are a result of both rna synthesis and degradation that are dynamically controlled and can vary up to 100-fold during the cell cycle or cellular differentiation . in eubacteria like escherichia coli , mrnas are generally degraded by endonucleolytic cleavage , followed by 3-to-5 exonucleolytic rna decay through the so - called rna degradasome consisting of ribonuclease e ( rnasee ) , 3-exoribonuclease polynucleotide phosphorylase ( pnpase ) , rna helicase ( rhlb ) and enolase . in eukaryotes , most cytoplasmic mrna degradation begins with shortening of the poly(a ) tail by deadenylases followed by removal of the 5 cap structure by the decapping enzymes , dcp1 and dcp2 . the decapped intermediates are then degraded either by an exonuclease ( xrn1p ) in the 5 to 3 direction , or by the cytoplasmic exosome in the 3 to 5 direction . in addition , eukaryotes own specialized pathways that target mrnas containing premature termination codons ( nonsense - mediated decay pathway , nmd ) that lack translational termination codons ( non - stop decay pathway , nsd ) or that bear stalled ribosomes ( no - go decay ) . degradation of specific mrnas can also be initiated by endonucleolytic cleavage through sequence - specific endonucleases , or in response to mirnas or sirnas . numerous cis - acting elements located in the 5-utr , the coding sequence ( cds ) or in the 3-utr of mrnas can function as binding sites for rna - binding proteins that regulate decay . for instance , au - rich elements ( ares ) , conserved sequences found in the 3-utr of nearly 5% of all human genes , interact with specific are - binding proteins that stabilize the rna or promote mrna degradation by recruiting the rna decay machinery . global analysis of mrna decay rates following arrest of transcription has been performed in all three kingdoms of life : bacteria [ 6870 ] , archea and eukaryotes including yeast [ 52 , 72 ] , plants , and human cells . in eubacteria and archea , two main characteristics seem to be evolutionarily conserved : adjusted decay rates for functionally related groups of messages , and the inverse correlation between the half - lives and the relative abundances of transcripts . as seen in the archaebacterium sulfolobus , transcripts encoding proteins involved in growth - related processes , such as transcription , trna synthesis , translation and energy production , generally decay rapidly ( t1/24 min ) , whereas those encoding products necessary for maintaining cellular homeostasis are relatively stable ( t1/2>9 min ) . short half - lives of highly abundant mrnas imply high - turnover rates and thus , enable cells to rapidly reprogram gene expression upon changes in environmental conditions [ 68 , 71 ] . interestingly , the half - life and abundance of distinct classes of transcripts appear to depend on particular rna degradosome components . this finding suggests the existence of structural features or biochemical factors that distinguish different classes of mrna targeted for degradation . this may also apply to specific growth phases , as seen in streptococcus where certain mrnas become sensitive to stationary - phase - induced pnpase . evidence for the existence of coordinated rna decay regulons in eukaryotes was obtained from global investigation of mrna decay profiles in yeast and human cells . here , transcription was shut - off using cells that bear a temperature - sensitive allele of rna polymerase ii or through chemical inactivation , and the decay of thousands of genes was monitored with dna microarrays over a time course [ 52 , 72 , 74 ] . strikingly , mrna half - lives among components of macromolecular complexes in yeast were significantly correlated . for instance , the transcripts for the four histone mrnas were among the least stable with closely matched , rapid decay rates ( t1/2=72 min ) ; the 131 mrnas coding for ribosomal proteins had average decay rates ( t1/2=226 min ) , and the four components of the trehalose phosphate synthetase complex were amongst the longest lived messages ( t1/2=10515 min ) . the examination of decay rates in human cells revealed similar mrna - turnover patterns among orthologous genes , indicating the presence of evolutionary conserved programs of rna stability control . transcripts encoding metabolic proteins have a tendency for longer half - lives , whereas transcripts encoding transcription factors or ribosome biogenesis factors are relatively short lived [ 52 , 72 ] . interestingly , it appears to be a universal feature that average transcript half - lives are roughly proportional to the length of the cell cycle : cell - cycle lengths of 20 , 90 , and 600 min correspond to median mrna half - lives of 5 , 21 and 600 min for e. coli , s. cerevisiae and human cells , respectively . dna microarrays have also been applied to investigate specialized decay pathways , such as nmd and nuclear exosome - mediated decay . mutants for nmd factors upf1 , nmd2 and upf3 alter the mrna levels of an overlapping set of 600 messages ( 10% of the transcriptome ) in yeast [ 77 , 78 ] . however , mrna levels in nmd strains may also be the result of indirect effects because transcription factors are also targeted through nmd and therefore , guan et al . dissected direct from indirect targets of nmd by profiling global rna decay rates in nmd strains . about half ( 300 transcripts ) are likely to be direct nmd targets decayed through 5 to 3 degradation by xrn1p . nmd - sensitive transcripts tend to be both non - abundant and short - lived , with one third of them coding for proteins that are connected to two central themes : first , replication and maintenance of telomeres , chromatin - mediated silencing and post - replication events related to the transmission of chromosomes during the cell division cycle ; and , second , synthesis and breakdown of plasma membrane components , including transport of macromolecules and nutrients , and cell wall proteins . several genes , located downstream of independently transcribed snorna genes , were overexpressed in exosome mutants . such read - through transcripts into downstream orfs are normally rapidly degraded by the exosome and , hence , could explain their enrichment in exosome mutants . global mrna turnover in mutant cells was monitored through gene expression analysis expecting adverse effects on subsets of messages . examined the effects of deletions of genes encoding deadenylase components ccr4p and pan2p and putative rna - binding proteins pub1p and puf4p after inhibition of transcription by chemicals and/or heat stress . this examination showed that ccr4p , the major yeast mrna deadenylase , contributes to the degradation of transcripts encoding both ribosomal proteins / rrna synthesis and ribosome assembly factors largely mediating the transcriptional response to heat stress . pan2p and puf4p also participate in degradation of these mrnas , while pub1p preferentially stabilized transcripts encoding ribosomal proteins . a second study focused on pub1p , a yeast rna - binding protein thought to destabilize mrnas through binding to au - rich sequences in 3-utrs . global decay profiles in pub1 mutants revealed a significant destabilization of proteins involved in ribosomal biogenesis and cellular metabolism , whereas genes involved in transporter activity demonstrated association with the protein , but displayed no measurable changes in transcript stability . therefore , in this case , the direct targets only partially related to the functional outcome under specific physiological conditions . this could be mediated through additional rna protein interactions forming a network through combinatorial binding . finally , foat et al . combined a computational and experimental approach to identify transcripts that are destabilized under specific environmental conditions ( sugar sources ) by yeast mrna stability regulators . for puf3p , which was known to primarily associate with mrnas coding for mitochondrial proteins , they computationally inferred and experimentally verified target destabilization in the presence of glucose , as some of these mrnas were up - regulated in puf3 mutants grown in a non - repressing carbon source , but down - regulated in a repressing carbon source . mammalian cells have evolved a variety of specific mrna decay programs that play important roles in medically relevant processes such as inflammation , hypoxia and cancer pathogenesis . for example , the expression of diverse cytokines is differentially regulated after t cell activation , and glucocorticoids inhibit inflammation through destabilization of proinflammatory transcripts like cyclooxygenase-2 . for instance , in resting t lymphocytes , the majority of transcripts are stable with half - lives of more than 6 h , but a small proportion ( 3% ) of expressed transcripts exhibits rapid decay with half - lives of less than 45 min . these short - lived transcripts encode a variety of regulatory proteins such as cell surface receptors , transcription factors and regulators of cell growth and apoptosis . focused on the massive degradation of transcripts occurring during meiotic arrest at the germinal - vesicle ( gv ) stage , and found that degradation is apparently not promiscuous but preferentially affects specific groups of messages . in particular , transcripts involved in processes associated with meiotic arrest at the gv stage and the progression of oocyte maturation , such oxidative phosphorylation , energy production , and protein synthesis , were rapidly degraded , whereas those encoding participants in signaling pathways maintaining the oocyte in the mii - arrested state were among the most stable . in conclusion , these studies exemplify that stimulus - dependent transcript destabilization is an important mechanisms for controlling gene expression in a coordinated manner . the presence of these motifs in mrnas often correlates with shifts in the distribution of decay rates ; however , their sole presence can not reliably predict turnover behavior . are - binding proteins may therefore differentially determine the fate of mrna depending on the cellular and environmental context . tristetraprolin ( ttp ) , a well - known are - binding protein , has several characterized physiological target mrnas including tumor necrosis factor ( tnf)- , granulocyte - macrophage colony - stimulating factor , and interleukin-2. micro - array analysis of rna obtained from wild - type and ttp - deficient fibroblast cell lines identified 250 transcripts with altered decay rates , some of them containing conserved ttp binding sites . the rna - binding protein t - cell intracellular antigen 1 ( tia-1 ) functions as a post - transcriptional regulator of gene expression and aggregates to form stress granules following cellular damage . tia-1 regulates mrnas for proteins involved in inflammatory responses such as tnf- and cyclooxygenase 2 . immunoprecipitation ( ip ) of tia-1-rna complexes , followed by microarray - based identification and computational analysis of bound transcripts revealed at least 300 potential targets , many of them bearing an u - rich motif . in conclusion , global analysis of mrna turnover underlines the importance of rna decay in the control of mrna levels and strongly suggests the presence of specific rna turnover programs . mrna decay certainly involves combinatorial interactions of rbp enabling stimulus - dependent decay programs through the integration of diverse signals . besides temporal control , rna decay may also occur spatially restricted , as seen with drosophila ire1 , a protein activated during the unfolded protein response in the endoplasmic reticulum directing the decay of specific subset of mrnas , many of which encode plasma - membrane proteins . moreover , still rather unexplored is the role of p - bodies and stress granules as storage place for untranslated mrna and site for mrna degradation at least the recent observation that are containing mrnas are localized to specific cytoplasmic granular structures containing exosome subunits that are distinct from p - bodies or stress granules , support the idea of specialized structures for storage or degradation of distinct groups of mrnas . putative rna - binding proteins comprise 3 11% of the proteomes in bacteria , archea and eukaryotes . the large number of rbps in all kingdoms of life may merely reflect the ancient origin of rna regulation , which is possibly the most evolutionary conserved part of cell physiology . rbps often contain distinct rna - binding domains that specifically interact with sequences or structural elements in the rna . approximately one hundred protein domains associated with rna metabolism have been described to date , half of them believed to have originated at early stages in evolution , whereas others , such as the rna recognition motif ( rrm ) , are exclusively present in eukaryotes and therefore may have been acquired later in evolution . a successful approach to globally identify the in vivo rna targets of rbps involves immunoprecipitation or affinity purification of epitope - tagged proteins followed by the analysis of associated rnas with dna microarrays or by sequencing ( fig . although low - density arrays were used to identify the bound mrnas , each rbp was associated with a distinct subset of the mrnas present in total cell lysate . moreover these results led to the proposal that groups of mrnas encoding functionally related proteins are organized as so - called post - transcriptional operons . in analogy to prokaryotic operons , this model predicts that specific rbps may coordinate groups of mrnas coding for functionally related proteins in eukaryotes . cis - acting elements in the mrna may provide the means to mimic the coordinated regulatory advantages of clustering genes into polycistronic operons [ 16 , 92 ] . a prime example for the coordination of functional related transcripts by specific rbps is represented by the pumilio - fem-3 binding ( puf ) proteins [ 80 , 93 ] . puf proteins comprise a conserved family of structurally related rbps that negatively regulate gene expression of specific mrnas . applying dna microarrays to identify their rna targets revealed that each of the five yeast puf proteins associated with distinct groups of 40 to 220 different mrnas with striking common themes in the functions and subcellular localization of the proteins they encode : puf3p binds nearly exclusively to cytoplasmic mrnas that encode mitochondrial proteins ; puf1p and puf2p interact preferentially with mrnas encoding membrane - associated proteins ; puf4p preferentially binds mrnas encoding nucleolar ribosomal rna - processing factors ; and puf5p is associated with mrnas encoding chromatin modifiers and components of the spindle pole body . the results were further corroborated by the identification of distinct sequence motifs in the 3-untranslated regions of the mrnas bound by puf3 , puf4 , and puf5 proteins . a physiological relation between puf3p and its mrna targets has also been observed as suggested from its association with mrna - encoding mitochondrial proteins , puf3 mutant cells showed a slow - growth phenotype on non - fermentable carbon sources indicative of a functional connection to mitochondrial physiology . genome - wide identification of rnas associated with the orthologous puf protein from drosophila melanogaster , called pumilio , revealed distinct clusters of mrnas in embryos and in ovaries of adult flies . subgroups of these pum - associated mrnas had commonalities , such as function in the anterior - posterior patterning system , and the subunits of the vacuolar h - atpase . moreover , a characteristic sequence motif was present in 3-utrs of pumilio - bound mrnas resembling the one previously identified for the yeast puf3 protein . . hence , the data obtained from the yeast and drosophila studies provided an additional source for considering their evolution . for instance , conservation of amino acid residues in the rna - binding domain ( the pum - homology domain ) between homologous puf proteins correlated with identified core motifs in 3-utr of mrna targets . however , the proteins encoded by the mrna targets appeared not to be particularly conserved . this discordance suggested that acquisition or loss of rbp binding motifs in utrs of genes may provide a surprisingly fluid evolutionary mechanism to modify post - transcriptional regulatory connections . ribonomic studies have now been conducted for more than 30 specific rbps ( table 1 ) . each of the analyzed rbps has a unique rna binding spectrum comprised of 201000 distinct transcripts that often share functionally related themes . occasionally , sequence or structural elements could be identified among mrna targets using bioinformatics tools , and novel physiological consequences were discovered ( e.g. , ) . the ribonomics approach has recently been implemented on the argonaute ( ago ) protein family to discover novel mrnas that potentially undergo mirna dependent regulation [ 96 , 97 ] . although the number of detectable ago - associated mrnas was low ( 90 messages ) compared to the thousands of genes expected to undergo mirna dependent regulation , the comparison of ago - associated mrnas in wild - type and mirna mutants may provide a tool to decipher mirna - specific targets . besides specific rbps general rna - binding proteins for the identification of messages expressed in particular tissues or cell - types . affinity - tagged poly(a ) binding protein ( pabp ) was expressed with tissue - specific promoters to identify muscle- or ciliated sensory neuron - specific transcripts in the worm caenorhabditis elegans [ 98 , 99 ] , and mrnas in photoreceptor cells of flies . the method was also used to measure gene expression of endothelial cells that were co - cultured with breast tumor cells . a similar approach with tagged ribosomal proteins may become another tool to determine gene expression in specific cells [ 102 , 103 ] . table 1global identification of rna targets for specific rna - binding proteins ( rbps).organismrbpprocesstargetsfunctional relation among proteins encoded by targetsrna motifreferenceyeastmex67rna export1142cell wall components , translation factors , membrane proteinsno(saccharomyces cerevisiae)yra1rna export1002cell cycle - regulated , cell wall , carbohydrate metabolismnoshe2 , she3 , myo4rna localization22cell cycle , membraneorf/3-utr[33 , 34]scp160translational regulation?>50membrane - associated factors?nolhp1rna biogenesis1000noncoding rna , ribosomal proteins and biogenesis , unfolded protein responsenopuf1mrna decay / translation40membrane - associated , plasma membranenopuf2mrna decay / translation146membrane - associated , plasma membranenopuf3mrna decay / translation220mitochondrion ( ribosome)3-utrpuf4mrna decay / translation205nucleolar factors ( rrna/ trna processing)3-utrpuf5mrna decay / translation224nuclear factors ( chromatin modifiers , spindle - pole body)3-utrpub1mrna decay384ribosome biogenesis , transporter3-utrcth2mrna decayiron metabolism3-utrnpl3mrna exportribosomal subunits3-utrnab2mrna exporttranscription3-utrnab4/hrp1mrna export , polyadenylationmetabolism ( amino acid , alcohol , ergosterol ) , energy pathways3-utrmammalselav/ humrna stability / translation60cell cycle regulators , transcription factors3-utr[119 global identification of rna targets for specific rna - binding proteins ( rbps ) . the application of genomic tools to study post - transcriptional gene regulation suggests additional levels of coordination and regulation that are beyond the traditional view of equally treated cellular mrnas that are similar processed , exported , and eventually translated in the cytoplasm [ 1416 ] . the decay , localization and translation of mrna seem to undergo coordinate control by regulatory programs , which may be embedded in a multifaceted post - transcriptional regulatory system . the properties of this system are controlled by rna - binding proteins or non - coding rnas ( e.g. , micrornas ) that coordinate functionally related sets of mrnas through binding to sequence elements in the rna . considering the hundreds of rbps encoded in eukaryotic genomes , post - transcriptional control may be comparable in its richness and complexity to transcriptional regulatory systems . this provides a means to link rna regulation to other cellular regulons such as signal transduction pathways allowing rapid and efficient reprogramming of gene expression in response to changing physiological conditions . further analysis of rbps and their target rnas may finally lead to a map of the proposed post - transcriptional regulatory system . however , besides the architecture , it will also be important to study the plasticity and dynamics of this regulation by measuring how it reacts in response to environmental or developmental changes , and how it is perturbed in certain diseases [ 35 , 105 ] . finally , a major challenge will be to connect the different levels of gene expression systems though large - scale data integration .

objectivethe purpose of this study is to assess the utility of web - based mobile technology monitoring tool , for ensuring linkages , and tracking of hiv - exposed child until 18 months of age.methodsthe early infant diagnosis ( eid ) follow - up system was designed as a tool for reminding the field level staff for follow - up of hiv - exposed babies . using java swing framework , software was developed which generates automatic advance sms alerts regarding patient information to the counsellor of the respective integrated counselling and testing center and district supervisor , 7 days prior to due dates . simultaneously , system generated e - mail is sent to district program officer for monitoring and updating the line-list.resultsbefore the introduction of eid follow - up system in june 2013 , only 55.9% ( 637/1139 ) of the hiv - exposed babies born were tested at 6 weeks for dna - polymerase chain reaction during april 2011march 2012 . however , after its introduction , 68.4% ( 1117/1631 ) of them were tested during april 2012march 2013 . correspondingly , the 18 months confirmatory hiv testing in eligible babies increased from 45.6% ( 934/2044 ) to 54.7%(1118/2044 ) during the same period.conclusionthe replicable technology driven initiative would help in strengthening the follow - up mechanisms and reach every hiv - exposed child for eid . abstract.post-transcriptional regulation of gene expression plays important roles in diverse cellular processes such as development , metabolism and cancer progression . whereas many classical studies explored the mechanistics and physiological impact on specific mrna substrates , the recent development of genome - wide analysis tools enables the study of post - transcriptional gene regulation on a global scale . importantly , these studies revealed distinct programs of rna regulation , suggesting a complex and versatile post - transcriptional regulatory network . this network is controlled by specific rna - binding proteins and/or non - coding rnas , which bind to specific sequence or structural elements in the rnas and thereby regulate subsets of mrnas that partly encode functionally related proteins . it will be a future challenge to link the spectra of targets for rna - binding proteins to post - transcriptional regulatory programs and to reveal its physiological implications .

fetus in fetu ( fif ) was first described by meckel in the early 19th century , and it was defined as a mass containing a vertebral axis often associated with other organs or limbs around this central axis in 19351 ) . since then , this disease entity has been rarely reported . with advance in prenatal diagnosis techniques , more than half of the cases have been detected prenatally . in this report fif differentiating a fif from other cause of meconium peritonitis is discussed with review of literature . a 31-year - old primi pregnant woman with minimal prenatal care and no history of fertility medications or in vitro fertilization came to our institution at 33 weeks ' gestation . obstetric sonography at 26 weeks ' gestation revealed a single fetus of appropriate size for gestational age , but bowel wall edema and small bowel obstruction were suspected . sonography at 29 weeks ' gestation showed a large amount of amniotic fluid and the aforementioned findings were not changed . sonography at 33 weeks ' gestation which was performed at our institution showed that a markedly dilated bowel and the bowel obstruction were still suspicious . the amount of amniotic fluid was increased , and a 6.86.4 cm pseudocyst and a 4.393.22 cm calcified heteroechgenic mass were found ( fig . our clinical diagnosis was a giant cystic type of meconium obstruction associated with peritonitis induced by perforation and atresia of the small bowel . the weekly ultrasonographic follow - ups showed no significant interval changes . at 38-weeks ' gestation , a female newborn weighing 4,120 g was delivered by an elective cesarean section , with apgar scores of 2 , 4 and 9 at 1 , 5 and 10 minutes . the baby had a grossly distended abdomen and did not present any evidence of dysmorphic features . the patient had respiratory distress after birth which was probably induced by elevated abdominal pressure due to the abdominal mass and ascites and necessitated assisted ventilation . laboratory tests showed no abnormalities except for a slightly decreased albumin level of 3.1 g / dl and an alpha - fetoprotein level of 5,845 ng / ml . abdominal radiographs showed a large abdominal mass containing multiple calcifications in the lower abdomen ( fig . abdominal ultrasonography showed a huge solitary and cystic mass which filled most of the abdomen . abdominal computed tomography ( ct ) showed a well - demarcated intraabdominal mass measuring 15.610.2 cm , with a heterogeneous enhancing solid portion , fatty component and a cystic portion containing multiple calcification or ossification lesions . a teratoma arising from both ovaries was the most likely diagnosis , and she underwent an elective laparotomy at 5 days of age . during surgery , we observed a small amount of ascites with a mass attached to the colon and mesocolon which was thought to be a splenic flexure ( fig . 3 ) . the mass did not have a cystic sac and had a buttock - like lesion with skin and hair follicles , fatty tissue , a finger and a coccyx . a tubular structure was also identified along the mesenteric border of the sigmoid colon , which was suspected of an intestinal duplication cyst . the mass measuring 1595 cm had a smooth surface partially covered with skin or thin membrane and 2 finger - like lesions ( fig . 4 ) . on section , most of the mass consisted of yellowish fat tissue , and the middle portion of the mass had a tubular structure which looked like an intestine . microscopic examination showed a well developed gastrointestinal tract , liver , pancreas and finger as well as 3 germinative layers , but the vertebral axis was not identified . meckel first coined the term " fetus in fetu " in the late 18th century to describe an encapsulated fetoid tumor within a fetus . after then , willis1 ) stated in 1935 that the presence of an axial skeleton distinguishes between fif and a teratoma , which remains a clinical definition . gonzalez - crussi4 ) defined fif in a narrower sense as " highly organotypic development and presence of a vertebral axis with arrangement of tissue around this axis " . thirty - one cases of fif were reported before 1900 , and thereafter only 11 cases were reported between 1900 and 19563 ) . its incidence is very low and is estimated to be 1 per 500,000 birth2 ) . the incidence of fif has increased and the incidence seems to be higher than in previous reports . with advances in prenatal diagnosis techniques , more than half of the cases have been detected prenatally . our case was also detected prenatally , but it was misdiagnosed as cystic meconium peritonitis . meconium peritonitis represents aseptic peritonitis caused by the spreading of meconium into the fetal peritoneal cavity . cyst occurs when the site of perforation is not sealed and a thick - wall cyst is formed within the intestinal loops . the cystic type of meconium peritonitis is thought to be a life - threatening condition . therefore , rapid deterioration of fetal status can be possible in this type . in this regard meconium pseudocyst must be antenatally differentiated from other intra - abdominal cystic images such as urinary bladder distention , ovarian cyst , intestinal duplication , hematoma , teratoma and fetal infection5 ) . fif can be presented prenatally as an abdominal cystic mass with diffuse calcification or ossification by ultrasonography . ct or mri has sometimes been used as a confirmatory diagnostic tool to differentiate between these diseases . however , if the vertebral axis - like structure is seen within a mass on prenatal imaging , it is also helpful in differential diagnosis . it is still controversial whether the presence of the vertebral axis should be included in the diagnostic criteria for fif . some previously reported cases without the vertebral axis were diagnosed as fif as in our case . it is thought to occur at the very early stage of embryo development before the ventral fusion of the lateral body walls . the spectrum is diverse from conjoined symmetric twins to a form of organized teratoma , but fif is thought to be a different category from teratoma based on its organo - level development , rarity of malignant potential and the possession of the same karyotype with the host . chromosome analysis was performed on 29 cases , which showed the same chromosomes as the host 's . miura et al.6 ) reported that in 2 cases undergoing dna methylation assay , 46.7% and 66.7% of clones from the mass had an unmethylated paternal allele . they suggested that 2 isolated blastocysts originated from one zygote , and one of the two was implanted into the other blastocyst during the process of methylation . in this regard , presence of vertebral axis to diagnosis of fif may not be a prerequisite factor . pathological confirmation is essential for a final diagnosis , and genetic testing can provide unambiguous evidence for a fetus in fetu . several cases have been reported in older children with a delay in diagnosis and 1 case in a 39-year - old man , was and they showed no malignant potential7 ) . the prognosis of intracranial fif is not good . only 1 case survived , and 2 cases were stillborn , 2 cases were terminated fatally due to intracranial hypertension , and 2 cases were terminated with their parents order . hopkins et al.8 ) reported a retroperitoneal fif presenting as a right abdominal mass which proved to be a teratoma with malignant components requiring chemotherapy8 ) . there are 2 other cases with local recurrence as a teratoma without malignant potential in the literature ; they were located in the retropenitoneum and recurred at 1 and 2 years of age , respectively9 , 10 ) . for this reason , it is necessary to regularly follow up patients with fif for the surveillance of recurrence . we report a case of fif which was misdiagnosed as cystic meconium peritonitis prenatally . abdominal cystic mass should be differentiated from various conditions . although fif is considered as a rare disease , it has been increasingly reported in the literature . thus , fif should also be included in the differential diagnosis of cystic masses in fetus . during october 1 , 2010july 31 , 2011 , the centers for disease control and prevention ( cdc ; atlanta , georgia , usa ) asked all state public health laboratories to submit pandemic ( h1n1 ) 2009 virus positive respiratory specimens and virus isolates for antiviral susceptibility testing . laboratories were asked to provide the first 5 specimens of any type / subtype collected during each 2-week period for virus isolation . comprehensive antiviral testing , including neuraminidase inhibition ( ni ) assay , was performed on all isolates , and sequencing was performed on all isolates with elevated 50% inhibitory concentration values . cdc also requested that laboratories provide 5 additional specimens every 2 weeks for pyrosequencing to identify the h275y substitution in the neuraminidase , a change associated with oseltamivir resistance ( 3 ) . in addition to ( or instead of ) participating in the national surveillance , state laboratories in california , maine , maryland , minnesota , new york , texas , and washington performed pyrosequencing on state surveillance specimens to detect the h275y substitution . we included those state - specific data in the national surveillance data for this report . state health departments used a standard case form to collect demographic and clinical information for patients with oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infection and their ill close contacts . oseltamivir resistance was determined by use of an ni assay or pyrosequencing for the h275y substitution . for ni testing on isolates , we used the na - fluor kit ( applied biosystems , foster city , ca , usa ) as described ( 4 ) . we performed pyrosequencing , as described ( 5 ) , on all oseltamivir - resistant pandemic ( h1n1 ) 2009 isolates identified by ni assay to confirm the presence of the h275y substitution . we performed pyrosequencing for h275y , without the ni assay , to screen pandemic ( h1n1 ) 2009 clinical specimens ( 5 ) . for the national surveillance , ni testing was performed at cdc and pyrosequencing was performed at cdc and at state laboratories in wisconsin , new york , and california . state laboratories followed pyrosequencing protocols provided by cdc ; when possible , cdc confirmed results for resistant viruses by use of pyrosequencing and ni assay . most oseltamivir - resistant viruses in this report were included in the weekly fluview report prepared by cdc ( 6 ) . we tested a total of 3,652 pandemic ( h1n1 ) 2009 virus isolates and specimens from every state and the district of columbia ; 35 ( 1.0% ) isolates / specimens from a total of 18 states were oseltamivir - resistant ( figure ) . overall , 8 ( 1.3% ) of 609 isolates tested by ni assay and 27 ( 1.0% ) of 3,043 specimens tested by pyrosequencing were resistant to oseltamivir . the state - specific prevalence of oseltamivir - resistant pandemic ( h1n1 ) 2009 viruses varied ; however , the number of viruses and specimens tested also varied markedly between states , and several states submitted only a few specimens . forty - four states submitted > 20 specimens for antiviral resistance surveillance . the prevalence of oseltamivir resistance among these specimens ranged from 0% to 5.6% . none of the 609 pandemic ( h1n1 ) 2009 isolates tested by ni assay were resistant to zanamivir . the ranges of 50% inhibitory concentration values for oseltamivir - resistant and -susceptible isolates were 166.17230.37 nmol / l and 0.100.80 geographic distribution of oseltamivir - resistant pandemic ( h1n1 ) 2009 viruses in the united states , october 1 , 2010july 31 , 2011 . numerators are number of oseltamivir - resistant viruses identified by state public health laboratories ; denominators are number of pandemic ( h1n1 ) 2009positive specimens submitted by each state for susceptibility testing . the median age of the 35 patients with oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infections was 33 years . of 34 patients with available information , 26% reported receiving oseltamivir before providing a specimen for antiviral susceptibility testing ( table 1 ) . among 33 patients with a completed case form , 67% had at least 1 preexisting chronic medical condition , 24% had an immunocompromising medical condition , 42% required hospitalization , and 9% died . most patients with oseltamivir - resistant virus infection for whom housing information was available lived in a single - family household . two siblings from 1 household had oseltamivir - resistant virus infection ; neither child had received oseltamivir . * the median age of patients was 33 y ( range 1 mo78 y ) . nine patients began oseltamivir treatment prior to specimen collection ; none received oseltamivir chemoprophylaxis . includes hiv / aids , malignancy , autoimmune disorder , solid organ transplant , stem cell transplant , and history of taking immunosuppressive therapy in the past year . includes morbid obesity , obstructive sleep apnea , chronic liver disease , and neurologic or developmental disorders . all oseltamivir - resistant pandemic ( h1n1 ) 2009 viruses were identified from specimens collected during december 2010april 2011 ; the prevalence of resistance did not change significantly over time ( test for trend , p = 0.18 ) ( table 2 ) . in addition , the proportion of patients with oseltamivir - resistant virus infections who did not have exposure to oseltamivir before specimen collection did not change significantly over time ( test for trend , p = 0.48 ) ; however , the number of specimens tested each month was small . the number of oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infected patients was small during the 200910 ( april 2009september 2010 ) and 201011 influenza seasons . however , the prevalence of oseltamivir - resistant virus infected patients was slightly higher during 201011 compared with 200910 ( 35/3,652 [ 1.0% ] vs. 37/6,740 [ 0.5% ] , respectively , p = 0.02 by test ) ( 2 ) . also , during 201011 , compared with 200910 , more patients with oseltamivir - resistant virus infection had no history of oseltamivir exposure before specimen collection ( 25/34 [ 73.5% ] vs. 4/35 [ 11.4% ] , respectively ; p<0.0001 by test ) . during the 201011 us influenza season , the prevalence of oseltamivir - resistant pandemic ( h1n1 ) 2009 viruses remained low , and most persons with oseltamivir - resistant virus infection had no prior oseltamivir exposure . this is a notable difference from surveillance findings both globally and in the united states during the 200910 season , when most patients with oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infection had a history of oseltamivir exposure , and many were severely immunocompromised , a condition that may increase the risk for resistance developing during oseltamivir therapy ( 2,7 ) . these data suggest a low level of community transmission of oseltamivir - resistant pandemic ( h1n1 ) 2009 virus in the united states during the 201011 influenza season . the united kingdom also reported that the proportion of oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infected patients without prior oseltamivir exposure was higher during 201011 than 200910 ( 8) . the increase during the 2010 - 11 influenza season in the proportion of patients with oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infections without prior oseltamivir exposure causes concern in light of the recent history of oseltamivir resistance among seasonal influenza a ( h1n1 ) viruses that circulated before pandemic ( h1n1 ) 2009 virus emerged . in the united states before the 200708 influenza season , the prevalence of oseltamivir resistance among seasonal influenza a ( h1n1 ) viruses was < 1% ( 9,10 ) . however , during the 200708 season , the prevalence of oseltamivir resistance among seasonal influenza a ( h1n1 ) viruses increased to 12% , and by the 200809 season , resistance dramatically increased to > 99% ( 9,11,12 ) . no association was found between this increase in oseltamivir resistance and prior oseltamivir use ( 11,13 ) . oseltamivir resistance in pandemic ( h1n1 ) 2009 and seasonal influenza a ( h1n1 ) viruses was conferred by the h275y substitution in the neuraminidase . however , unlike seasonal influenza a ( h1n1 ) viruses , which retained susceptibility to the m2-blocking adamantanes ( amantadine and rimantadine ) , > 99% of circulating pandemic ( h1n1 ) 2009 viruses are inherently resistant to adamantanes ( 14 ) . thus , inhaled zanamivir or investigational drugs , such as intravenous zanamivir , are the only treatment options for patients with oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infection . our conclusions are limited by the small number of patients with oseltamivir - resistant pandemic ( h1n1 ) 2009 virus infection . variability in state surveillance and the number of specimens tested from each state may also have limited the representativeness of our data . despite these shortcomings , our findings emphasize the importance of ongoing surveillance for oseltamivir - resistant pandemic ( h1n1 ) 2009 viruses in the united states and globally and of close monitoring for changes in the epidemiology of oseltamivir resistance among pandemic ( h1n1 ) 2009 viruses . updated information about antiviral resistance in influenza viruses in the united states is available at www.cdc.gov / flu / professionals/.

fetus - in - fetu ( fif ) is a rare congenital condition in which a fetiform mass is detected in the host abdomen and also in other sites such as the intracranium , thorax , head , and neck . this condition has been rarely reported in the literature . herein , we report the case of a fetus presenting with abdominal cystic mass and ascites and prenatally diagnosed as meconium pseudocyst . explorative laparotomy revealed an irregular fetiform mass in the retroperitoneum within a fluid - filled cyst . the mass contained intestinal tract , liver , pancreas , and finger . fetal abdominal cystic mass has been identified in a broad spectrum of diseases . however , as in our case , fif is often overlooked during differential diagnosis . fif should also be differentiated from other conditions associated with fetal abdominal masses . during october 2010july 2011 , 1.0% of pandemic ( h1n1 ) 2009 viruses in the united states were oseltamivir resistant , compared with 0.5% during the 200910 influenza season . of resistant viruses from 201011 and 200910 , 26% and 89% , respectively , were from persons exposed to oseltamivir before specimen collection . findings suggest limited community transmission of oseltamivir - resistant virus .

the incidence of cutaneous melanoma ( cm ) is rapidly increasing in europe , especially in the caucasian population . incidences are known to vary amongst white populations , reaching 15 cases per 100,000 inhabitants per year in scandinavia and 57 cases per 100,000 inhabitants per year in mediterranean countries [ 24 ] . fortunately , these data are matched by active research directed toward a greater identification of risk factors linked with the onset of melanoma . in this regard , the presence of naevi assumes particular importance as an independent and very high risk factor for the development of melanoma . clinically dysplastic naevi and an excess number of melanocytic naevi are among the most serious known risk factors for cutaneous melanoma . in the last decade , a plethora of investigations aimed at identifying risk factors for the development of melanoma have been conducted . more recently , working on 200 patients affected by melanoma and 200 healthy control patients , nikolaou et al . confirmed that the presence of atypical naevi is an important risk factor for the development of cm , tripling the odds ratio ( or ) . according to carli et al . , the greatest difference between subjects with or without atypical naevi is related to their number of common naevi : the authors demonstrated that more than 30 naevi are encountered in 41.5% of melanoma cases , but in only 9% of healthy control subjects ( or 8.0 ; confidence interval ( ci ) 6.310.3 ) . even if atypical clinical features of naevi constitute a very high risk factor for cm , their partial correspondence with histologic dysplasia indicates that the total number of naevi remains the most important factor in the development of cm . these data confirm the major risk of dysplasia or the development of cm when the total number of naevi exceeds 100 , and the presence of an elevated number of naevi needs to be carefully evaluated in the panorama of risk factors for the development of melanoma [ 13 , 14 ] . as a result , numerous studies have been undertaken in order to establish a classification of naevi and to define their physiological and pathological evolution , the majority of which have aimed to correlate the macroscopic appearance of these structures with their histopathological characteristics [ 15 , 16 ] , dividing them into dermal , composed , and junctional . kincannon and boutzale further divided acquired naevi into five categories according to their rough macroscopic appearance and their relationship to the skin surface , noticing that the papillomatous variant is most commonly associated with such phenotypes as blue or green eye colour and fair , light brown , or red hair . these authors underlined the importance of any possible correlations between naevi and phenotype characters and also drew up a more accurate and detailed classification of naevi , even though it was restricted almost exclusively to dermal naevi . gallagher and kwan showed that naevi have a preferred body distribution in the two genders which is already evident in the pediatric age : in males naevi are more numerous on the head , neck , and trunk , whereas females show a greater distribution on their limbs . naevi are much more numerous in children intermittently exposed to the sun , highlighting the importance of sun exposure during childhood in the genesis of benign acquired naevi . however , the same distribution pattern in photoprotected locations raises the possibility that hormonal differences play a consistent role in the development of melanocytic naevi [ 19 , 20 ] . the most famous and widely accepted classification of acquired melanocytic naevi was published by ackermann and magana - garcia who classify naevi into four types , of which clark naevi constitute the predominant type among commonly acquired melanocytic naevi . when their diameter exceeds 5 mm and at least two other signs of clinical activity are present , such as irregular or poorly defined edges , uneven colouring , or erythematous patch variations in skin pattern , they are defined as atypical , whereas when histology shows the presence of specific architectural features they are diagnosed as dysplastic naevi . however , although ackerman 's classification continues to be a reference , all pigmented melanocytic naevi are grouped under the single denomination of clark naevi which does not enable the analytical investigation of naevi in the field of melanoma prevention : further steps in this direction have only led to the conviction that the presence of a small number of clark naevi has weak meaning in young patients with no melanoma familiarity . on the other hand , when clark naevi arise in older subjects , between the ages of 40 and 50 with no familiarity , their meaning is less clear but it is considered as an anomaly of the normal formation and growth process of melanocytes . several variants , whose denominations can be found in the literature , are mentioned and defined by means of clinical criteria in saurat 's book . this publication includes the most common naevi present in the population ; sutton or halo naevus is also mentioned and congenital pigmented naevi are divided into small ( < 1.5 cm ) , medium ( 1.520 cm ) , and large ( > 20 cm ) . further progress in this direction can be achieved by making a precise systematic table of the various subspecies of naevus , free of any excess or overlapping . we have conducted this case - control study with the aim of observing different numbers of moles and different mole typology associations between patients affected by melanoma and control volunteers . the analysis of naevus variants is not only of commercial value but can be of considerable practical value in the self - analysis of naevi , which is of great importance in the prevention of cm . this analysis makes it easier for patient and physician to know the number and distribution of the naevi , thus enabling the timely observation of any anomalous cellular activity in progress , of any lesions at risk or of a still undetected early clinical melanoma . in addition , this study may provide a further contribution to the determination of a naevic map in the patient undergoing a routine dermatological checkup without suspect lesions , evaluating the possibility of identifying one or more categories of naevus variants for every individual in order to identify further guide lines for the study of melanoma risk factors . naevus variants can improve the interaction between doctors and their patients , who will be able to furnish more accurate information on the naevi and their evolution , thus providing doctors with better guidelines for diagnosis and therapy . in this way , they will be able to better orient themselves in the identification of risk naevus / naevi , examining mole variant associations and their numerosity , according to the total number of naevi and the phenotypic features of the people concerned . one of the aims our study is to try to identify the phenotypic features of individual naevus variants with a view to establishing different risk patterns for mc , which may in turn suggest different follow - up procedures . 64 cases and 183 controls were selected amongst patients at the dermatology department of federico ii university , naples between october 2009 and february 2011 . the cases and the controls ( 102 males and 145 females ) had an average age of 34.8 years ( minimum 2 and maximum 82 ) . the 64 cases were equally distributed between the two sexes ; of the 183 controls 70 were males and 113 females . the cases enrolled were patients affected with nodular or superficial melanoma , the thickness of the lesion had been determined in each case ( staging model ajcc 1988 ) , and the time elapsed from the excision was calculated as between 0 and 10 years . patients with melanoma that had developed on a malignant freckle were excluded , as these melanomas rarely break out in association with a preexisting naevus . the controls were first - degree relatives of cases living in the same household for the same period . all patients were caucasian and homogeneous in respect of socioeconomic indicators and occupation ( table 2 ) . before the naevus count , a trained interviewer compiled a form for each case and control . the interview lasted about 15 minutes and included the following data : residence , phenotype characteristics such as eye colour ( brown , hazel , green , and blue ) , hair colour ( black , brown , fair , and red ) ; phototypes for groups and subgroups from i to iv , ( since v and vi are practically absent in our area for ethnic reasons ) , melanoma familiarity , sunburn history subdivided by age ( juvenile 015 years old , and adult over 15 years old ) , and use of sun beds . the naevi were evaluated after being classified into one of three major groups : the common acquired melanocytic naevi ( nmac ) ( > 2 mm ) , congenital naevi , and atypical naevi . the location of each one was reported : head or neck , arm , upper arm , hands , chest , abdomen , upper back , lower back , thigh , and feet . sun freckles or ephelides were recorded by a number code ( l = none , 2 = from 1 to 5 , and 3 = more than 5 ) . those naevi which were identifiable as atypical were reported separately from the total count . in order to minimise possible errors in the naevus count and according to data reported in the literature , a series of clinical criteria identifying the variants was established ( table 1 ) , based on clinical morphological criteria , that considered the following : relationship to the skin surface , this was used by the dermatologists to identify every naevus lesion . for each variant , the presence of naevic elements and their number was reported , as well as the total number of variants present and the total number of naevi > 2 mm , regardless of their variant group membership . univariate analysis was carried out , followed by a chi - square test to evaluate the degree of association between several variables . finally , the or and the 95% confidence limit were adjusted for age , gender , number of naevi , and some known risk factors for cm , such as phototype i - ii , melanoma familiarity , and past sunburn history . more than 70% of the sample had homogeneous phenotypes : phototype ii or iii ; dark hair ; dark eyes ( 9% of the subjects phototype i , 40% phototype ii ; 35% phototype iii ; 14% phototype iv ) . the sample was also homogeneous in relation to the total number of naevi : 42% of cases and 45% of controls , thus nearly half the sample reported a total number of naevi ranging between 10 and 50 elements . 67% of cases and 80% of controls were negative for childhood sunburn : more than 75% of the sample did not present a childhood sunburn risk factor . with regard to our first aim , that is , the distribution of the single types in the sample , our study showed that the small brown naevus was present in more than 70% of controls and of cases . the raised brown naevus , on the other hand , was found in 93% of control subjects and in only 7% of cases . the target naevus was identified in 7.8% of the cases and in 16.1% of the controls . the association small brown + small black is equally distributed between the two groups : 27% of the cases and the controls . the presence of the target naevus varies in the two groups when it is the only type present and , when in association with others , more predominantly in cases ( target : 16% of cases and 8% of controls ; target + small brown : 16% of cases and 7% of controls ; target + small brown + small black : 10% of cases and 3% of controls ; target + small brown + small black + large brown : 8% of cases and 2% of controls ) . the presence of the small brown does not seem to be associated with cm ( small brown or 2.05 ; ci 1.04.42 ) . the risk increases when there is also the target variant ( target or 3.65 ; ci 1.49.7 ) . the raised brown naevus , on the other hand can be considered as protective for the development of cm ( raised nevus or 0.2 ; ci 0.030.8 ) . it can be seen that among subjects with an equal number of naevi , a greater number of variants corresponds to a greater cutaneous melanoma risk . the results were relative to the variants indicated in table 3 . with an equal number of naevi , the combination of only two variants was found to be of slight significance : ( small black + large brown or 3.5 ; ci 1.39.2 ) ; ( target + large brown or 3.6 ; ci 1.111.2 ) ; ( small black + large brown + large black or 5.5 ; ci 1.421.7 ) ; ( target + small brown + small black or 5.9 ; ci 1.523.2 ) indicating that apart from the number of naevi , naevus variants might influence the degree of risk . on the other hand the or does not change when the small brown variant is added to the association of small black and small brown ( small brown + large brown + small black ( or 3.5 ; ic 1.39.5 ) , small black + large brown ( or 3.5 ; ic 1.39.2 ) ) . analysing these data , it can be seen that a further significant element is provided by the specific impact of the different variants : the data indicate that the presence of the very common small brown naevus , does not modify the or , whereas the target naevus requires more careful evaluation ( or 3.65 ; ci 1.49.7 ) . moreover , the association of the target naevus with other variants , increases the degree of risk [ target + small brown or 5.25 ; ci 1.815.4 ] ; [ target + small brown + small black or 5.9 ; ic 1.523.2 ] ; [ target + small brown + small black + large brown or 5.0 ; ic 1.122.4 ] . our study indicates that some variants and/or variant combinations , do not significantly increase risk . ( fried egg or 0.4 ; ci 0.081.86 ; p = 0.23 ) ; ( unna or 0.94 ; ci 0.461.90 ; p = 0.8 ) ; ( meischer or 1.26 ; ci 0.52.9 ; p = 0.6 ) . the self - assessment of pigmented lesions remains an important step in the prevention of skin melanoma . in an australian study , carried out to determine the correspondence between the patient 's and the dermatologist 's naevus check , it was shown that the number of pigmented lesions is always underestimated by the patient , but agreement increases for atypical melanocytic naevi ; however , the role of the atypical naevus has been the subject of much debate in the literature and elsewhere , and , of course , it is not so easy to diagnose clinically . our study aims to introduce the differentiation of naevus variants among the monitoring criteria of risk factors for cutaneous melanoma . the results show that even if the total number of naevi represents a risk factor for the development of cutaneous melanoma , orientation in the naevus panorama of a specific subject requires the identification of several naevus variants . the most common naevus variants , such as dermal naevi and homogeneously pigmented naevi ( small and large brown or small and large black naevi ) can not alone represent a risk marker ; however the combination of two or more variants becomes a significant risk marker , and their combination in a person thus enables the identification of groups of subjects as reference targets potentially at risk of developing cutaneous melanoma . on the other hand , people presenting the target naevus in association with other naevus variants , have a major rearrangement and activity of pigmented lesions , and therefore require a more rigorous follow - up procedure . the greater variability of this variant is perhaps also expressed through its greater clinical inhomogeneity . this introduces a further aspect of our results : it is important to consider carefully not only the non - uniformity of a single type but also the general non - uniformity of the whole naevus panorama . the data actually available in the literature indicate that when the total number of naevi present in a subject increases , the risk of developing cutaneous melanoma also increases . a further datum emerges from our study : when two subjects present a similar number of naevi , the greater risk for cutaneous melanoma is linked to the greater number of naevus variants . subjects having a moderate number of lesions but presenting clinical inhomogeneous pigmentation are included in the group of subjects at risk . conversely , subjects with an elevated number of naevi but with a homogeneous panorama and low variability , can be observed with greater tranquillity , without however neglecting other risk factors . the classification of naevus in variants can be an indicator of clinical predictivity particularly in subjects who present naevi and especially an elevated number of naevi . furthermore , some variants considered singularly can play a key role in determining the clinical picture of the subject and are to be evaluated with great attention . consider , for example , naevi such as the small brown naevus whose presence or absence appears to be totally uninfluential , whereas the presence of others , such as the bullseye naevus , can greatly modify the dermatologist 's attitude toward the patient . head and neck squamous cell carcinoma ( hnscc ) is one of the most prevalent and lethal cancers worldwide ( argiris et al . , 2008 ) . despite recent advances in early detection , surgical techniques , radiation and chemotherapeutic regimes ( temam et al . 2009 ) , the survival rate and quality of life for patients with hnscc has only marginally improved over the past few decades ( haddad and shin , 2008 ; masuda et al . , 2011 ) . there is a desperate need for better biomarkers , to identify patients at high - risk for treatment failure , as well as novel concepts for a more effective and less toxic therapy for patients with advanced tumors ( hunter et al . head and neck squamous cell carcinoma has a strong epidemiologic background ( rodriguez et al . , 2004 ; dsouza et al . , 2007 ; furniss et al . , 2009 ) including environmental and lifestyle factors . the major risk factors for hnscc are tobacco and alcohol abuse ; however , the escalating incidence of oropharyngeal carcinoma ( oropharyngeal squamous cell carcinoma , opscc ) are not explained by increases in either smoking and alcohol consumption suggesting that atypical behavioral and environmental factors are also involved ( ernster et al . , 2007 ) . importantly , a subgroup of opsccs has causally been linked to infection with high - risk types of human papillomavirus ( hpv ) , especially hpv16 ( gillison et al . , 2008 ; pai and westra , 2009 ) . hpv infection has been established as a causative agent for almost 70% of opsccs and 2025% of all hnsccs ( gillison et al . , 2000 ; , 2005 ; kreimer et al . , 2005 ; reimers et al . , 2007 ; hoffmann et al . , , hpv - related tumors have been shown to be a new tumor entity in itself , with distinct clinical and histopathological features , including younger median age , oropharyngeal primary anatomical site , small tumor size , high lymph node involvement , as well as a lack of typical risk factors , and increasing incidence over the last decades at variance with decreasing total hnscc incidence . furthermore , hpv - related tumors do not show significant keratinization and they have a prominent basaloid morphology . the differences between hpv - related and hpv - negative tumors have been reviewed in several excellent recent reviews ( argiris et al . , 2008 ; psyrri and dimaio , 2008 ; leemans et al . , 2011 ) . intriguingly , patients with a hpv - positive tumor have improved performance status and a better prognosis compared to hpv - negative patients , even though the former patients were more likely to present with regional lymph node metastasis at the time of diagnosis ( curado and hashibe , 2009 ; pai and westra , 2009 ) . in general , hpv - positive patients are highly curable with ionizing radiation with or without chemotherapy , and have better overall and disease - free survival compared to patients diagnosed with hpv - negative tumors ( figure 1 ) ( fakhry et al . , 2008 ; ang et al . , 2010 ) . however , in several studies hpv dna - positive opsccs were heterogeneous in both biological and clinical behavior , possibly due to differences in viral load and/or viral oncogene expression ( paz et al . , 1997 ; andl et al . , 1998 ; , 2001 ; van houten et al . , 2001 ; mellin et al . , 2002 ; braakhuis et al . , 2004 ; lindquist et al . , 2007 ; reimers et al . hpv - related tumors are distinct from non - hpv - related tumors in biological and clinical features , including response to radio- and chemotherapy and clinical outcome . functional genome and transcriptome studies strongly suggest that hpv - related hnsccs comprise a disease entity that differs from hpv - negative tumors in regard to distinct molecular characteristics . in the past , several groups used comparative genomic hybridization ( cgh ) to identify genomic imbalance in the pathogenesis of hnsccs , focusing on hpv - related and hpv - negative tumors ( dahlgren et al . , 2003 ; smeets et al . , 2006 ; klussmann et al . , 2009 ; wilting et al . , 2009 ) . despite tumor - associated genomic alterations , which are common in both groups , these studies highlighted that per tumor , the total amount of chromosomal alterations , as well as amplifications was significantly lower in the hpv - related than in the hpv - unrelated tumors ( table 1 ) . this finding is consistent with the assumption that due to the inactivation of the tumor suppressor proteins p53 and prb by the viral e6 and e7 oncoproteins , respectively , the number of required genetic alterations for a malignant phenotype is lower in hpv - related carcinogenesis . consequently , a better clinical outcome of patients with hpv - related tumors could simply be a reflection of less genetic aberrations at the time of treatment . however , specific hpv - related genetic alterations have also been identified ( table 1 ) some of which were connected with improved survival . for example , 16q loss , which was predominantly identified in hpv - related opscc , was a strong indicator of favorable outcome and none of these patients had a tumor recurrence ( klussmann et al . , 2009 ) . additionally , hpv - negative tumors demonstrate a loss at 18q12.123 , in contrast to a gain in hpv - positive tumors ( smeets et al . , 2006 ) . it will be a major challenge in the future to identify candidate genes at the affected genomic sites , and to investigate their contribution to treatment resistance and tumor cell survival in both patient cohorts as well as preclinical model systems . alteration in the genome of hnsccs : genomic aberrations characteristic for hpv - related opscc . to uncover the genetic origin and the mutational landscape of hnscc , two recent studies applied gene copy number and whole exome sequence analysis on specimens of tumor normal pairs , including samples from patients with hpv - related tumors ( agrawal et al . , 2011 ; stransky et al . , 2011 ) . in both studies , the majority of tumors exhibited a mutational profile consistent with tobacco exposure , and there were obvious differences in the genetic landscapes of hpv - related and hpv - negative hnsccs . for instance , tumors that were negative for hpv had more mutations than hpv - positive tumors , independently of the smoking status . this finding is in line with the data derived by cgh analysis and further confirms major differences in the pathogenesis of hpv - related and hpv - negative tumors . furthermore , both studies observed an inverse correlation between the hpv status and tp53 mutations , as already shown previously ( wiest et al . , 2002 ; westra et al . , in addition to genomic alteration and gene mutation , aberrant dna methylation is widely recognized as a mechanism in the progression of hnscc ( hasegawa et al . , 2002 ; schmezer and plass , 2008 ) , but our knowledge on virus - induced changes in dna methylation of the host genome as well as other modes of epigenetic alterations , as part of the divergent carcinogenic pathway , is limited . nevertheless , the identification of distinct epigenetic profiles in hpv - related and hpv - negative tumors should provide clues to novel drug targets for development of individualized therapeutic strategies . currently , there is a limited number of studies which address the differences in epigenetic alteration between hpv - related and hpv - negative tumors . furthermore , the published data has largely evaluated changes in promoter methylation of a limited number of candidate genes , most of which were selected based on functional relevance in several human malignancies ( table 2 ) . a more recent study by sartor et al . ( 2011 ) applied an integrative approach combining genome - wide methylation and gene expression profiling to characterize the molecular differences between hpv - related and hpv - negative tumor cell lines . they found a higher overall dna methylation in hpv - positive compared to hpv - negative cell lines . this hpv - dependent difference was consistent with findings in primary tumor samples and in line with data from richards et al . moreover , an inverse correlation between gene promoter methylation and expression of affected genes was found in hpv - positive cells ( sartor et al . , 2011 ) . although differentially methylated loci found in tumor cell lines were significantly correlated with primary tumor dna methylation levels , a recent study demonstrated major changes in the dna methylation pattern between cultured cells and primary tumors ( hennessey et al . , 2011 ) . although these findings may direct alterations in the epigenome to different disease pathways involved in hpv - related versus hpv - negative tumors , critical validation in larger hnscc tumor sample sets is required . genes / proteins , specific gene(s ) or protein(s ; or total number ) of genes investigated in each study ; t , total number of tumors / patients included in the study ; cl , number of different cell lines included in the study ; hpv , consideration of hpv status in the study . global gene expression profiling comparing hpv - related and hpv - negative tumors revealed distinct transcriptome signatures , reflecting underlying heterogeneous somatic , genetic , and epigenetic alterations . as it would have been expected , all published studies identified differentially expressed genes that regulate dna replication and cell cycle progression ( slebos et al . , 2006 ; 2009 ) , while some also describe aberrant regulation of genes involved in viral defense and immune response ( schlecht et al . it is worth to mention that some differentially expressed genes are already known to modulate resistance to radiation and chemotherapeutic drugs , but experimental evidence of their impact on the favorable outcome of patients with hpv - related tumors are still missing . moreover , dysregulation of genes associated with processes of natural killer cell - mediated cytotoxicity , activation of the toll - like receptor , jak stat signaling pathways ( jung et al . , 2010 ) , as well as immune response and inflammation ( schlecht et al . , 2007 ) , are of particular interest , assumpting that the mechanisms underlying the clinical differences of hpv - related and hpv - negative tumors may involve the combined effects of immune surveillance to viral - specific tumor antigens , an intact apoptotic response to radiation , and the absence of widespread genetic alterations ( pai and westra , 2009 ) . yet , the correlation between altered cytokine and interferon signaling and the hpv status further supports the conclusion that improved survival of patients with hpv - related tumors may not only depend on tumor cell intrinsic features , but also could be due to different activation of stromal cells within the tumor microenvironment ( figure 2 ) . intrinsic and extrinsic features of hpv - related hnscc tumors potentially involved in the molecular mechanisms underlying improved disease course . hpv - related tumors have a better survival likely due to the combined effects of phenomena occurring in epigenome , genome , and transcriptome . these events drive alternations in intracellular signaling networks and along with complex interactions of malignant cells with components of tumor microenvironment including immune cells , they orchestrate the improved response to treatment and the favorable outcome . micrornas ( mirnas ) are small non - coding rnas , which represent another mode of gene regulation by influencing mrna translation or decay . several studies reported altered expression of distinct mirnas in the pathogenesis of hnscc ( babu et al . , 2011 ) . two recent studies characterized the influence of hpv on global mirna expression in hnscc cell lines ( wald et al . , 2011 ) , and tumor biopsies of hnscc patients ( lajer et al . , 2011 although , the overlap on common deregulated mirnas is only minor ( which may be due to the use of cell lines versus tumor biopsies ) increasing our understanding of the virus - targeted mirnas may provide further insight into molecular mechanisms for the distinct clinical behavior of hpv - related tumors . several intrinsic hallmarks of tumor cells have been described , including : ( i ) sustaining proliferative signaling , ( ii ) evading growth suppressors , ( iii ) resisting apoptosis , ( iv ) enabling unlimited replication , ( v ) inducing angiogenesis , and ( vi ) activating invasion and metastasis ( hanahan and weinberg , 2000 , 2011 ) . however , which of these capabilities differ between hpv - related and hpv - positive tumor cells and their likely contribution to the differential clinical outcome , is still a matter of debate . for instance , while patients with hpv - related hnscc respond better to radiotherapy , there is controversial data concerning whether hpv - related tumor cells are indeed inherently more radiosensitive than hpv - negative cells ( pai and westra , 2009 ) . infection by hpv results in over - expression of the viral oncoproteins and thereby affects many cellular pathways as well as genome stability . the oncogenic potential of hpv involves the expression of the viral e6 and e7 oncoproteins , which disrupts the tumor suppressive p53 and prb signaling pathways , respectively , and thereby modulate cell cycle progression and survival of infected keratinocytes ( zur hausen , 2002 ) . continued expression of viral oncogenes is necessary for neoplastic progression and the malignant phenotype of an hpv - associated tumor . transcriptionally active viral dna has been shown to be biologically and clinically relevant ( figure 1 ) ( wiest et al . , 2002 ; hpv - related hnsccs have less frequent p53 or prb mutations , which often occur in hpv - negative tumors ( marur et al . , 2010 ) . the tumor suppressor p53 acts as a central mediator of the cellular stress response , including dna damage induced apoptosis and cellular senescence by chemotherapy and ionizing radiation . therefore , the presence of wild - type p53 in hpv - related tumors could , at least in part , confer accelerated chemo- and radiosensitivity compared to hpv - negative tumors with p53 mutations . ( 2006 ) found that the clinical behavior of hnscc patients critically depends on the hpv and the p53 status . they provided clinical evidence that only patients with hpv - related tumors and wild - type p53 exhibit an improved survival , while patients with hpv - related tumors and mutant p53 have an unfavorable outcome , similar to hpv - negative hnscc patients . however , in a more recent publication pang et al . ( 2011 ) showed that expression of the viral e6 oncogene sensitizes tumor cells to radiation - induced apoptosis , and that the e6i , and not the e6ii isoform , is sufficient to mediate this effect . interestingly , e6i was shown to be a weak inhibitor of p53 transactivator activity , and the study supports a role for e6i in the enhanced responsiveness of hpv - positive opsccs to p53-independent radiation - induced cell death . the finding that nuclear survivin is associated with hpv - negative compared to hpv - related opscc and correlated with a poor clinical outcome further suggests a fundamental role in cell death regulation ( preuss et al . , 2008 ) . survivin is an inhibitor of apoptosis ( iap ) and nuclear survivin is believed to control cell division , whereas cytoplasmic survivin is considered cytoprotective ( stauber et al . , 2007 ) . hnscc cases with nuclear survivin and aurora - b expression exhibited marked malignant behaviors whereas both survivin and aurora - b knockdown inhibited cell growth and tumor formation ( qi et al . , 2010 ) . furthermore , survivin also plays a critical role in mediating resistance to radiation in part through suppression of apoptosis . more recently , gubanova et al . investigated whether the mechanism underlying increased sensitivity of hpv - positive tumors to radiation may be connected to the impaired ability of the cancer cells to respond to dna damage . they found that expression of smg-1 ( suppressor with morphogenetic effect on genitalia ) is diminished in hpv - positive tumors due to gene promoter hypermethylation ( gubanova et al . , 2012 ) . smg-1 belongs to a family of phosphoinositide 3-kinase ( pi3-kinase)-related kinases ( pikks ) involved in the maintenance of genome integrity via genotoxic stress response pathways and plays an important role in the dna damage response network ( shiloh , 2003 ; brumbaugh et al . , 2004 ; gewandter et al . , low smg expression correlated with positive hpv status and improved patient survival , whereas depletion of smg-1 in hnscc cells resulted in their increased sensitivity to radiation ( gubanova et al . , the impact of hpv e6 and e7 oncoproteins in in vitro cell sensitization to radiation remains controversial , since other studies have suggested that hpv16-positive cervical cancer cells ( padilla et al . , 2002 ) and hnscc cells ( hoffmann et al . , 2008 ) as well as hpv - negative cells with ectopic e6 and e7 expression ( hampson et al . , 2001 ) are resistant to treatment with cytotoxic drugs and radiation . it is worth noting that increased expression of another iap molecule , namely ciap2 , was reported to be significantly associated with hpv16-related hnsccs ( mansour et al . , 2012 ) . viral e6 oncoprotein has been shown to induce ciap2 expression through either the nf-b or egfr / pi3k / akt signaling pathways . ciap2 confers less sensitivity to apoptosis in hpv16 e6-immortalized human oral keratinocytes ( yuan et al . , 2005 ; james et al . , 2006 ) , and resistance to cisplatin in hpv - infected lung cancer ( wu et al . , 2010 ) , or 5-fu treatment of hnscc cell lines ( nagata et al . , 2011 ) . furthermore , high ciap2 expression was associated with poor clinical outcome , at least in patients with oral squamous cell carcinoma ( nagata et al . , 2011 ) . although there is a growing body of in vivo and in vitro data supporting the assumption that hpv - related tumors have a better survival due to a higher sensitivity to radiation and chemotherapy , it is difficult to conclusively attribute the improved clinical outcome only to the intrinsic features of the hpv - infected cells . it is more likely that a complex paracrine interaction among intrinsically mediated key pathways and the tumor microenvironment , including cells of the immune system , may determine the accelerated sensitivity to either radiation or chemotherapy ( figure 2 ) . accordingly , hpv - positive tumors have been postulated to be more susceptible to immune surveillance of tumor - specific antigens ( argiris et al . , 2008 ) , but so far only a few studies have explored endogenous host immunity to viral proteins in hnscc patients , in order to elucidate pathogenetic and treatment response mechanisms , and to gain insights into novel immunotherapeutic strategies . cytotoxic therapies including radiation induce potentially immunogenic cell death , releasing tumor - associated antigens , as a danger signal to the immune system ( shinohara and maity , 2009 ) . the efficacy of radiotherapy is determined not only by tumor cell properties , but also endothelial cells and infiltrating immune cells within the tumor environment ( shinohara and maity , 2009 ) . high levels of tumor - infiltrating lymphocytes ( tils ) have been identified as a favorable prognostic biomarker in various cancers ( pages et al . , 2010 ) , including hnscc ( brandwein - gensler et al . , 2006 ; uppaluri et al . , 2008 ) . however , their exact role on hnscc , especially in the context of hpv - related tumors remains to be elucidated . in this context , hoffmann et al . found that the frequency of hpv16-e7-specific t cells in the serum did not differ between hnscc patients and healthy controls . however , higher frequencies of t cells specific for hpv16-e7 epitopes were observed in hnscc patients with hpv - related tumors ( hoffmann et al . , 2006 ) , which is in line with previous findings ( albers et al . , 2005 ) the fact that hpv16-e7 epitopes are apparently immunogenic raises the question as to why antigen - specific t cells in hnscc patients are unable to eliminate hpv - related tumors without treatment . one possible explanation was provided by the finding that e7-specific cytotoxic t cells obtained by in vitro stimulation only recognize human leukocyte antigen hla - a201 positive and hpv16-transformed hnscc cells after pretreatment with ifn - gamma ( albers et al . , 2005 ) . further analysis demonstrated that this cell line had little or no expression of critical components of the hla class i antigen - processing machinery , which were subsequently up - regulated upon ifn - gamma treatment . in addition , the antigen - processing machinery components were also found to be downregulated in hpv - related tumors compared to adjacent normal epithelium . it currently remains uncertain as to whether impaired antigen presentation in hpv - related tumors may be reversed by radiation or chemotherapy and thereby induces an antitumor immune response . in a retrospective study , rajjoub et al . ( 2007 ) reported a positive correlation between large numbers of tils ( cd3 ) and lower incidence of metastasis at presentation , regardless of hpv status . however , when the hpv status was taken into account , the correlation between a cd3 count and a lower rate of metastasis was maintained in hpv - positive but not in hpv - negative patients ( rajjoub et al . , 2007 ) , further supporting the crucial role of the adaptive immunity in the favorable clinical outcome of hpv - related tumors . in line with this assumption , the percentage of circulating cd8-positive t cells was significantly higher in patients with hpv - related tumors as compared to hpv - negative hnscc patients ( wansom et al . , 2010 ) . furthermore this prospective study revealed a positive correlation between a higher percentage of cd8-positive t cells and the response to induction chemotherapy and complete tumor response after chemotherapy . ( 2012 ) reported that the degree of t cell infiltration in tumor tissue is associated with improved survival but is independent of the hpv status , similar to previous findings ( rajjoub et al . they concluded that more comprehensive studies with larger numbers of patients combined with functional analysis of individual subsets may be necessary to detect significant differences in local immunity in hpv - related hnscc . there is increasing evidence that radiotherapy modulates immune response and that the underlying mechanism behind the better prognosis of hpv - positive tumors may be enhanced immune response following radiotherapy ( vu et al . , 2010 ) . ( 2009 ) found that both hpv - positive human and murine transformed cell lines cells were even more resistant to radiation and cisplatin treatment in vitro compared with hpv - negative counterparts . however , hpv - positive cells were more sensitive to radiation in a syngeneic mouse tumor model , and complete clearance was found at 20 gy , in contrast to hpv - negative cells , which showed persistent growth ( spanos et al . , 2009 ) . intriguingly , neither radiation nor cisplatin therapy cured hpv - positive tumors in immune - incompetent mice , strongly supporting a crucial role of the immune system in the therapeutic sensitivity of hpv - positive tumors . in line with this assumption , williams et al . ( 2009 ) showed in a syngeneic mouse tumor model that only hpv - positive tumor cells induce an antigen - specific antitumor response , which was mediated by cd4- and cd8-positive t lymphocytes . innate immune cells may also contribute to the favorable clinical outcome of patients with hpv - related opsccs . as basal keratinocytes are the primary target of hpv infection ( feller et al . , 2009 ) , innate immunity represents the first line of defense against invading viruses . keratinocytes express several pathogen recognition receptors ( prrs ; pries et al . , 2008 ) , and their engagement by pathogen - associated or danger - associated molecular patterns leads to direct activation of the pro - inflammatory transcription factors , and/or activation of an anti - viral type i interferon ( ifn ) response ( rincon - orozco et al . several reports highlighted a major impact of viral oncoproteins on the regulation of pro - inflammatory mediators and anti - viral responses , suggesting that hpv has developed strategies to evade the innate host immunity ( schlecht et al . s100a9 were downregulated in hpv - immortalized cells ( delloste et al . , 2008 ) . vice versa , the s100a8/s100a9 protein complex has been shown to inhibit phosphorylation of the viral e7 oncoprotein by the casein kinase ii and may play an important role in hpv - infected epithelial cells to counteract the oncogenic activity of e7 ( tugizov et al . , 2005 ) . s100a8 ( mrp8 ) and s100a9 ( mrp14 ) belong to the large family of s100 proteins , which are low molecular weight proteins sharing conserved structural motifs that consist of two ef - hand ca - binding domains . s100a8 and s100a9 form a functional heterodimer ( also known as calprotectin ) and have been identified as important endogenous damage - associated molecular pattern proteins that critically contribute to pathological conditions of acute and chronic inflammation , and may therefore represent a key player in the setting of inflammation - associated carcinogenesis ( gebhardt et al . , 2006 ; it is worth mentioning , that inflammatory signals and the establishment of a pro - inflammatory environment are increasingly linked to the induction and promotion of tumorigenesis ( balkwill and mantovani , 2001 ; coussens and werb , 2002 ; grivennikov et al . , 2010 ) . one of the most extensively studied signaling network in inflammation - associated cancer is the nf-b pathway ( ben - neriah and karin , 2011 ) , and there is increasing evidence for its crucial role in the pathogenesis of hnscc including hpv - related tumors ( molinolo et al . , 2009 ) . analysis of hpv16-transformed cell lines revealed that functional components of the nf-b signaling pathway were sequestered in the cytoplasm , or became inactivated by viral e6 and e7 expression , accompanied by reduced expression of cytokines and chemokines ( guess and mccance , 2005 ; havard et al . , 2005 ) . however , it will be a challenge for the future to address the question of whether changes in pro - inflammatory signaling pathways , such as nf-b , and the establishment of an activated pro - tumorigenic environment are causally linked to the increased sensitivity to therapy of patients with hpv - related tumors and their improved survival . currently , hpv - positive opscc dominates the head and neck oncology landscape , and its escalating incidence has a strong impact on clinical and translational research . although it is well accepted that hpv - related tumors represent a distinct histopathological and biological entity with a favorable clinical outcome , we have just now begun to establish the molecular principles underlying improved survival . as a consequence of modern genome - wide approaches , our understanding of the fundamental mechanisms and biological processes involved in the pathogenesis of hpv - related and hpv - negative hnsccs has rapidly increased . however , most of the functional genomics data was derived from primary tumor samples largely devoid of the important information on differences in response to current treatment options . furthermore , the use of several cell culture and mouse tumor models have produced conflicting results which neither mimic the complex interaction between the tumor and its activated tumor microenvironment nor the etiology of hpv - related tumors . hence , there is an urgent need for an appropriate and more sophisticated preclinical model system(s ) . the mechanisms underlying the clinical differences between hpv - related and hpv - negative tumors may involve the combined effects of tumor cell intrinsic features and paracrine interactions between tumor cells and stromal cells of the activated tumor microenvironment , especially cells of the adaptive and innate immunity . unraveling the molecular nature of improved clinical outcome of hpv - related hnscc will not only support personalized risk assessment but will also unravel new concepts for a targeted therapy for hnscc patients with hpv - negative tumors . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

background . the incidence of cutaneous melanoma is rapidly increasing in europe . active research is directed toward the identification of naevi as a risk factor . objective . the aim of our case - control study was to observe different numbers of moles and different mole typology associations in order to evaluate clinical predictivity and to establish a new classification for some naevus variants . methods . a case - control study was carried out , enrolling 64 cases affected by melanoma and 183 controls , between october 2009 and february 2011 . each patient was interviewed and subjected to clinical examination . the resulting data were analysed using the statistical elaboration program spss 16.0 . results . the association of target naevus with other variants increases the degree of risk ( target + small brown odds ratio 5.25 ; confidence interval 1.815.4 ) ; ( target + small brown + small black + large brown odds ratio 5.0 ; confidence interval 1.122.4 ) . therefore , other variants and/or other variant combinations do not significantly increase risk . conclusion . people presenting two naevus variants in association with other naevus variants seem to run a major risk . the general nonuniformity of the whole naevus panorama should be carefully considered . human papillomavirus ( hpv ) infection is well established as an etiological agent responsible for a number of pathologies affecting the stratified epithelia of skin and anogenital sites . more recently , the infection by ( mucosal ) high - risk hpv types has also been found to be causally associated with squamous cell carcinoma in the head and neck region ( hnscc ) , especially in the oropharynx . intriguingly , hpv - related oropharyngeal squamous cell carcinomas ( opscc ) represent a distinct clinical entity compared to hpv - negative tumors with particular regard to treatment response and survival outcome . the association between hpv infection and opscc may therefore have important implications for the prevention and/or treatment of opscc . the improved survival of patients with hpv - related tumors also raises the question , as to whether a better understanding of the underlying differences may help to identify new therapeutic concepts that could be used in targeted therapy for hpv - negative and improved therapy for hpv - positive cancers . this review summarizes the most recent advances in our understanding of the molecular principles of hpv - related opscc , mainly based on functional genomic approaches , but also emphasizes the significant role played by the tumor microenvironment , especially the immune system , for improved clinical outcome and differential sensitivity of hpv - related tumors to current treatment options .

crohn 's disease ( cd ) is a form of inflammatory bowel disease ( ibd ) affecting the entire gastrointestinal ( gi ) tract [ 14 ] . cd is characterized by transmural inflammation of gi wall in a noncontiguous pattern anywhere from the mouth to the anus [ 5 , 6 ] . it is believed that , in genetically predisposed individuals , interaction between local microbiota and the intestinal mucosa can cause chronic inflammation and ulceration [ 79 ] . a key pathological feature of cd is a granulomatous inflammatory response , characterized by the presence of epithelioid and multinucleated giant cells as well as macrophage infiltration [ 10 , 11 ] . it is believed that the onset and development of the inflammatory reaction depend on persistence of an inciting agent , resulting in a chronic inflammatory milieu and perpetuating a complex immune reaction subsequent to the local granulomatous response . persistent inflammation and immune activation are believed to be the leading causes of tissue necrosis and fibrosis . newly diagnosed cd usually presents with diarrhea , abdominal pain , fever , fatigue , stomatitis , and weight loss . in more advanced cases , strictures and fistulas may develop , changing the clinical presentation to include severe abdominal pain , distension , bloating , vomiting , perianal fistulae , and abscesses . cd can be diagnosed at any age ; however disease progression and clinical presentation frequently differ in young and adult patients . cd in children and adolescents tends to have a more severe clinical manifestation often with development of strictures or fistulae [ 1 , 3 , 14 ] . in contrast , later onset of cd is often characterized by a milder course with a lower frequency of complications [ 13 , 14 ] . furthermore , it has been shown that the childhood - onset cd is more likely to require immunosuppressive therapy and has a higher frequency of surgery as compared to cd diagnosed in adulthood [ 4 , 13 ] . additionally , juvenile and adolescent onset cd has more frequent involvement of the small bowel , while adult cd is generally more likely to be colon - associated [ 1519 ] . an altered immune response , together with genetic and environmental factors , may govern the timing of cd onset . there is a higher frequency of cd family history in patients diagnosed before the age of 20 relative to those diagnosed later in life , suggesting that genetic predisposition plays a role in early onset [ 13 , 20 ] . for example , it has been shown in adult cd that higher levels of serum antibody reactivity towards increasing amount of microbiota in small intestine correlate with a greater frequency of complications [ 21 , 22 ] . a similar observation has been made in a pediatric cohort [ 23 , 24 ] . furthermore , cd patients have been shown to have increased numbers of circulating th17 lymphocytes and upregulation of il17 transcriptional activity in intestinal mucosa . this indicates that the progression of cd is characterized by an exacerbated th17 response , which may explain the continuing nature of the disease . immune mechanisms are believed to play a role in the pathogenesis of adult and juvenile cd , with cytokines being essential to establish and maintain the immune response . however , little is known about differences in serum cytokine profiles between juvenile and adult cd cases . we have found that all cd patients have upregulated serum cxcl10 levels , regardless of the age of diagnosis and the stage of the disease . also , we have demonstrated upregulation of il-1b and il-6 in juvenile cd , with no changes detected in levels of gm - csf and ifn-. in contrast , the adult cd serum profile was characterized by upregulation of gm - csf and ifn- , as well as il-6 and il-1b , when compared to controls . it has been shown that gm - csf and ifn- promote a th1 immune response , while il-6 is essential for activation of th17 lymphocytes [ 2628 ] . therefore , this suggests that the mechanisms of pathogenesis differ in adult and juvenile cd patients . clinical surplus serum samples from 64 patients diagnosed with cd were utilized in this study ; 12 juvenile patients ( 6 male , 6 female ; average age 14.0 2.1 ) hospitalized in the volga region federal medical research center of the ministry of public health ( nizhny novgorod , russia ) and 52 adult patients ( 26 male , 26 female , average age 35.1 13 ) hospitalized in the department of gastroenterology of republican clinical hospital ( kazan , russia ) . diagnosis of cd was established in all patients based on clinical presentation and was verified by upper gi endoscopy . additionally , a small number of samples ( 6 ) were collected from juvenile patients during their period of clinical improvement . age and gender matched control samples were collected from 10 juvenile ( 5 male , 5 female ) as well as from 23 adult ( 13 male , 10 female ) controls . all serum samples were stored at 80c until use . the study was approved by the ethics committee of kazan state medical university ( protocol 23 ) and the ethics committee of volga region federal medical research center of the ministry of public health nizhny novgorod ( protocol 4 ) . serum cytokine levels were analyzed using single - plex sets for il-1b , il-2 , il-5 , il-4 , il-6 , il-8 , il-10 , il-12p40 , gm - csf , ifn- , cxcl10 , and tnf- ( bio - rad , hercules , ca , usa ) following the manufacturer 's instructions . serum aliquots ( 50 l ) were used for analysis , with a minimum of 50 beads acquired per analyte . data collected was analyzed with masterplex ct control software and masterplex qt analysis software ( hitachi software , san bruno , ca , usa ) . statistical analysis was performed using the statistica 7.0 software package ( statsoft , tulsa , ok , usa ) . levels of 12 cytokines were analyzed in juvenile cd serum collected during both the acute stage and remission . acute serum was characterized by upregulation of il-1b , il-6 , cxcl10 , and tnf- levels compared to controls ( table 1 ) . in contrast , no changes in il-2 , il-5 , il-4 , il-10 , il-12p40 , gm - csf , and ifn- were found in acute juvenile patients compared to controls . median values of il-8 in acute serum were upregulated ( 1415.30 [ 997.12654.4 ] versus 2777.00 [ 611.07382.5 ] ) ; however , differences were not statistically significant from controls . during remission , serum levels of cxcl10 ( p < 0.05 ) and il-1b ( p < 0.05 ) remained significantly upregulated in juvenile cd patients compared to controls ( table 1 ) . the median value of il-1b during remission was higher than that in acute patients ( 832.75 [ 481.91016.0 ] versus 550.00 [ 192.0784.3 ] ) , suggesting an upward trend in cytokine production ( p = 0.067 ) . il-6 and tnf- serum levels were significantly higher during the acute stage but then declined in remission and remained comparable to controls . similarly , in acute stage patients , median values of il-8 were upregulated during remission ( 1415.30 [ 997.12654.4 ] versus 4 194.0 [ 411.97 220.9 ] ) ; however , differences were not statistically significant from controls . several leukocyte surface markers were examined including cd3 ( lymphocytes ) , cd4 ( t - helper ( th ) lymphocytes ) , cd8 ( cytotoxic t lymphocytes ( ctl ) ) , cd16 ( natural killer ( nk ) cells ) , cd19 ( b lymphocytes ) , and cd56 ( nk cells ) ( table 2 ) . increased numbers of cd16+cd56 + leukocytes were characteristic for acute stage juvenile cd compared to remission ( 0.26 0.08 versus 0.14 0.02 10/l ; p < 0.02 ) ( table 2 ) . when the cd4/cd8 ratio was analyzed , two groups of acute stage patients could be identified having a ratio higher or lower than 2.0 . since cytokines play a central role in differentiation and proliferation of leukocyte cd4 and cd8 subsets , we analyzed serum cytokines in the cd4/cd8 subgroups of juvenile cd patients ( table 3 ) . acute stage patients with a cd4/cd8 ratio above 2.0 were characterized by upregulated serum levels of il-1b , cxcl10 , tnf- , and il-10 . additionally , high cd4/cd8 ratio patients had a greater il-8 median value compared to those with a cd4/cd8 ratio below 2.0 ( 4161.50 [ 2956.08372.8 ] versus 1760.00 [ 257.46014.1 ] ) ; however , these differences were not significant . interestingly , cxcl10 levels were significantly higher in juvenile cd patients with a cd4/cd8 ratio below 2.0 than those with a cd4/cd8 ratio above 2.0 . serum cytokine profiles in adult cd patients depended on the stage ( acute or remission ) of the disease . for example , a subset of serum cytokines , including il-1b , il-6 , gm - csf , ifn- , and cxcl10 , were upregulated in the acute stage of cd relative to controls ( table 4 ) and remained upregulated as in remission . however , serum levels of tnf- and il-10 , unchanged in acute stage the disease , were downregulated during remission ( table 4 ) . it should be noted that the il-12 median value was 20-fold higher in adult cd patients in either acute stage or remission than controls , though not statistically significant . although these differences were not significant , there was a strong trend towards upregulation in adult serum , both during the acute stage and the remission . interestingly , in contrast , the serum level of il-12 in juveniles in both the acute stage and the remission did not differ from controls . further , in serum of acute stage , adults with cd both gm - csf and ifn- were upregulated , whereas these cytokines remained unchanged in acute juvenile cd patients . this strongly suggests that several cytokines , including gm - csf , ifn- , and il-12 , play a role in the pathogenesis of cd in adult patients . cd is a chronic inflammatory bowel disease affecting the integrity and function of the gi tract . the disease has a bimodal age distribution , with 2025% of cases diagnosed early in life ( childhood and adolescence ) , while 70% of cases are diagnosed in adulthood . clinical symptoms of cd include abdominal pain , diarrhea , weight loss , and fever . in addition to the common gi symptoms , juvenile cd is often associated with failure to thrive that can include retarded growth , malnutrition , pubertal delay , and bone demineralization [ 31 , 32 ] . studies using animal models have shown that cd pathogenesis is closely associated with a th1 cytokine profile , while activation of the th2 cytokines is more prevalent in the pathogenesis of ulcerative colitis [ 33 , 34 ] . activation of t cell - mediated immunity is linked to the development of epithelioid granulomas , which are pathognomonic to cd . the serum cytokine profile of cd patients is also indicative of activation of a th1 and a th17 type immune response . for example , upregulation of il-17a , il-17f , il-21 , il-22 , il-26 , and ccl20 has been demonstrated in cd cases pointing to activation of th17 type immunity . increased serum levels of tnf- are a hallmark of cd suggesting a fundamental role for this cytokine in disease pathogenesis [ 3739 ] . the role of cytokines in cd was supported by prehn et al . who demonstrated enhanced production of ifn- , the key cytokine for activation of a th1 type immune response , by mucosal t lymphocytes upon stimulation with tnf- . therefore , these data suggest that cytokines play an important role in activating and maintaining the th1 and th17 type immune response , which is indicative of cd . although the role of cytokines in cd pathogenesis is well established , little is known about age differences in cytokine activation in cd patients . juvenile cd is usually characterized by severe clinical manifestations , often including development of strictures or fistulae requiring surgical intervention , whereas adults frequently have a milder clinical presentation with a lower likelihood of developing complications [ 4 , 13 ] . therefore , we sought to determine whether the cytokine profiles in juvenile and adult cd differed . serum levels of 12 cytokines were evaluated during both the acute stage and the remission in juvenile and adult cd patients . an interesting observation was that there was upregulation of cxcl10 cytokine in all patients regardless of age and stage of the disease . although increased cxcl10 serum levels and enhanced expression of cxcl10 in inflammatory bowel tissue has previously been shown in adult cd , here we present the first evidence for upregulation of this chemokine in the serum from juvenile cd cases . cxcl10 is a chemokine known to exclusively recruit activated t lymphocytes and nk cells [ 4143 ] . additionally , it has been demonstrated that cxcl10 targets th1 lymphocytes [ 42 , 43 ] . the essential role of cxcl10 in the gut lymphocyte infiltration and inflammation has been established by grip and janciauskiene . these authors demonstrated that reduced plasma levels of cxcl10 correlate with decreased level of the prominent inflammation marker , c reactive protein . in another study , hyun et al . demonstrated that a reduction in cxcl10 serum levels impairs activation and reduces recruitment of th1 lymphocytes into the gut mucosa and lymphoid tissue therefore , our data support the notion that upregulation of cxcl10 can facilitate th1 lymphocyte infiltration and perpetuate epithelial inflammation in the gut . furthermore , increased serum levels of cxcl10 were found in all patients regardless of the stage of the disease , suggesting chronic th1 lymphocyte activation , which is ongoing even during clinical remission . an interesting observation regarding serum il-8 levels in juvenile and adult cd patients was that , although not significant , the serum level of il-8 was higher ( 1.9 and 2.9 times ) in acute and remission juvenile cd compared to controls . in contrast , serum il8 levels in adult cd remained similar to age - matched controls . il-8 is a pleotropic cytokine , known as the prototype neutrophil attractant , which also activates neutrophils by upregulating phagocytosis and respiratory burst . since inflamed tissue is often infiltrated by activated neutrophils , this suggests a contribution of il-8 to both local and systemic inflammation . although high circulating il-8 is characteristic of inflammation , a lack of change in serum il-8 in some cases of gastroduodenitis has been documented . further investigation demonstrated that although il-8 levels in serum were unchanged , there were increased levels of il-8 transcripts within intestinal tissue [ 50 , 51 ] , suggesting local production of il-8 . therefore , this suggests that lack of changes in il-8 in serum may not always reflect ongoing in situ inflammation . therefore , we propose that in juvenile cd that high serum il-8 may reflect systemic inflammation , whereas , in adult cd , the lack of changes in circulating il-8 level indicates that inflammation could be restricted to bowel tissue . another finding was that the serum level of tnf- was significantly upregulated in juvenile cd compared to controls , while it remained unchanged in adult cases . these data suggest that juvenile cd , relative to adult , is characterized by a stronger inflammatory milieu in the gut . additionally , increased serum il-6 was detected all cd cases , regardless of patient 's age , relative to controls . il-6 is a b cell differentiating factor that , when combined with tgf , promotes the differentiation of th17 lymphocytes . moreover , il-6 is considered an essential cytokine for th17 differentiation of naive t cells . th17 lymphocytes are believed to be pathogenic and are often associated with the severe tissue damage and development of autoimmunity . the mechanisms of th17-associated tissue damage are linked to a breach in the integrity of the intestinal epithelium due to increased production of inflammatory cytokines and matrix metalloproteinases , as well as inhibition of proliferation of human gut epithelial cells [ 56 , 57 ] . interestingly , juvenile cd is characterized by upregulation of the il-6 , while levels of ifn- were unaffected . however , the opposite pattern of cytokine activation was found in adult cd , where only ifn- levels were significantly upregulated . ifn- is secreted by ctl and often used as a marker of the th1 type immune response . the th1 ( il12-ifn- ) and th17 ( il-6-il-17 ) pathways seem to be mutually exclusive , with ifn- and il-17 acting as reciprocal inhibitory cytokines . however , a subpopulation of th17 lymphocytes producing il-17 and ifn- was identified in cd gut tissue by annunziato et al . . the authors suggested that il-17/ifn- positive th17 are important for cd pathogenesis , as these cells were able to support b cell proliferation , had low cytotoxic activity , and were poorly responsive to autologous regulatory t cells . therefore , we hypothesize that the th17 response is characteristic for juvenile and adult cd . however , upregulation of il-17 and ifn- in adult cd suggests that there are more complex mechanisms of pathogenesis relative to juvenile cd . we propose that the th17 ( il-17/ifn- ) , or a combination of th17 and th1 immune responses , play a leading role in pathogenesis of adult cd . studies using tissue biopsies from juvenile and adult cd will help to better understand the role of these th subsets in cd pathogenesis . it has been suggested that gm - csf can alter the course of intestinal inflammation . have shown that gm - csf activated macrophages protect mice from t cell - induced colitis . these authors suggested that gm - csf - activated macrophages facilitate differentiation of th2 lymphocytes by upregulating production of il-4 , il-10 , and il-13 in lamina propria resident leukocytes , which may decrease local inflammation , improve bacterial clearance , and promote healing . in another study , have shown that the presence of anti - gm - csf antibody can serve as marker for the severity of cd . further , these authors demonstrated that elevated anti - gm - csf antibody levels were associated with intestinal stricture , penetration , and resection in cd patients . a similar observation was published by gathungu et al . , where cd patients with elevated anti - gm - csf antibody exhibited increased bowel permeability as compared to those with lower levels of this antibody . it has been suggested that gm - csf neutralization promotes ileac disease by modulating expression of ccl25 by intestinal epithelium and ccr9 by t lymphocytes via nod2-dependent and -independent pathways . therefore , increased gm - csf levels in adult cd may contribute to inhibition of bowel inflammation and prevention of tissue damage . adult cd was characterized by upregulation of serum gm - csf and ifn- , a combination with strong anti - inflammatory potential . for example , it has been shown that treatment of an lps challenge with gm - csf and ifn- inhibited an inflammatory response by lowering neutrophil counts and increasing the number of suppressive neutrophils ( cd16 ( bright)/cd62l ( dim ) ) in healthy volunteers . the gm - csf / ifn- combination may have a profound effect on leukocyte polarization . both cytokines play a key role in developing m1 type macrophages , which are associated with th1 type immune response . . therefore , increased serum gm - csf and ifn- may reflect activation of th1 type immune response in adult cd . our data suggest that juvenile and adult cd are characterized by unique serum cytokine profiles , which may reflect age - dependent difference in disease pathogenesis . contaminated sites are extensively present in europe where approximately 250,000 sites require cleanup interventions , as listed by the european environment agency . several thousands of these sites are located in italy , and a total of 57 sites , defined in 2009 as national priority contaminated sites ( npcss ) , qualify for remediation because of contamination documented in qualitative and/or quantitative terms , and because of a potential health impact . the site of taranto , located in apulia region ( southern italy ) , includes two municipalities and 216,618 inhabitants at 2001 census . this site is of interest because of several polluting sources , such as a large steel plant , a refinery , the harbor , and both controlled and illegal waste dumps . previous environmental and epidemiological investigations in the area have provided evidence of environmental contamination [ 29 ] . these studies have documented severe air pollution originating mainly from the steel industry , that is , particulate matter , heavy metals , polycyclic aromatic hydrocarbons , and organ - halogenated compounds . epidemiological studies showed an increased mortality / morbidity from respiratory , cardiovascular diseases and several cancer sites [ 1013 ] . npcss ) funded by the italian ministry of health studied mortality for 63 causes among residents of 44 npcss included in the national environmental remediation programme . the distinguishing feature of sentieri project is that the epidemiological evidence evaluation has been carried out before the study to minimize the risk for researchers to be data - driven when performing and interpreting the specific epidemiologic investigation . sentieri dealt with the complexity of the relation between area contamination and health effects by examining , for each combination of causes of death / environmental exposures , the epidemiological evidence ( 19982009 ) and then building a matrix of the a priori evaluation of the strength of the causal association . the environmental exposures were classified as chemicals , petrochemicals and refineries , steel plants , power plants , mines and/or quarries , harbour areas , asbestos or other mineral fibers , landfills , and incinerators labelled on the basis of the legislative decrees defining the sites ' boundaries . a standardized procedure was set up to collect the available epidemiological literature , which was reviewed on the basis of explicit criteria and led to classify each cause of death / environmental exposures combination in terms of strength of causal association . the evaluation was categorized as sufficient to infer the presence of a causal association ( s ) , limited to infer the presence of a causal association ( l ) , and inadequate to infer the presence or the absence of a causal association ( i ) . the rationale , scope , methods , and details of the a priori evidence evaluation can be found in , and the procedures and results of the evidence evaluation have been published in italian . with specific reference to the environmental exposures in taranto npcs , this procedure led to classify the presence of a steel industry , a refinery , a harbour area , and a number of landfills and waste dump sites as associated , with limited evidence , with an increased risk of lung cancer , pleural mesothelioma , nonmalignant respiratory diseases , congenital malformations , and perinatal conditions . in this context , residence near steel industry was evaluated as specifically associated with the occurrence of both acute and chronic respiratory diseases in adults and children , based on studies by [ 1723 ] . it should be underlined , however , that air pollution from particulate matter has been causally linked by who with several health effects , including all - cause mortality and cardiovascular and respiratory morbidity . sentieri analyzed mortality at municipality level in the period 19952002 , computing standardized mortality ratios ( smr ) both crude and adjusted for a deprivation index . while sentieri strengths are the a priori epidemiological evidence evaluation and the mortality analysis of all npcss adopting the same analytical approach and adjusting for deprivation , there are several limitations that should be noted , such as its ecological design and the use of mortality data at municipal level for a short period of time . with the aim of overcoming the above limitations , this paper presents an epidemiological profile of taranto npcs residents analyzing different health indicators available at municipality level , that is , cause - specific mortality ( 20032009 ) , mortality time trend ( 19802008 ) , and cancer incidence ( 2006 - 2007 ) . a cohort study of the resident population examined mortality ( 19982008 ) and morbidity ( 19982010 ) in the districts close to the steel plant . details on the codes used during the study period for the 9th and 10th revisions of the international classification of diseases ( icd-9 and icd-10 ) and on the demographic data of the two municipalities included in taranto npcs are presented in appendix a. mortality in taranto npcs residents was initially studied for the period 19952002 and then updated for the years 20032009 ( note that the period 2004 - 2005 was not available from istat ) . the analysis considered 63 single or grouped causes ( all ages , both genders ) ; 0 - 1 and 014 age classes were also analyzed for a selection of causes ( both genders combined ) . standardized mortality ratios both crude ( smrs ) and adjusted for deprivation together with 90% confidence intervals ( 90% cis ) were computed using regional rates for comparison . in sentieri project the deprivation index ( di ) was constructed using the 2001 national census variables representing the following socioeconomic domains : education , unemployment , dwelling ownership , and overcrowding . the strengths and weaknesses of sentieri i d , its correlation with 2001 national deprivation index , its efficacy in representing deprivation in different categories of demographic dimensions , together with suggestions about the use of socioeconomic indices in small area studies of environment and health are discussed in pasetto et al . the analysis was performed for the population 099 years separately for men and women ; directly standardized death rates ( sdrs ) per 100,000 were calculated ( standard : italian population at 2001 census ) together with their 90% ci . the population size of taranto npcs is relatively small ; therefore the study period was divided into three - year periods to obtain stable values of the indicators . the overall mortality was analyzed together with specific diseases , selected on the basis of the a priori evidence evaluation of their link with environmental exposures in taranto npcs . the selected diseases were all cancers ( in particular lung cancer ) , circulatory diseases ( in particular ischemic heart disease ) , and respiratory diseases ( in particular , the acute , and chronic ones ) . we also analyzed the overall infant mortality ( i.e. mortality from all causes during the first year of life ) without gender distinction , as not informative in this age group . for cancer incidence ( 2006 - 2007 ) , standardized incidence ratio ( sir ) and 90% ci were calculated for both genders ; the incidence rates of italian south and islands cancer registries macroarea ( 20052007 ) and of taranto province , excluding npcss municipalities ( 2006 - 2007 ) , were used for comparison . a cohort study design was applied to evaluate cause - specific mortality and hospitalization in relation to residence in specific districts close to the industrial sites . a cohort of residents ( all subjects living in taranto , massafra , and statte as january 1 , 1998 , and subsequently entered in these municipalities up to 2010 ) was enrolled from the municipal register . individual follow - up for vital status assessment at 31.01.2010 was performed using municipality data . this cohort population is different from the base population analyzed for mortality in 20032009 ( see previous paragraph for details ) . the socioeconomic position level ( sep ) of the census block of residence and the district of residence were assigned to each participant ( five categories from low to high sep ) , on the basis of the addresses geocoded at the beginning of the follow - up . occupational history for all cohort members was traced through the national insurance company ( inps ) database ( people employed in 1974 and subsequently ) , and the subcohort of individuals employed in industries located in the area was identified . mortality / morbidity information was retrieved from regional health databases ( 19982008 for mortality , 19982010 for hospital admissions ) . the associations of district of residence with mortality / morbidity were estimated by calculating mortality and morbidity hazard ratios ( hr , ci 95% ) using the proportional cox models . all models considered age ( temporal axis ) , calendar period , and area - based socioeconomic status , and they were calculated separately for men and women . tables 13 show mortality results in the periods 19952002 and 20032009 . in table 1 , mortality , from the main causes of death , is displayed for descriptive purposes ; tables 2 and 3 present the results for the causes selected on the basis of the a priori evidence evaluation , the distinguishing feature of sentieri project . table 1 shows that in both periods , for both genders , for all causes and all neoplasms , there was an excess of mortality ranging between 7% and 15% ; adjustment for deprivation did not substantially change smrs values . in both periods , among males and females , the observed mortality was above expected for circulatory , respiratory , and digestive systems diseases ; also in these cases , accounting for socioeconomic factors did not essentially change the study results . for diseases of the genitourinary system , table 2 presents the results for the causes of death for which sentieri classified the epidemiological evidence of causal association with the environmental exposures in taranto npcs as limited . from now on lung cancer showed a 20% excess in the first period , confirmed in the second one ; among females the excesses were , respectively , about 30% ( 19952002 ) and 20% . correspondingly , in the two periods excesses for pleural tumors were 193% and 167% among males , 90% and 103% among females . excesses for acute respiratory diseases among males were 49% ( 19952002 ) and 37% ( 20032009 ) , and for females 38% and 14% , respectively . the observed mortality for chronic respiratory diseases in 19952002 was as expected in both genders , while in 20032009 a 10% excess was present for males . asthma mortality was not increased , but the observed number of death is small . table 3 displays the results combined for males and females , again for causes with limited epidemiological evidence of causal association with the environmental exposures in taranto npcs . the mortality from congenital anomalies showed a 17% excess in 19952002 , while in 20032009 it was below expectation . for mortality from perinatal conditions , an heterogeneous group of diseases affecting fetus or newborn spanning from pregnancy and delivery complications to digestive or hematological disorders , the excess was 21% and 47% , in the first and second periods , respectively . in the age class 014 less than 3 deaths from acute respiratory diseases and asthma were observed . some noteworthy results should be considered ( results not shown in tables ) . among males , the observed deaths were above expected in 19952002 and 20032009 for dementia ( resp . , 105 and 102 deaths ) , hypertensive diseases ( resp . , 307 and 287 deaths ) , ischemic heart diseases ( resp . , 1032 and 679 deaths ) , and cirrhosis ( resp . , 266 and 156 deaths ) . in 20032009 excesses were reported for melanoma ( 50% , 26 deaths ) , non - hodgkin lymphoma ( 34% , 45 deaths ) , and myeloid leukemia ( 35% , 37 deaths ) . time trends for mortality from all causes and selected causes among adults and overall infant mortality are presented in figures 117 and in appendix b ( see tables s2 , s3 , and s4 in supplementary material available online at http://dx.doi.org/10.1155/2013/753719 ) . since many decades , overall mortality in italy and in apulia has been declining ( resp . , 44% and 45% ) . this favorable trend was also observed in taranto npcs , where the regular fall showed a slackening in the last three - year period . since the early 90s , the sdrs observed in taranto npcs were higher than those observed in apulia , which in turn were lower than the italian ones ; in the most recent three - year period the sdr observed in taranto site was higher than that in apulia and italy ( figure 1 ) . in italy , mortality from all neoplasms has been falling throughout the study period , while in taranto npcs and in apulia , the trend has been moderately increasing for the same study period . in taranto npcs sdrs tend to be higher than in apulia , which in turn are lower than in italy ( figure 2 ) . mortality from lung cancer has been declining in italy , apulia , and taranto npcs throughout the years 19802008 ; sdrs in the study site are higher than in italy and apulia , since 19951997 , this differential shows a reduction ( figure 3 ) . mortality from circulatory diseases in italy , apulia , and taranto site decreased , and the mortality rates almost halved during the study period ; yet , in the most recent time , taranto 's mortality rates are higher than in italy ( figure 4 ) . mortality trends from ischemic heart disease have been declining in italy , apulia , and taranto site ; yet , since the end of the 1980s , taranto 's sdrs are higher than in apulia ( figure 5 ) . mortality trends from respiratory diseases ( overall , acute , and chronic ) showed a decrease in italy , apulia , and taranto . the rates in taranto site are higher than those in the italian ones over the whole study period , the exception being the last decade , when only mortality due to acute respiratory was higher in taranto ( figures 6 , 7 , and 8) . overall mortality among women in italy , apulia , and taranto site showed a long - term decreasing trend : from 1980 to 2008 the decrease was respectively 45% , 46% , and 38% ; yet , since the beginning of the 2000s , mortality rates in taranto are higher compared to those in apulia and italy ( figure 9 ) . mortality from cancer ( all sites ) has been decreasing in italy , remaining stable in apulia , and increasing in taranto site ( figure 10 ) . in contrast with the overall cancer mortality , lung cancer mortality has been rising steadily ; in the study period the increase was 59% in italy , 44% in apulia , and 78% in taranto npcs , where sdrs were higher than in apulia ( figure 11 ) . mortality from circulatory diseases showed a declining trend in italy , apulia , and taranto site ( figure 12 ) . mortality from ischemic heart disease declined as well , but the rates observed in taranto npcs were higher than in apulia and italy throughout the study period ( figure 13 ) . finally , mortality from respiratory diseases ( overall , acute , and chronic ) showed a decline in italy , apulia , and taranto site , but the rates in taranto are higher than in italy ( figures 14 , 15 , and 16 ) . the results indicate that some differential between taranto and other areas are emerging in recent years ; it may be attributed to the presence for many years of pollutants in the environment , given that no remediation has been performed in the taranto area . infant mortality showed a steady decline both in italy and apulia ; sdrs in taranto were decreasing , but they stayed higher in taranto than in apulia and italy ( table s4 and figure 17 ) . in taranto npcs ( table 4 ) excesses were observed among both males and females when using for comparison both the rates of south and islands macroarea and of the province ( taranto and statte excluded ) for all tumors and a number of tumor sites ( stomach , colon - rectum , liver , pancreas , lung , mesothelioma , skin melanoma , kidney and other unspecified genitourinary organs , and leukemia ) . among males an increase was present for prostate cancer and among females for breast cancer . for most sites , the excesses were confirmed when province rates ( taranto and statte excluded ) were used for reference , thus supporting a major health impact among residents in taranto npcs . the study area was divided into 9 districts in taranto city and 2 municipalities ( massafra and statte ) . we considered that the districts located close to the industrial zone were the most areas affected by environmental pollution , especially considering the prevailing winds from northwest . the exposed districts were ( 1 ) tamburi ( we also included in this category the small districts of isola , porta napoli , and lido azzurro ) , ( 2 ) borgo , ( 3 ) paolo vi , and ( 4 ) statte . all the other districts were considered the reference zone ( italia - montegragnano , san vito - lama - carelli , salinella , solito , corvisea , talsano , tre carrare - battisti , and massafra ) . a total of 321,356 people ( 157,031 males and 164,325 females ) were enrolled in the cohort . at the time of the enrolment ( january 1 , 1998 ) , 84.9% of the subjects were already resident in the study area , and 39.1% of them had been residing at the same address for more than 20 years . as far as socioeconomic status is concerned , 35% of the cohort members were in the low sep category and 21.4% in the high sep category . the social distribution in the different districts was heterogeneous , with elevated proportion of high social level ( 62.2% ) in some districts in the reference area ( san vito , lama , carelli ) and low social level in tamburi ( 69.4% ) and paolo vi ( 64.3% ) . in the tamburi and paolo vi districts there was a higher proportion of subjects with previous employment at the steel industry than in other areas . a total of 3,384,302 person years were accumulated for the cohort . at the end of follow - up ( december 31 , 2010 ) , 76.6% of the cohort members were alive and resident in the study area , 14.6% had moved , and 28,171 individuals ( 8.8% ) had died . only 2.3% of the study subjects were born abroad , and most of the cohort members were born in taranto ( 81.6% ) and in southern italy ( 93.5% ) . an analysis of the mortality differences by socioeconomic status ( low versus high category ) shows ( data not in tables ) in both genders a higher mortality for all causes ( hazard ratio hr 1.25 , 95% ci 1.191.31 among males and hr 1.18 , 95% ci 1.131.24 among females ) , cardiovascular diseases ( hr 1.14 , 95% ci 1.041.25 among males and hr 1.21 , 95% ci 1.111.32 among females ) , respiratory diseases ( hr 1.89 , 95% ci 1.552.29 among males and hr 1.38 , 95% ci 1.111.72 among females ) , and digestive diseases ( hr 1.46 , 95% ci 1.191.79 among males and hr 1.56 , 95% ci 1.241.95 among females ) . socioeconomic differences in mortality were observed among males for all neoplasms ( hr 1.26 , 95% ci 1.151.37 ) and cancer of the stomach ( hr 1.69 , 95% ci 1.102.59 ) , larynx ( hr 3.32 , 95% ci 1.557.09 ) , lung ( hr 1.40 , 95% ci 1.201.64 ) , and bladder ( hr 1.55 , 95% ci 1.082.23 ) . the results for hospitalizations confirm the increased risks among subjects in the low socioeconomic category when compared with those in the highest socioeconomic group . tables 5 and 6 show , respectively , for males and females , cause - specific mortality in the exposed subareas , that is , tamburi , borgo , paolo vi , and statte , compared with mortality observed in the reference ones . after adjusting for socioeconomic status , the exposed subareas ( tamburi , borgo , paolo vi , and statte ) have a higher mortality for all causes in comparison with the reference in males ( in particular , paolo vi and tamburi ) . the most notable increases in mortality among males were in paolo vi , with 42% excess for all malignant neoplasms ( especially lung cancer , + 76% ) , diseases of the cardiovascular ( + 28% ) , respiratory ( + 64% ) and digestive ( + 47% ) systems . in tamburi , an excess was observed among males for all malignant neoplasms ( + 11% ) and cardiovascular diseases ( + 10% ) , specifically ischemic heart diseases ( + 20% ) . among females , in paolo vi , excesses are present for all cancers ( + 23% ) , in particular lung , pleural and liver cancer , cardiovascular diseases ( + 18% ) , chronic obstructive pulmonary disease ( copd ) , and digestive system diseases . in tamburi , excesses were present among females for cardiovascular diseases ( + 15% ) , in particular ischemic heart diseases , copd ( + 39% ) , and renal diseases ( + 57% ) . the hospitalization analysis confirms the mortality results , documenting the major health impact on residents in tamburi and paolo vi areas , where excesses were observed among males for a number of causes such as lung cancer ( 29% and 61% in tamburi and paolo vi , resp . ) , neurological ( 26% and 43% in tamburi and paolo vi , resp . ) , cardiovascular ( 18% and 32% in tamburi and paolo vi , resp . ) , respiratory ( 36% and 52% in tamburi and paolo vi , resp . ) , and renal diseases ( 35% both in tamburi and paolo vi ) . among females , similar excesses were observed for cardiovascular diseases ( 15 and 31% in tamburi and paolo vi , resp . ) , respiratory diseases ( 28% and 39% in tamburi and paolo vi , resp . ) , digestive diseases ( 18% and 25% in tamburi and paolo vi , resp . ) , and renal diseases ( 47% and 35% among males in tamburi and paolo vi , resp . ) . in paolo vi , pleural ( 235% ) and breast cancer ( 33% ) the health impact of residence in taranto npcs was investigated using different epidemiological approaches : geographical ( mortality and cancer incidence at municipality level ) , historical ( mortality time trends at municipality level ) , and residential cohort studies ( mortality and hospital discharge records at individual level ) . we adopted an analysis at small - area scale which includes a mix of small area and individual based data . the design issues relevant to this type of investigation have been thoroughly examined by elliott and savitz , some of them are briefly examined in the following paragraphs . in environmental epidemiology , exposure ascertainment is a key phase because the exposure / s affecting the study population should ideally be described in detail , while in most instances the available exposure information is indirect and qualitative . in ecological investigations , the exposure / s can be a single event from a point emission source of some contaminants ; more often the contaminants are heterogeneous mixture progressively polluting different matrices in the area . for example , in sentieri project the sources of environmental exposures were abstracted from the legislative decrees defining sites ' boundaries and fixed on the basis of the possible sources of contamination ( e.g. , chemical industry , steel plants , and landfills ) . a further limitation lies in the implicit assumption that all residents in the area under investigation experience the same exposures , while exposure variability is likely to be substantial , due to many factors ( e.g. , concentration of contaminants and their diffusion to soil and water , distance of residence from polluting sources ) . the possible consequences of such nondifferential exposure misclassification are complex , and the direction of the resulting bias is not predictable . in addition , information exposure source / s with possible health impact , such as concurrent air pollution from road traffic and exposures in the occupational setting , are often not available . finally , vital statistics are accessible for a given administrative area whose boundaries hardly correspond to the distribution of environmental pollutants , so that the misclassification of exposure ( and loss of statistical power ) is common . a more detailed description of these limitations of ecological study design is available [ 15 , 28 , 29 ] . exposure ascertainment is a critical issue in ecological investigations as well as in studies based on individuals , as cohort of residents are . in this case residential history , overtime information on exposure / s in different residences and different environmental matrices , daily variability , and seasonal variability should be available to classify subjects in different exposure categories . obtaining individual - based measures of exposure as described above is clearly infeasible , and modeling of exposures , ranging from simple measures such as distance from a point source or distance to nearest road to more complex estimation , for example , dispersion modeling around a point source , is used . the possible exposure misclassification in such approaches has been discussed in . as far as outcome measures are concerned , many studies of environmental healthin polluted areas consider mortality , based on death records . however , the analysis of hospital discharge records , ad hoc registry data of specific pathologies ( e.g. , cancer , congenital malformations ) can give a better picture of the health profile of residents in npcss . the key issue is that , whatever the outcome under investigation , databases need to be validated for use in epidemiological studies . reporting the event of death is usually exhaustive ; therefore the overall mortality can be analyzed with confidence . in italy , validity of cause of death certification has been documented for specific diseases [ 3134 ] . the validity of hospital discharge records ( hdrs ) , indicators of hospital activity , has not been systematically evaluated , although in italy some critical aspects of this novel utilization of hdrs have been examined . another crucial aspect in environmental health studies is that factors such as socioeconomic status , occupational exposures / s , and individual lifestyles can have an etiologic role on the health effects under study thus possibly confounding the exposure - disease relationships . for a review of adjustment for socioeconomic status using census data in ecological studies of environment and health refer to pasetto et al , 2010 . to account for deprivation in sentieri project , mortality data both crude and adjusted were analyzed using an ad hoc built deprivation index . also in individual - based studies , the analyses can be adjusted for socioeconomic factors , usually with an aggregate indicator based on residence address . in the present cohort of residents in taranto npcs sep was assigned to each participant on the basis of the geocoded addresses at the beginning of the follow - up . . the ecological components of the present investigation are affected by this limitation , while for the cohort study individual occupational history was traced through the national insurance company ( inps ) database , and the subcohort of individuals employed in industries located in the area was identified indicating a high proportion of past employment at the steel plant among residents in tamburi and paolo vi . again on this topic , with specific reference to pleural mesothelioma , it should be noted that the italian national mesothelioma registry analysis of residential asbestos exposure showed that steel mills and iron foundries were the second most frequent sources of asbestos in the neighborhood ( after asbestos - cement factories ) , ranking equal to asbestos textiles production . furthermore a case - control study in the area of taranto ( hdr , 19982002 ) adjusted for occupational exposures , showed an increase in malignant pleural neoplasms among residents close to the steel mill ( or 1.62 , 95% ci 0.377.10 , 11 cases ) and the coke plant ( or 2.18 , 95% ci 0.3115.31 , 9 cases ) . another aspect to consider in environmental epidemiology is that large populations are needed to study many of the health concerns of greatest interest . in ecological investigations , the reference populations should be selected balancing the need for comparability of study and reference populations for factors other than the environmental exposure / s with possible health impact ( socioeconomic status and lifestyle factors as diet and tobacco use ) and the requirement of sufficiently numerous populations to have stable reference rates also for rare diseases . these needs are satisfied in the present investigation where national , macroregional , and regional populations were used for comparison in the mortality , time trend , and cancer incidence analysis . in the cohort study an internal comparison was carried out , and the reference population was composed of residents in districts distant from the industrial area . for chronic diseases including most cancers , latency effects are important , such that exposures experienced many years previously , or accumulated exposures , may be crucial . in taranto npcs , the mortality ( 19952009 ) and the time trends analyses ( 19802008 ) show consistent results and cover a time span which should encompass latency effects . analogously in the cohort of residents , most subjects were present at enrolment in 1998 ( 85% ) , and half of them had a residence duration of 10 or more years . a brief comment on the use of 90% ci in our analyses is needed . in this respect we refer to sterne and smith , who affirm that confidence intervals for the main results should always be included , but 90% rather than 95% levels should be used . ci should not be used as a surrogate means of examining significance at the conventional 5% level . interpretation of ci should focus on the implications ( clinical importance ) of the range of values in the interval . in taranto npcs , mortality data at municipality level analyzed , in the context of sentieri project , time trend analysis and cancer incidence results coherently showed , in both genders , excess risks for a number of causes of death , among them : all causes , all cancers , lung cancer , cardiovascular , and respiratory diseases , both acute and chronic . for these causes , an etiologic role of environmental exposure present in taranto npcs can be supported on the basis of a priori evaluation of the epidemiological evidence completed in sentieri . in the cohort study among residents in the districts nearer to the industrial area , excess mortality / morbidity risks were shown for natural cause , cancers , cardiovascular , and respiratory diseases . these excesses were also observed in low socioeconomic position groups compared to high ones , some of them could be explained on the basis of previous employment of residents in industries active in the study area . as discussed above the present results for taranto npcs are based on sound study design and valid data , which make a low potential for bias and help to strengthen etiologic inference . it should not be disregarded the fact that most diseases showing an increased risk have multifactorial etiology , therefore interventions of proven efficacy , such as smoking cessation , food education , measures for cardiovascular risk reduction , and breast cancer screening programs , should be planned . to build a climate of confidence and trust between citizens and public institutions , study results and public health actions are to be communicated objectively and transparently .

crohn 's disease ( cd ) is a chronic inflammatory bowel disease that can be diagnosed at any age . there are two major patient groups based on diagnosis of this disease , before or after the age of 20 ( juvenile / adolescent or adult ) , with disease progression in adults usually milder than in juvenile cd patients . immune mechanisms have been suggested to play an important role in cd pathogenesis , with cytokines governing the development of the immune response . upregulation of inflammatory cytokines in serum of juvenile and adult cd patients has been documented ; still little is known about age - dependent differences in serum cytokine profiles of cd patients . we applied multiplex technology to analyze serum levels of 12 cytokines in juveniles and adults . we show that during the acute stage of the disease all cd patients have high serum levels of cxcl10 , which remains upregulated during remission . increased serum levels of tnf- and il-6 during the acute stage was characteristic of juvenile cd patients , whereas adult cd patients had upregulated levels of gm - csf and ifn-. taken together , these results demonstrate age - dependent differences in cytokine profiles , which may affect the pathogenesis of cd in patients at different ages of disease onset . the national environmental remediation programme in italy includes sites with documented contamination and associated potential health impacts ( national priority contaminated sites npcss ) . sentieri project , an extensive investigation of mortality in 44 npcss , considered the area of taranto , a npcs where a number of polluting sources are present . health indicators available at municipality level were analyzed , that is , mortality ( 20032009 ) , mortality time trend ( 19802008 ) , and cancer incidence ( 2006 - 2007 ) . in addition , the cohort of individuals living in the area was followed up to evaluate mortality ( 19982008 ) and morbidity ( 19982010 ) by district of residence . the results of the study consistently showed excess risks for a number of causes of death in both genders , among them : all causes , all cancers , lung cancer , and cardiovascular and respiratory diseases , both acute and chronic . an increased infant mortality was also observed from the time trends analysis . mortality / morbidity excesses were detected in residents living in districts near the industrial area , for several disorders including cancer , cardiovascular , and respiratory diseases . these coherent findings from different epidemiological approaches corroborate the need to promptly proceed with environmental cleanup interventions . most diseases showing an increase in taranto npcs have a multifactorial etiology , and preventive measures of proven efficacy ( e.g. , smoking cessation and cardiovascular risk reduction programs , breast cancer screening ) should be planned . the study results and public health actions are to be communicated objectively and transparently so that a climate of confidence and trust between citizens and public institutions is maintained .

simultaneous bipolar dislocation of the clavicle is uncommon and unusual , although it has been documented as panclavicular dislocation , floating clavicle , and complete dislocation of the clavicle . a review of the recent literature indicated that most bipolar clavicular dislocations have been treated non - operatively . we report the case of a 23 year polytrauma patient with bipolar dislocation of the right clavicle . patient presented to us 2 weeks post injury and had an unstable corao - clavicular joint . on radiographic assessment a simultaneous dislocation of the coraco - clavicular and sterno - clavicular joints of right side was diagnosed . in view of the unstable condition of the right clavicle , young age of the patient , requirement of high physical activity a decision for operative treatment for clavicle dislocation although bipolar dislocation of the clavicle are rare a high index of suspicion will avoid missed diagnosis . open reduction and internal fixation is a good option in young patients and gives good short term result . simultaneous bipolar dislocation of the clavicle is uncommon and unusual , although it has been documented as panclavicular dislocation , floating clavicle , and complete dislocation of the clavicle [ 1 - 13 ] . porral reported the first case of a dislocation of both ends of the clavicle in 1831 . a review of the recent literature indicated that most bipolar clavicular dislocations have been treated non - operatively . we report the case of a 23 year old polytrauma patient with bipolar dislocation of the right clavicle treated surgically . he sustained contusion of both lungs , multiple fractures of the 1st to 6th ribs left hand , and of the 3rd to 6th ribs right hand , fracture of the transverse processus of the 1st and of the 8th to 10th dorsal vertebrae , a fracture of the sacrum left hand , and a bipolar dislocation of the right clavicle . first aid was performed at a primary center and patient was transferred to our hospital after 2 weeks . the anteriorly dislocated sternoclavicular joint could be reduced passively , but was unstable , and the protruding anterior deformity quickly recurred . on physical examination the lateral end of the clavicle was tender and was palpable in a posteriorly displaced position . on the radiograph of the right shoulder and of the thorax a type four dislocation of the right acromioclavicular joint according to rockwood and an anterior dislocation of the right sternoclavicular joint fractures of the spine and the sacrum were treaded conservatively , the patient was mobilized using analgesics and physiotherapy . in view of the unstable condition of the right clavicle , young age of the patient , requirement of high physical activity a decision for operative treatment for clavicle dislocation was taken . at 3 weeks after the accident , open reduction of both the acromioclavicular and the sternoclavicular joint and internal fixation by cerclage wires was performed ( fig.2 ) . 18 months after injury , the patient was referred to the senior one of us ( t.s . ) for evaluation . the patient had no complaints of pain or discomfort and both shoulders had a full range of motion . the patient could perform all activities of daily living and had returned to his former work . a - anteroposterior view of the right shoulder shows a dislocation of the acromioclavicular joint . b- anteroposterior view of both sternoclvicular joints shows a widening of the joint space of the right sternoclavicular joint . c - ct scan of the thorax and both sternoclavicular joints confirms widening of the joint space of the right sternoclavicular joint . simultaneous bipolar dislocation of the clavicle is uncommon and unusual [ 1 - 13 ] . the predominant mechanism of injury causing acromioclavicular separation is a downward blow to the acromion , which tears the coracoclavicular and acromioclavicular ligaments and leads to a superior displacement of the distal pole of the clavicle . the least common mechanism is a direct blow on the anterior aspect of the medial end of the clavicle , causing it to dislocate posterior to the manubrium . a medially directed force on the lateral aspect of the shoulder can result in the more common anterior or superior dislocation of the medial end of the clavicle . however , the treatment of an isolated dislocation of the acromioclavicubar or sternoclavicular joint is still controversial . slings , braces , shoulder spicas , and ligament repair or reconstruction , as well as internal fixation . because of the rarity of simultaneous dislocation of both poles of the clavicle , little is known about the treatment of this injury . a review of the recent literature indicated that most bipolar clavicular dislocations have been treated non - operatively . gearen et al . treated bipolar clavicular dislocation in a young patient with a shoulder - spica cast in order to maintain the reduction of the clavicle at the acromioclavicular joint that had been obtained by manipulation . cook and horowitz reported the case of a patient who had a bipolar clavicular dislocation with a grade ii acromioclavicular separation whom they treated non - operatively with closed reduction . several days after treatment , the clavicle redislocated , but this went unreported . at follow - up beckman reviewed 16 cases of patients who had biterminal dislocation of the clavicle that was treated by closed manipulation . ten patient 's obtained a good functional result . in one shoulder that could not be manipulated because of the patient s poor general condition , symptoms involving the brachial plexus developed ; a total claviculectomy provided a good functional result . the reasons for the remaining poor results were not stated . the acromioclavicular joint has been well described . the conoid ligament medially and the trapezoid laterally connect the clavicle to the coracoid process and are strong stabilizers of the acromioclavicuhar joint . a posterior dislocation of the acromioclavicular joint can be detected clinically by palpation of the end of the clavicle posterior to the acromion and within the substance of the trapezius . the displacement ( that is , the widening of the acromioclavicular joint space ) may be seen on an anteroposterior radiograph like in our case . an axillary lateral radiograph or a computed ct scan can confirm the diagnosis by showing the posterior displacement of the clavicle from the acromion . the sternoclavicular joint connects the large medial end of the clavicle with a small sternal site of articulation . the joint is stabilized by a dense fibrocartilaginous disc , as well as by the rhomboid ligament between the clavicle and the first rib , the capsular ligaments anteriorly and posteriorly between the clavicle and the manubrium , and the interclavicuhar ligament superiorly . the injuries may be classified as type i- sprain with ligaments intact and no instability , type ii - persistent subluxation , and type iii- dislocation with gross disruption of the ligament [ 12 , 15 ) . in the current case a 23 year old polytrauma patient suffered biterminal dislocation of the right clavicle . the sternoclavicular joint showed an anterior dislocation , the acromioclavicular joint showed a grade iv dislocation according to rockwood . in our case there were several additional injuries especially serial fractures of the ribs . in respect to the young age of the patient with high physical demands and the polytrauama condition we performed open reduction of both joints with internal stabilization . review of the literature did not show satisfying results with conservative treatment of this rare injury and we believe conservative treatment should be reserved for elderly and multimorbid patients . a - postoperative radiograph of the right coraco - clavicular joint shows stabilization with a k - wire and cerclage wire . bipolar clavicular dislocations are rare and a high index of suspicion should be excercised in polytrauma patients with unipolar clavicle dislocations to avoid missed diagnosis . although a conservative treatment is reported we believe operative treatment should be offered to young patients especially in cases with high physical demands . neural stem cells ( nscs ) are multipotent cells that can give rise to various cell types in the central nervous system ( cns ) , including neurons , astrocytes , and oligodendrocytes ( alvarez - buylla et al . , 2002 ) . in adult mammals , neurogenesis is limited to two brain regions ; the subventricular zone ( svz ) by the lateral ventricles and the subgranular zone ( sgz ) of the dentate gyrus in the hippocampus ( reviewed in zhao et al . , 2008 ) . nscs in the svz , so called type - b cells , are of astrocytic lineage and generate transient amplifying type - c cells . neuroblasts then migrate along a defined pathway , the rostral migratory stream , to mature and form interneurons in the olfactory bulb ( doetsch et al . , 1999 ) . they generate type 2 intermediate proliferating progenitors that subsequently give rise to type 3 neuroblasts migrating into the subgranular layer , to become mature functional glutamatergic granule neurons ( kempermann et al . , 2004 ) . although the functional role of adult neurogenesis still is debated , it is undoubtedly a complex and highly regulated process . recently , micrornas ( mirnas ) have been suggested as players of neurogenesis , controlling expression of key regulatory genes ( gao , 2010 ; shi et al . , 2010 ; luikart et al . , 2011 ) . mirnas are one of the central determinants of mrna abundance , functioning as standby mediators of mrna regulation . they are small ( 2123 nucleotides long ) , non - coding endogenously expressed rna that bind to complementary mrna targets , resulting in a decrease in target mrna activity ( bartel , 2009 ) . since the discovery of the first mirna in 1993 , they have been identified in animals , plants , and viruses and more than 1000 mirna sequences have so far been found in humans ( www.mirbase.org ) . many are conserved across species . some have a general expression pattern , others are specifically expressed in certain tissues or cell types and expression can be spatially and temporally restricted . several labs have utilized conditional deletion of dicer , a key enzyme in mirna biogenesis , as a tool to study the role of mirnas in neurogenesis . disruption of the function of dicer during brain development results in gross anatomical changes and in some cases it is embryonic lethal ( giraldez et al . , 2005 ; davis et al . , 2008 ; de pietri tonelli et al . , nscs can be cultured in the absence of dicer but are unable to generate neurons or astrocytes upon differentiation ( andersson et al . , 2010 ) . although , dicer knockout ( ko ) studies indicate that mirnas are important regulators of neurogenesis and nsc self - renewal , they are difficult to interpret . not only because the simultaneous deficiency of all mirnas , but the long half - life of mature mirna makes conditional dicer ko experiments difficult to control from a temporal aspect , moreover there are concerns that non - mirna related functions of dicer contribute to the observed phenotypes ( konopka et al . . for example , when dicer was conditionally deleted in the retinal pigmented epithelium , cell death occurred via a mechanism that is independent on mirna , rather depending on the accumulation of retrotransposon transcripts ( kaneko et al . , 2011 ) . with this in mind , studies of the functional role of individual mirnas are a necessity . in 2007 there were four simultaneous reports of mirna ko mice ( rodriguez et al . , 2007 ; thai et al . , 2007 ; van rooij et al . , 2007 ; zhao et al . , 2007 ) , which enabled loss - of - function studies of specific mirnas . however , for several mirna , including many of those involved in neurogenesis , a classic ko strategy is complicated to apply due to single mirna species being located in multiple copies in separate regions of the genome , in clusters or located within other genes . one example of such a mirna family is mir-124 , which is transcribed as three different primary transcripts from three independent loci . mir-124 - 1 , mir-124 - 2 , and mir-124 - 3 all produce the same mature mirna ( griffiths - jones , 2006 ) . another example is mir-9 which is expressed from three independent loci , mir-9 - 1 , mir-9 - 2 , and mir-9 - 3 . the mature sequence is identical , and conserved in vertebrates and mammals . with let-7 the situation is even more complex with 12 human let-7 genes encoding for nine distinct , but closely related , mature forms of the mirna ( griffiths - jones , 2006 ) . recently , a ko mouse for mir-124 - 1 was described showing that reduction of mir-124 has severe consequences for neuronal survival and axonal outgrowth ( sanuki et al . , 2011 ) . while this study demonstrates the importance of mir-124 , it also highlights the problems of using a classic ko strategy . ( 2011 ) that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124 - 1 ko mouse . likewise , a ko mice that have two of the three copies of mir-9 deleted has been reported ( shibata et al . , these two reports suggest that conditional deletion of all three copies of either mir-9 or mir-124 is necessary in order to fully understand the role of these mirnas in adult neurogenesis . although challenging and time consuming , such a strategy may be feasible for mir-9 and mir-124 . however , for let-7 such a strategy appears unlikely to be successful given the large size of the let-7-family . adding to this , in situ analysis of the expression profile of individual mirna is difficult due to the small size of the mature mirna , which leads to poor resolution obtained in the brain with current histological techniques . as such , the study of the functional role of individual mirnas in vivo is complicated and makes interpretation and comparison between different studies challenging . we describe the current status of the field , existing attempts to study loss of mirna function , and point out technical limitations that need to be circumvented in order to move the field forward . it is fairly straightforward to profile mirna - expression patterns from bulk rna samples , either at single species resolution using for example northern blot or pcr - techniques , or at a global level using mirna arrays , pcr - array , deep sequencing of small rnas or other more specialized platforms . these methods all have their innate differences and parallel analysis of the same samples using different techniques may give significantly different results ( see , e.g. , hebert and nelson , 2011 for a discussion on this matter ) . since there is currently no gold standard for transcriptional profiling of mirna , the use of independent techniques to verify results is therefore necessary . nevertheless , these approaches have revealed the complexity of mirna - expression patterns among different cell types and have allowed identification of a number of candidate mirnas that appear to be enriched in cultured nscs . however , the technical difficulties of purifying populations of nscs and progenitors from in vivo material , using for example fluorescence activated cell sorting , make these approaches problematic to transfer to the in vivo setting ( see table 1 ) . histological approaches to study mirna expression in brain tissue have to a great extent relied on in situ hybridization ( ish ) techniques . due to the small size of the mirna it is not possible to use standard ish protocols ; an additional fixation step of the mirna is needed and probe hybridization must be optimal ( pena et al . , 2009 ) . locked nucleic acid ( lna ) modified oligonucleotides is preferable to use , since the melting temperature of the lna probe / mirna duplex is increased , resulting in stringent hybridization conditions , which in turn increases specificity and sensitivity ( reviewed in obernosterer et al . , 2007 ; wheeler et al . , 2007 ) . to do so , additional probes that target all the various precursor transcripts need to be used ( obernosterer et al . , however , this can be technically challenging when analyzing mirnas with multiple precursor transcripts ( such as mir-9 or mir-124 ) . furthermore , the results from this method are of limited resolution , thereby making it difficult to distinguish between two adjacent cells . in addition , ish is also problematic to use in combination with other labeling techniques that are routinely used in nsc - research . we have in our lab not been able to adopt protocols that allow the use of mirna ish in combination with , for example brdu - labeling , which is widely used in this field . this is primarily due to the stringent treatment of the tissue that is necessary for ish , which is incompatible with the tissue treatments for brdu - labeling . the problem of in situ mirna - expression analysis is a major concern for the study of mirna in the nervous system where it is essential to understand the cellular localization with regards to functionality . more recently , mirna reporter or sensor vectors have been used to visualize the expression pattern of endogenous mirna in cells . these are gene transfer vectors that contain a reporter gene ( i.e. , gfp ) along with binding sites for specific mirna ( mansfield et al . . in the case that a cell is actively expressing the specific mirna , the expression of the reporter gene will be suppressed by the binding of the mirna to the complimentary binding sites . thus , this system reports the absence of the target mirna , and cells that do not express it will be gfp - positive . this technique is highly specific , simple , and robust and makes it possible to study mirna expression as cells differentiate . injection of a mir-124 reporter vector into the brain will separate reporter gene expression between different cell types such as neurons and astrocytes ( colin et al . , 2009 ) . the system has been used to segregate differentiated neural cells in pluripotent cell cultures , based on the expression of mir-292 that is expressed in embryonic stem cells but not in nscs ( sachdeva et al . , 2010 ) , as well as the opposite where a mirna let-7a reporter was used to select undifferentiated cells from more differentiated cells ( di stefano et al . , 2011 ) . in nscs in vivo , sensor vectors have been used to track the expression of mir-132 in the dg ( luikart et al . the use of sensor vectors has the advantage that they measure the activity of the mirna rather than expression per se allowing easy determination if the mature mirna is present . also , the use of a fluorescent reporter gene allows excellent morphological analysis of cells in vivo . still , the technique is time - consuming including the generation of viral vectors followed by experimentation in cell culture or in vivo models . however , the versatility of the technique opens up the possibility of generating transgenic reporter animals , making it possible to visualize the expression pattern of a specific mirna throughout an organism over time ( see , e.g. , gentner et al . , 2010 ) . although this approach remains to be tested in the cns , it may be an attractive alternative in order to achieve sensitive , high - quality expression analysis of mirnas in vivo . as mentioned above , generation of ko mice for individual mirnas is often complicated since many mirnas are present in several copies or in clusters while some are present within introns of genes . to circumvent this issue several knockdown or inhibition approaches an early approach to analyze specific mirnas was by using anti - mirna oligonucleotide ( amo ) which are nucleic acids that are antisense to the mirna , thus hindering the interaction between the mirna and target mrna . this technique was first used to inactivate mir-2 and mir-13 in drosophila , in the search for mirna - target genes ( boutla et al . , 2003 ) . it was soon found that unmodified oligonucleotides are ineffective since the cellular machinery degrades them . 2-o - methyl amo is a simple chemical modification where the methyl group reduces the chances of endonucleolytic cleavage , and improves binding affinity to the mirna ( weiler et al . , 2006 ) . another variant is 2-o - methoxyethyl amo , which are fully modified oligonucleotides , with higher affinity and specificity than 2-o - methyl amo variants ( davis et al . , 2006 ; esau et al . , recently , lna ( locked nucleic acid ) modified oligonucleotides allow for even further stabilization of the mirna / target duplex structure improving silencing and also making it possible to the use small oligonucleotides that enables targeting of entire mirna - families ( vester and wengel , 2004 ; obad et al . , 2011 ) . anti - mirna oligonucleotide targets single mirnas with high specificity , as they are completely complementary to the mature sequence of the mirna ( boutla et al . , 2003 ) . despite the successful knockdown of mirna in vitro and in vivo using this method , it has several limitations . first , a direct measurement of the down regulation of mirna is difficult , because amo binds to the mirna and sequesters it from its target rather than inducing its degradation ( krutzfeldt et al . , 2005 ; davis et al . , 2009 ) . therefore the only possible way to confirm the decrease of mirna is to use indirect methods , whereby one can measure the level of expression of a reporter gene containing a target sequence of the mirna or analyzing upregulation of endogenous target genes . secondly it is not possible to identify the cells in which the amos are active as they do not carry a reporter . on top of that , the level of amo should preferably be kept constant to allow a continuous sequestration of the mirna . oligonucleotide antagomirs are chemically modified ; cholesterol conjugated single stranded rna analogs complementary to a target mirna ( krutzfeldt et al . , 2005 ) . the modification includes a partial phosphorothioate backbone in addition to 2-o - methoxyethyl to inhibit ago-2-mediated cleavage . in vivo , antagomirs have been given through intravenous injection where they appear to efficiently target mirna in various tissues . however , antagomirs do not cross the blood brain barrier , which means that in the brain , antagomirs have the same limitations as amos have . microrna sponges are transcripts expressed from strong promoters , containing multiple , tandem binding sites to a selected member of the mirna seed family of interest ( ebert et al . , 2007 ) . the binding site is imperfect , containing a bulge , for preventing rna interference cleavage and degradation of the sponge rna through endonucleolytic cleavage by ago-2 . the main advantage of mirna sponges is the possibility to achieve stable expression from integrated transgenes in vivo ( gentner et al . , 2009 ) . this can be used for studying long - term effects of mirna loss - of - function and also allows for stably expressing cell lines to be generated . the use of vector - mediated delivery also enables the incorporation of a reporter gene in order to identify the modified cells . another advantage is that sponges complementarily bind to the seed sequence of the mirna , which means that one sponge can target an entire family of mirnas . in summary , these features make sponge vectors an attractive approach to study mirna function in vivo in nscs . recently ; this technology was employed to demonstrate that mir-132 affects the integration of new - born neurons in the adult hippocampus ( luikart et al . , 2011 ) . a previous report used a retrovirus expressing the cre - recombinase to delete a floxed mir-132 gene allowing a side - by - side comparison of sponge vs. conditional ko ( magill et al . , 2010 ; luikart et al . , 2011 ) . the sponge recapitulates some , but not all , defects detected following a complete deletion of mir-132 suggesting that the use of a sponge reduces rather than eliminates levels of mir-132 ( magill et al . , 2010 ; luikart et al . , 2011 ) . as with the use of amos , the sponge vectors also have other limitations , including the difficulty of validating the down regulation of a specific mirna and the only possible way to confirm the decrease of mirna is to use indirect methods as described above . taken together , it is evident that is technically challenging to perform loss - of - function studies of mirnas in vivo in nscs . the problem of validating the inhibition , the use of transient systems together with the appearance of potential off - target effects makes the interpretation of several studies challenging . an example of this are two studies performed in the developing chick , one report suggested that mir-124 plays no role in neurogenesis while another found that mir-124 modestly promotes neurogenesis ( cao et al . , 2007 ; visvanathan et al . , 2007 ) . direct delivery of mirna - duplexes or the use of various plasmid based approaches and viral vectors have been effectively used to overexpress mirna . there are several relatively simple designs of vectors enabling stable expression that all appear to work efficiently ( krutzfeldt et al . , 2006 ; the statement that gain of function studies are easier to perform than loss of function studies is reflected in the literature and a great extent of the insight gained in the mirna field is from overexpression studies . in the last part of this review we discuss in detail three mirnas that have been functionally implicated in neurogenesis ; mir-9 , mir-124 , and the let-7 family . we give an overview of the current understanding of how these mirnas influence neurogenesis and also highlight the technical shortcomings that still prevent a full understanding of the role of these mirnas in vivo . in addition to the three above - mentioned mirna , there is a growing literature of other mirnas , including for example mir-125b , mir-132 , mir-137 , and mir-184 that influence neurogenesis ( le et al . , 2009 ; liu et al . , 2010 ; magill et al . , 2010 ; szulwach et al . , 2010 ; luikart et al . , 2011 ; sun et al . , 2011 ) . several of the technical problems that limit our understanding of mir-9 , mir-124 , and the let-7 family also hold true for other mirnas . mir-124 is perhaps the best characterized brain - specific mirna , and accounts for 2548% of all brain mirna ( lagos - quintana et al . , 2002 ) . mir-124 is expressed in neurons and have been proposed to suppress non - neural transcripts to promote neural identity ( visvanathan et al . , 2007 ; de pietri tonelli et al . , 2008 ; maiorano and mallamaci , 2009 ; farrell et al . , 2011 ; liu et al . , 2011a ; yoo et al . , 2011 ) several studies suggest that mir-124 is not expressed in other cells of the cns such as astrocytes ( see , e.g. , smirnova et al . , 2005 ) while ponomarev et al . ( 2010 ) also found it to be expressed in microglia and downregulated in activated microglia , being a key regulator of microglia quiescence . mir-124 is not expressed in nscs but is suggested to regulate the temporal progression of neurogenesis in svz . it is upregulated in the transition between type - c and type - a cells and further upregulated as the neuroblasts exit the cell cycle ( cheng et al . , 2009 ) . delivery of mir-124 to mouse or human cells in vitro causes the global expression profile of mrna to shift toward that of the brain . ( 2008 ) used a dna plasmid with primary mir-124 transcript whereas yoo et al . ( 2011 ) delivered mir-124 with a lentiviral vector harboring a mir-124 precursor , all resulting in promoting a neural phenotype . in addition , transfection of mir-124 duplexes into glioblastoma cells inhibits proliferation and induces differentiation ( silber et al . , 2008 ) . in vitro loss of function , by using antisense 2-o - methyl amo , results in delayed neurite outgrowth ( yu et al . , 2008 ) and upregulation of non - neural transcripts ( conaco et al . , 2006 ) . blocking mir-124 in svz cell populations in vivo by delivering antisense 2-o - methyl amo by a micro - osmotic pump into the ventricle , maintains neural progenitors as dividing precursors ( cheng et al . , 2009 ) . on the contrary , injecting a retrovirus overexpressing mir-124 into the svz promotes precocious neural maturation ( cheng et al . , 2009 ) . although mir-124 has been reported to be an important regulator of neurogenesis both in the developing and the adult brain , contradictory findings have been published suggesting mir-124 to be less important for neurogenesis in the developing spinal cord ( cao et al . based on in vitro mir-124 suppression or overexpression experiments , numerous mir-124 target genes have been found and validated . mir-124 has been shown to suppress several components of the re1 silencing transcription factor ( rest ) pathway ( conaco et al . , 2006 ; rest is a master regulator of neuronal phenotype ( lunyak and rosenfeld , 2005 ) and together with co - repressors it recruits histone deacetylases to suppress non - neural genes . mir-124 and rest act reciprocally ; mir-124 represses rest in neurons to promote expression of neural genes , whereas rest downregulates mir-124 in non - neural cells to inhibit expression of neural genes ( conaco et al . , 2006 ) . another mir-124 target is ptbp1 ( makeyev et al . , 2007 ) , a repressor of alternative splicing in non - neural cells demonstrating that mir-124 promotes a neuronal transcriptome by altering splicing . these targets suggest that mir-124 expression maintains a neuronal transcriptome by repressing non - neuronal genes at several levels . other targets include jagged1 in the notch signaling pathway ( cheng et al . , 2009 ; liu et al . , these targets suggest that mir-124 also plays a role in regulating the exit of a self - renewing state of nsc . however , these are only a few examples since computational algorithms suggest more than 1000 mir-124 targets ( griffiths - jones , 2006 ) . the large number of mir-124 target genes together with the observation that overexpression of mir-124 can induce a neuronal gene program suggest that mir-124 plays a crucial role in establishing and maintaining a neuronal transcription network . in light of this , it is surprising that the in vivo phenotypes found when blocking mir-124 using antisense technology is subtle , characterized mainly by a delay in differentiation or without detectable malformations ( cao et al . , 2007 ; however , a recent report using a classic ko strategy shows that deletion of mir-124 - 1 leads to major developmental phenotypes including small brain size , defective axonal outgrowth , and cell death confirming a crucial role for mir-124 in neurogenesis ( sanuki et al . , 2011 ) . as mentioned above , it is clear that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124-ko mouse , which highlights the need for generation of a conditional triple - mir-124-ko mouse . although this will be challenging and time consuming , such a strategy is necessary in order to understand the role of mir-124 during brain development and will assist the understanding of mir-124 in adult neurogenesis . however , since the use of retroviral or lentiviral vectors allow targeting of nscs in vivo , the application of stable inhibition vectors using for example mirna sponges to reassess the role of mir-124 in adult nscs is an interesting alternative . another well - studied brain - specific mirna involved in neurogenesis is mir-9 that is expressed in nscs and upregulated upon neural differentiation ( krichevsky et al . , 2006 ; de pietri tonelli et al . , 2008 ; zhao et al . , 2008 ; laneve et al . , 2010 ; bonev et al . , 2011 ; shibata et al . , 2011 ; initial studies , done in zebrafish , showed that mir-9 directs late organizer activity of the midbrain hindbrain boundary ( mhb ; leucht et al . , 2008 ) . the mhb is an organizing center in the vertebrate neural tube essential for proper development of the midbrain and anterior hindbrain ( wurst and bally - cuif , 2001 ) . in human cells , mir-9 was found to have an important role in migration and proliferation of nscs . knockdown experiments in neurospheres , using an lna antisense probe , led to reduced proliferation , and an increased migration ( delaloy et al . , 2010 ) . in contrast , zhao et al . found reduced proliferation accompanied with increased differentiation of mouse nscs when overexpressing mir-9 by rna duplexes . when they knocked down mir-9 with 2-o - methyl amo , proliferation increased ( zhao et al . , 2009 ) . in mouse , mir-9 has been shown to have a regional diversity along the anterior / posterior - axis ; knockdown in hindbrain leads to a failure of cell cycle , promoting proliferation of neural progenitor cells , whereas cells lacking mir-9 in the forebrain undergo p53-dependent apoptosis ( bonev et al . , , mir-9 has reciprocal actions with rest ; rest inhibits mir-9 - 2 in undifferentiated neuroblastoma cells , rest and creb inactivation triggers mir-9 - 2 activation ( laneve et al . , 2010 ) . other mir-9 targets include stathmin that increases microtubule instability ( delaloy et al . , 2010 ) and tlx that regulate stem cell fate ( zhao et al . , 2009 ) . tlx is suppressed by mir-9 to negatively regulate stem cell proliferation and accelerate neural differentiation ( zhao et al . , 2009 ) . hairy1 has also been suggested to mediate the effects of mir-9 on proliferation ( bonev et al . , 2011 ) . mir-9 expression pattern during mouse development has been investigated by ish ; it is expressed in the developing medial pallium although it is most abundant in cortex . when a mir-9 amo was electroporated into e11.5 cerebral cortex , deficiencies in differentiation of cajal retzius cells and early born neurons were seen , suggested to be due to the increased expression of the target gene foxg1 ( shibata et al . , 2008 ) . generation of a mir-9 - 2 and mir-9 - 3 double ko mouse , that is the two most abundant forms during telencephalon development , resulted in major phenotypic brain defects . cortical layers and vz were reduced , lateral ventricles expanded , the proliferative zones hyperplasic and differentiated structures reduced . in addition , mice suffered from growth retardation and died within 1 week , demonstrating the importance for mir-9 in neurogenesis ( shibata et al . , 2011 ) . these reports clearly demonstrate that mir-9 influence nscs , perhaps by regulating self - renewal and migration . still , much remains unclear regarding the functional role of mir-9 in nscs in vivo . as with mir-124 , it will be interesting to follow the generation of a conditional triple - mir-9-ko mouse or the application of stable mirna sponges to study the functional role of mir-9 . let-7 is one of the first mirna discovered in c. elegans , the first known human mirna and it is conserved over species . there are 12 human let-7 genes encoding for nine distinct mature forms of the mirna , let-7a through to let-7i . increased let-7 expression is seen in early neurogenesis and neural differentiation while it is decreased in the adult brain . ish shows induction of let-7 already at e9.5 in the developing cns ( wulczyn et al . , 2007 ) . let-7 expression closely resembles the expression of other brain - enriched mirnas ( wulczyn et al . , 2007 ) . in utero electroporation of let-7b duplexes injected into the lateral ventricles of e13.5 mice causes a reduction in cell cycle progression in nscs ( zhao et al . , overexpression of let-7b in nscs causes reduced proliferation and an increase in neural differentiation ( zhao et al . , 2010 ) . suppression of let-7 , using a mirna - sponge , causes an increase in levels of lin28 protein ( rybak et al . , 2008 ) . lin28 is a protein that specifically binds and blocks processing of let-7 , thereby inducing pluripotency ( rybak et al . , 2008 ; balzer et al . , 2010 ) lin28 is expressed broadly throughout the neural tube early during development where neural differentiation has not begun ( balzer et al . , 2010 ) , at this stage it co - localizes with sox2 , a maker for nscs . it has also been shown that let-7b suppresses the expression of tlx ( zhao et al . , 2010 ) . these reports suggest that the let-7-family serves as key regulators of nsc proliferation and accelerated neural differentiation ( wulczyn et al . , 2007 ; . however , the large size of the let-7 family posses a technical hurdle for the generation of loss - of - function mutants , which limits our understanding of the role of let-7 . in the future it appears likely that the use of sponge - vectors allowing stable inhibition of the entire family may be the most feasible choice to study the functional role of let-7 in nscs in vivo . it is likely that we have so far only begun to realize the complexity of mirna - mediated regulation of nscs . the multitude of mirna complimentary targets in the genome implicates the complexity of mirna gene regulation . therefore studies of mirna - target regulation in specific cell types at various developmental time points are essential . other questions that need to be answered are ; can several mirnas act to suppress the same mrna simultaneously and do they have compensatory , collaborative or competitive effects ? the development of new biotechnological tools such as mirna sponges and transgenic reporter systems will enable new types of studies that will clarify the functional properties of individual mirnas . in the coming years it will be extremely interesting to follow this field as it matures and unravels the full role of mirnas in nscs . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

introduction : simultaneous bipolar dislocation of the clavicle is uncommon and unusual , although it has been documented as panclavicular dislocation , floating clavicle , and complete dislocation of the clavicle . a review of the recent literature indicated that most bipolar clavicular dislocations have been treated non-operatively.case report : we report the case of a 23 year polytrauma patient with bipolar dislocation of the right clavicle . patient presented to us 2 weeks post injury and had an unstable corao - clavicular joint . on radiographic assessment a simultaneous dislocation of the coraco - clavicular and sterno - clavicular joints of right side was diagnosed . in view of the unstable condition of the right clavicle , young age of the patient , requirement of high physical activity a decision for operative treatment for clavicle dislocation was taken . patient was treated surgically with open reduction and tension band wiring with good result.conclusion:although bipolar dislocation of the clavicle are rare a high index of suspicion will avoid missed diagnosis . open reduction and internal fixation is a good option in young patients and gives good short term result . in adult mammals , neural stem cells ( nscs ) are found in two niches of the brain ; the subventricular zone by the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus . neurogenesis is a complex process that is tightly controlled on a molecular level . recently , micrornas ( mirnas ) have been implicated to play a central role in the regulation of ncss . mirnas are small , endogenously expressed rnas that regulate gene expression at the post - transcriptional level . however , functional studies of mirnas are complicated due to current technical limitations . in this review we describe recent findings about mirnas in nscs looking closely at mir-124 , mir-9 , and let-7 . in addition , we highlight technical strategies used to investigate mirna function , accentuating limitations , and potentials .

torus hyperplasia is a very rare condition also known as focal pyloric hypertrophy ( 1 ) . this lesion is caused by a circular muscle hypertrophy affecting the lesser curvature near the pylorus . pathogenesis of the lesion is unclear , some considering it as a persistence of infantile pyloric hypertrophy to adulthood , others noting that it develops within a few weeks in adult ( 1 , 2 ) . some have speculated that the lesion results from chronic gastritis or from repeated spastic contractions of the area secondary to visceral - visceral reflexes that could be established as a result of local irritation stimulating afferent fibers and starting a reflex arc involving efferents that locally innervate the circular pyloric musculature ( 1 , 3 ) . in summary , focal hypertrophic stenosis of the pyloric antrum in adult is classified as primary or secondary types ; 1 ) primary type being an idiopathic torus hyperplasia uncertain of origin , and 2 ) secondary type from a result of muscular thickening due to inflammation , ulceration , or carcinoma ( 4 ) . after the first report on 1946 , torus hyperplasia has been reported as a rare disease ( 5 ) . although there are some typical findings ( i.e. , recognizable radiologically by narrowing and enlongation of the pyloric cancal and endoscopically by appearances resembling those of the cervix so called characteristic pyloric " cervix sign " ) in this benign disease ( 6 , 7 ) , it is difficult to confirm without resection . moreover , it is hard to predict the prevalence of the disease because of its ' rarity ( 3 ) . herein , we report a 56-yr - old man who was diagnosed as torus hyperplasia with appendiceal mucocele after visiting our hospital because of abdominal discomfort . to the best of our knowledge , this is the first case of torus hyperplasia with appendiceal mucocele which is found incidentally . a 56-yr - old man with a history of hypertension presented with abdominal discomfort for one month . he reported no bowel habit change , nausea , vomiting , or fever , but complained of anal pain . he had a family history since his father died of advanced gastric cancer . on arrival , his blood pressure was 128/87 mmhg , heart rate 80 beats / min , respiratory rate was 20/min , and body temperature was 36.0. the physical examination revealed no abnormalities except rectal examination . his abdomen was soft and non - tender with normal bowel sound . on rectal examination , upper gastrointestinal endoscopy showed a 1.0 cm sized irregular submucosal lesion proximal to the pylorus to the distal antrum on the lesser curvature . central erosion was noticed on the surface of the lesion due to previous biopsy ( fig . 1 ) . cushion sign revealed negative finding , and biopsy revealed no significant abnormality . on colonoscopy examination which was performed on the same day with upper gastrointestinal endoscopy , histological section revealed marked hypertrophy of the distal circular pyloric musculature without an evidence of neoplasia ( fig . the present case revealed marked hypertrophy of the distal circular pyloric musculature on histological finding , and was diagnosed as torus hyperplasia after the resection . it was consistent with adult idiopathic hypertrophic pyloric stenosis , a benign disease resulting from hypertrophy of the circular fibers of the pyloric canal of unknown etiology . since the patient had no predisposing factors for the development of secondary pyloric stenosis , we considered this lesion as congenital in origin . although the endoscopic finding revealed typical cervix sign , we were not able to differentiate from true neoplastic lesion before the resection . despite the recent progress in radiology and endoscopy , it is very hard to define hypertrophic stenosis in adults ( 8) . on endoscopic finding , torus hyperplasia reveals as submucosal tumor since it arise from circular muscle consisting the pyloric canal which makes it hard to diagnose through endoscopic biopsy ( 6 , 8) . in addition , it is usually misdiagnosed as carcinoma of the antrum ( 6 ) . although recent progress of endoscopic ultrasonography and endoscopic resection had been made , it is hard to diagnose only with the aid of endoscopic procedures . most of these lesions are found incidentally like our case because it cause minimal clinical signs or is asymptomatic . in the absence of the symptoms , no clinical treatment is required , and resection is advocated only when stenosis gives rise to symptoms or when a malignancy is suspicious ( 6 , 8) . although endoscopic dilatation or laparascopic hypertrophy have been reported as alternative treatments ( 9 , 10 ) , distal gastrectomy with gastroduodenostomy is still the main therapy for final diagnosis and treatment . second , the patient had a fear of gastric cancer since his father died of this malignancy . third , elective operation for appendiceal mucocele was planned for suspicious malignant potential . in summary , we have experienced a rare disease , tours hyperplasia which was difficult to differentiate from gastric cancer mimicking submucosal tumor . our experience including typical cervix sign on endoscopic finding would help other endoscopists for their diagnosis . the incidence of non - hodgkin lymphoma ( nhl ) is rising worldwide and is higher in developed countries than in africa and asia . b cell non - hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis c virus ( hcv ) infection . the prevalence of hcv infection in patients with b cell non - hodgkin lymphoma is approximately 15% . the hcv envelope protein e2 binds human cd81 , a tetraspanin expressed on various types of cells , including lymphocytes , and activates b cell proliferation . infection and replication of hcv were observed in b cells [ 6 , 7 ] although the direct effects , particularly in vivo , have not been clarified . to determine the direct effect of hcv infection on b cells in vivo , we crossed transgenic mice with an integrated full - length hcv genome ( rz ) under the conditional cre / loxp expression system , with mice expressing the cre enzyme under transcriptional control of the b lineage - restricted gene cd19 . to investigate the mechanism of development of lymphoproliferation or b cell non - hodgkin lymphoma in hcv patients , we also developed a transgenic mouse model that conditionally expressed hcv cdna ( nucleotides 2943435 ) , including the viral genes that encode the core , e1 , e2 , and ns2 proteins , by using the cre / loxp system ( in core - ns2 [ cn2 ] mice ) [ 10 , 11 ] . conditional transgene activation of the hcv cdna ( core , e1 , e2 , and ns2 ) protects mice from fas - mediated lethal acute liver failure , by inhibiting cytochrome c release from mitochondria . persistent hcv protein expression is established by targeted disruption of interferon regulatory factor-1 ( irf-1 ) , and high incidences of lymphoproliferative disorders are noted in irf-1 cn2 mice . previously , transgenic mice that expressed the hcv core protein were established using a promoter derived from hepatitis b virus , whereas mice that expressed structural or complete viral proteins were established using promoters derived from the albumin gene . these mice were immunotolerant to the transgene and did not develop hepatic inflammation . however , they developed age - related hepatic steatosis and hepatocellular carcinomas . in contrast , the cn2 mice used in the present study were not immunotolerant to the hcv gene and developed hepatitis after the onset of hcv gene expression . however , the expression of hcv in these mice was usually lost after 21 days . therefore , an animal model of persistent hcv protein expression is required for examining the effects of chronic hcv infection in vivo . ifn signaling mediates tumor - suppressor effects and antiviral responses and is regulated by key transcription factors of the interferon - regulatory factor ( irf ) protein family , including irf1 , irf2 , irf3 , irf7 , and irf9 . targeted disruption of irf-1 results in aberrant lymphocyte development and a marked reduction in the number of cd8 t cells in the peripheral blood , spleen , and lymph nodes . the mechanisms by which hcv infection induces ifn resistance and influences the development of lymphomas are poorly understood . therefore , we established an irf-1 cn2 mouse model of persistent hcv expression , which allowed us to investigate the effects of hcv on lymphatic tissue tumor development . the full - genome hcv expression was induced by the cre / loxp system with cd19cre ( figure 1(a ) ) . the incidence of b cell lymphomas in rzcd19cre mice was 25.0% ( 22.2% in male and 29.6% in female mice ) and was significantly higher than the incidence in the hcv - negative groups . most were cd45r positive and located in the mesenteric lymph nodes ( figure 1(c ) ) . indeed , the expression of the hcv or hcv proteins induces the spontaneous development of b cell lymphomas , irrespective of the integrated site in the mouse genome . serum concentrations of il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , il-9 , il-10 , il-12(p40 ) , il-12(p70 ) , il-13 , il-17 , eotaxin , g - csf , gm - csf , ifn- , kc , mcp-1 , mip-1 , mip-1 , rantes , tnf- , il-15 , fgf - basic , lif , m - csf , mig , mip-2 , pdgf , vegf , alanine aminotransferase ( alt ) , and aspartate aminotransferase ( ast ) were not significantly different in the presence or absence of b cell lymphomas . interestingly , the average sil-2r level was significantly higher in the sera from rzcd19cre mice with b cell lymphomas ( 830.3 533.0 pg / ml ) than in that from tumor - free control groups , including the rzcd19cre , rz , cd19cre , and wild - type ( wt ) mice ( 499.9 110.2 pg / ml ; p < 0.0057 ) ( figure 1(d ) ) . the difference in the average sil-2r levels between the sera from groups with tumors other than b cell lymphomas ( 430.46 141.15 pg / ml ) and that from tumor - free control groups was insignificant ( p > 0.05 ) . moreover , all rzcd19cre mice with a relatively high level of sil-2r ( > 1000 pg / ml ) presented with b cell lymphomas . a significant increase in sil-2r was also observed in mxcre / cn2 - 29 mice that expressed the hcv cn2 gene and had b cell lymphomas , compared with tumor - free control ( cn2 - 29 ) mice . to examine whether sil-2r was derived from lymphoma tissues , we quantified il-2r concentrations in splenocytes , peripheral blood lymphocytes ( pbls ) , and b cell lymphoma tissues . the concentration of il-2r was significantly higher in splenocytes from rzcd19cre mice than in splenocytes from cd19cre mice . moreover , the concentration of il-2r in b cell lymphoma tissues from rzcd19cre mice was higher than that in splenocytes . these results strongly suggest that b cell lymphomas directly contribute to the elevated serum concentrations of sil-2r in rzcd19cre mice . they also strongly support the possibility that persistent expression of hcv could directly induce transformation of b cells . rzcd19cre mice are immunotolerant to hcv , because hcv is expressed in b cells before birth . in contrast , cn2 mice express hcv after they are administered the recombinant adenovirus that expresses the cre enzyme ( figures 2(a ) and 2(b ) ) . the expression of hcv in these mice is usually lost after 21 days ( figure 2(b ) ) through removal of hcv - expressing hepatocytes by the immune response . to establish persistent hcv expression , we disrupted irf-1 by crossing of rzcd19cre mice with irf-1 mice . irf-1 plays a significant role in the th1-type immune response and its absence is expected to decrease the elimination of hcv - expressing cells . as expected , hcv expression in irf-1 cn2 mice persisted for more than 500 days ( figure 2(c ) ) . a significant percentage of the mice that expressed the hcv core protein ( irf-1 cn2 mice ) showed polyclonal lymphoid growth disturbances , including splenomegaly , expanded lymph nodes , adenocarcinoma in the abdomen or leg , and lymphoma of the liver or peyer 's patches ( figures 2(d ) and 2(e ) ) . in contrast , hepatocytes with abundant expression of hcv proteins rarely developed into hepatocellular carcinomas . hematoxylin and eosin ( h&e ) staining of splenomegalic tissue showed extensive hyperplasia of the white pulp zones , in which the cortical zones contained lymphoid follicles and scattered germinal centers although mitotic figures were rarely observed . these results indicate that persistent expression of hcv proteins frequently induces lymphoproliferative disorders in addition to liver hyperplasia , which is consistent with the phenotype of patients with hepatocellular carcinoma . the average ratio of t cells to b cells in the lymph nodes and spleens of cn2 mice was significantly higher than that in wt mice . the majority of cd3 lymphocytes and a few cd8 lymphocytes expressed cd4 on their surfaces . these results confirm that hcv protein expression induces lymphoproliferative disorders that involve excessive expansion of both t cells and b cells . the cell population that showed negative results for t cell receptor ( , , , and isoforms ) staining was smaller in irf-1 cn2 mice than that in the other mice . the disruption of irf-1 inhibited fas - induced apoptosis , presumably by decreasing the levels of caspase-6 and caspase-7 messenger rna . these results suggest that the reduced expression of effector caspases delays fas - mediated apoptosis in irf-1 mice and prevents the elimination of hcv - expressing cells in vivo . the cn2 mice showed significantly increased levels of serum il-2 , il-10 , and il-12 ( figure 3(a ) ) . notably , the cn2 mice with proliferative disturbances in the lymph nodes and spleen had dramatically elevated levels of these cytokines , suggesting that altered cytokine production is involved in aberrant lymphocyte proliferation or differentiation . in contrast , the irf-1 cn2 mice did not show elevated levels of serum il-12 , but had significantly higher levels of serum il-2 and il-10 than did irf-1 mice ( figure 3(b ) ) . thus , the disruption of irf-1 negated the increase in the il-12 level , but augmented the increases in the levels of il-2 and il-10 in cn2 mice . these results indicate that il-2 and il-10 play key roles in the induction of the lymphoproliferative phenotype in irf-1 cn2 mice . a significantly positive correlation was found between the cytokine levels and spleen weights of cn2 gene - expressing mice with the irf-1 background ( r = 0.43 , p < 0.05 , and r = 0.53 , p < 0.05 , resp . ) . these results indicate that il-2 and il-10 are involved in lymphoproliferation in viral protein - expressing mice . bcl-2 is an integral inner mitochondrial membrane protein , overexpression of which blocks the apoptotic death of pro - b - lymphocyte death . bcl-2 transgene expression increases the oncogenic potential and is linked with b cell neoplasm and t(14;18 ) translocation . we therefore examined the level of bcl-2 protein and found that it was upregulated in the lymph nodes of irf-1 cn2 mice after 400 days ( figure 3(c ) ) . il-10 treatment in the presence of il-2 greatly inhibited fas - induced apoptosis in irf-1 cn2 mice compared with other groups ( table 1 ) . furthermore , irf-1 disruption accelerated the resistance of splenocytes to fas - induced apoptosis in the presence of il-2 , il-10 , and/or il-12 . in particular , il-2 plus il-10 treatment produced the strongest upregulation of the bcl-2 mrna levels in splenocytes of irf-1 cn2 mice . this indicates that il-2 , il-10 , and/or il-12 contribute to upregulation of bcl-2 expression , which subsequently inhibits fas - induced apoptosis . these results indicate that aberrant cytokine expression and disruption of ifn signaling affect bcl-2 expression synergizing with hcv proteins , which is associated with the inhibition of caspase expression . these results indicate that the hcv core and e2 proteins are responsible for il-2 , il-10 , and il-12 expression . core protein expression and il-10 stimulation most strongly induced bcl-2 expression ( table 2 ) . from these results , core protein contributes significantly to the induction of bcl-2 in the presence of cytokines . our results show that the conditional expression of hcv proteins induces inflammation and lymphoproliferative disorders . furthermore , established animal models will probably provide critical information for the elucidation of the molecular mechanism(s ) underlying the spontaneous development of b cell non - hodgkin lymphoma after hcv infection . therefore , irf-1-inducible genes probably play essential roles in suppressing hcv - induced lymphoma and in eliminating hcv protein - expressing cells . the overexpression of apoptosis - related proteins ( including bcl-2 ) and/or aberrant cytokine production are the primary events in hcv - induced lymphoproliferation . the hcv gene has the potential to induce b cell lymphomas in rzcd19cre mice , without inducing host immune responses against hcv gene product . this is in agreement with the results of a previous study , which indicate that viral elimination reduces the incidence of malignant lymphoma in patients infected with hcv . the incidence of b cell lymphoma in the hcv transgenic mouse strain ( mxcre / cn2 - 29 ) is high , and this strongly suggests that development of b cell lymphomas occurs via expression of the hcv transgene . the level of il-2r was higher in splenocytes of rzcd19cre mice than in those of cd19cre mice ; however , the differences in the serum concentrations of sil-2r between rzcd19cre mice without b cell lymphomas and other control groups ( rz , cd19cre , and wt ) were insignificant . these results indicate that hcv increases il-2r expression in b cells ; proteolytic cleavage of il-2r increased after b cell lymphoma development in the rzcd19cre mice . the detailed mechanism by which hcv expression induces il-2r remains unclear , but hcv core protein induces il-10 expression in mouse splenocytes . il-10 upregulates the expression of il-2r ( tac / cd25 ) in normal and leukemic b lymphocytes . therefore , through il-10 , the hcv core protein might induce il-2r in b cells of the rzcd19cre mouse . this leads to lymphoproliferative diseases resulting from reduced apoptosis ( i.e. , lower levels of caspase-1 , caspase-6 , and caspase-7 expression ) . hcv cn2 transgenic ( tg+ ) mice are resistant to fas - induced apoptosis because of the inhibition of cytochrome c release from mitochondria . mice with disruption of irf-1 have several defects in their innate and adaptive immunities , including lineage - specific defects in thymocyte development , and the development of immature t cells into mature cd4 cells but not cd8 t cells [ 16 , 23 ] . irf-1 controls the positive and negative selection of cd8 thymocytes and is required for the development of the th1-type immune response . the absence of irf-1 induces th2-type immune response [ 16 , 25 ] . the number of natural killer cells is dramatically reduced in irf-1 mice . this defect may markedly increase viral protein expression and inhibit tumor surveillance mechanisms , leading to the development of non - hodgkin lymphoma . hypermutation of the immunoglobulin genes in b cells induced by hcv infection is the cause of the lymphomagenesis observed in hcv infection [ 16 , 26 ] . this finding may provide a more direct insight into lymphoma production , because hcv - induced hypermutation causes genetic instability and chromosomal aberrations , possibly resulting in neoplastic transformation . in addition , the antiapoptotic phenotype resulting from sustained viral protein expression may enhance the survival of lymphocytes and inhibit activation - induced cell death to turn off the activated lymphocytes . the dysregulated cytokine profiles and sustained lymphocyte survival may alter the fates of regulatory t cells and dendritic cells . in summary , the mouse model of b cell lymphoma and lymphoproliferative disorder represents a powerful tool to address the molecular mechanism of lymphoma development by hcv .

primary or idiopathic hypertrophy of the pyloric muscle in adult , so called torus hyperplasia , is an infrequent but an established entity . it is caused by a circular muscle hypertrophy affecting the lesser curvature near the pylorus . since most of the lesions are difficult to differentiate from tumor , distal gastrectomy is usually preformed to rule out most causes of pyloric lesions including neoplastic ones through a pathological study . a 56-yr - old man with a family history of gastric cancer presented with abdominal discomfort of 1 month duration . upper gastrointestinal endoscopy showed a 1.0 cm sized irregular submucosal lesion proximal to the pylorus to the distal antrum on the lesser curvature . on colonoscopy examination , a 1.5 cm sized protruding mass was noticed on the appendiceal orifice . gastrectomy and cecectomy were done , and histological section revealed marked hypertrophy of the distal circular pyloric musculature and an appendiceal mucocele . to the best of our knowledge , this is the first case of torus hyperplasia with appendiceal mucocele which is found incidentally . b cell non - hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis c virus ( hcv ) infection . the mechanism by which hcv infection leads to lymphoproliferative disorder remains unclear . our group established hcv transgenic mice that expressed the full hcv genome in b cells ( rzcd19cre mice ) . we observed a 25.0% incidence of diffuse large b cell non - hodgkin lymphomas ( 22.2% in male and 29.6% in female mice ) within 600 days of birth . interestingly , rzcd19cre mice with substantially elevated serum - soluble interleukin-2 receptor -subunit ( sil-2r ) levels ( > 1000 pg / ml ) developed b cell lymphomas . another mouse model of lymphoproliferative disorder was established by persistent expression of hcv structural proteins through disruption of interferon regulatory factor-1 ( irf-1_/_/cn2 mice ) . irf-1_/_/cn2 mice showed extremely high incidences of lymphomas and lymphoproliferative disorders . moreover , these mice showed increased levels of interleukin ( il)-2 , il-10 , and bcl-2 as well as increased bcl-2 expression , which promoted oncogenic transformation of lymphocytes .

we studied 22 samples , from dogs , cattle , wildlife , and humans in cear , obtained from 1997 to 2003 ( table ) . samples were antigenically characterized by using a monoclonal antibody ( mab ) panel against the viral nucleoprotein ( 35 ) . isolates were injected into the brains of suckling mice , and brain impressions were made for mab typing ( 3,5 ) . antigenic variant-2 ( agv2 ) , maintained by dogs , was found in all c. thous , p. crancrivourous , and human cases and in all dog isolates with the exception of brdg5360 , which was positive with all the mabs . antigenic variant-3 ( agv3 ) , epidemiologically associated with vampire bats , desmodus rotundus , was identified in 3 bovine samples . a previously reported profile , representing an agv that circulates in marmosets in cear ( 5 ) , was detected in sample brsg5696 . * the cear viruses were analyzed genetically through a comparative phylogenetic study based on a 320-bp fragment of the nucleoprotein gene , from position 1157 to 1476 , as compared with sadb19 ( 58 ) . these isolates were also compared with rabies virus variants circulating among domestic animals and wildlife from the americas . the viral rna was extracted from infected tissues , and the cdna was obtained by reverse transcription pcr techniques , using primers 21 g and 304 , and was sequenced with primer 304 ( 7,9 ) . the phylogenetic analyses were made by using the pileup program of the wisconsin package version 10.1 ( 10 ) and the programs dnadist , neighbor , seqboot , and consense of the phylip package ( 11 ) . rabies virus isolates by geographic localization and neighbor - joining tree showing a comparison of the groups formed by cear state , brazil , samples isolated from 1997 to 2003 . bootstrap values of > 50% obtained from 100 resamplings of the data using distance matrix methods are shown in the nods . lineage a was represented by a sample from a dog from maranguape , which was obtained in 2001 . lineage b was formed by all the c. thous isolates , a sample from a human bitten by a p. cancrivorous raccoon in fortaleza in 1997 , and a virus from a p. crancrivorous raccoon collected in maranguape during 2001 ( intrinsic identity 96.5%100% ) . this lineage showed its highest percentage of identity with lineage c ( intrinsic identity 90.6%92.8% ) . lineage c consisted of 9 human samples collected in 5 different counties from 2000 to 2003 and an isolate obtained from a dog in 2000 . lineage d included 3 bovines collected in 3 geographically distant counties during 2000 and 2001 ( intrinsic identity 97.5%98.4% ) . lineage e was represented by the only sample collected from a c. j jacchus marmoset , these last 2 lineages were related distantly to all the others . when compared with representatives of rabies variants maintained by terrestrial and bat species in the americas ( figure 2 ) , lineages a , b , and c continue to segregate as independent lineages with high statistical support . cear bovine samples representing lineage d clustered with d. rotundus and d. rotundus related cases from latin america ( intrinsic identity 94%97.8% ) . neighbor - joining tree showing a comparison of cear samples ( groups a , b , c , d , e ) with isolates obtained from the americas . bootstrap values of > 50% obtained from 100 resamplings of the data using distance matrix methods are shown in the nods . the sequences from latin america used in the comparison were identified as as follows : group i , dogs and terrestrial wildlife from mexico , venezuela , colombia , dominican republic , and peru ( mxsk , skunk from mexico ; mxdg and mxmx , dog from mexico ; vedg , dog from venezuela ; codg , dog from colombia ; drmg , mongoose from dominican republic ; mxgm , bobcat from mexico ; pefx , fox from peru ) and terrestrial wildlife from the united states ( caussk , skunk from california ; txuscy , coyote from texas ; wiussk , striped skunk from wisconsin ; arussk , striped skunk from arkansas ; azusfox , gray fox from arizona ; txusfx , gray fox from texas ) ; and group ii , terrestrial wildlife from the united states ( flrac , raccoon from florida ; parac , raccoon from philadelphia ; ksussk , striped skunk from kansas ; arussk , striped skunk from arkansas ) . the antigenic variant and endemic cycle to which it belongs are shown in the tree . the only sample representative of lineage e segregated with 2 isolates from humans bitten by c. j. jacchus and a sample collected from a marmoset kept as a pet ( 5 ) . a thorough description of rabies epidemiology depends on a comprehensive surveillance program and application of accurate molecular methods to discriminate among different variants and the emergence of new foci . antigenic and limited sequencing analyses were used to better understand the emergent epidemiologic events in wildlife in cear , brazil . these analyses allowed identification of 5 potential cycles in this region , despite antigenic homogeneity . lack of antigenic and genetic relationships of sample brdg5693 , representing lineage a , with the rest of the isolates from cear and the known terrestrial rabies vectors from the americas shows that this virus is a variant not previously described . this virus was geographically and temporarily associated with samples brpcr5698 and brcth5692 , obtained in maranguape during 2001 . these circumstances demonstrated the existence of at least 2 overlapping endemic cycles in this area . lineage b was formed mainly by isolates from c. thous , which indicates the existence of an emerging rabies cycle in this species . the epidemiologic situation in cear was complicated because of overlapping distributions of dog and c. thous rabies cases ( figure 1 ) . tree topology and genetic relationships between dog and c. thous variants suggested that the canine virus was introduced in c. thous populations because of spillover events , which gave rise to an emergent cycle . a similar event was described between domestic dogs and canis adustus ( jackal ) in zimbawe . in this case , the variant circulating in dogs was introduced into the c. adustus population by spillover events , with the consequent emergence of an independent cycle ( 13 ) . recently , the hoary fox has been identified as a rabies reservoir in brazil ( 14 ) . cancrivorous raccoon and another sample collected from this species suggested the risk of establishing c. thous variant in p. cancrivorous . epidemiologic data which indicates that humans had been exposed to dog bites , results of molecular characterization , and inclusion of a dog isolate in the c lineage strongly incriminate the dog as the reservoir of this variant . identification of the source of infection by using classic surveillance alone is complicated by the presence of multiple cycles of transmission . genetic comparison of samples from lineage d with viruses representing bats viruses from the americas helped to identify d. rotundus as the source of livestock infection . the close genetic relationship of sample brsg5696 with rabies isolates obtained from c. j. jacchus and human cases bitten by marmosets further supported c. j. jaccuss as the most important vector of this variant . this finding indicates that this species plays an important role for disease maintenance in nature . methods for antigenic and genetic identification of rabies samples isolated in the americas have contributed effectively to the development of health programs , as well as recognition of possible wild reservoirs of urban rabies . the emergence of new cycles in latin american wildlife indicates the need to strengthen surveillance programs in these species and research development for the evaluation of the feasibility of oral vaccination interventions . toxoplasma gondii ( t. gondii ) is an obligate intracellular protozoan parasite that causes toxoplasmosis , a usually asymptomatic disease in most immune - competent individuals but potentially severe in immune - compromised hosts . successful control of this pathogen depends on the early production of the proinflammatory cytokines il-12 and ifn- , required for the development of a protective cell - mediated immune response against the parasite [ 25 ] . although neutrophils and macrophages produce il-12 in response to t. gondii infection or its soluble antigens ( stag ) [ 69 ] , in vivo studies indicate that cd8 and cd11b dendritic cell ( dc ) populations may be the most important sources of this proinflammatory cytokine during acute infection [ 1012 ] . the strong il-12-driven response against t. gondii results in the systemic production of ifn- by natural killer ( nk ) cd4 and cd8 t cells , which promotes the effector mechanisms to control both acute and chronic infections [ 2 , 1318 ] . mif is a pleiotropic inflammatory cytokine that plays a role in the outcome of a variety of protozoan infectious diseases [ 1923 ] . we have previously demonstrated that balb / c mif mice were more susceptible to t. gondii infection , with the virulent rh strain and the avirulent me49 strain . the increased susceptibility in mif mice was associated with deficient serum levels of inflammatory cytokines ( il-12 and ifn- ) , as well as impaired il-12 production and reduced expression of costimulatory molecules in cd11c cells from spleen or mesenteric lymph nodes . these data indicated a deficient innate immune response on infected mif mice presumably due to a deficient dc maturation . however , as macrophages are able to express cd11c and different dcs subsets share the expression of this marker , it is difficult to discern whether mif has a role in the maturation of dc subsets involved in the resistance to t. gondii . therefore , the present study aimed to assess the role of mif in the maturation of conventional cd8 and cd11b dcs during the acute infection with avirulent t. gondii me49 strain . we demonstrate that serum mif increased rapidly during the acute infection with t. gondii and promoted maturation of cd11b but not cd8 dcs . adoptive transfer of bone marrow ly6c monocytes suggested that systemic mif levels are required for conversion of this monocyte subset into tipdcs . mif mice displayed reduced ifn- natural killer ( nk ) cells compared to infected wt mice , suggesting that mif promotes the recruitment of ifn--producing nk cells . finally , administration of rmif to t. gondii - infected mif mice restored the numbers of tipdcs and inflammatory cytokines along with the resistance to the parasite . our results uncover a new role for mif in the development of cell - mediated immunity to t. gondii by favoring tipdc differentiation during acute toxoplasmosis . all experiments in this study were performed strictly according to the guidelines for the care and use of laboratory animals adopted by the us national institutes of health and the mexican regulation of animal care ( nom-062-zoo-1999 , 2001 ) . protocols were also approved by the institutional animal care and use committee ( iacuc ) , the research institute at nationwide children 's hospital . adult 6- to 8-week - old female mice ( balb / c background ) were purchased from harlan laboratory ( mxico or usa ) . mif mice were developed as previously described and backcrossed for more than 10 generations to a balb / c genetic background . all animals were maintained in a pathogen - free environment and established as breeding colonies in the animal facility at fes - iztacala , universidad nacional autnoma de mxico ( unam ) , or in the transgenic mouse facility at the research institute at nationwide children 's hospital animal facility . cysts of the avirulent t. gondii me49 strain were harvested from brains of c57bl/6 mice that had been inoculated i.p . with 40 cysts 2 months earlier . for experimental infections , mif or wt mice received via intraperitoneal ( i.p . ) 100 cysts or pbs as a control . control inoculations with uninfected brains failed to elicit detectable inflammatory responses or significant increases in cytokine levels . to assess acute disease progression , mice were killed by co2 and the peritoneal fluids were recovered under sterile condition using 3 ml of warm pbs and gentle massage at days 5 and 7 after toxoplasma infection of mif and wt mice . tachyzoites were counted by trypan blue exclusion with a neubauer hemocytometer under olympus bx51 microscope ( 100 ; olympus american , melville , ny ) equipped with a digital video camera . serum was obtained from infected or uninfected mif and wt mice during the acute phase of infection with t. gondii ( 1 , 2 , 3 , 5 , and 7 days after infection ) . the whole blood was centrifuged for 10 min at 2000 g at 4c . mif levels were determined by elisa according to the manufacturer 's guidelines ( neobiolab ) . serum ifn- , il-6 , il-12p70 , il-4 , and il-10 were measured using a bio - plex pro assay ( bio - rad ) or cytometric bead array ( cba th1/th2 , bd biosciences ) according to the manufacturer 's protocols . single - cell suspensions were obtained from peritoneal exudate cells ( pecs ) by peritoneal wash with 5 ml ice - cold pbs . pecs pellets were washed twice with complete rpmi 1640 media and filtered with 70 m nylon mesh ( fisher scientific ) . two milliliters of ack lysing buffer ( gibco , life technologies ) was added to lyse red blood cells . harvested cells were incubated in saturated doses of anti - mouse fc receptor in 100 l of ice - cold facs buffer ( 1% bovine serum albumin/0.01% nan3 in pbs ) for 15 min . after washing , 3 10 cells were stained with various combinations of antibodies in ice - cold facs buffer for 15 min and further collected on a lsr ii cytofluorometer ( becton dickinson , bd ) . cells were gated according to size ( ssc - a ) and forward scatter ( fsc - a ) . blue - fluorescent reactive dye l23105 ( life technologies ) was used to discard dead cells . absolute cell numbers were calculated with the total cell count multiplied successively by the percentages for the appropriate gates obtained through flow cytometry . peritoneal cells free of red blood cells were washed with complete rpmi 1640 media and adjusted 3 10 cells / ml . two ml per well of the cell suspension was incubated in 24-well plate , stimulated with 5 ng / ml of phorbol 12-myristate 13-acetate ( pma ) ( sigma ) plus 500 ng / ml of ionomycin ( sigma ) and 10 g / ml of brefeldin - a ( biolegend ) at 37c , 5% co2 for 4 hours . cells were recovered and placed into facs tubes containing 1 ml of ice - cold facs buffer and centrifuged at 1500 rpm for 5 min . after surface staining , cells were washed and fixed using 2% paraformaldehyde in pbs for 10 min , rt , darkness . cells were washed with intracellular staining buffer ( pbs , 1% fcs , and 0.025% saponin ) and resuspended with 100 l of the same buffer containing the indicated mabs for cytokines for 10 mins , rt , darkness . cells were washed , resuspended in 500 l of buffer facs , and analyzed in facsaria fusion cytometer ( becton dickinson , bd ) . the following mabs were used : anti - cd11c alexa fluor 700 ( n418 ) or anti - dec-205 pe - cy7 ( 205yekta ) , anti - mhcii efluor 450 ( m5/114.15.2 ) , anti - cd40 pe ( 1c10 ) , anti - cd86 fitc ( ct - cd8b ) , anti - cd86 brilliant violet 605 ( gl1 ) , anti - cd11b alexa fluor 647 ( m1/70 ) , anti - ly6c alexa fluor 488 ( hk1.4 ) , anti - f480 alexa fluor 488 ( bm8 ) , anti - ly6c efluor 450 ( hk1.4 ) , and anti - inos pe ( cxnft ) from ebioscience ; anti - cd8 alexa fluor 700 or percp ( 53 - 6.7 ) , anti - cd11b alexa fluor 700 ( m1/70 ) , anti - f4/80 brilliant violet 605 ( bm8 ) , anti - ly6 g percp ( 1a8 ) , anti - cxcr4 brilliant violet 421 ( 1276f12 ) , anti - cxcr2 pe / cy7 ( 5e8/cxcr2 ) , anti - tnf- brilliant violet 650 ( mp6-xt22 ) , anti - cd3 fitc ( 145 - 2c11 ) , anti - cd49 apc ( dx5 ) , and anti - ifn- pecy7 ( xmg1.2 ) from biolegend ; and anti - mhc - ii pe ( af6 - 120.1 ) , anti - cd8 percp ( 53 - 6 ) , anti - cd11c apc ( n418 ) , anti - cd74 fitc ( in-1 ) , anti - il-12p40/70 v450 ( c15.6 ) , and anti - il-12p40/70 apc ( c15.6 ) from bd bioscience . , bone marrow cell suspensions were isolated by flushing femurs and tibias from 6- to 8-week - old mif or wt balb / c mice with complete rpmi 1640 supplemented with 10% fetal calf serum ( fcs ) , 100 units of penicillin / streptomycin , 2 mm glutamine , 25 mm hepes buffer , and 1% nonessential amino acids ( all from gibco , brl grand island , ny , usa ) . bone marrow monocyte cells ( bm - mncs ) were isolated using a density - gradient centrifugation method . bm - mncs were incubated with a mixture of antibody microbeads according to the manufacturer 's protocol ( miltenyi biotec , auburn , ca ) . the negative fraction was collected ( putative monocytes ) and stained for cell sorting with a bd influx sorter . isolated populations of ly6c monocytes were obtained and stained with the cell trace proliferation dye efluor 450 ( cat . number 65 - 0842 - 85 ; ebioscience ) and transferred ( 2 10 cells ) intravenously ( i.v . ) to t. gondii - infected or uninfected mif or wt balb / c mice . a group of 13 mif and 13 wt mice were infected via i.p . with 100 cysts of t. gondii . mice received ( via i.p . ) 0.5 g per mouse of rmif ( biolegend # 599504 ) daily during the first 5 days after t. gondii infection . three mice of this group were selected randomly and sacrificed on days 3 and 5 after infection to analyze the presence of tipdcs and production of serum ifn- ( as previously described ) . uninfected mice that did not receive rmif were used as a control . the rest of the group , 7 mif and 7 wt mice , were monitored until day 25 to register the mortality and number of cysts per brain . the total number of cysts was determined by counting cysts under olympus bx51 microscope ( 100 ; olympus american , melville , ny ) equipped with a digital video camera ( 40x ) in a 10 l aliquot and multiplied by 200 . statistical comparisons were performed by either student 's t - test or one - way anova followed by tukey 's multiple comparisons for data that had a normal distribution . although mif plays a critical role in the resistance to t. gondii infection , it is not known whether this protective role starts during the acute phase of infection with the parasite . therefore , we first evaluated the systemic levels of inflammatory cytokines from acutely infected wt and mif mice with the avirulent me49 strain from t. gondii . as shown in figure 1(a ) , serum mif from infected wt mice increased as early as 1 day after infection , peaked at 3 and 5 days , and decreased at day 7 . the absence of mif led to deficient levels of il-12p70 , ifn- , il-6 , and tnf- in sera ( figures 1(b)1(e ) ) , but the anti - inflammatory cytokine il-10 was similar between groups ( figure 1(f ) ) . these data suggest that the early upregulation of mif favors the prompt inflammatory immune response to acute toxoplasmosis . under conditions of infection , dcs mature by exposure to microbial agents or inflammatory mediators and increase their antigen processing capacity as well as their expression of costimulatory molecules and cytokine production . recent studies in our laboratory suggest a role for mif in inducing dc maturation from spleen or mesenteric lymph nodes during experimental toxoplasmosis . because dcs are essential for resistance to acute toxoplasmosis [ 1012 , 31 ] , we evaluated the role of mif in the maturation of conventional cd8 and cd11b dc subsets in peritoneal exudate cells as the early primary response in the i.p . t. gondii infection . in figure 2(a ) , we show the gating strategy used for identification of cd11b ( cd11c mhcii cd11b ) and cd8 dcs ( cd11c mhcii dec205 cd8 ) subsets . the absolute numbers of both cd11b ( figure 2(b ) ) and cd8 ( figure 2(c ) ) dcs increased at the site of infection ( peritoneal cavity ) until reaching a peak by day 5 after infection . comparable numbers of these cell populations were observed between wt and mif mice . nevertheless , mif mice displayed fewer il-12p70 cd11b dcs ( figure 2(d ) ) , compared to wt mice at 3 ( p = 0.0028 ) and 5 ( p = 0.0006 ) days after infection . similar numbers of il-12p70 cd8 dcs were observed between mif and wt infected mice ( figure 2(e ) ) . in addition , mif cd11b dcs displayed reduced cd86 expression ( figures 3(a ) and 3(b ) ) at 3 ( p = 0.0498 ) and 5 ( p = 0.0194 ) days after infection , but no differences were observed in their expression of cd40 ( figure 3(d ) ) . on the other hand , the expression of costimulatory molecules in cd8 dcs was unaltered in the absence of mif ( figures 3(a ) , 3(c ) , and 3(e ) ) . similar results were observed on dcs from the spleen ( figure s-1 in supplementary material available online at http://dx.doi.org/10.1155/2016/9101762 ) . these findings suggest a role for mif in selectively inducing maturation of cd11b dcs , a population that has proven to be crucial in the control of parasite replication and induction of the adaptive immune response against t. gondii . previous studies have demonstrated that both inos and tnf- are essential factors in the control of t. gondii infection . inos - deficient mice succumb during the chronic stage of infection with t. gondii , due to uncontrolled parasite replication , while tnf-r - deficient mice are susceptible to both acute and chronic toxoplasmic encephalitis [ 4 , 3235 ] . monocyte - derived tnf-/inos - producing cd11b dcs ( tipdcs ) can be induced during parasitic or microbial infections , and they exert potent antimicrobial functions required to control the infection [ 3639 ] . however , the role of mif in inducing tipdcs during acute t. gondii infection has not been addressed . therefore , using the same strategy for identification of cd11b ( cd11c mhcii cd11b ) and cd8 ( cd11c mhcii dec205 cd8 ) dcs described above , we determined tnf- and inos production on cd11b and cd8 dcs from infected wt and mif mice . our data revealed a population of tipdcs that gradually increased in both wt and mif mice during the acute phase of infection ( figure 4(a ) ) . however , infected mif mice displayed reduced numbers of tipdcs ( p = 0.0049 ; figures 4(a ) and 4(b ) ) and tnf- inos cd11b ( p = 0.0090 , figures 4(a ) and 4(c ) ) dcs at 5 days after infection . no differences were observed in inos tnf- cd11b dcs ( figures 4(a ) and 4(d ) ) . cd8 dcs showed a gradual increase in tnf- ( figures 4(e)4(h ) ) , but not inos ( figures 4(f ) and 4(h ) ) , production during acute t. gondii infection . comparable numbers of tnf--producing cd8 dcs were observed between wt and mif mice ( figure 4(g ) ) . these findings demonstrate a novel role for mif in inducing tipdcs during acute toxoplasmosis and the ability of cd11b but not cd8 dcs to mature into tipdcs . the chemokine - like inflammatory mediator mif is a ligand for the single - pass type ii membrane protein cd74 and a noncognate ligand for the chemokine receptors cxcr2 , cxcr4 , and cxcr7 . because the role of mif in promoting dc maturation was only observed on cd11b dcs , we determined whether this phenomenon was associated with a differential expression of mif receptors on cd11b and cd8 dcs from infected wt mice . in basal conditions , wt cd11b and cd8 dcs show similar expression of cd74 , cxcr4 , and cxcr2 ( figures 5(a ) , 5(b ) , and 5(c ) ; day 0 ) . however , during the course of t. gondii infection , wt cd11b dcs express more intensively the receptors cd74 ( 3 days , p = 0.0013 ; 5 days , p = 0.0014 ; figure 5(b ) ) , cxcr4 ( 5 days , p = 0.0014 ; figure 5(c ) ) , and cxcr2 ( 3 days , p = 0.0241 ; 5 days , p = 0.0341 ; figure 5(d ) ) , compared to wt cd8 dcs ( figures 5(a)5(d ) ) . these results suggest that the selective role of mif in the maturation of cd11b dcs may be related to the high expression of mif receptors on this dc subset . two major subsets of circulating monocytes have been characterized in mice based on their phenotypical and functional characteristics . one subset expresses a high level of ly6c and is referred to as inflammatory monocytes due to its early response and selective migration into inflamed tissues [ 42 , 43 ] . the second subset , termed resident or patrolling monocytes , expresses a low level of ly6c and is primarily distributed in blood vessels and tissues under the steady state , where it provides surveillance functions [ 4245 ] . because inflammatory ly6c monocytes are able to differentiate into tipdcs [ 36 , 39 ] and newly recruited ly6c monocytes infection with t. gondii [ 4650 ] , we analyzed the activation of ly6c and ly6c monocytes in acutely infected mif and wt mice . we found that resident ly6c monocytes and f4/80 macrophages ( ms ) patrolled the peritoneal cavity in basal conditions ( figure s-2b , day 0 ) . however , upon infection with t. gondii , newly recruited monocytes ( ly6c and ly6c ) populated the peritoneal cavity at 3 and 5 days after infection , and few or no resident ms remained ( figure s-2b , 3 and 5 days after infection ) . no significant differences were observed in the absolute numbers of monocytes or resident ms between infected mif and wt mice ( figure s2c - e ) . figure 6 shows that mif ly6c monocytes had reduced numbers of both tnf- inos ( p = 0.0330 ; figures 6(a ) and 6(b ) ) and inos tnf- ( p = 0.0473 ; figures 6(a ) and 6(c ) ) cells , when compared to wt ly6c monocytes . no differences were observed in ly6c tnf- inos monocytes between mif and wt infected mice ( figures 6(a ) and 6(d ) ) . interestingly , the ly6c subset ( figure 6(e ) ) showed no differences between mif and wt infected mice in tnf- inos ( figure 6(f ) ) , inos tnf- ( figure 6(g ) ) , and tnf- inos monocytes . these data suggest that mif promotes the activation of inflammatory ly6c monocytes and possibly their differentiation into tipdcs during acute t. gondii infection . to address whether mif influences differentiation of inflammatory ly6c monocytes into tipdcs during the acute t. gondii infection , we determined the fate of adoptively transferred bone marrow ly6c monocytes from mif or wt mice ( donors ) into t. gondii - infected ( recipients ) mif or wt mice ( figure s3 ) . we first analyzed the numbers of transferred ly6c monocytes differentiated into cd11b dcs ( cd11c mhcii cd11b cell trace , figure 7(a ) ) in the peritoneal cavity . no significant differences were observed in the total number of monocyte - derived cd11b dcs between wt and mif recipient mice after the transfer with wt or mif ly6c monocytes ( figures 7(b ) and 7(c ) , resp . ) . moreover , we sought the tipdcs subset in the population of cd11b dcs and found that tipdcs were missing in both healthy wt and mif recipient mice ( figures 7(a ) , 7(d ) , and 7(e ) , day 0 ) . however , after 5 days of t. gondii infection , wt and mif recipient mice showed a subset of ly6c monocytes expressing markers for tipdcs ( figure 7(a ) , day 5 ) . interestingly , wt and mif ly6c monocytes displayed similar ability to differentiate into tipdcs after being transferred to wt recipient mice ( figures 7(d ) and 7(e ) , wt recipient mice ) . however , both wt and mif ly6c monocytes were less able to differentiate into tipdcs when transferred into recipient mif mice compared to recipient wt mice ( figure 7(d ) , p = 0.0049 ; figure 7(e ) , p = 0.0047 ) . these results indicate that ly6c monocytes differentiate into cd11b dcs in mif - independent manner ( figures 7(b ) and 7(c ) ) , but endogenous mif levels could be required to induce maturation of monocyte - derived cd11b dcs into tipdcs during acute toxoplasmosis . on the other hand , ly6c monocytes displayed high expression of mif receptors cxcr4 ( figure s4 : ( a)-(b ) ) and cd74 ( figure s4 : ( c)-(d ) ) , compared to ly6c monocytes at 5 days after infection or resident ms ( f4/80 ) from uninfected mice , which could explain the selective role of mif in inducing activation and differentiation of ly6c monocytes into tipdcs . no differences were observed in cxcr2 expression ( figure s4 : ( e ) and ( f ) ) . early studies by goldszmid et al . have demonstrated that natural killer ( nk ) cell - derived ifn- is responsible for driving the differentiation of mononuclear phagocytes into macrophages and il-12 producing cd11b dcs during experimental toxoplasmosis . in this context , the role of mif in regulating nk cell - derived ifn- at the site of infection with t. gondii could be essential for the differentiation of ly6c monocytes into tipdcs . therefore , we analyzed the ifn- production in peritoneal nk cells from t. gondii - infected wt and mif mice ; in figure 8(a ) , we show the gating strategy used . we observed that either wt or mif nk cells ( cd3 cd49b ) displayed low percentages of ifn- cells under steady state ( figure 8(b ) ) . however , upon t. gondii infection , wt and mif nk cells ( cd3 cd49b ) drastically increased their ifn- production at 2 and 3 days after infection ( figure 8(b ) ) . interestingly , mif nk cells showed a reduced percentage of ifn- cells , compared to wt nk cells ( figure 8(b ) ) . this observation was in line with a higher number of tachyzoites observed in the peritoneum of mif compared with wt mice at days 5 and 7 after t. gondii infection ( figure s5 ) . the data suggest that mif induces ifn--producing nk cells during the acute infection with t. gondii . these findings could be related to a recent study in a murine model of skin inflammation , demonstrating a role for mif in inducing the recruitment of ifn--producing nkt cells . therefore , mif could promote the migration and/or activation of nk cells during acute t. gondii infection , a crucial event in the differentiation of ly6c monocytes into tipdcs . because our results indicated that systemic mif levels are critical for resistance and induction of tipdcs during acute toxoplasmosis , we confirmed these data by administration ( via i.p . ) of rmif into mif mice ( 0.5 g per mouse ) the rmif administration fully abolished the susceptibility of infected mif mice , which reached a survival rate of 100% , whereas untreated mif mice displayed a survival rate of 40% ( p = 0.0114 ) ( figure 9(a ) ) . the enhanced resistance induced by rmif administration was associated with decreased numbers of cysts in the brain from mif mice ( p = 0.0234 ; figure 9(b ) ) after 25 days after infection . in addition , serum ifn- levels were significantly recovered in mif mice that received rmif compared to mif that did not receive rmif ( p = 0.0107 ; figure 9(c ) ) . strikingly , rmif administration reconstituted the absolute numbers of tipdcs at 5 days after t. gondii infection ( figure 9(d ) ) . altogether , these data demonstrate that induced mif levels during acute toxoplasmosis have a crucial role in the resistance and the development of the protective cell - mediated immune response against t. gondii . we have previously demonstrated that mif favors immunity to t. gondii by inducing inflammatory cytokines ( il-12 and ifn- ) essential to control both acute and chronic infections with this parasite . moreover , mif has a role in inducing maturation of cd11c cells , which indicates that mif promotes cell - mediated immune responses against t. gondii infection . however , it is not known whether this inflammatory mediator induces maturation of specific dcs subsets required to control acute toxoplasmosis . cd8 and cd11b dcs have prominent roles as the major sources of the inflammatory cytokine il-12 , required for ifn--mediated resistance to t. gondii [ 1012 , 31 ] . therefore , in the present study , we sought to determine the role of mif in the maturation of cd11b and cd8 dcs during acute t. gondii infection . we found that mif was one of the first cytokines increased in serum from infected wt mice , and in agreement with previous studies , the lack of this inflammatory mediator reduced the levels of other inflammatory cytokines , such as il-12 , ifn- , and tnf- , with all of them involved in immunity against t. gondii [ 32 , 5355 ] . interestingly , cd11b but not cd8 dcs from infected mif mice showed impaired maturation characterized by deficient il-12p70 production and low expression of the costimulatory molecule cd86 . therefore , as cd11b dcs have a crucial role in the control of acute toxoplasmosis , an important mechanism by which mif promotes resistance to t. gondii may be through inducing maturation of cd11b dcs . additionally , cd11b dcs displayed high expression of mif receptors cxcr4 , cd74 , and cxcr2 compared to cd8 dcs , suggesting that cd11b dcs are more sensitive to paracrine / autocrine mif stimulus than cd8 dcs during acute toxoplasmosis . parasitic or microbial infections with trypanosoma brucei [ 38 , 39 ] or listeria monocytogenes , respectively , induce potent inflammatory immune responses that generate inos / tnf--producing cd11b dcs ( tipdcs ) , which derive from inflammatory monocytes recruited in the site of infection . t. gondii infection induces monocyte - derived il-12 producing cd11b dcs , which accumulate in the peritoneal cavity and have a crucial role in the protective immune response to the parasite . nevertheless , the production of inos and tnf- , essential factors in the control of t. gondii infection [ 4 , 3235 ] , has not been addressed on this dc subset [ 32 , 49 , 57 , 58 ] . here infection with t. gondii , a population of tipdcs gradually increased in the peritoneal cavity from infected wt and mif mice . interestingly , infected mif mice displayed reduced numbers of tipdcs ( 5 days after infection ) compared to infected wt mice , indicating a novel role for mif in inducing tipdcs during experimental toxoplasmosis . however , it is important to note that although the term tipdcs has been widely accepted to designate inos / tnf--producing dcs , previous reports have argued that it does not necessarily refer to a distinct dc subset , but rather to common dcs responding to their environment with inos and tnf- production [ 36 , 37 ] . therefore , as comparable numbers of cd11b dcs were detected in both infected mif and wt mice , but reduced numbers of tipdcs were observed in the absence of mif , these data suggest that mif induces maturation of cd11b dcs into tipdcs , rather than recruitment of tipdcs . furthermore , a small population of cd8 dcs was detected during acute t. gondii infection ( figure 4(b ) ) , corroborating a previous report . cd8 dcs displayed mif - independent maturation , characterized by increased tnf- but not inos production , which also indicates that cd11b but not cd8 dcs are able to become tipdcs during acute toxoplasmosis . previous studies have demonstrated that nk cell - derived ifn- is required for local differentiation of inflammatory monocytes into both macrophages and mature il-12 producing dcs . in contrast , maturation of cd8 dcs does not require priming by ifn- [ 31 , 59 ] but is critically dependent on tlr stimuli . interestingly , the role of mif inducing the recruitment of ifn--producing nkt cells in a murine model of psoriasis has been recently demonstrated . therefore , we analyzed ifn--producing nk cells from t. gondii - infected wt and mif mice . we found that t. gondii - infected mif mice displayed a lower percentage of ifn- nk cells compared to infected wt mice , which could alter the activation and local differentiation of inflammatory monocytes into tipdcs . inflammatory monocytes are known as the main precursors of tipdcs during parasitic and microbial infections [ 36 , 39 ] . a variety of studies indicate that ly6c monocytes are required to control cerebral toxoplasmosis , and depletion of inflammatory monocytes ( ly6c ) but not neutrophils ( ly6 g ) increases the susceptibility of c57bl/6 mice to oral t. gondii infection [ 4649 ] . therefore , we analyzed the activation state and ability of inflammatory monocytes to differentiate into tipdcs during acute toxoplasmosis . infection with t. gondii , newly recruited monocytes ( ly6c and ly6c ) populated the peritoneal cavity of infected mice in a mif - independent manner . however , the absence of mif reduced the activation of ly6c monocytes as indicated by significant lower inos / tnf- production ( well - established markers for m1 polarization ) . in contrast , this was not observed on ly6c monocytes , suggesting a role for mif in promoting the activation of ly6c monocytes into a m1 profile . furthermore , adoptive transfer of bone marrow ly6c monocytes revealed that mif monocytes were as capable as wt monocytes of converting into tipdcs when transferred to infected wt mice . however , this ability was reduced in both , wt and mif monocytes , when transferred into infected mif mice , demonstrating that the presence of mif in ly6c monocytes has a limited role in the conversion of this population into tipdcs , compared to systemic mif levels released / produced by other cell types during acute t. gondii infection . finally , administration of rmif to infected mif mice over the first 5 days of infection with t. gondii fully restored the resistance of mif mice and reconstituted the absolute numbers of tipdcs as well as serum ifn- levels . enhanced resistance by rmif administration was associated with reduced parasite loads in brain , which indicated a better control of t. gondii infection . our data demonstrate a novel role for mif promoting the resistance to t. gondii - infection by inducing classical activation of inflammatory monocytes ly6c and their conversion into tipdcs . in addition , mif induced ifn--producing nk cells during the acute phase of infection with t. gondii , required for differentiation of inflammatory monocytes into tipdcs . further experiments are required to detail the mechanism by which mif regulates ifn- production in nk cells . the administration of rmif into mif mice fully restored the resistance to acute toxoplasmosis , which highlights the crucial role of this inflammatory mediator in the development of the protective cell - mediated immune response to t. gondii and indicates a potential use of rmif as a therapeutic tool on immune - compromised hosts to control toxoplasmosis and probably other protozoan infectious diseases .

rabies viruses circulating in cear , brazil , were identified by molecular analysis to be related to variants maintained by dogs , bats , and other wildlife . most of these viruses are associated with human rabies cases . we document the emergence of a rabies virus variant responsible for an independent epidemic cycle in the crab - eating fox ( cerdocyon thous ) . macrophage migration inhibitory factor ( mif ) mediates immunity against toxoplasma gondii infection by inducing inflammatory cytokines required to control the parasite replication . however , the role of this inflammatory mediator in the cell - mediated immune response against this infection is still poorly understood . here , we used t. gondii - infected wt and mif/ mice to analyze the role of mif in the maturation of cd11b+ and cd8+ dendritic cells ( dcs ) . we found that mif promotes maturation of cd11b+ but not cd8+ dcs , by inducing il-12p70 production and cd86 expression . infected mif/ mice showed significantly lower numbers of tnf and inducible nitric oxide synthase- ( inos- ) producing dcs ( tipdcs ) compared to infected wt mice . the adoptive transfer of ly6chigh monocytes into infected wt or mif/ mice demonstrated that mif participates in the differentiation of ly6chigh monocytes into tipdcs . in addition , infected mif/ mice display a lower percentage of ifn--producing natural killer ( nk ) cells compared to wt mice , which is associated with reducing numbers of tipdcs in mif/ mice . furthermore , administration of recombinant mif ( rmif ) into t. gondii - infected mif/ mice restored the numbers of tipdcs and reversed the susceptible phenotype of mif/ mice . collectively , these results demonstrate an important role for mif inducing cell - mediated immunity to t. gondii infection .

implantation always involves penetration to the skin and damage of smaller or larger blood vessels through the surgical procedure and the implanted scaffold material will have direct contact with human blood . scaffolds as medical implants require assessment of hemocompatibility to predict potential procoagulation or immune system activity [ 1 , 2 ] . antithrombogenic biomaterial is being extensively studied in order to fabricate artificial organs and biomaterials in contact with blood . a significant goal for the application of antithrombogenic biomaterial is to prevent thrombus formation on the material surface . in general , the factors , which influence the hemocompatibility of a material , include the chemical structure of the surface , the hydrophobicity / hydrophilicity , surface charge , surface roughness and the thrombus formation when the surface is in contact with blood . the design of a suitable biomaterial that offers improved blood biocompatibility is highly desirable for blood - contacting devices . the fabrication and design of submicron to nanoscale structural architectures , which geometrically or topologically mimic the native state of extra cellular matrix , have received much attention in regenerative medical applications . electrospinning has recently emerged as a leading technique for the formation of nanofibers because it can produce fibers with diameters ranging from several microns to tens of nanometer depending upon the solution and process parameters . nanofibrous scaffolds have attracted lot of attention as scaffolds for tissue engineering applications due to their structural similarity to extra cellular matrix , having very large surface area to volume ratio , flexibility in surface functionality , superior mechanical properties , and its high porosity [ 5 , 6 ] . zein is a protein present in corn in the seeds of maize in large amounts . due to its biodegradability and biocompatibility , researchers have provided proofs that zein was also used as a biomaterial in various biomedical applications . the proposed uses of zein in biomedical applications are as carriers for drug delivery [ 810 ] , food packaging , and scaffolding materials for cell / tissue culture [ 7 , 12 ] . however the mechanical strength of the electrospun zein scaffold was found to be very low . in an effort to improve the tensile properties of zein nanofiber , we have incorporated zein with the swcnt . due to the unique properties of cnts such as mechanical , electrical , thermal , optical , and structural properties , currently researchers have been being explored its applications in biomedical engineering and medical chemistry [ 13 , 14 ] . researchers have focused on utilizing these remarkable characteristics for engineering applications such as polymeric composites , materials for energy storage , electronics , catalysis , and vaccine delivery . a recent report on safety issues and toxic effects of cnts argued that exposure to pristine cnts causes minimal cytotoxicity at higher concentrations , while chemically functionalized cnts did not show any toxicity for drug delivery applications . furthermore , the cell growth on cnts - based materials has been shown to change cell behavior , such as migration and viability due to the influence of electric fields . however , it has been shown that cnts can be discharged from the body without any side effect or mortality . in recent years , considerable attention has been focused on preparing nanocomposites wherein cnts are dispersed in different polymeric matrices [ 2325 ] . the dispersion of nanometer - sized materials in the polymer medium was suggested as an effective method to increase the mechanical and electrical properties of the system [ 2628 ] . these composites may consist of synthetic polymers [ 29 , 30 ] , naturally derived biopolymers or a combination of both . the reinforcement of naturally derived polymers with cnts is another promising area that is just beginning to be explored . up to date , the nanoscale fillers and polymer composite reinforced with electrospun nanofibers have been developed mainly for providing some outstanding physical ( e.g. , optical and electrical ) and chemical properties and provides superior structural properties such as high modulus and strength to weight ratios . the rigidity and cylindrical shape of cnts make their surfaces good supports for protein crystallization which can improve the mechanical properties of the electrospun nanofibers . supronowicz and webster et al . , have observed that the potential use of composites of synthetic polymers and cnt in neural and orthopedic tissue engineering applications [ 34 , 35 ] . the inert nature and biocompatible chemical surfaces to cells and tissues , multiwalled carbon nanotubes polyurethane composite and poly(carbonate urethane ) have been demonstrated to have excellent antiadhesion to platelets . inspired by these promising results , we aim to develop a novel nanofibrous blood - compatible scaffold incorporating cnts for its reduced thrombogenicity effect and with improved mechanical strength . in addition , electrospinning method offers the synthesis polymer / carbon nanotubes ( cnts ) nanocomposite fibers without compromising the structural integrity of the individual cnts [ 38 , 39 ] . in this study , we have attempted the coelectrospinning method to prepare swcnts - zein nanofiber with different swcnts content ( ranging from 0.2 wt% to 1 wt% ) through a solution - based method , which has the potential of providing much better dispersion of swcnts on the polymer matrix yielding a composite with uniform structure . we are reporting here the effect of swcnts content on the morphology , mechanical properties thermal stability , and its excellent antiadhesion to platelets of zein - swcnt nanocomposite scaffolds . we found that zein - swcnt composite containing 22 wt% zein and 0.8 wt% swcnt is very good candidate for biomaterial applications . the solvent used trifluoro ethanol ( tfe ) was purchased from kanto chemical co. ( tokyo , japan ) used as such without further purification . zein was dissolved in tfe ( 100% ) to form a homogeneous solution and maintained under constant stirring for 24 hrs . zein ( 22 wt% ) was added to the swcnt - tfe mixture and further sonicated for an hour and then stirred for another hour . to fabricate the ultrafine composite nanofibrous scaffold we have used nanon electrospinning setup ( nanon-01a , mecc co. , ltd . , fukuoka , japan ) . for electrospinning , 10 ml of each kind of zein - swcnts solution was loaded into a 10 ml glass syringe and injected through an 18 g stainless - steel blunt - ended needle . the syringe was then placed in a syringe pump at a flow rate of 0.5 mlh . the electric field 2.0 kvcm ( expressed in terms of voltage / distance ) between the collection plate ( cathode ) and the needle tip ( anode ) was applied . the collector covered with aluminum foil placed at 10 cm on which fibers were collected . the morphology and diameters of electrospun fibers were determined using scanning electron microscope ( jsm-7400f , jeol , japan ) . the sample was sputter coated with a thin platinum layer using an autosputter fine coater ( e-1030 ion sputter , hitachi , japan ) before imaging . according to the sem images , additionally , zein - swcnt composite fibers were examined in a tem ( jem 2100 , jeol , japan ) . the samples were prepared by drawing out small fibrils with the aid of tweezers and placed on carbon - coated copper grids . the accelerating voltage used was 120 kv . to measure the thermal properties of the electrospun nanocomposite fibers , the thermal stability was analyzed in a thermogravimetric analyzer ( tga ) ( dtg-60h , shimadzu , japan ) . the runs were performed in the temperature range of 30c to 1000c and consisted of a ramp at a steady rate of 10c min with continuous nitrogen flow . the tensile properties of electrospun nanocomposite fibrous scaffolds , approximately 8 0.03 25 mm ( l w h ) , were characterized using a universal testing machine ( instron 3345 , uk ) equipped with a 500 n load cell . the ends of the rectangular specimens were mounted vertically on two mechanical gripping units of the tensile tester , leaving a 40 mm gauge length for mechanical loading at an extension rate of 1 mm min . the weights of the completely dried films were determined directly with an analytical balance ( wdry ) . strips of zein - swcnt - based scaffold ( 1 cm 2 cm ) were immersed into deionized water at 37c in an incubator for 24 h. the resultant swollen scaffold was gently blotted with filter paper to remove excess surface water and weighed again ( wwet ) . the water uptake ( wt ) of the scaffold is expressed as the percentage of weight obtained using ( 1 ) : ( 1)wt%= wwetwdrywdry100 . to measure the water retention ability , the wet scaffolds were transferred to centrifuge tubes with filter paper at the bottom , centrifuged at 500 rpm for 3 m and weighed immediately ( wwet ) . the percentage of water retention ( wr ) of the scaffolds at equilibrium was calculated using following ( 2 ) : ( 2)wr%=wwet(r)wdrywdry100 , where wwet(r ) is the wet weight after a predetermined time , wdry is the original weight of the sample . the nanocomposite scaffolds ( zein nanofiber ; zein-0.2 wt% swcnt ; zein-0.5 wt% swcnt ; zein-0.8 wt% swcnt ; zein-1 wt% swcnt ) were cut into small pieces and the samples were equilibrated in normal saline water for 30 min at room temperature . rabbit blood obtained from nihon seibutsu zairyo centre , japan . 200 l was added to each scaffold after taking dry weight of each scaffold . after a predetermined time period , 4 ml of saline water added to each sample to stop hemolysis and the samples were kept at constant temperature ( 35c ) for 1 h. positive and negative controls were produced by adding 200 l of rabbit 's blood to 4 ml of distilled water and saline water respectively . was measured at 545 nm using spectrophotometer ( v-650 spectrophotometer , jasco , japan ) . the percent of hemolysis was calculated using ( 3 ) : ( 3)% hemolysis ( hp ) = od of test sampleod of ve controlod of + ve controlod of ve control100 . for platelet adhesion studies , nanocomposite scaffolds of 1.5 1.5 cm size were used . experiments were carried out with anticoagulated rabbit blood bought from the nihon seibutsu zairyo centre , japan . an anticoagulated blood was centrifuged at 2500 rpm for 5 min to obtain platelet - rich plasma ( prp ) . to perform the platelet adhesion examination , then , a sample of 20 l of prp was carefully dropped on the scaffold center . after incubation for 30 min at room temperature ( about 37c ) , the scaffold was carefully rinsed several times in phosphate buffer solution ( ph-7.4 ) to remove non - adhering platelets . adherent platelets on the scaffolds were preserved with 2.5% glutaraldehyde / pbs solution for 30 min , followed by dehydration procedure using a series of ethanol - water mixtures ( 0 , 30 , 50 , 70 , 90 , 100 vol % of ethanol ) for 30 min , respectively . for electron microscopy , samples were then air dried and coated with platinum and examined in a scanning electron microscope ( jsm-7400f , jeol , japan ) . swcnt - zein solutions with varying swcnt concentrations were prepared under sonication without phase separation and maintained homogenous solution . due to the methods of dispersion of the swcnts by sonication and mechanical stirring , the swcnt clusters break down . the addition of zein forms a coating around each individual cnt and keeps them from aggregating or binding together . solutions with 0 wt% , 0.2 wt% , 0.5 wt% , 0.8 wt% , and 1 wt% swcnt content are spun according to the spinning conditions . continuous fibers were obtained using a zein concentration of 24 wt% and electrospinning at a distance of 10 cm under electric field strength of 2 kvcm . as can be seen from figure 1(a ) , the absence of beads and the uniform fibers with smooth surface morphology was obtained for all experimental materials ( figures 1(b)1(e ) ) . among them , the electrospun pure zein nanofibers figure 1(a ) had an average fiber diameter of 230 nm and diameter distribution for all loadings of swcnt in the zein fibers ( figures 1(b)1(e ) ) are all below the 300 nm range . however , at low concentration of nanotubes , relatively good dispersion was achieved , but as the concentration of nanotubes increased from 0.5 wt% to 0.8 wt% and 1 wt% swcnts promoted the creation of web - like structures in comparison with the zein nanofibers without swcnts . random nanofiber structures and web - like structures can be obtained ( figures 1(d ) and 1(e ) ) , whereas the web - like structures consisted of regularly distributed very fine nanofibers with diameters of just about 2040 nm , which were strongly embedded in the zein nanofibers . the web formation could be a result of the inclusion of carbon nanotubes into the fibers . during electrospinning the swcnts are expelled from the polymer jet under extremely high force and velocity , which causes opening of the cluster to form the web - like structure . the zein - swcnt electrospun web like structure is very similar to the structure previously reported with using bombyx mori silk nanofibers containing swcnts . we do believe that these nanowebs were created because of strong secondary electric fields occurring between individual swcnts or their agglomerated form during the electrospinning process . tem analysis of the zein - swcnt nanofibers prepared by the electrospinning process is provided in figure 2 with the aim of analyzing the creation of the nanoweb fiber . the high resolution tem micrograph shows the close inspection on electrospun composite fibers which indicates that the swcnts are embedded in the nanofibers without any sign of agglomerates . in many regions of the electrospun nanofibers ; the embedded nanotubes appeared to be well oriented along the fiber axis . such alignment is obviously associated with the extreme high longitudinal strain rate of jet during electrospinning process , which may cause disentanglement or pulling out of curved nanotubes under high shear force . . similar orientation of cnts in the electrospun nanofibers has also been observed on other composite systems and it confirms that the original dispersion contains individual cnts rather than aggregates or bulk [ 4345 ] . thermogravimetric curves of electrospun zein nanofiber and nanocomposite ( zein - swcnt ; 0.21 wt% ) are shown in figure 3 . thermal analysis shows that the improvement in thermal stability in case of zein - swcnts nanocomposite than that of pure zein nanofiber . the initial weight loss observed for all samples at 80c this can be attributed to the breakdown of the amino acid residues , as well as cleavage of the peptide bonds . the initial degradation temperature is at 230c and full degradation occurs at 455c . from their degradation curves , it seems like the degradation of the swcnt composite fibers occurs in two distinct phases . the electrospun zein fibers on the other hand , have an initial degradation temperature of 280c and final degradation temperature of 370c at which the rate of mass loss is greatest . the 1 wt% swcnt - zein composite fiber has the highest thermal stability , with onset degradation at 250c and full degradation occurring at 470c . du et al . reported that the tensile strength , fracture toughness , and hardness tests showed improved properties up to 7 wt% of cnts . when the cnt content exceeds 7 wt% the mechanical properties of the composites decreases . the tensile strength of pmma by the addition of 3 wt% functionalized cnts increased by 21% , which was low compared to a 90% increase in the modulus of pmma by the addition of 2 wt% nonfunctionalized swcnts . figure 4 illustrates the typical stress strain curves of zein and zein - swcnts composite nanofibrous scaffold . pure zein nanofiber shows a tensile strength of 1.4 mpa , while the tensile strength of zein - swcnts nanofiber increased with increasing swcnts content ( 0.2 ; 0.5 and 0.8 wt% ) to 3.2 mpa . moreover , the zein-0.8 wt% swcnts sample exhibited the maximum strength ( about 7.7 mpa ) . subsequently , further increasing the swcnts content ( 1 wt% ) lead to the decrease of tensile strength ( 6.8 mpa ) of zein - swcnts nanofiber scaffold . it was observed that zein nanofibrous scaffold with swcnts content higher than 1 wt% gives poor mechanical properties , which could be explained due to poor dispersions of the cnt within the fiber , result in forming defects of the scaffold . the above results indicate that the mechanical properties of zein nanofiber can be improved by the addition of swcnts . the tensile tests have revealed that the scaffolds containing swcnts were able to withstand a higher stress than the corresponding unreinforced scaffolds . this can be explained taking into account the tem images , which have clearly shown that the swcnts are aligned within and along the fiber orientation , thereby improving the mechanical properties . nanomaterials have high surface energy and are easy to aggregate , which lead to the poor dispersion of nanomaterials in polymer matrix results in poor mechanical properties . thus , good dispersion of swcnts in a polymer matrix the composite exhibited significant mechanical properties increased by incorporating the cnts . the composite materials were expected to be used as the scaffolds ; hence their water retention ability of the materials to keep water inside the matrix has an important role for the absorption of body fluid and for a transfer of cell nutrients and metabolites through the materials . scaffolds showing higher degree of water uptake will have a larger surface area / volume ratio thus allowing the scaffold to have the maximum probability of cell growth in 3d scaffold . the increase in water uptake the results in figure 5 give the water uptake ratios of five groups of scaffolds . the prepared 3d nanocomposite scaffolds based on zein showed significant water uptake ability ; the values were over 100% , and the highest value reached 128 1.09% for zein nanofiber . after 24 h of water absorption we have observed only 12% difference in the equilibrium water uptake capacity between native ( 128 1.09% ) and composite nanofiber ( 109 0.45% ) with 1 wt% of swcnts . these results demonstrate that the presence of swcnts has led to decrease on the water uptake capacity of the scaffold . the percent water retention values of the nanocomposite scaffolds were measured and showed in figure 5 . we found that the values of water retention were still higher than 75% even if swcnt was used , implying that the scaffolds could retain water whose weight was more than their own . water retention meant that the scaffolds could absorb water to some extent and the tight aggregation of nanofibers might make the scaffold stable in sizes and shapes . in this research work , the hemolysis percentage ( hp ) represents the extent of red blood cells hemolysis when they come in contact with sample . when the polymeric scaffold in contact with blood it must not induce thrombosis , thromboembolisms , antigenic responses , destruction of blood constituents , plasma proteins , and so forth . thus , biocompatibility , especially blood compatibility , is the most important property with regard to biomedical materials . results obtained for hemolysis of rabbit blood with nanocomposite scaffolds were shown in table 1 . the positive reference ( 100% lysis ) was blood / water mixture and the negative reference ( 0% lysis ) was a blood / saline mixture . each absorbance data point was obtained by measuring three samples and also the deviations of the three tests were determined . this results well within the permissible limit set by autian , who reported that a value of up to 5% hemolysis is permissible for biomaterials . the maximum hp value ( 4.0 0.29% ) when the swcnts content of the zein was altered , no significant difference was observed in the hemolysis assay , indicating good character of antihemolysis among all the zein - swcnts nanofiber scaffolds ( table 1 ) . it may be conclude from the hemolysis percentage that the zein - swcnt composite scaffold may be suitable as biomaterials for specific implant clinical application purpose . to determine the hemocompatibility of the zein - swcnt composite nanofiber scaffolds , as is well known , platelet adhesion has generally been applied to evaluate the hemocompatibility of materials . when a foreign material comes into contact with blood , the initial blood response is the adsorption of blood proteins , followed by platelet adhesion and the activation of coagulation pathways , leading to thrombus formation . the morphologies of activated platelets can be divided into 5 categories including dendritic , dendritic spread , spread , fully spread , and nonviable . these criteria were used to assess the activation state of the platelets that adhered to the surface of zein - swcnt in the current study . figure 6 shows the electron micrographs of platelet adhesion on zein and zein - swcnt composite nanofiber scaffolds . as can be seen from the photomicrographs , the both platelet adhesion and activation are observed on the zein nanofiber ( figure 6(a ) ) . the promotion of activation and adhesion of platelets was obviously due to the presence of nanofibers . we have observed a trend in the direction of decrease in platelet adhesion and absence of activation while increasing the swcnts and found very low in 1 wt% swcnt - zein composite ( figure 6(e ) ) , which might be due to a lower water adsorption . however , on zein nanofiber scaffold with a higher swcnt content , some of the platelets have extended , but many retain a discoid shape which is similar to the original shape of the platelet and indicates an unactivated state . changes in surface chemistry or roughness of sample surface will have an effect on the adsorption characteristic . surfaces with negatively charged groups were reported to be antithrombogenic , whereas positively charged surfaces are thrombogenic . we hypothesize that the decrease in the platelet adhesion on the zein - swcnt composite is influenced by the surface chemistry as well the surface morphology , where hydrophilic repulsion occurs and prevented the direct contact between the platelets with the zein - swcnt composite surface . these results may suggest that zein - swcnt nanofibrous composite containing 22 wt% zein and 0.8 wt% swcnt could be a potential candidate for anti - thrombogenicity , which would be particularly useful for artificial blood prostheses . we have developed nanofibrous nanocomposite scaffolds which were prepared by electrospinning of zein and zein - swcnts solutions . microscopy images show good dispersion of swcnts and the composite exhibited significant mechanical properties as well as thermal stability of electrospun zein - swcnt nanocomposites . we have demonstrated that when the swcnts content of the zein was altered , no significant difference was observed in the hemolysis assay , while it did alter the response in platelet adhesion with the absence of platelet activation on zein - swcnt composite . this suggested that platelet activation could be efficiently suppressed on this nanostructured composite with its excellent antiadhesion and subsequently low platelet activation . the developed zein - swcnt composite with homogeneous microstructure and improved mechanical properties can be used as a potential biomaterial for implant applications . the purpose of this study was to investigate the efficacy and safety of fixed dose combination ( fdc ) of olanzapine 5 mg and fluoxetine 20 mg in indian patients with severe or treatment resistant depression . this was an open , non - comparative study of seven weeks duration with an initial placebo run in period of one week . one hundred and forty - four patients completed the study as per protocol and 151 patients were safety evaluable . one hundred and eleven patients ( 77% ) received one tablet of fdc of olanzapine 5 mg / fluoxetine 20 mg once daily for 6 weeks , in patients ( 14% ) , the dose was stepped up at the end of 2 weeks to 2 tablets of fdc of olanzapine 5 mg/ fluoxetine 20 mg once daily for a further 4 weeks and 13 patients ( 9% ) required dose to be stepped up at the end of 4 weeks to 3 tablets of fdc of olanzapine 5 mg and fluoxetine 20 mg once daily for last 2 weeks . one hundred and thirty four patients ( 93% ) responded to fdc of olanzapine and fluoxetine therapy ( a responder was defined as a patient with 50 % reduction over baseline in hdrs total score at the end of therapy).statistically significant ( p < 0.0001 ) reductions in hdrs total score , madrs total score and cgi severity scores were seen with olanzapine/ fluoxetine combination . one hundred and four patients ( 72% ) were remitters ( hdrs total score of < 7 ) after 6 weeks of therapy . treatment with fdc of olanzapine 5 mg / fluoxetine 20 mg was highly effective and well tolerated in indian patients with severe or treatment resistant depression .

design of blood compatible surfaces is required to minimize platelet surface interactions and increase the thromboresistance of foreign surfaces when they are used as biomaterials especially for artificial blood prostheses . in this study , single wall carbon nanotubes ( swcnts ) and zein fibrous nanocomposite scaffolds were fabricated by electrospinning and evaluated its antithrombogenicity and hydrophilicity . the uniform and highly smooth nanofibers of zein composited with different swcnts content ( ranging from 0.2 wt% to 1 wt% ) were successfully prepared by electrospinning method without the occurrence of bead defects . the resulting fiber diameters were in the range of 100300 nm without any beads . composite nanofibers with and without swcnt were characterized through a variety of methods including scanning electron microscopy , transmission electron microscopy , thermogravimetric analysis , and tensile mechanical testing . the water uptake and retention ability of composite scaffolds decreased whereas thermal stability increased with an addition of swcnts . hemolytic property and platelet adhesion ability of the nanocomposite ( zein - swcnts ) were explored . these observations suggest that the novel zein - swcnts composite scaffolds may possibly hold great promises as useful antithrombotic material and promising biomaterials for tissue engineering application . objective : the purpose of this study was to investigate the efficacy and safety of fixed dose combination ( fdc ) of olanzapine 5 mg and fluoxetine 20 mg in indian patients with severe or treatment resistant depression.design : this was an open , non - comparative study of seven weeks duration with an initial placebo run in period of one week.method : one hundred and fifty three patients were enrolled . one hundred and forty - four patients completed the study as per protocol and 151 patients were safety evaluable . one hundred and eleven patients ( 77% ) received one tablet of fdc of olanzapine 5 mg / fluoxetine 20 mg once daily for 6 weeks , in patients ( 14% ) , the dose was stepped up at the end of 2 weeks to 2 tablets of fdc of olanzapine 5 mg/ fluoxetine 20 mg once daily for a further 4 weeks and 13 patients ( 9% ) required dose to be stepped up at the end of 4 weeks to 3 tablets of fdc of olanzapine 5 mg and fluoxetine 20 mg once daily for last 2 weeks . results : one hundred and thirty four patients ( 93% ) responded to fdc of olanzapine and fluoxetine therapy ( a responder was defined as a patient with 50 % reduction over baseline in hdrs total score at the end of therapy).statistically significant ( p < 0.0001 ) reductions in hdrs total score , madrs total score and cgi severity scores were seen with olanzapine/ fluoxetine combination . one hundred and four patients ( 72% ) were remitters ( hdrs total score of < 7 ) after 6 weeks of therapy.adverse experiences were reported by thirty one patients ( 20.5% ) . majority of them were mild in intensity . no serious adverse event was recorded with study therapy . three patients were withdrawn from the therapy due to adverse event.conclusion : treatment with fdc of olanzapine 5 mg / fluoxetine 20 mg was highly effective and well tolerated in indian patients with severe or treatment resistant depression .

for ensuring post - endodontic restorations which provide tooth function and form with adequate force distribution , placement of fiber posts to the root canal are used commonly.1 moreover , the placement of a post is often needed for the restoration of a root canal treated tooth in order to support the core material.2,3 due to the requirement for aesthetics , circular fiber posts have recently gained popularity and these materials are developed gradually.2 the lack of adaptation of the circular posts to the canal walls are filled with resin cements . but , the major factor for clinical performance of the post supported restorations is cement thickness.4 adhesive failures associated with fiber posts are common and usually happens along the resin cement that fills the post - dentin interface.5 although the ideal thickness of the resin cement needed to improve retentive bond strength is unknown , the fit of the fiber posts into the post spaces should be as tight as possible.6 ovoid shaped fiber posts have recently been introduced because of the clinical problems in oval - shaped canals , especially in canines and mandibular premolars.7 oval fiber posts are produced to mimic the oval - shaped canals , thus it can be a better fit to the post - spaces.7,8 the present study evaluated whether the push - out bond strength to dentin varies between oval and circular fiber posts , and how the resin cement thickness around oval and circular fiber posts cemented into oval shaped canals differed . the first null hypothesis of the current study was that the push - out bond strength is not significantly different circular versus oval fiber posts in ovoid root canals . the second null hypothesis of the current study was that the resin cement thickness is not significantly different circular versus oval fiber posts in ovoid root canals . eighteen human mandibular first premolars with similar size that was free of restoration and caries , and that had been stored in normal saline solution immediately after extraction , were used in this study . first , the root canals were determined as ovoid shape when the ratio of the bucco - lingual to mesio - distal root canal diameter , calculated at 5 mm from the apex , was 2 , as described by wu et al.9 in addition , all teeth were scanned with a cone - beam computerized tomography ( newtom 5 g fp , verona , italy ) to identify the oval - shaped root canals . external debris on the teeth was removed with a cavitron ( cavitron ; dentsply , york , pa , usa ) and the root surfaces were gently scraped with curettes to clean periodontal ligament remnants . the crowns of the teeth were removed at the cemento - enamel junction with a slow - speed diamond saw ( isomet ; buehler , lake bluff , il , usa ) under water cooling . the root canal lengths were standardized by cutting the roots to a uniform length of 14 mm . the root canals were prepared with protaper ni - ti rotary instruments ( dentsply maillefer , ballaigues , switzerland ) up to protaper f3 instrument . irrigation was performed using 5 ml of 2.5% naocl between each instrument during the cleaning and shaping of the root canal . the final irrigation was performed using 5 ml of 17% ethylenediaminetetraacetic acid ( edta ) for 1 minute , followed by copious irrigation with distilled water . after drying with paper points , the root canal treatments were done with a cold lateral condensation technique using gutta - percha ( dentsply maillefer , petropolis , rj , brazil ) and resin sealer ( ah plus ; dentsply detrey gmbh , konstanz , germany ) . the pulp chambers of the roots were temporarily filled with cavit ( 3 m espe ag , seefeld , germany ) , and the roots were stored at 37 , 100% humidity for 7 days . after storage , roots were randomly separated into two groups for oval and circular fiber posts ( n=9 ) and the procedures mentioned below were done by one operator in a standardized procedure . tochigi , japan ) was used to remove the gutta - percha and the post spaces were all prepared with special preparation drills ( d.t . # 0.5 and d.t . # 2 , bisco inc . , schaumburg , il , usa ) to a depth of 10 mm from the cemento - enamel junction under continuous water cooling from a handpiece ( 25 ml / min ) . group 2 : a # 2 gates glidden bur was used to remove the gutta - percha ( mani inc . ) and an oval - shaped medium - grit ( 76-m ) diamond - coated ultrasonic tip ( ellipson tip ; rtd / satelec , merigcac , france ) that was mounted on an ultrasonic unit ( satelec p5 newtron xs ; acteon group , mount laurel , nj , usa ) at medium power until a 10-mm post space was obtained . incessant water cooling from a handpiece ( 25 ml / min ) was used at the entire stages of the preparations . the oval fiber posts ( with an apical width and thickness dimensions are 1.1 and 0.5 mm , respectively ; whereas the bucco - lingual dimension is 1.9 mm , and mesio - distal dimension is 1.1 mm cervically ) and circular fiber posts ( with an apical diameter of 1 mm ; and cervical diameter of 1.8 mm ) were then luted with dual - polymerizing luting agent ( panavia f 2.0 , kuraray , japan ) to the post spaces . the posts were cleaned properly with alcohol , rinsed with distilled water , and air - dried . the ed primer ii ( kuraray , japan ) was mixed at a ratio of 1 : 1 , applied to the dentin walls of the post spaces using a microbrush ( microbrush x , microbrush corp , grafton , wi , usa ) for 30 seconds , and slowly air - dried ; the excess was then removed with paper points . panavia f 2.0 was mixed for 20 seconds and placed in the post spaces using a lentulo spiral filler ( dentsply - maillefer ) . cement was polymerized for 40 seconds using a light - polymerizing unit ( 3 m espe , elipar s10 , germany ) by placing the light tip perpendicular to the post for 40 seconds . then , the roots were stored at 37 , 100% humidity for 7 days before testing . each root was cut horizontally with a slow - speed diamond saw ( buehler ) to obtain six 1-mm - thick specimens ( two apical , two middle , and two cervical ) . afterwards , all specimens were examined with an operation microscope ( zeiss , opmipico , germany ) to determine any artifacts arised by the slicing process , and no artifacts were observed . a digital image of each specimen was taken to measure the resin cement thicknesses using a metallographic optical microscope ( leica , germany ) with 5 magnification . eight points were determined in each digital image and resin cement thicknesses were measured between the canal wall and the fiber - post perimeter at these points . following the resin cement thickness measurement procedures , the push - out tests were done at a cross - head speed of 0.5 mm / min with a universal testing machine ( instron , canton , ma , usa ) . due to the tapered design of the posts , three different sizes of plungers were used for the push - out tests . the diameter of the plunger was 1 mm for the cervical two specimens , 0.5 mm for the two middle specimens , and 0.3 mm for the two apical specimens . the force ( f ) required to dislodge the fiber posts was transformed into tension ( in mpa ) by dividing the dislodgement force in newtons by the interfacial area of the post fragment . transformation to mpa was calculated differently for the different geometrical shape of the circular and oval fiber posts . for circular posts that were considered truncated cones , the method used to transform to mpa was similar to that used by costa et al.10 the upper and lower diameters of the specimens were measured individually , and the following formula was used : mpa = f / sl . sl was calculated using the following equation : sl=(r+r)g ; where sl = sealer adhesion area ; =3.14 ; r = mean radius of the cervical post ( mm ) ; r = mean radius of the apical post ( mm ) ; g = slice thickness ( mm ) . the above formula could not be used because the oval post specimen shape is not considered a truncated cone . the oval post presents a section made up of a rectangle and two equal circular segments . therefore , a fragment of an oval post was considered as a geometric solid figure made up of a truncated rectangular based pyramid ( thicker lines ) and two equal parts of a truncated cone . therefore the lateral surface area was calculated using a method similar to that used by coniglio et al.7 two digital images of each specimen were taken from apical and cervical sides using a metallographic optical microscope with 5 magnification . then , an image processing software program ( photoshop cs5 , adobe , san jose , ca , usa ) was used to determine points ' axis and ordinate as pixel . r1 , r2 , oh , o'h ' hd , h'd ' , ab , a'b ' distances stated in the formula were translated to mm from the pixel format : sl={r12arccos[1/r1oh]+r22arccos[1/r2oh]}[(r1-r2)+ h]+(hd+hd ' ) [[(ab - ab')/2]+ h ] ; where o = the center of the circle of the oval post section ; h= the intersection of the major ( cf ) and minor axis ( de ) of the oval post ; a , b , d , e= the corners of the oval post section ; r1=mean radius of the cervical side of the post ( mm ) ; r2=mean radius of the apical side of the post ( mm ) ; and o was calculated with an analytical geometry rule which is the center of the circle from three identified points ( a , b , c points ) . the symbol ( ' ) indicates the equivalent segments on both sides of the specimen . two separate kruskal - wallis anova on ranks were applied to compare the push - out and resin cement thickness data in each canal third . mann - whitney u - tests were done to detect differences between the oval and circular post groups for each canal third . statistical analyses were performed using sigmastat software , version 12.0 ( systat software , san jose , ca , usa ) , and the statistical significance was determined as p<.05 . table 1 is presented to show push - out test results . in terms of the push - out test results of the cervical , middle and apical specimens of the oval and circular fiber - post groups , comparisons in terms of root segments for the oval fiber - post group revealed that the bond strengths in the apical specimens were lower than in the cervical specimens and the middle specimens ( p<.05 ) . the bond strength results in the cervical and middle specimens did not reveal statistically significance ( p>.05 ) . when the bond strengths were evaluated in terms of the root segments for the circular fiber - post group , significantly higher bond strength values were measured in the cervical compared with the apical specimens ( p<.05 ) . there were no significant differences between the bond strengths in the cervical and middle specimens and in the middle and apical specimens ( p>.05 ) . the resin cement thicknesses of the oval posts were higher than those of the circular post group in the cervical , middle and apical specimens ( p<.05 ) ( table 2 ) . circular posts had a significantly higher resin cement thickness in the cervical third than in the apical and middle thirds ( p<.05 ) . in addition , the resin cement layer of the oval posts was thicker in the cervical and apical specimens than in the middle specimen ( p<.05 ) . according to the chi - square test for the failure mode comparisons within the groups no significant differences were revealed ( p>.05).the most frequent type of failure mode in all groups was adhesive failure between cement and dentine . to reflect the root canal morphology after drilling the root canals in oval shaped teeth and to eliminate possible disadvantages of circular fiber posts , oval fiber posts were developed with a minimally invasive diamond - coated ultrasonic tip.8,11 the anatomical , oval ellipson fiber post is formed with a high percentage loading 64% vol . ( 80% wt . ) of pretensed long continuous unidirectional quartz fibers with epoxy resin , and is radiopaque . ellipson post has an elastic modulus of 13 gpa , and a flexural strength of 1600 mpa , and has no risk of corrosion . the ellipson tip for the minimally invasive oval preparation with ultrasonic , is stainless steel with a diamond coated tip of 76 micron grain , a diameter of 1.8 mm and has depth gauge markings . this tip can be used to simultaneously remove root canal filling material , clean the canal and shape the canal to adapt the oval ellipson fiber post.12 to assess the bond strength between fiber posts and the root canal dentin , conventional shear and tensile tests , microshear , microtensile , and pull - out and push - out tests have been used.13,14,15,16 it is suggested that the bond strength is better obtained by the push - out test than with the conventional shear test because the fracture occurs parallel to the dentin bonding interface in the push - out test , which makes it a true shear test.13 chen et al.17 stated that , the stresses concentrate at the filler - loading pin and filler - dentin interfaces in push - out method . additionally , as premature failures occur during specimen preparation and because of the large data distribution observed in microtensile tests , the push - out test is considered more reliable.15,16,18 the shear stress achieved with the push - out tests is comparable to the stress , under clinical conditions , at the interface between the dentin and the luting cement , as well as between the post and the luting cement.19 according to a previous study , investigators showed the pin diameter , specimen thickness , and elastic modulus of the filling material all affected the push - out bond strength values.17 in the present study , luting resin cement , specimen thickness and pin diameter was set standard ; also , oval and circular posts have the same elastic modulus . this situation might be one of the factors that lead to similar push - out results between the groups . to determine the interfacial area of the circular and oval fiber posts , which have different geometrical shapes , two distinct formulas were applied to calculate the push - out bond strength in megapascals . the interfacial area of the circular fiber - post specimens was calculated as described by costa et al.,10 whereas a specific formula that was introduced by coniglio et al.7 was modified in the present study to calculate the interfacial area of the oval fiber - post specimens . in this study , the push - out bond strengths and resin cement thicknesses of two post systems were evaluated . the push - out bond strengths of the oval and circular fiber posts were not significantly different . therefore , the results obtained support the first null hypothesis that the push - out bond strengths of the oval and circular fiber posts would not differ significantly in ovoid root canals . these results are in agreement with those of coniglio et al.,7 who found that the oval tip - oval post system resulted in comparable push - out bond strength values to the circular drill - post system . the results of the study do not support the second hypothesis that the resin cement thickness would not differ significantly whether oval and circular fiber posts were used in ovoid root canals . muoz et al.20 reported no significant difference in resin cement thicknesses between oval and circular fiber posts , whereas coniglio et al.8 showed that the resin cement thicknesses of oval fiber posts were significantly less than those of circular fiber posts in ovoid root canals . in a recent study , rengo et al.21 evaluated the volume of luting cement around the oval and circular fiber posts by means of ct imaging in ovoid root canals ; and found that there were significantly higher cement thicknesses around oval post group than the circular post group as our study . in the present study , the resin cement thicknesses of the oval fiber posts were significantly greater than those of the circular fiber posts . in our study , the cervical diameter of the circular fiber posts was 1.8 mm , whereas the oval fiber posts had a bucco - lingual dimension of 1.9 mm , and a mesio - distal dimension of 1.1 mm cervically . further , the apical diameter of the circular fiber posts was 1 mm , and the apical width and thickness dimensions of the oval fiber posts were 1.1 and 0.5 mm , respectively . the explanation for the smaller resin cement thickness of the circular fiber posts in our study may be related to the wider mesial - distal dimension of circular fiber posts compared to oval fiber posts . in addition , the vibration of the medium grit diamond - coated ultrasonic tip used for oval fiber posts might have resulted in a wider post space than the original dimension of the post . therefore , using the fine grit diamond - coated ultrasonic tip could be recommended for achieving lower resin cement thicknesses.8 the differing results of muoz et al.20 and coniglio et al.8 may be related to the circular fiber post diameters that were not explained clearly in these studies . additionally , in these studies the post space lengths were 11 and 8 mm , respectively , whereas our study used 10-mm post - space lengths . furthermore , in our study root canals were prepared using protaper system up to f3 instrument . however , muoz et al.20 and coniglio et al.8 prepared narrower root canals in their study , with protaper f2 and mtwo 25/0.6 as the master apical files , respectively . in the present study , for both oval and circular fiber posts , the bond strengths in the apical specimens were significantly lower compared to the cervical specimens . factors possibly affecting the lower bond strengths to root dentin in the apical root specimens could be the non - uniform adaptation of the luting agent or its incomplete polymerization , both related to the difficult access of postspace walls during handling.22 the oval and circular posts used in the current study were both of quartz fiber . the only criterion that may affect the push - out bond strength is resin cement thickness . previous studies reported that , regardless of the anatomy of the posts , the resin cement thickness did not affect the push - out bond strength.8,23,24 in the light of the results of the current study , the resin cement thickness in the oval fiber - post group was greater than in the circular fiber - post group , and resin cement thickness did not affect the push - out bond strength . when considering failure modes after push - out tests , there were no significant difference between groups ; in addition , adhesive failure between dentin and cement were the most frequent type ; and this shows the weak link between the root dentin and luting cement . the situation might be a result of debris that have remained on canal walls and reduced the bonding . according to the results , it can be stated that resin cement thickness does not affect the push - out bond strength . pediatric soft - tissue sarcomas ( stss ) account for approximately 7% of all childhood tumors . out though surgery is the mainstay of treatment , advent of neoadjuvant chemotherapy ( nact ) has reduced the incidence of recurrence / metastasis . we present a case of ifs with early and unusual distant metastasis , localized on fluorodeoxyglucose positron emission tomography - computed tomography ( fdg pet - ct ) scan . the patient , a 4-year - old girl child , is a known case of ifs , primarily involving right proximal tibia and staging whole body pet / magnetic resonance ( mr ) scan not suggestive of disease elsewhere . she was detected with local relapse 3 months later for which she underwent chemotherapy followed by right above knee amputation . on physical examination , she had a palpable mildly tender lump in right proximal thigh . an mr imaging of the right thigh was obtained , which showed a neoplastic lesion involving right proximal shaft of femur . the maximum intensity projection [ figure 1 ] was suggestive of significant tracer uptake in right facial , lower lumbosacral , and bilateral proximal thigh regions . the axial ct and fused pet - ct images demonstrated fdg avid hypermetabolic foci in soft - tissue lesions involving right zygoma with intraorbital extension ( maximum standardized uptake value [ suvmax 9 ] ) [ figure 2a ] , right proximal femur [ suvmax 6.1 , figure 2b ] , sacrum with involvement of right sacroiliac joint [ suvmax 16 , figure 2c ] , and right suprascapular region [ suvmax 9 , figure 2d ] . maximum intensity projection of f-18 fluorodeoxyglucose positron emission tomography - computed tomography showing increased tracer uptake in regions pointed by arrowheads transaxial computed tomography and fusion positron emission tomography - computed tomography images on f-18 fluorodeoxyglucose positron emission tomography - computed tomography scan showing fluorodeoxyglucose avid hypermetabolic foci in soft - tissue lesions involving ; ( a ) right zygomatic bone with intraorbital extension , ( b ) right proximal femur , ( c ) sacrum and right sacroiliac joint , and ( d ) right suprascapular region subsequently , biopsy was obtained from sacral and right proximal femoral lesions . the tumor cells were arranged in interlacing fascicles and herringbone pattern [ figure 3a ] , with pleomorphic nuclei and brisk mitotic activity of > 10/10 hpf . microscopic images of hematoxylin and eosin stained biopsy slides obtained from sacral and right proximal femur lesions showing ; ( a ) arrangement of tumor cells in interlacing fascicles and herringbone pattern , with ( b ) multiple blood vessels dispersed within the tumor tissue ifs has incidence of 5 per million in the pediatric age group and majority of the patients are < 2 years old . although its etiology is unknown , a fusion transcript etv6-ntrk3 resulting from a chromosomal translocation t(12;15 ) is commonly associated . the extremities account for the primary in almost 74% of the cases , followed by head and neck region . most common site of origin is around knee joint followed by thigh and hip regions . the pathologic appearance of ifs includes spindle cells with many blood vessels arranged in hemangiopericytoma - like fascicular pattern . advent of nact allowed conservative surgeries and decreased local as well as metastatic relapses . in a large study comprising 56 infants by orbach et al . seventy - three percent of these were found to be chemosensitive and 22% maintained stable disease . performed a retrospective study with 11 patients who were treated with surgery alone or nact + surgery . rest , all were followed up for varying periods of 18 months to 13.5 years and were found to be disease free . three patients who had primary in extremities achieved complete remission and were disease free even after 2 years of follow - up . one infant had a neck primary and developed metastases to brain , lungs , and kidneys , which responded to chemotherapy . in addition , studies have proved that ifs carries overall 5-year survival rate of > 85% . overall , 1743% of cases present with local recurrence within 1 year after curative treatment . further , ifs originating from extremities is found less likely to cause distant metastases ( 8% ) than axial ifs ( 26% ) . who classification of stss categorizes ifs in intermediate rarely metastasizing neoplasm , which is defined as the ones which have a documented ability to metastasize but seldom do . as a group , they have < 2% chances of distant spread which can not be predicted on the basis of histomorphology . the more common sites of distant metastasis are lungs and lymph nodes although distant seeding of kidneys and brain have been documented . it has been well documented in adult stss for staging , response evaluation , and recurrence setting . in a meta - analysis , the sensitivity and specificity of fdg pet - ct in identifying primary as well as metastatic disease in stss were found to be 92% and 73% . chen et al . reported a case of ifs with local recurrence demonstrated by fdg pet - ct . the lesion was found to be low - grade fdg avid ( suvmax < 2.5 ) . our case of this rare pediatric neoplasm originating from an extremity uniquely presented with distant soft - tissue metastasis within 1 year of receiving nact and surgery . the fdg avidity of metastatic lesions was unusually high unlike reported in the literature so far . our findings revealed fdg pet - ct as a potentially useful tool in detection of metastatic disease recurrence in ifs cases . furthermore , metabolic parameters such as suvmax can be evaluated as prognostic tools to assess the tumor aggressiveness .

purposethe aim of this study was to evaluate whether the push - out bond strength varies between oval and circular fiber posts , and to examine the effect on the resin cement thicknesses around the posts.materials and methodseighteen mandibular premolar roots were separated into two groups for oval and circular fiber posts systems . post spaces were prepared and fiber posts were luted to the post spaces . roots were cut horizontally to produce 1-mm - thick specimens . resin cement thicknesses were determined with a metallographic optical microscope and push - out tests were done.resultsno significant differences were observed in terms of push - out bond strength between the oval and circular fiber posts ( p>.05 ) the resin cement thicknesses of the oval posts were greater than those of the circular posts group in the coronal , middle and apical specimens ( p<.05).conclusionin the light of these results , it can be stated that resin cement thickness does not affect the push - out bond strength . infantile fibrosarcoma ( ifs ) is a rare soft - tissue sarcoma originating from extremities and occasionally from axial soft tissue . the prognosis is good with favorable long - term survival . it is rarely metastasizing tumor , the chances being lesser with ifs originating from extremities . use of neoadjuvant chemotherapy ( nact ) as a treatment regime further reduces the chances of local relapse and distant metastasis . the organs commonly affected in metastatic ifs are lungs and lymph nodes . we report an unusual case of an ifs originating from extremity , which received nact , yet presented with an early metastatic disease involving soft tissues and sparing lungs and lymph nodes , as demonstrated on fluorodeoxyglucose positron emission tomography - computed tomography .

we studied 1,297 patients with type 1 diabetes from the nationwide finnish diabetic nephropathy study ( finndiane ) . patients were required to have an onset of diabetes before 35 years of age and initiation of permanent insulin treatment within 1 year of diagnosis . patients were classified into four groups based on the urinary albumin excretion rate ( aer ) or the presence of end - stage renal disease ( esrd ) ( table 1 ) . patients with normal aer were required to have a duration of diabetes over 15 years . serum concentration of mbl was determined in 1,064 patients ( available in the supplemental methods at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1495/dc1 ) . for nondiabetic control subjects , 701 finnish blood donors ( 49% men ; mean age 46.1 years ) from all over the country were applied and compared with an age- and sex - matched subgroup of patients with type 1 diabetes ( n = 701 ; 49% men ; mean age 43.9 years ) . the power of our study sample was calculated using the genetic power calculator ( http://pngu.mgh.harvard.edu/purcell/gpc/ ) ( 21 ) . clinical characteristics of the patients grouped by their nephropathy status data are means sd , median ( interquartile range ) , or percent unless otherwise indicated . patients were classified into three groups based on the aer in at least two of three consecutive overnight or 24-h urine collections . a fourth patient group consisted of patients on dialysis ( n = 34 ) and patients who had received a kidney transplant ( n = 144 ) and were thus classified as having esrd . the 24-h aer and serum creatinine values were derived from the last central laboratory measurements . therefore , single values may exceed or fall behind the thresholds for the classification due to effects of treatment . estimated glomerular filtration rate ( egfr ) was calculated using the cockcroft - gault formula adjusted for body surface area ( ref . < 0.0001 versus microalbuminuria . p < 0.01 versus microalbuminuria . p < 0.0001 versus macroalbuminuria . dbp , diastolic blood pressure ; dnp , diabetic nephropathy ; sbp , systolic blood pressure . we selected 17 snps using the hapmap database release 21a ( ceu ) ( http://www.hapmap.org/ ) and the haploview tagger program ( http://www.broad.mit.edu/mpg/haploview/index.php ) ( 22 ) from a 23-kb region covering the 6.3-kb mbl2 gene and the 5 untranslated region and 3 untranslated region . snps were chosen to capture all known hapmap snps having a minor allele frequency ( maf ) > 0.01 in the ceu population with pairwise r 0.8 . additionally , three functional snps ( allele c , allele d , and variant comparisons between groups were performed using the mann - whitney u test because mbl concentrations were not normally distributed . these analyses were done using the spss v. 15.0 software ( spss , chicago , il ) . allele frequencies and genotype distributions for single snps were compared between the control subjects and the matched subset of patients with type 1 diabetes as well as between patients with macroalbuminuria and patients with type 1 diabetes but normal aer . we also compared patients having esrd with patients with normal aer and compared a combined group of patients with macroalbuminuria or esrd with patients with normal aer . an allelic association test and three genotypic tests ( general , dominant , and recessive models ) were performed for each snp . additionally , analyses with adjustment for potential confounders ( age , sex , a1c , diabetes duration , and history of smoking ) were performed . the plink analysis program , version 1.00 , was applied in all analyses ( http://pngu.mgh.harvard.edu/purcell/plink/ ) ( 23 ) . for haplotype association analyses , we selected 17 snps using the hapmap database release 21a ( ceu ) ( http://www.hapmap.org/ ) and the haploview tagger program ( http://www.broad.mit.edu/mpg/haploview/index.php ) ( 22 ) from a 23-kb region covering the 6.3-kb mbl2 gene and the 5 untranslated region and 3 untranslated region . snps were chosen to capture all known hapmap snps having a minor allele frequency ( maf ) > 0.01 in the ceu population with pairwise r 0.8 . additionally , three functional snps ( allele c , allele d , and variant comparisons between groups were performed using the mann - whitney u test because mbl concentrations were not normally distributed . these analyses were done using the spss v. 15.0 software ( spss , chicago , il ) . allele frequencies and genotype distributions for single snps were compared between the control subjects and the matched subset of patients with type 1 diabetes as well as between patients with macroalbuminuria and patients with type 1 diabetes but normal aer . we also compared patients having esrd with patients with normal aer and compared a combined group of patients with macroalbuminuria or esrd with patients with normal aer . an allelic association test and three genotypic tests ( general , dominant , and recessive models ) were performed for each snp . additionally , analyses with adjustment for potential confounders ( age , sex , a1c , diabetes duration , and history of smoking ) were performed . the plink analysis program , version 1.00 , was applied in all analyses ( http://pngu.mgh.harvard.edu/purcell/plink/ ) ( 23 ) . for haplotype association analyses , the 17 tag snps genotyped captured the 68 hapmap snps having an maf>0.01 with a mean pairwise r of 0.97 . all snps were in hardy - weinberg equilibrium ( p > 0.01 ) both in the healthy control subjects ( supplementary table 1 ) and in type 1 diabetic patients ( data not shown ) . the frequencies of the exon 1 variants were 0.13 ( b ) , 0.06 ( c ) , and 0.006 ( d ) in the healthy subjects , which is in line with the previously published frequencies in the finnish population ( 17 ) . of the patients with diabetes , 30.6% were heterozygous carriers of one of these variants ( a / o genotype ) and 5.8% were homozygotes or compound heterozygotes ( o / o genotype ) . the median serum concentration of mbl was higher in patients with macroalbuminuria ( 1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) ; p = 0.019 ( fig . 1 ) . supplementary table 2 shows the distribution of the mbl2 diplotypes and the corresponding median mbl concentrations . as expected , the carriers of the exon 1 variants and the individuals homozygous for the x promoter polymorphism had clearly reduced mbl concentrations , with a median of 397 g / l . when the patients were stratified both by nephropathy status and mbl2 diplotype category , it was evident that patients with macroalbuminuria had significantly higher mbl concentrations than patients with normal aer for two of the six ( ya / ya : p = 0.0002 ; ya / yo : p = 0.003 ) diplotype categories ( supplementary table 2 and fig . box plot diagram showing the distribution of serum mbl concentrations , for patients with type 1 diabetes , stratified by nephropathy status . black horizontal lines , median values ; , interquartile ranges ; , outliers . 1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) , p = 0.019 . the median concentration was 1,645 g / l ( 5313,220 ) in patients with microalbuminuria and 1,359 g / l ( 3432798 ) in patients with esrd , p = 0.17 and p = 0.45 , respectively , compared with patients with normal aer . distribution of serum mbl concentrations in patients with normal aer and patients with macroalbuminuria , stratified by the mbl2 diplotype . there were no significant differences in allele frequencies or genotype distributions between the patients with type 1 diabetes and nondiabetic control subjects ( supplementary table 3 ) . although nominal evidence of allelic association ( p = 0.010.04 ) was seen for four snps ( rs930507 , rs2384045 , rs11003132 , and rs11003137 ) , the p values did not remain significant after correction for the number of tests performed . men and women were also tested separately , but no significant p values were achieved ( data not shown ) . association analyses showed no significant differences in allele frequencies or genotype distributions between patients with macroalbuminuria and patients with type 1 diabetes but normal aer ( supplementary table 4 ) . when patients with esrd were compared with patients with normal aer , the maf of this snp was 0.19 in patients with normal aer and 0.29 in patients with esrd ( p = 0.00009 ) . the genotype distribution of this snp was also significantly different between these groups ( p = 0.0002 ) , with homozygous individuals being more common among esrd patients ( 10 vs. 3% ) . the same snp had a p value of 0.006 in the genotypic test when a combined group of patients with macroalbuminuria or esrd was compared with patients with normal aer . the analyses were also performed with adjustment for potential confounders ( supplementary table 4 ) . all snps , except rs10824793 , were associated with mbl concentration ( supplementary table 5 ) . the snps showing the strongest associations were rs7899547 ( p = 3.0 10 ) , rs1031101 ( tagging the b allele ; p = 8.0 10 ) , rs2384045 ( p = 2.2 10 ) , and rs920727 ( p = 7.6 10 ) . the promoter variants h / l ( rs11003125 ) and p / q ( tagged by rs920724 ) had p values of 8.8 10 and 6.1 10 , respectively , whereas the alleles c and d had p values of 1.5 10 and 9.0 10 , respectively . the analyses were also performed with adjustment for potential confounders ( supplementary table 5 ) . the haplotypes constructed using the haploview program were studied for association with type 1 diabetes and diabetic nephropathy . the distribution of the mbl2 diplotypes , diplotype categories , and the high- and low - mbl genotypes in each patient group is presented in table 2 . there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer ( 0.60 ) and macroalbuminuria ( 0.61 ; p = 0.39 ) . similarly , when the distribution of the six diplotype categories was analyzed , no differences were seen ( p = 0.63 ) . distribution of the mbl2 genotypes within the different patient groups data are n or n ( percent ) . the mbl2 haplotypes were determined based on the co - occurrence of the three promoter variants ( h / l , rs11003125 ; x / y , rs7096206 ; and p / q , rs7095891 ) and the three exon 1 variants ( b , rs1800450 ; c , rs1800451 ; and d , rs5030737 ) . diplotypes were grouped into six categories ( ya / ya , ya / xa , xa / xa , ya / yo , xa / yo , and yo / yo ) based on the x / y polymorphism and the presence of any of the exon 1 variants ( b , c , and d ) , collectively designated with o , and further into low- and high - mbl genotypes . there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer and macroalbuminuria . however , the frequency of the high - mbl genotypes within the group of esrd patients ( 0.54 ) was lower than in patients with normal aer ( 0.60 ) , although this difference was not significant ( p = 0.10 ) . the 17 tag snps genotyped captured the 68 hapmap snps having an maf>0.01 with a mean pairwise r of 0.97 . all snps were in hardy - weinberg equilibrium ( p > 0.01 ) both in the healthy control subjects ( supplementary table 1 ) and in type 1 diabetic patients ( data not shown ) . the frequencies of the exon 1 variants were 0.13 ( b ) , 0.06 ( c ) , and 0.006 ( d ) in the healthy subjects , which is in line with the previously published frequencies in the finnish population ( 17 ) . of the patients with diabetes , 30.6% were heterozygous carriers of one of these variants ( a / o genotype ) and 5.8% were homozygotes or compound heterozygotes ( o / o genotype ) . the median serum concentration of mbl was higher in patients with macroalbuminuria ( 1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) ; p = 0.019 ( fig . 1 ) . supplementary table 2 shows the distribution of the mbl2 diplotypes and the corresponding median mbl concentrations . as expected , the carriers of the exon 1 variants and the individuals homozygous for the x promoter polymorphism had clearly reduced mbl concentrations , with a median of 397 g / l . when the patients were stratified both by nephropathy status and mbl2 diplotype category , it was evident that patients with macroalbuminuria had significantly higher mbl concentrations than patients with normal aer for two of the six ( ya / ya : p = 0.0002 ; ya / yo : p = 0.003 ) diplotype categories ( supplementary table 2 and fig . box plot diagram showing the distribution of serum mbl concentrations , for patients with type 1 diabetes , stratified by nephropathy status . black horizontal lines , median values ; , interquartile ranges ; , outliers . the median serum concentration of mbl was higher in patients with macroalbuminuria ( median 1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) , p = 0.019 . the median concentration was 1,645 g / l ( 5313,220 ) in patients with microalbuminuria and 1,359 g / l ( 3432798 ) in patients with esrd , p = 0.17 and p = 0.45 , respectively , compared with patients with normal aer . distribution of serum mbl concentrations in patients with normal aer and patients with macroalbuminuria , stratified by the mbl2 diplotype . there were no significant differences in allele frequencies or genotype distributions between the patients with type 1 diabetes and nondiabetic control subjects ( supplementary table 3 ) . although nominal evidence of allelic association ( p = 0.010.04 ) was seen for four snps ( rs930507 , rs2384045 , rs11003132 , and rs11003137 ) , the p values did not remain significant after correction for the number of tests performed . men and women were also tested separately , but no significant p values were achieved ( data not shown ) . association analyses showed no significant differences in allele frequencies or genotype distributions between patients with macroalbuminuria and patients with type 1 diabetes but normal aer ( supplementary table 4 ) . when patients with esrd were compared with patients with normal aer , significant evidence of association the maf of this snp was 0.19 in patients with normal aer and 0.29 in patients with esrd ( p = 0.00009 ) . the genotype distribution of this snp was also significantly different between these groups ( p = 0.0002 ) , with homozygous individuals being more common among esrd patients ( 10 vs. 3% ) . the same snp had a p value of 0.006 in the genotypic test when a combined group of patients with macroalbuminuria or esrd was compared with patients with normal aer . the analyses were also performed with adjustment for potential confounders ( supplementary table 4 ) . all snps , except rs10824793 , were associated with mbl concentration ( supplementary table 5 ) . the snps showing the strongest associations were rs7899547 ( p = 3.0 10 ) , rs1031101 ( tagging the b allele ; p = 8.0 10 ) , rs2384045 ( p = 2.2 10 ) , and rs920727 ( p = 7.6 10 ) . the promoter variants h / l ( rs11003125 ) and p / q ( tagged by rs920724 ) had p values of 8.8 10 and 6.1 10 , respectively , whereas the alleles c and d had p values of 1.5 10 and 9.0 10 , respectively . the analyses were also performed with adjustment for potential confounders ( supplementary table 5 ) . the haplotypes constructed using the haploview program were studied for association with type 1 diabetes and diabetic nephropathy . the distribution of the mbl2 diplotypes , diplotype categories , and the high- and low - mbl genotypes in each patient group is presented in table 2 . there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer ( 0.60 ) and macroalbuminuria ( 0.61 ; p = 0.39 ) . similarly , when the distribution of the six diplotype categories was analyzed , no differences were seen ( p = 0.63 ) . distribution of the mbl2 genotypes within the different patient groups data are n or n ( percent ) . the mbl2 haplotypes were determined based on the co - occurrence of the three promoter variants ( h / l , rs11003125 ; x / y , rs7096206 ; and p / q , rs7095891 ) and the three exon 1 variants ( b , rs1800450 ; c , rs1800451 ; and d , rs5030737 ) . diplotypes were grouped into six categories ( ya / ya , ya / xa , xa / xa , ya / yo , xa / yo , and yo / yo ) based on the x / y polymorphism and the presence of any of the exon 1 variants ( b , c , and d ) , collectively designated with o , and further into low- and high - mbl genotypes . there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer and macroalbuminuria . however , the frequency of the high - mbl genotypes within the group of esrd patients ( 0.54 ) was lower than in patients with normal aer ( 0.60 ) , although this difference was not significant ( p = 0.10 ) . our study showed no evidence of association between the mbl2 snps and type 1 diabetes , although weak association signals ( uncorrected p = 0.010.04 ) were seen for four snps . importantly , using tag snps , we have captured information on the whole gene and its surroundings , whereas the other studies addressed only the functional exon 1 variants . in a danish study ( 6 ) , genotypes producing high mbl concentrations were more common in patients having diabetic nephropathy than in patients with normal aer . we found no evidence of such an association , although our study sample was about double the size ( 6 ) . furthermore , we thoroughly studied association between both single mbl2 snps and haplotypes and nephropathy , but all the results were negative . in accordance with previous studies ( 6,7 ) , we showed that the median serum mbl concentration was significantly higher in patients with macroalbuminuria compared with the patients with normal aer . this result persisted even when the patients were stratified by the mbl diplotype categories , although the difference was significant in only two of them . thus , it seems that the high serum mbl concentration in patients with nephropathy is likely to reflect some still unknown pathogenic event such as chronic low - grade inflammation . further studies are needed to resolve whether elevated mbl concentrations are a marker associated with some other contributing factor or a consequence of nephropathy or diabetic microvascular complications in general . the minor allele of the snp rs920727 was more common in esrd patients than in the other patients 0.29 vs. 0.190.20 ) . furthermore , the frequency of the high - mbl genotypes was somewhat lower in the esrd patients than in the other groups ( 0.54 vs. 0.600.61 ) , potentially explaining the relatively low mbl concentration in the esrd patients . however , we do not consider these differences as an indication of a causal association with esrd phenotype . most likely they are due to chance or even signs of selective mortality . supporting this hypothesis , evidence exists that mortality of patients with type 2 diabetes is higher among individuals with an mbl concentration 1,000 g / l ( 24 ) . moreover , most of the esrd patients in our study have received a kidney transplant , and graft rejection is less common for the group of patients with low mbl concentrations ( 25 ) . many of the snps showing strong association with mbl concentration ( including the esrd - associated rs920727 ) did not belong to the group of snps forming the common mbl2 haplotypes . some of these variants may have an independent effect on the mbl concentration or tag other still unknown functional variants . however , this somewhat surprising association pattern can mainly be explained by ld between the associated and haplotype - forming snps ( supplementary table 6 ) . our study sample has a reasonably high power to detect associations with relatively common snps ( population frequency > 20% ) with modest effects . the previously described higher median mbl concentration in patients with macroalbuminuria ( 6,7 ) was evident also in our sample . we have thoroughly covered the common variation within the mbl2 gene in carefully characterized finnish type 1 diabetic patients . we conclude that although most of the mbl2 snps studied were associated with the mbl concentration , neither any single snp nor any of their haplotype combinations confer risk of type 1 diabetes or diabetic nephropathy . gwas2 included 450 unrelated generalized vitiligo patients ( cases ) of non - hispanic / latino european ancestry ( eur ) from north america and europe , who met strict clinical criteria for generalized vitiligo . controls for gwas2 were 3182 eur individuals not specifically known to have any autoimmune disease or malignant melanoma , for whom genome - wide genotypes were obtained from the database of genotypes and phenotypes ( dbgap ; phs000092v1 , phs000125v1 , phs000138v2 , phs000168v1 , and phs000206v3 ) , or from the illumina icontroldb . the replication study included 1440 unrelated eur generalized vitiligo cases and 1316 unrelated eur controls from north america and europe , principally spouses of vitiligo patients from the gwas2 study and the replication study itself . there was no overlap of cases and controls between the gwas1 , gwas2 , and replication cohorts . cases and controls provided clinical history regarding vitiligo and other autoimmune diseases as described previously for gwas1 , and controls having known relatives with vitiligo or reporting any known autoimmune diseases or melanoma were excluded . this study was approved by each institutional review board and was conducted according to declaration of helsinki principles . genomic dna was prepared from saliva specimens using a dna self - collection kit per the manufacturer 's instructions ( oragene , dna genotek ) . for genome - wide genotyping , dna concentrations were assayed by both fluorescence staining ( picogreen method , invitrogen ) and ultraviolet a260 spectrophotometry ( nanodrop , thermo scientific ) . for genotyping specific snps in the replication study , dna concentrations were assayed by nanodrop . genome - wide genotyping for gwas2 cases was performed for 657,366 snps using illumina human660w - quad beadchips per the manufacturer 's instructions . gwas2 controls obtained from dbgap and illumina icontroldb had been genotyped using illumina human 610-quad and human 1mv1 beadchips . genotyping of 46 snps in the replication study was performed using a custom illumina goldengate assay per the manufacturer 's instructions . quality control filtering of genome - wide genotype data in gwas2 was carried out as described for gwas1 using illumina genomestudio , version 3 and plink , version 1.07 . cases were excluded on the basis of snp call rates < 98.5% ( n = 16 ) , discordance between reported and observed sex ( n = 0 ) , and/or inadvertent subject duplication ( n = 0 ) . beyond prior quality control procedures , controls were excluded on the basis of snp call rates < 95% ( n = 0 ) . additional cases ( n = 6 ) and controls ( n = 356 ) were excluded on the basis of cryptic relatedness based on pair - wise identity - by - descent estimation ( pi - hat > 0.05 ) of the entire ( gwas1 + gwas2 ) summary dataset , in which case the individual with lower snp call rate was excluded . snps were excluded on the basis of genotype missing rate 2% overall ( n = 97,301 ) in cases plus controls , observed minor allele frequency < 0.01 ( n = 23,064 ) , significant deviation ( p < 10 ) from hardy - weinberg equilibrium in the control dataset ( n = 5,241 ) , and/or significant difference ( p < 10 ) in genotype missing rate in cases versus controls ( n = 35,939 ) . data for snps with p values < 10 were reviewed with respect to genotype clusters and allele calls in cases , and minor allele frequency in controls compared to data in public data sources , and were excluded if there were apparent data quality problems ( n = 2 ) . to control for population stratification , we performed principle components analysis using eigensoft , version 4.2 , and excluded as outliers cases ( n = 10 ) and controls ( n = 16 ) whose ancestry was 6 standard deviations from the mean on one of the top ten eigenvectors . in the replication study we successfully determined genotypes for 46 snps that showed suggestive ( p < 10 ) or significant p values in the genome - wide meta - analysis of gwas1 and gwas2 , using a custom illumina goldengate assay . snp genotypes were subjected to quality control filters similar to those in gwas1 and gwas2 , as appropriate . for gwas2 , after quality control filtering of subjects and snp data and removal of genetic outliers , we compared allele frequencies of the remaining 495,821 snps in the final 418 cases and 2810 controls using the unadjusted cochran - armitage trend test implemented in plink and the adjusted cochran - armitage trend test implemented in eigensoft , in which both phenotypes and genotypes of subjects were adjusted for ancestry using the top ten eigenvectors . the unadjusted cochran - armitage trend tests and the adjusted cochran - armitage trend tests yielded genomic inflation factors of 1.054 and 1.050 , respectively , indicating that residual population stratification was negligible . odds ratios and 95% confidence limits were calculated by logistic regression analysis by use of plink . to fully assess association at the 24 novel suggestive loci , we imputed genotypes for each locus using mach , ver.1.0 based on patterns of haplotype variation in the 1000 genomes project european ancestry samples ( release aug 4 , 2010 ) . we retained imputed snps with r > 0.3 and minor allele frequency > 0.01 for further analyses . for the replication study , after quality control filtering , we compared allele frequencies for genotyped snps in the remaining 1377 patients and 1284 controls using the cochran - armitage trend test . odds ratios and 95% confidence limits were calculated by logistic regression analysis . to obtain combined ors and p values for gwas1 and gwas2 , and for the combined gwas1 , gwas2 , and replication studies , we performed meta - analysis using a cochran - mantel - haenszel test . a breslow - day test was used to test for heterogeneity of ors of the same snp in different study cohorts . calculation of linkage disequilibrium between snps in regions of association was carried out using haploview , version 4.2 . as a test of the independent effect of a given locus conditioned on the effect of another locus , we compared the fit of a model containing both loci to a model containing only the conditioning locus , assuming a multiplicative genotypic effect for the high - risk allele of each locus . we estimated the contribution of known vitiligo susceptibility loci to the total variance in vitiligo liability as the difference between the variance accounted for by all snps genome - wide and the variance remaining after removing snps in known vitiligo susceptibility loci , using gcta to estimate the two components of variance . we estimated heritability of vitiligo liability assuming a vitiligo prevalence in the eur population of 0.0038 and sibling risk 0.06 . to gain insights into potential functional relationships among proteins encoded by vitiligo risk loci , we carried out functional interaction network analysis using the search tool for the retrieval of interacting genes ( string ) 9.0 . input data were all known vitiligo susceptibility loci ( including ticam1 , and selecting btnl2 to represent the mhc class ii gene region ) . in addition , we iteratively tested inclusion of all proteins encoded by genes in the 16q24.3 and 22q13.2 association regions as well as fam76b as potentially representing the 11q21 association region as a means of possible gene identification . gwas2 included 450 unrelated generalized vitiligo patients ( cases ) of non - hispanic / latino european ancestry ( eur ) from north america and europe , who met strict clinical criteria for generalized vitiligo . controls for gwas2 were 3182 eur individuals not specifically known to have any autoimmune disease or malignant melanoma , for whom genome - wide genotypes were obtained from the database of genotypes and phenotypes ( dbgap ; phs000092v1 , phs000125v1 , phs000138v2 , phs000168v1 , and phs000206v3 ) , or from the illumina icontroldb . the replication study included 1440 unrelated eur generalized vitiligo cases and 1316 unrelated eur controls from north america and europe , principally spouses of vitiligo patients from the gwas2 study and the replication study itself . there was no overlap of cases and controls between the gwas1 , gwas2 , and replication cohorts . cases and controls provided clinical history regarding vitiligo and other autoimmune diseases as described previously for gwas1 , and controls having known relatives with vitiligo or reporting any known autoimmune diseases or melanoma were excluded . this study was approved by each institutional review board and was conducted according to declaration of helsinki principles . genomic dna was prepared from saliva specimens using a dna self - collection kit per the manufacturer 's instructions ( oragene , dna genotek ) . for genome - wide genotyping , dna concentrations were assayed by both fluorescence staining ( picogreen method , invitrogen ) and ultraviolet a260 spectrophotometry ( nanodrop , thermo scientific ) . for genotyping specific snps in the replication study , genome - wide genotyping for gwas2 cases was performed for 657,366 snps using illumina human660w - quad beadchips per the manufacturer 's instructions . gwas2 controls obtained from dbgap and illumina icontroldb had been genotyped using illumina human 610-quad and human 1mv1 beadchips . genotyping of 46 snps in the replication study was performed using a custom illumina goldengate assay per the manufacturer 's instructions . quality control filtering of genome - wide genotype data in gwas2 was carried out as described for gwas1 using illumina genomestudio , version 3 and plink , version 1.07 . cases were excluded on the basis of snp call rates < 98.5% ( n = 16 ) , discordance between reported and observed sex ( n = 0 ) , and/or inadvertent subject duplication ( n = 0 ) . beyond prior quality control procedures , controls were excluded on the basis of snp call rates < 95% ( n = 0 ) . additional cases ( n = 6 ) and controls ( n = 356 ) were excluded on the basis of cryptic relatedness based on pair - wise identity - by - descent estimation ( pi - hat > 0.05 ) of the entire ( gwas1 + gwas2 ) summary dataset , in which case the individual with lower snp call rate was excluded . snps were excluded on the basis of genotype missing rate 2% overall ( n = 97,301 ) in cases plus controls , observed minor allele frequency < 0.01 ( n = 23,064 ) , significant deviation ( p < 10 ) from hardy - weinberg equilibrium in the control dataset ( n = 5,241 ) , and/or significant difference ( p < 10 ) in genotype missing rate in cases versus controls ( n = 35,939 ) . data for snps with p values < 10 were reviewed with respect to genotype clusters and allele calls in cases , and minor allele frequency in controls compared to data in public data sources , and were excluded if there were apparent data quality problems ( n = 2 ) . to control for population stratification , we performed principle components analysis using eigensoft , version 4.2 , and excluded as outliers cases ( n = 10 ) and controls ( n = 16 ) whose ancestry was 6 standard deviations from the mean on one of the top ten eigenvectors . in the replication study we successfully determined genotypes for 46 snps that showed suggestive ( p < 10 ) or significant p values in the genome - wide meta - analysis of gwas1 and gwas2 , using a custom illumina goldengate assay . snp genotypes were subjected to quality control filters similar to those in gwas1 and gwas2 , as appropriate . for gwas2 , after quality control filtering of subjects and snp data and removal of genetic outliers , we compared allele frequencies of the remaining 495,821 snps in the final 418 cases and 2810 controls using the unadjusted cochran - armitage trend test implemented in plink and the adjusted cochran - armitage trend test implemented in eigensoft , in which both phenotypes and genotypes of subjects were adjusted for ancestry using the top ten eigenvectors . the unadjusted cochran - armitage trend tests and the adjusted cochran - armitage trend tests yielded genomic inflation factors of 1.054 and 1.050 , respectively , indicating that residual population stratification was negligible . odds ratios and 95% confidence limits were calculated by logistic regression analysis by use of plink . to fully assess association at the 24 novel suggestive loci , we imputed genotypes for each locus using mach , ver.1.0 based on patterns of haplotype variation in the 1000 genomes project european ancestry samples ( release aug 4 , 2010 ) . we retained imputed snps with r > 0.3 and minor allele frequency > 0.01 for further analyses . for the replication study , after quality control filtering , we compared allele frequencies for genotyped snps in the remaining 1377 patients and 1284 controls using the cochran - armitage trend test . odds ratios and 95% confidence limits were calculated by logistic regression analysis . to obtain combined ors and p values for gwas1 and gwas2 , and for the combined gwas1 , gwas2 , and replication studies , we performed meta - analysis using a cochran - mantel - haenszel test . a breslow - day test was used to test for heterogeneity of ors of the same snp in different study cohorts . calculation of linkage disequilibrium between snps in regions of association was carried out using haploview , version 4.2 . as a test of the independent effect of a given locus conditioned on the effect of another locus , we compared the fit of a model containing both loci to a model containing only the conditioning locus , assuming a multiplicative genotypic effect for the high - risk allele of each locus . we estimated the contribution of known vitiligo susceptibility loci to the total variance in vitiligo liability as the difference between the variance accounted for by all snps genome - wide and the variance remaining after removing snps in known vitiligo susceptibility loci , using gcta to estimate the two components of variance . we estimated heritability of vitiligo liability assuming a vitiligo prevalence in the eur population of 0.0038 and sibling risk 0.06 . to gain insights into potential functional relationships among proteins encoded by vitiligo risk loci , we carried out functional interaction network analysis using the search tool for the retrieval of interacting genes ( string ) 9.0 . input data were all known vitiligo susceptibility loci ( including ticam1 , and selecting btnl2 to represent the mhc class ii gene region ) . in addition , we iteratively tested inclusion of all proteins encoded by genes in the 16q24.3 and 22q13.2 association regions as well as fam76b as potentially representing the 11q21 association region as a means of possible gene identification .

objectivemannose - binding lectin ( mbl ) is an essential component of the acute - phase immune response and may thus play a role in the pathogenesis of type 1 diabetes and diabetic nephropathy . the serum concentration of mbl is mainly genetically determined , and elevated concentrations have been associated with both type 1 diabetes and diabetic nephropathy . previous genetic studies have not been conclusive due to the small number of patients and polymorphisms studied . we investigated whether mbl2 polymorphisms are associated with type 1 diabetes or diabetic nephropathy and whether patients with nephropathy have elevated mbl concentrations as indicated previously . furthermore , we studied the association between mbl2 polymorphisms and mbl concentration.research design and methodswe genotyped 20 mbl2 single nucleotide polymorphisms ( snps ) in a large , well - characterized finnish case - control sample consisting of 1,297 patients with type 1 diabetes with or without nephropathy and 701 nondiabetic individuals . the serum concentration of mbl was available for 1,064 patients.resultswe found that 19 snps were associated with the mbl concentration ( p = 3 10817 104 ) . mbl concentrations were higher in patients with macroalbuminuria compared with patients without nephropathy even when the patients were stratified by the mbl2 genotypic background in accordance with previous studies . however , no evidence of association between any of the snps or their haplotype combinations and type 1 diabetes or diabetic nephropathy was observed.conclusionsalthough most of the mbl2 snps studied were associated with the mbl concentration , no common variations ( neither single snps nor their haplotype combinations ) confer risk of type 1 diabetes or diabetic nephropathy . in previous linkage and genome - wide association studies we identified 17 susceptibility loci for generalized vitiligo . by a second genome - wide association study , meta - analysis , and independent replication study , we have now identified 13 additional vitiligo - associated loci , including oca2-herc2 , a region of 16q24.3 containing mc1r , a region of chromosome 11q21 near tyr , several immunoregulatory loci including ifih1 , cd80 , clnk , bach2 , sla , casp7 , cd44 , ikzf4 , sh2b3 , and a region of 22q13.2 where the causal gene remains uncertain . functional pathway analysis shows that most vitiligo susceptibility loci encode immunoregulatory proteins or melanocyte components that likely mediate immune targeting and genetic relationships among vitiligo , malignant melanoma , and normal variation of eye , skin , and hair color .

now they need more attention because as the average life expectancy , elderly population , and subsequent resulting osteoporosis continue to increase , orthopaedic surgeons will get more such cases [ 14 ] . intertrochanteric fractures in the elderly are associated with high rates of mortality , ranging from 15 to 20% , as they are at a high risk for deep vein thrombosis ( dvt ) , urinary tract infections , and pulmonary embolism if they fail to mobilize or ambulate early . surgical stabilization fulfills the aim of early mobilization and facilitates union in an anatomical position . due to this , operative stabilization of these fractures is now the gold standard treatment . although other options are available , the standard approach is to use a dynamic hip screw ( dhs ) with a 4-holed side plate in stable fractures in most centers [ 68 ] . traditionally a wide surgical exposure is necessary for this procedure which comes with its drawbacks like a large skin incision , considerable soft tissue trauma , significant blood loss , and pain . to avoid these problems minimally invasive it has theoretical advantages of decreased blood loss , better cosmesis , less pain , and rapid rehabilitation . we conducted a prospective comparative study of conventional ( open ) dhs and minimally invasive dhs at our center to test the utility of this new approach . this study was carried out in the department of orthopaedic surgery , sohar hospital , oman , between 2008 and 2011 . a total of 60 patients with intertrochanteric femoral fractures were selected for this study and all of them gave informed written consent for the same . all the fractures were reduced and fixed with 135 degree dynamic hip screws with 4-hole side plate , 30 using conventional ( open ) technique ( cdhs ) and 30 with minimally invasive technique ( midhs ) . patients in both the groups were matched with respect to age , preoperative hemoglobin level , and morbidity . in particular , we also measured the difference between pre- and postoperative hemoglobin levels ( hemoglobin drop ) , which is an indicator of blood loss the operating time was measured from beginning of skin incision to skin closure . in all cases of both conventional dhs group and all patients were rehabilitated using the same standard postoperative hip fracture management protocol by starting mobilization and weight bearing within 24 hours of surgery . the length of hospital stay was noted for each case and complications were also recorded for both the groups . the surgical technique for both the procedures is described below [ 3 , 4 , 7 ] . a longitudinal skin incision 1015 cm in length was made over the lateral aspect of upper thigh , starting from the middle of the greater trochanteric prominence and extending down the lateral aspect of femoral shaft . fracture was reduced and its confirmation done with fluoroscope . following fixation of the fracture in the standard fashion , a drain was used as per surgeon 's preference , and the incision was closed in layers . all fractures in this study received adequate closed reduction under c arm guidance ( anatomical to 10 of valgus on anteroposterior radiograph and anatomical on lateral radiograph ) prior to the start of operation . the incision was placed under fluoroscopic guidance by the identification of the site on the hip that corresponded to the position of the fracture . the size of the incision was not longer than 5 cm in any case . the iliotibial band and vastus muscles were split through one incision . after the insertion of a guide wire , after this barrel plate was also introduced through the same incision , turning the barrel from 180 to 90 as shown in figure 1 . the guide wire was then reintroduced through the side plate barrel and then rotated until the side plate lied suitably under the soft tissues . the barrel was then engaged in the lag screw and advanced in the conventional fashion . the side plate screws were then placed in the usual manner through side plate holes by retracting the skin and subcutaneous tissue with a right angled soft tissue retractor . compared to patients in the conventional group , those in the minimally invasive group had shorter operating times ( mean 50 versus 40 minutes ) and a higher proportion of cases whose drain was removed within 24 hours ( 26% versus 71% ) . the drain output and decrease in haemoglobin level were not significantly different in the 2 groups . there was no intraoperative and 30-day mortality in both the groups , but the 6-month mortality was 6.6% ( 2/30 ) in conventional group while being 0% in the minimally invasive group . while one - year mortality of 13.3% ( 4/30 ) was found in the conventional dhs group , 3.33% ( 1/30 ) was found in minimally invasive group . both groups have been compared in table 1 and figures 3 , 4 , 5 , 6 , 7 , 8 , 9 , and 10 . hip fractures are a common cause of morbidity and mortality in the elderly population and are associated with considerable health expenditure . although many internal fixation devices provide sufficient stabilization , the surgical treatment of intertrochanteric femoral fractures is still challenging . the dynamic hip screw , which provides rigid fixation and allows early mobilization as it enables optimal collapse and compression of the fracture site , is the most common extramedullary device used for intertrochanteric fractures and has reasonable results [ 6 , 8 ] . in addition , when compared with sliding hip screws , no definite evidence exists of a reduced failure rate with intramedullary nails in unstable intertrochanteric fractures . therefore , the routine use of intramedullary devices has not been recommended for the treatment of intertrochanteric fractures , and the dynamic hip screw is still the standard type of fixation for intertrochanteric fractures [ 6 , 11 ] . however , insertion of this conventionally requires a 1015 cm incision splitting the vastus lateralis , causing considerable bleeding and damage to the overlying soft tissues , and intertrochanteric fractures often occur in the elderly , who commonly have multiple comorbid conditions that may be worsened by the surgical trauma associated with a major operation . in order to find less invasive techniques to simplify surgery and lower complication rate by reducing surgical time and blood loss , some authors used custom made implants or new devices ( e.g. , per cutaneous compression plate , pccp ) which require the purchase of additional instruments and implants by the hospital [ 3 , 4 , 7 ] . on the other hand , the minimally invasive dhs technique uses the existing instruments with which the operating team is familiar and confident , with no need to purchase new instruments . several authors have shown that the same advantages can be gained by modifying the surgical approach while using existing fixation devices , thus requiring neither a new plating system nor training of operating theatre staff to familiarize with them . therefore , the development of the minimally invasive dynamic hip screw technique , which causes less tissue damage and bleeding and shorter operative times and provides good fixation , may result in better outcomes , especially in elderly patients . a reduced operative time , especially in elderly patients with comorbid conditions or poor cardiopulmonary reserve , is desirable because it reduces the risks of general anesthetic . this , along with reduced surgical trauma , may be significant in reducing postoperative morbidity and mortality in such patients . with regard to surgical blood loss , a previous angiographic study revealed that the average distance from the vastus lateralis ridge to the first significant perforating branch was 9.3 cm . therefore this area is relatively a safe vascular zone . in the minimally invasive dynamic hip screw technique , a 35 cm incision is made , and the incision point is approximately 4 cm below the vastus lateralis ridge . therefore , blood loss decreases due to less soft tissue dissection and less fracture exposure and because an incision is made in the safe vascular zone [ 3 , 4 ] . because decreased blood loss is thought to be an explanation of reduced cardiovascular complications , which decrease the need for blood transfusion , this may have great clinical significance [ 9 , 12 ] . wong et al . proposed a novel and precise technique for determining the guide wire entry site which was the first step of their midhs surgical technique based on the concept of an isosceles right triangle ( in such a triangle the two angles along the hypotenuse are equal to 45 and the external angle is 180 45 = 135 which is the same as the angle of the guide plate , as demonstrated in figure 2 . by placing a guide plate with guide wire on the anterior hip and placing the plate along the lateral border of the proximal femur , under fluoroscopic guidance , an ap line was traced along the femoral head and neck region along the guide wire and another along the side plate , with the two lines meeting at a point e. a lateral radiograph was then obtained , a guide wire was placed along the centre of femoral head and neck , and a lateral line was traced . a vertical line dropped from the previously mentioned point intersects this lateral line at another point i. fluoroscopy is used to measure the distance of this point from skin to bone . this distance was then used to finally mark the entry site at the same distance along the lateral line distally . a 2.5 cm long incision was then made from the guide wire entry site distally . quantified the surgical trauma in conventional and minimally invasive surgical techniques for pertrochanteric fracture surgery based on measurement of markers of inflammation ( interleukins ) . significantly higher level of interleukin 6 was found in the postoperative period in patients operated by conventional technique than in those operated by minimally invasive technique . results from the literature and their study indicate that il-6 levels enable measurement of not only local tissue trauma / invasiveness but also the subsequent systemic response . by measuring the invasiveness of the intervention ( second hit / additional tissue trauma ) based on levels of interleukins , it could become possible not only to detect local damage but also to obtain an independent predictor of risk / outcome in elderly patients with pertrochanteric fracture . however a study by lee et al . showed a relatively high incidence of avascular necrosis of the femoral head with the minimally invasive technique . vascular insult to the femoral head may therefore be considered as one potential drawback of this technique . zhou et al . carried out a meta - analysis of studies on minimally invasive versus conventional dhs [ 3 , 4 , 7 , 9 ] and found that there was a lower rate of serious postoperative complications in the minimally invasive dynamic hip screw group compared with the conventional dynamic hip screw group ( relative risk , 0.35 ; 95% confidence interval ( ci ) , 0.16 , 0.78 ) . also the average operative time ( weighted mean difference , 16.32 ; 95% ci , 28.78 to 3.86 ) , hemoglobin decrease ( weighted mean difference , 1.44 ; 95% ci , 1.98 to 0.89 ) , and length of stay ( weighted mean difference , 3.72 ; 95% ci , 5.44 to 2.01 ) were lower in the minimally invasive dynamic hip screw group , while the postoperative harris hip score ( weighted mean difference , 1.42 ; 95% ci , 0.23 to 2.60 ) was higher in the minimally invasive dynamic hip screw group . recent studies [ 15 , 16 ] showed that stable pertrochanteric had successful fixation with two - hole dhs plate . it appears that the widespread use of four - hole side plate dhs in stable pertrochanteric fractures is merely based on tradition and needs to be reconsidered in the present era of evidence - based medicine . biomechanical studies have also demonstrated equivalent peak load to failure when comparing the two- and four - hole dhs plates . therefore , using the two - hole dhs may be at least as safe as the four - hole dhs in nonosteoporotic patients , and the surgical exposure required will be smaller . it may also be economical , both financially and in the use of operating time . in osteoporotic bone , postoperatively , elderly patients are at a high risk for dvt , urinary tract infections , and pulmonary embolism if they fail to mobilize or ambulate early . reduced operative time , less bleeding , and less postoperative pain may promote earlier ambulation in the minimally invasive dynamic hip screw group , which may contribute to a more favorable outcome in terms of serious postoperative complications [ 7 , 13 ] . also it may fasten overall patient recovery , thereby facilitating early discharge and , therefore , having significant financial implications for hospitals . because intertrochanteric fractures usually occur in elderly patients who may have cognitive deficits from age or have medical diseases , it is important for patients to return to preinjury activity levels as soon as possible to avoid complications . the minimally invasive dynamic hip screw , as it offers faster rehabilitation , is especially beneficial for older patients [ 4 , 7 , 13 ] . further studies with larger number of patients , proper randomization , blinding procedure , and robust analysis will give more conclusive results . to conclude , the minimally invasive dynamic hip screw and the conventional dynamic hip screw both are effective , simple , and safe for the treatment of intertrochanteric fractures . compared with the conventional dynamic hip screw , the minimally invasive dynamic hip screw usually has a shorter operative time , less hemoglobin decrease , and a shorter length of stay , which benefit patients and reduce hospital costs and may therefore be recommended in place of conventional technique . nucleic acids are highly attractive class of therapeutics due to their potential to regulate any selected gene of interest . given their capacity to modulate conventionally undruggable targets , oligonucleotides ( ons ) have been extensively investigated as potential therapeutics to treat cancer , viral infections , genetic diseases , and immunological disorders . despite the clear therapeutic potential of ons , their poor permeability across cellular membranes ( due to their intrinsic polyanionic nature and high molecular weight ) and susceptibility to degradation by ubiquitous nucleases hamper their clinical translation . recent advances in the development of various delivery vehicles ( e.g. , polymer- , lipid- , peptide- , nano / microparticles- , or viral - based ) have helped overcoming some of the on delivery problems ; however , issues such as systemic toxicity , low concentration at target sites and pharmaceutical complexity of the delivery systems still represent obstacles to the clinical translation of on therapeutics . the conception of stable ons with enhanced affinity via various chemical modifications is one of the most remarkable achievements in this field . for example , the combination of phosphorothioate ( ps ) backbone modification with 2-o - methyl ( ome ) and 2-o-(2-methoxyethyl ) ( moe ) moieties in the sugar units or bicyclic ribonucleosides are the most widely used chemical strategies under clinical investigation . another approach is modification with 2-deoxy-2-fluoro - arabinonucleic acid ( fana ) , which upon binding to the target mrna induces its rnase h - mediated degradation . several chemically stabilized ons are already marketed ( e.g. , mipomersen for homozygous familial hypercholesterolemia ) or in late - phase clinical trials . nucleic acid therapy could be especially beneficial for several disorders of the gastrointestinal ( gi ) system that currently lack appropriate treatments such as inflammatory bowel diseases , colon cancer , and familial adenomatous polyposis . the delivery of nucleic acids directly to the gi mucosa is ideal to achieve high local concentrations while minimizing systemic exposure and subsequent side - effects . indeed , a group of carrier - free ons targeting gi mucosa for inflammatory bowel disease therapy is progressing through clinical trials , although high doses of ons are required to obtain the positive therapeutic effects . therefore , a safe and efficient delivery approach to the gi mucosa would be desirable to facilitate the cellular uptake of on and to decrease dosing . two major strategies are currently investigated to improve the on delivery efficacy to the intestinal tissue : particle - based systems ( e.g. , polyplex- or lipoplex - based ) and single - molecule - based conjugates ( e.g. , free on or cell - penetrating peptide - on conjugates ) . on incorporated in particles can be taken up primarily by m cells in the peyer 's patches of the intestine and eventually by mucosal macrophages via phagocytosis . therefore , particle - based systems would be suitable for the targeting of disease - related genes expressed mainly in the immune cells , such as tnf-. in contrast , single - molecule - based system can be taken up not only by lamina propria immune cells but also by epithelial cells . considering that the dysregulated expression of genes causing intestinal disease is often localized on the gut epithelium , single - molecule - based systems could open promising therapeutic avenues for previously inaccessible epithelium - specific targets . in addition , these systems possess the advantage of reduced carrier toxicity and immunogenicity , as well as simpler characterization processes , compared with particle - based systems . recently , we have shown that the conjugation of a long - chain lipid moiety such as docosanoyl ( dsa ) group to chemically modified ons enabled significant target mrna knockdown in a prostate cancer cell line in the absence of transfection reagents . their binding to serum proteins , however , suggested that amphiphilic on conjugates might constitute an effective delivery approach primarily for topical applications or liver targeting . however , their applicability to the intestinal mucosa remains questionable since the harsh gi environment containing digestive enzymes coupled with the known difficulties transfecting differentiated epithelium in vitro suggests that on delivery will be limited . in this work , dsa was conjugated to various chemically modified ons targeting a model bcl-2 mrna , and the transfecting properties of the resulting conjugates were evaluated in vitro . the integrity and transfection efficiency of the most potent conjugate with lowest toxicity were assessed under various conditions mimicking the intestinal environment ( inclusion of food - derived fats and digestive enzymes ) . in a previously published study , we reported that lipophilic docosanoic acid ( l ; dsa ) conjugated to ons modified with 2-f - arabinonucleosides were more potent than nucleic acids derivatized with cholesterol or docosahexaenoic acid to transfect prostate cancer cells . in a search for novel and safe amphiphilic ons capable of modulating gene expression in intestinal cells , we synthesized and characterized a set of conjugates by linking dsa to different single - stranded antisense ons and double - stranded small interfering rnas ( sirnas ) targeting the mrna of the oncoprotein bcl-2 ( table 1 ) as previously described . the antisense ons were based on a single parent dna sequence ( oblimersen ) and were modified by incorporating ome , moe - ribonucleosides , or 2-f - modified arabinonucleosides connected via ps linkages ( l - dna , l - ome , l - moe , and l - fana , respectively ) . these chemical modifications of nucleotides are commonly employed to enhance the binding affinity of ons for target mrnas as well as to increase their stability against nuclease digestion . typically , antisense ons ' mode of action involves recruitment of rnase h , which recognizes the thus formed dna : rna heteroduplex leading to a processive cleavage of the mrna strand in a catalytic fashion . since oligoribonucleotides and their 2-o - modified analogues generally do not support rnase h binding , a gapmer design , in which a central dna stretch is flanked by modified ribonucleotides ( ome and moe ) at both termini , is preferred for preserving an rnase h binding site , while providing the on with higher nuclease resistance . in contrast to 2-o - modified ribonucleotides , the fana modification can be incorporated throughout an on sequence , since upon binding to complementary mrna , the fana nucleotide adopts a conformation similar to that of dna and therefore supports rnase h recognition . since an altimer design in our previous work , it was chosen for the fana - modified on in this study . in order to test whether dsa is capable of facilitating the intracellular delivery of double - stranded sirnas , it was conjugated to the unmodified phosphodiester sirna ( l - sirna ) and its fully phosphorothioated counterpart ( l - ps sirna ) . as shown in figure 1a , b , both single- and double - stranded l - ons decreased target mrna levels in colon carcinoma cells in the absence of transfecting reagent . l - on conjugates showed the following order of efficacy ( based on statistically significant differences at various concentrations ) : l - fana > l - ome > l - sirna > > l - dna , where l - dna did not exhibit a statistically significant reduction . there was no significant difference between the efficacies of l - fana and l - moe or l - ps sirna and l - ome at any of the concentrations tested . l - moe , l - ps sirna , and l - ome caused a small but significant decrease in cell viability 24 hours after transfection ( figure 1a , dot plot ) . however , the difference in cell viability upon treatment with the different l - on conjugates was negligible for longer incubation times ( 2.5 days post - transfection ) ( see supplementary figure s1 ) . l - on conjugates downregulated their target mrna expression in a dose - dependent manner , and the ec50 values of l - ps sirna , l - ome , l - moe , and l - fana were 0.86 , 0.69 , 0.47 , and 0.28 mol / l , respectively ( figure 1b ) . these results indicate that the silencing potency and effect on cell viability of the l - on conjugate depends on the type of on used and on its chemical modification . dsa coupled to the unmodified sirna was less efficient than l - ps sirna in the absence of transfection reagent . since l - ps sirna possessed slightly lower silencing potency compared with l - sirna upon the lf - mediated transfection ( see supplementary figure s2 ) , the superior silencing properties of carrier - free l - ps sirna could have been due to its higher resistance to extra- and intracellular nucleases and/or higher uptake of ps - modified oligonucleotides . we selected the l - fana conjugate as a promising candidate for further investigation based on a combination of lowest ec50 with a favorable cytotoxicity profile . we further investigated the target mrna silencing effect of l - fana by transfecting two human colorectal carcinoma cell lines , hct-116 and caco-2 , with l - fana and its control groups . at a concentration of 1 mol / l , l - fana downregulated bcl-2 mrna in hct-116 and caco-2 cells by 81 and 82% , respectively ( figure 2 ) . monitoring the bcl-2 mrna silencing over time showed that the knockdown efficacy of l - fana was constant from 1 to 3.5 days post - transfection ( see supplementary figure s3 ) . transfection of free fana and negative control conjugate with an irrelevant nucleic acid sequence ( l - fananc ) did not change the bcl-2 mrna expression levels , indicating that target mrna inhibition is caused by a sequence - specific antisense mechanism and that lipid conjugation contributes to the improved intracellular delivery of fana . in order to assess whether sedimentation influenced the transfection efficiency , experiments with cells in inverted position recent studies with nanoparticles demonstrated that cellular uptake can be dramatically reduced in the inverted configuration ( cells on top ) , as opposed to the conventional in vitro setup where cells are on the bottom of the culture plate and sedimentation can artificially promote internalization . indeed , we observed that transfection efficiency of sirna / lf lipoplex was significantly reduced using the inverted setup , whereas the silencing efficiency of l - fana conjugate was the same in both cell setups . these results indicated that l - fana was predominantly taken up by cells via sedimentation - independent uptake routes , which was in line with the outcome of our previous work on peptide - nucleic acid conjugates . in the case of the sirna / lf lipoplex , it is likely that the rapid onset of silencing was a consequence of the fast uptake of sedimented particles ( see supplementary figure s4 ) . this was in contrast to the behavior of l - fana , where increasing the exposure time to the oligonucleotide conjugate from 5 to 15 hours resulted in significantly higher knockdown efficacy ( 38 versus 65% , respectively , at 0.5 mol / l ; see supplementary figure s4 ) , further supporting a presumed sedimentation - independent uptake . an efficient gene silencing in intestinal epithelium first requires that the nucleic acid drugs be stable against the attack of intestinal enzymes . to this end , we tested whether the preincubation of l - fana conjugate in simulated intestinal fluid ( sif ) containing pancreatic digestive enzymes would impair its transfection efficacy ( figure 4 ) . importantly , l - fana conjugate was stable even after 15 hours of incubation with sif , as assessed by polyacrylamide gel electrophoresis analysis ( figure 4a ) , and the lipid moiety was not cleaved off upon incubation with pancreatic enzymes containing lipases ( figure 4b ) . for the silencing experiments , l - fana and its negative control ( l - fananc ) conjugates were incubated with pancreatic enzymes at ph 6.8 for 2 hours at 37c , and after the heat - inactivation of the digestive enzymes and dilution with cell culture medium , the mixture was directly added to the hct-116 cells . the difference between relative expression levels of target bcl-2 mrna after the treatment with l - fana with or without pancreatin preincubation was not statistically significant ( 31 and 19% , respectively ; figure 4c ) . this result shows that the preincubation with pancreatin did not strongly affect the silencing efficacy of the l - fana conjugate due to the enzymatic degradation or unspecific protein binding . treatments with pancreatin alone or pancreatin with l - fananc did not change the target mrna expression , indicating that the silencing effect was caused by l - fana in a sequence - specific manner . it was previously demonstrated that amphiphilic on conjugates can bind to lipid - containing particles ( e.g. , low - density lipoprotein or chylomicrons ) , implying the possibility of interaction of l - on conjugates with food - derived fats in the intestine . using a gel - based binding assay , we observed that l - fana indeed bound to soybean oil in a concentration - dependent manner , while the migration of unconjugated fana was not retarded in the presence of oil ( figure 4a and supplementary figure s5 ) . the decrease of silencing efficacy observed for l - fana in the presence of oil emulsion could be attributed to the interaction between the oil and the lipid moiety of the conjugate interfering with the cellular uptake ( figure 4c ) . however , the interaction of l - fana with the oil emulsion could be disrupted after the digestion of the oil by the sif ( figure 4a ) . importantly , the released l - fana from sif - digested oil emulsion fully preserved its silencing capacity ( figure 4c ) . this finding suggests that the interaction of amphiphilic ons with food - derived fat would not hamper their cellular uptake and subsequent silencing activity . intestinal epithelial cells which are differentiated have reduced proliferative and endocytic capacity which makes their in vivo transfection rather challenging . to compare the delivery of modified on into proliferating cells and difficult - to - transfect epithelial cell monolayers , the l - fana conjugate was tested on differentiated caco-2 cells . to illustrate the resistance to transfection of fully differentiated caco-2 cell monolayers , experiments were performed with bcl-2 targeting sirna complexed with lf ( figure 5a , b ) . mol / l elicited a small decrease of bcl-2 mrna expression ( 21% , figure 5a ) , which was not accompanied by its downregulation at the protein level ( figure 5b ) . we could not achieve dose - dependent bcl-2 mrna knockdown even with sirna concentrations as high as 0.4 mol / l , at which the control sirnanc possessing a target - unrelated sequence caused appreciable suppression of bcl-2 mrna , probably due to the toxicity caused by high concentrations of lf . in contrast , bcl-2 silencing was successfully achieved in proliferating caco-2 cells at a dose of 0.05 mol / l of sirna ( see supplementary figure s6 ) . strikingly , the single - molecule - based l - fana conjugate was able to knockdown bcl-2 mrna in differentiated caco-2 cell monolayers in a sequence - specific and dose - dependent manner ( figure 5a ) . at l - fana concentrations of 1.25 , 2.5 , and 5 mol / l the target bcl-2 mrna expression was reduced to 56 , 32 , and 26% , respectively . at 5 mol conjugation of dsa was found to be essential for successful delivery , as unconjugated fana did not have any effect on bcl-2 mrna expression even at 5 mol / l . consistent with mrna silencing , western blot analysis demonstrated that l - fana reduced efficiently the target bcl-2 protein level ( by 53% at 2.5 importantly , the treatment with relatively high dose of l - fana neither caused cytotoxicity ( figure 5c ) nor decreased the transepithelial electrical resistance ( teer ) of caco-2 monolayers ( figure 5d ) , which is a measurable indicator of the monolayer integrity . in comparison , the treatment with sodium decanoate , a widely used absorbefacient disrupting the tight junctions , caused a pronounced drop in teer . based on these results , it is likely that l - fana conjugates will be taken up primarily by epithelial cells in vivo with minimal systemic translocation via the paracellular route . in a previously published study , we reported that lipophilic docosanoic acid ( l ; dsa ) conjugated to ons modified with 2-f - arabinonucleosides were more potent than nucleic acids derivatized with cholesterol or docosahexaenoic acid to transfect prostate cancer cells . in a search for novel and safe amphiphilic ons capable of modulating gene expression in intestinal cells , we synthesized and characterized a set of conjugates by linking dsa to different single - stranded antisense ons and double - stranded small interfering rnas ( sirnas ) targeting the mrna of the oncoprotein bcl-2 ( table 1 ) as previously described . the antisense ons were based on a single parent dna sequence ( oblimersen ) and were modified by incorporating ome , moe - ribonucleosides , or 2-f - modified arabinonucleosides connected via ps linkages ( l - dna , l - ome , l - moe , and l - fana , respectively ) . these chemical modifications of nucleotides are commonly employed to enhance the binding affinity of ons for target mrnas as well as to increase their stability against nuclease digestion . typically , antisense ons ' mode of action involves recruitment of rnase h , which recognizes the thus formed dna : rna heteroduplex leading to a processive cleavage of the mrna strand in a catalytic fashion . since oligoribonucleotides and their 2-o - modified analogues generally do not support rnase h binding , a gapmer design , in which a central dna stretch is flanked by modified ribonucleotides ( ome and moe ) at both termini , is preferred for preserving an rnase h binding site , while providing the on with higher nuclease resistance . in contrast to 2-o - modified ribonucleotides , the fana modification can be incorporated throughout an on sequence , since upon binding to complementary mrna , the fana nucleotide adopts a conformation similar to that of dna and therefore supports rnase h recognition . since an altimer design in our previous work , it was chosen for the fana - modified on in this study . in order to test whether dsa is capable of facilitating the intracellular delivery of double - stranded sirnas , it was conjugated to the unmodified phosphodiester sirna ( l - sirna ) and its fully phosphorothioated counterpart ( l - ps sirna ) . as shown in figure 1a , b , both single- and double - stranded l - ons decreased target mrna levels in colon carcinoma cells in the absence of transfecting reagent . l - on conjugates showed the following order of efficacy ( based on statistically significant differences at various concentrations ) : l - fana > l - ome > l - sirna > > l - dna , where l - dna did not exhibit a statistically significant reduction . there was no significant difference between the efficacies of l - fana and l - moe or l - ps sirna and l - ome at any of the concentrations tested . l - moe , l - ps sirna , and l - ome caused a small but significant decrease in cell viability 24 hours after transfection ( figure 1a , dot plot ) . however , the difference in cell viability upon treatment with the different l - on conjugates was negligible for longer incubation times ( 2.5 days post - transfection ) ( see supplementary figure s1 ) . l - on conjugates downregulated their target mrna expression in a dose - dependent manner , and the ec50 values of l - ps sirna , l - ome , l - moe , and l - fana were 0.86 , 0.69 , 0.47 , and 0.28 mol / l , respectively ( figure 1b ) . these results indicate that the silencing potency and effect on cell viability of the l - on conjugate depends on the type of on used and on its chemical modification . dsa coupled to the unmodified sirna was less efficient than l - ps sirna in the absence of transfection reagent . since l - ps sirna possessed slightly lower silencing potency compared with l - sirna upon the lf - mediated transfection ( see supplementary figure s2 ) , the superior silencing properties of carrier - free l - ps sirna could have been due to its higher resistance to extra- and intracellular nucleases and/or higher uptake of ps - modified oligonucleotides . we selected the l - fana conjugate as a promising candidate for further investigation based on a combination of lowest ec50 with a favorable cytotoxicity profile . we further investigated the target mrna silencing effect of l - fana by transfecting two human colorectal carcinoma cell lines , hct-116 and caco-2 , with l - fana and its control groups . at a concentration of 1 mol / l , l - fana downregulated bcl-2 mrna in hct-116 and caco-2 cells by 81 and 82% , respectively ( figure 2 ) . monitoring the bcl-2 mrna silencing over time showed that the knockdown efficacy of l - fana was constant from 1 to 3.5 days post - transfection ( see supplementary figure s3 ) . transfection of free fana and negative control conjugate with an irrelevant nucleic acid sequence ( l - fananc ) did not change the bcl-2 mrna expression levels , indicating that target mrna inhibition is caused by a sequence - specific antisense mechanism and that lipid conjugation contributes to the improved intracellular delivery of fana . in order to assess whether sedimentation influenced the transfection efficiency , experiments with cells in inverted position recent studies with nanoparticles demonstrated that cellular uptake can be dramatically reduced in the inverted configuration ( cells on top ) , as opposed to the conventional in vitro setup where cells are on the bottom of the culture plate and sedimentation can artificially promote internalization . indeed , we observed that transfection efficiency of sirna / lf lipoplex was significantly reduced using the inverted setup , whereas the silencing efficiency of l - fana conjugate was the same in both cell setups . these results indicated that l - fana was predominantly taken up by cells via sedimentation - independent uptake routes , which was in line with the outcome of our previous work on peptide - nucleic acid conjugates . in the case of the sirna / lf lipoplex , it is likely that the rapid onset of silencing was a consequence of the fast uptake of sedimented particles ( see supplementary figure s4 ) . this was in contrast to the behavior of l - fana , where increasing the exposure time to the oligonucleotide conjugate from 5 to 15 hours resulted in significantly higher knockdown efficacy ( 38 versus 65% , respectively , at 0.5 mol / l ; see supplementary figure s4 ) , further supporting a presumed sedimentation - independent uptake . an efficient gene silencing in intestinal epithelium first requires that the nucleic acid drugs be stable against the attack of intestinal enzymes . to this end , we tested whether the preincubation of l - fana conjugate in simulated intestinal fluid ( sif ) containing pancreatic digestive enzymes would impair its transfection efficacy ( figure 4 ) . importantly , l - fana conjugate was stable even after 15 hours of incubation with sif , as assessed by polyacrylamide gel electrophoresis analysis ( figure 4a ) , and the lipid moiety was not cleaved off upon incubation with pancreatic enzymes containing lipases ( figure 4b ) . for the silencing experiments , l - fana and its negative control ( l - fananc ) conjugates were incubated with pancreatic enzymes at ph 6.8 for 2 hours at 37c , and after the heat - inactivation of the digestive enzymes and dilution with cell culture medium , the mixture was directly added to the hct-116 cells . the difference between relative expression levels of target bcl-2 mrna after the treatment with l - fana with or without pancreatin preincubation was not statistically significant ( 31 and 19% , respectively ; figure 4c ) . this result shows that the preincubation with pancreatin did not strongly affect the silencing efficacy of the l - fana conjugate due to the enzymatic degradation or unspecific protein binding . treatments with pancreatin alone or pancreatin with l - fananc did not change the target mrna expression , indicating that the silencing effect was caused by l - fana in a sequence - specific manner . it was previously demonstrated that amphiphilic on conjugates can bind to lipid - containing particles ( e.g. , low - density lipoprotein or chylomicrons ) , implying the possibility of interaction of l - on conjugates with food - derived fats in the intestine . using a gel - based binding assay , we observed that l - fana indeed bound to soybean oil in a concentration - dependent manner , while the migration of unconjugated fana was not retarded in the presence of oil ( figure 4a and supplementary figure s5 ) . the decrease of silencing efficacy observed for l - fana in the presence of oil emulsion could be attributed to the interaction between the oil and the lipid moiety of the conjugate interfering with the cellular uptake ( figure 4c ) . however , the interaction of l - fana with the oil emulsion could be disrupted after the digestion of the oil by the sif ( figure 4a ) . importantly , the released l - fana from sif - digested oil emulsion fully preserved its silencing capacity ( figure 4c ) . this finding suggests that the interaction of amphiphilic ons with food - derived fat would not hamper their cellular uptake and subsequent silencing activity . intestinal epithelial cells which are differentiated have reduced proliferative and endocytic capacity which makes their in vivo transfection rather challenging . to compare the delivery of modified on into proliferating cells and difficult - to - transfect epithelial cell monolayers , the l - fana conjugate was tested on differentiated caco-2 cells . to illustrate the resistance to transfection of fully differentiated caco-2 cell monolayers , experiments were performed with bcl-2 targeting sirna complexed with lf ( figure 5a , b ) . transfection of sirna / lf at a concentration of 0.2 mol / l elicited a small decrease of bcl-2 mrna expression ( 21% , figure 5a ) , which was not accompanied by its downregulation at the protein level ( figure 5b ) . we could not achieve dose - dependent bcl-2 mrna knockdown even with sirna concentrations as high as 0.4 mol / l , at which the control sirnanc possessing a target - unrelated sequence caused appreciable suppression of bcl-2 mrna , probably due to the toxicity caused by high concentrations of lf . in contrast , bcl-2 silencing was successfully achieved in proliferating caco-2 cells at a dose of 0.05 mol / l of sirna ( see supplementary figure s6 ) . strikingly , the single - molecule - based l - fana conjugate was able to knockdown bcl-2 mrna in differentiated caco-2 cell monolayers in a sequence - specific and dose - dependent manner ( figure 5a ) . at l - fana concentrations of 1.25 , 2.5 , and 5 / l the target bcl-2 mrna expression was reduced to 56 , 32 , and 26% , respectively . at 5 mol conjugation of dsa was found to be essential for successful delivery , as unconjugated fana did not have any effect on bcl-2 mrna expression even at 5 mol / l . consistent with mrna silencing , western blot analysis demonstrated that l - fana reduced efficiently the target bcl-2 protein level ( by 53% at 2.5 importantly , the treatment with relatively high dose of l - fana neither caused cytotoxicity ( figure 5c ) nor decreased the transepithelial electrical resistance ( teer ) of caco-2 monolayers ( figure 5d ) , which is a measurable indicator of the monolayer integrity . in comparison , the treatment with sodium decanoate , a widely used absorbefacient disrupting the tight junctions , caused a pronounced drop in teer . based on these results , it is likely that l - fana conjugates will be taken up primarily by epithelial cells in vivo with minimal systemic translocation via the paracellular route . intestinal delivery of nucleic acid drugs is a promising therapeutic strategy for treatment of several diseases but faces numerous challenges associated with the complexity of the gi environment . to identify a potent delivery platform , a set of l - on conjugates although all the l - on conjugates carried the same dsa lipid moiety , they induced different degrees of cytotoxicity . this could be attributed to the modification - dependent protein binding of ons or bcl-2-level - dependent cell death . superior silencing efficacy of l - fana and l - moe conjugates compared to those of l - dna and l - ome could result from a combination of factors including higher nuclease resistance and higher affinity for the complementary mrna . a fully phosphorothioated l - ps sirna conjugate showed significant bcl-2 silencing upon carrier - free transfection , while ps sirna without a dsa group did not ( see supplementary figure s2 ) , suggesting that the uptake of l - ps sirna was mediated by the lipid moiety . a phenomenon called gymnosis was recently reported in which target genes are sequence - specifically suppressed in cells by chemically modified ons , such as locked nucleic acids and fana , in the absence of transfection reagents or delivery moiety conjugation . for an efficient and nontoxic gymnotic transfection , prolonged exposure of proliferating cells ( 6 10 days ) to high ( mol / l ) concentrations of ons is required . considering that the average transit times in the small and large intestines of healthy humans are 3 hours and 27 hours , respectively ( as reviewed in refs . 57,58 ) , gymnotic on delivery is likely to be too slow for intestinal tissues , at least in the absence of frequent dosing . moreover , the renewal cycle of intestinal epithelial cells lasts barely 3 5 days , and therefore an intestinal on therapy needs an efficient delivery method to facilitate the target mrna and protein knockdown . in this study , dsa conjugation to ons promoted the delivery of single - stranded antisense ons and even double - stranded sirnas during limited exposure times . several groups have reported that the conjugation of neutral lipids , such as cholesterol and aliphatic fatty acids , to ons improves their uptake and silencing efficacy , both in vitro and in vivo . however , the mechanism of cellular uptake of amphiphilic ons and their trafficking remain poorly understood and appear to be system - dependent . for a successful in vivo delivery of on to intestinal epithelium , l - on conjugates must remain functional in the intestinal environment , and they should be able to silence target genes in difficult - to - transfect epithelial cell monolayers . one concern regarding their biological activity in the intestine was that amphiphilic on conjugates might bind to intestinal lipophilic content , potentially leading to reduced activity . here , in a model system , we demonstrated that free l - fana is released from oil phase after digestion by pancreatic enzymes and retains its silencing ability . the combination of the extreme stability of fana - modified ons toward nucleases and hydrolysis at low ph , together with the high stability of dsa under similar conditions found in the gi tract makes dsa - fana conjugates particularly suitable for gi applications . differentiated intestinal epithelial cells featuring microvilli and expressing tight junctions represent a difficult target for nucleic acid delivery . their reduced proliferation and endocytosis rate in comparison to undifferentiated cells are largely responsible for poor transfection efficacy . regarding particle - based delivery systems , the resistance to transfection observed with differentiated cells may be partially attributed to the structure of their apical membrane featuring microvilli , which limits access to the absorptive membrane . several studies have described a variety of attempts to overcome this barrier by , for example , pretreatment with membrane - disturbing agents , transfection of proliferating caco-2 in the suspension state followed by accelerated differentiation , formulation with small lipid nanoparticles , use of 1-integrin - mediated endocytosis or electroporation . as previously reported for sirna / lf lipoplexes , we were unable to efficiently downregulate target bcl-2 gene in polarized caco-2 cell monolayers . in contrast , the single - molecule - based l - fana conjugate was able to effectively silence the bcl-2 gene expression at both mrna and protein levels in differentiated monolayers . importantly , despite lower potency of carrier - free amphiphilic conjugates in comparison to sirna / lf lipoplexes in classical in vitro assays based on rapidly proliferating cancer cells , they by far outperform particulate delivery vehicles under more stringent conditions , such as the inverted setup or using differentiated cells . the involvement of cell surface receptors , such as fatty acid receptors , better access of l - fana to the adsorptive cell membrane due to the smaller size , or other uptake routes different from lf - mediated endocytosis may explain the efficient target silencing by l - fana conjugate in a nondividing polarized epithelium . although several studies have extrapolated the possible internalization routes of amphiphilic on conjugates in proliferating cells , it is possible that the uptake mechanism and intracellular trafficking can vary among different cell types and states of cell differentiation . the present report demonstrates that amphiphilic on conjugates can be efficiently delivered to colon carcinoma cells in the absence of delivery agents , causing target gene silencing . conjugation of dsa and chemical modification of the on backbone were essential for efficient delivery and enzymatic resistance , respectively . the most encouraging finding was that an l - on conjugate was able to silence bcl-2 mrna and protein in fully differentiated caco-2 cell monolayers . this well - defined single - molecule - based approach proved to be superior to conventionally employed lipoplexes , which were ineffective for transfecting the epithelium . to the best of our knowledge , this is the first study showing the efficient gene silencing in differentiated epithelial cells by an oligonucleotide conjugate in the absence of complexation , delivery device , or membrane - disturbing agents . we believe that l - fana represents a suitable approach to further improve the efficacy of on enteral treatments currently under clinical investigation . in addition , l - fana would also provide a carrier - free tool for molecular biologists studying gene functions in differentiated intestinal epithelium . future work will focus on evaluation of the proposed delivery platform in intestinal disease relevant targets in vivo . azzalin 's group at eth zurich and caco-2 cells were purchased from atcc ( manassas , va ) . dulbecco 's modified essential medium ( dmem ) with glutamax , opti - mem medium , fetal bovine serum ( fbs ) , nonessential amino acids , penicillin - streptomycin solution , trypsin , lipofectamine 2000 ( lf ) , phosphate - buffered saline ( pbs ; 1 mmol / l kh2po4 , 3 mmol / l na2hpo4 , 155 mmol / l nacl , ph 7.4 ) , and rnase - free distilled water were obtained from invitrogen ( carlsbad , ca ) . porcine pancreatin ( 4usp ) , ammonium persulfate , triethylammonium acetate ( teaa ) buffer 1 mol / l , intralipid ( 20% w / v of soybean oil , 1.2% of egg yolk phospholipids , 2.25% of glycerol , ph 68.9 ; mimic of high - fat meal ) , chloroform ( chcl3 ) , triton x-100 , naf , na3vo4 , tris , and skim milk powder were obtained from sigma - aldrich ( buchs , switzerland ) . duplex annealing buffer ( 100 mmol / l potassium acetate , 30 mmol / l hepes , ph 7.5 ) was purchased from integrated dna technologies ( idt , coralville , ia ) . methanol ( meoh ) , n , n , n,n-tetramethylethylenediamine ( temed ) , and phenylmethylsulfonyl fluoride ( pmsf ) were purchased from acros organics ( geel , belgium ) . ethylenediaminetetraacetic acid ( edta ) and polysorbate 20 were purchased from applichem ( darmstadt , germany ) . gel red nucleic acid gel stain was obtained from biotium ( hayward , ca ) . cell counting kit-8 ( cck-8 ) was obtained from dojindo molecular technologies ( rockville , md ) . thermanox coverslips , dna loading dye , nacl , and micro bca protein assay kit were purchased from thermo fisher scientific ( waltham , ma ) . low - binding microcentrifugation tubes ( dna lobind ) were purchased from eppendorf - vaudaux ( schnenbuch , switzerland ) . rneasy mini kit and specific primers for -splicing variant of human bcl-2 mrna ( hs_bcl2_1_sg ; qt00025011 ) and human -actin ( hs_actb_2_sg ; qt01680476 ) were obtained from qiagen ( valencia , ca ) . high - capacity cdna reverse transcription kit and power sybr green polymerase chain reaction ( pcr ) master mix were purchased from applied biosystems ( foster city , ca ) . complete edta - free protease inhibitors ' cocktail was purchased from roche diagnostics ( mannheim , germany ) . poly(vinylidene difluoride ) membranes ( pvdf ) were obtained from bio - rad laboratories ( hercules , ca ) . mouse antihuman bcl-2 monoclonal antibody and horseradish peroxidase ( hrp)-conjugated goat antimouse igg polyclonal antibody were purchased from dako ( glostrup , denmark ) . rabbit anti--actin polyclonal antibody and hrp - conjugated goat antirabbit igg polyclonal antibody were obtained from abcam ( cambridge , uk ) . immunocruz western blotting luminol reagent was purchased from santa cruz biotechnology ( dallas , tx ) . super rx x - ray films were obtained from fujifilm ( tokyo , japan ) . unmodified bcl-2 targeting sirna and negative control sirnanc with nontargeting sequence were synthesized by bioneer ( daejeon , south korea ) . all ons were synthesized according to the standard protocol for automated phosphoramidite solid - phase synthesis except for l - ome conjugate , which was provided by microsynth . dsa was conjugated via an aminohexanol - linker to the 5-end of ons in line with previously described method . all l - on conjugates were purified by reverse - phase hplc , analyzed by lc - ms , and quantified via uv spectrophotometry ( nanophotometer p 330 , implen , germany ) . the ons ' molar extinction coefficients at 260 nm were calculated using the software of idt website ( oligoanalyzer tool , www.idtdna.com/calc/analyzer ) . these modifications were assumed to have negligible effect on the extinction coefficients , as previously described . the complementary single strands of sirnas were combined in duplex annealing buffer at a concentration of 50 mol / l each , heated up to 95c for 1 minute , and cooled slowly to 4c overnight to ensure proper annealing . various ons and l - on conjugates were dissolved in rnase - free deionized water at a concentration of 100 hct-116 cells were maintained in dmem medium supplemented with glutamax containing 10% fbs , 100 units / ml penicillin , and 100 g / ml streptomycin at 37c in a 5%-co2 humidified atmosphere . the cells were seeded in a 24-multiwell plate at a density of 4 10 cells / well . for the inverted transfection , the cells were seeded on thermanox coverslips in a 24-multiwell plate at a density of 1 10 cells / well . caco-2 cells were maintained in dmem medium supplemented with glutamax containing 10% fbs , 100 units / ml penicillin , 100 g / ml streptomycin , and 1% of nonessential amino acids at 37c in a 5%-co2 humidified atmosphere . cells with passage number between 58 and 77 were seeded in a 12-multiwell plate at a density of 7 10 cells / well . for the differentiated caco-2 cell monolayer transfection , the cells were seeded in transwell inserts with polyester membrane with pore size of 0.4 m in a 12-multiwell plate at a density of 1.12 10 cells / well as previously described . the medium was exchanged every other day , and cells between 13 and 17 days of differentiation were used for the experiments . the differentiation of monolayers was monitored by measuring teer using an evom epithelial voltmeter with stx2 electrode ( world precision instruments , sarasota , fl ) . all the monolayers achieved teer higher than 1,000 cm after the 2 weeks of culturing , indicating the completion of differentiation process . teer values of individual wells measured just before the transfection with ons were set to 100% and their change was monitored for the three following days . all experiments were performed on mycoplasma - free cell lines ( regularly checked by mycoalert plus mycoplasma detection kit ) , and only cells in the exponential phase of growth were used for seeding . hct-116 cells were seeded in a 96-multiwell plate at a density of 7 10 cells / well the day before the experiment . alternatively , caco-2 cells were seeded in a 96-multiwell plate , and the culture medium was exchanged every other day for 2 weeks to obtain differentiated monolayers . the cells were treated with various concentrations of l - on conjugates in 50 l of serum - deficient opti - mem medium for 15 hours . the opti - mem medium containing no l - on was used as a control . the medium was exchanged for 100 l of dmem supplemented with 10% fbs , and the cells were further incubated for 24 hours , after which the cell viability was assessed using tetrazolium - based cck-8 reagent following the manufacturer 's instructions . screening of bcl-2 mrna silencing efficiencies of various l - ons . to assess the silencing efficiency of various l - on conjugates , the conjugates at various concentrations ( 0.25 1 given the absence of intact serum in the intestinal environment , all transfection experiments were performed in serum deficient opti - mem medium . the opti - mem medium containing no ons was used as a control . following overnight incubation ( 15 hours ) after 2.5 days of further incubation cells were washed with pbs , and total rna was isolated using rneasy mini kit ( abs260 /abs230 > 1.8 ) according to the previously optimized method . the expression levels of bcl-2 mrna relative to the internal control -actin mrna were quantified by two - step quantitative real - time pcr . briefly , cdna was synthesized from 1.2 g of total mrna using high - capacity cdna reverse transcription kit according to the manufacturer 's instructions . quantitative real - time pcr was performed using power sybr green pcr master mix and specific primers for human bcl-2 and -actin on a 7900ht fast real time pcr instrument ( applied biosystems ) according to the manufacturer 's instructions . briefly , the reaction mixtures were incubated at 50c for 2 minutes and 95c for 10 minutes followed by 40 cycles of denaturation ( 95c for 15 seconds ) and extension / detection ( 60c for 1 minute ) . relative mrna expression levels were calculated using the delta delta ct ( 2 ) method ( the fluorescence threshold was set to 0.4 ) . results are expressed as the bcl-2 mrna level change between on - treated and on - free medium treated cells . l - on potency ( effective concentration causing 50% of target mrna silencing , ec50 ) was calculated using a four - parameter logistic function to fit the dose - response data via sigmaplot 13.0 software . the transfection medium consisted of 250 l of opti - mem containing either 1 mol / l of carrier - free bcl-2 targeting l - fana conjugate or 0.05 mol / l of bcl-2 specific sirna complexed with lf according to the manufacturer 's instructions as a particle - mediated delivery control . hct-116 cells grown on the coverslips were washed with opti - mem , and the coverslips were transferred using tweezers into a new 24-multiwell plate for the transfection . for the upright transfection , the cells were placed on the bottom of the multiwell plate followed by the addition of the transfection medium . for the inverted transfection , the transfection medium was added to the empty well , and the cells grown on coverslips were carefully deposited upside down onto the surface of the medium . the coverslips floated on the surface of the medium due to the surface tension of the medium . after overnight incubation ( 15 hours ) , the cells were transferred to a new plate and further cultured for 2.5 days in 500 l of dmem supplemented with 10% fbs . subsequently , total rna was isolated and gene expression levels of bcl-2 mrna were assessed as described above . l - fana conjugates stability in sif and 0.5% soybean oil emulsion . to investigate the influence of digestive enzymes and food - derived fats present in intestine on the biological function of l - fana , its stability and efficacy were tested in simulated intestinal environment containing pancreatic enzymes and lipids . for polyacrylamide gel electrophoresis analysis , 1 l of l - fana ( 100 mol / l ) was mixed with 9 l of usp sif ( 2.5 g / l porcine pancreatin ( 4 usp ) , 50 mmol / l kh2po4 , ph 6.8 ) or water and incubated for 2 hours or 15 hours at 37c . samples were kept at 95c for 15 minutes to heat - inactivate the enzymes and mixed with 1 l of dna loading dye . alternatively , 1 l of l - fana was premixed with 1 l of 5% soybean oil emulsion in opti - mem ( prepared by diluting 20% intralipid with opti - mem ) for 30 minutes followed by incubation at 37c for 2 hours with 8 l of active / heat - inactivated sif or water . samples were then loaded onto 20% ( w / v ) polyacrylamide gel prepared in a tris - acetate - edta buffer ( tae : 40 mmol / l tris - acetate , 1 mmol / l edta , ph 8.0 ) by free - radical polymerization with ammonium persulfate / temed as an initiator . the gel was then immersed in tae buffer and electrophoresed at constant voltage of 150 v for 60 min . l - fana was revealed following manufacture 's protocol for gel red nucleic acid gel stain , and fluorescence was recorded on a chemidoc xrs ( bio - rad laboratories ) . for the lc - ms analysis , 2 l of l - fana ( 100 mol / l ) were mixed with 18 l of sif or water and incubated for 2 hours at 37c , then at 95c for 15 minutes , and centrifuged at 14000 g at room temperature for 10 minutes . the supernatants were mixed with 50 l of chcl3 , and shaken to extract possible hydrophobic contaminants from sif . after incubating the mixtures at room temperature for a few minutes , analytical liquid chromatography - mass spectrometry ( lc - ms ) was carried out on agilent lc - ms using a reverse phase column ( waters acquity ost c18 , 2.1 x 50 mm , 1.7 m ) with solvent a being 400 mmol / l hfip and 15 mmol / l teaa in water and solvent b being meoh . the flow was set at 0.3 ml / min , and a gradient was run at 65c from 5 to 90% b in 14 minutes . the influence of preincubation of l - fana conjugates with sif on the transfection efficacy was investigated . hct-116 cells were seeded one day prior to the experiment in a 24-multiwell plate at a density of 4 10 cells / well in dmem containing 10% fbs . two and a half microliters of l - fana were pre - incubated with 22.5 l of sif for 2 hours at 37c followed by heat - inactivation of enzymes for 15 minutes at 95c . the mixtures were diluted ten times with opti - mem to a final l - fana concentration of 1 mol / l and incubated with cells overnight ( 15 hours ) . after 2.5 days of further incubation , total rna was isolated , and gene expression levels of bcl-2 mrna were assessed as described above . to study the silencing activity of l - fana released from sif - digested oil emulsion , 2.5 l of l - fana ( 100 mol / l ) were preincubated with 6.25 l of 20% intralipid for 30 minutes at room temperature followed by incubation with 25 l of sif for 2 hours at 37c . the released l - fana was separated from cytotoxic oil digestion products by preparative polyacrylamide gel electrophoresis before transfection experiments . the l - fana band was excised , weighed , and immersed in 250 l of opti - mem . for l - fana extraction , samples containing gel pieces were flash - frozen in liquid nitrogen , heated at 90c for 15 minutes , and agitated at room temperature for 3 hours . the differentiated caco-2 cell monolayers grown in transwell inserts for 2 weeks were washed with opti - mem medium from apical and basal sides . the 500 l of opti - mem medium containing either naked on derivative ( 0.625 5 mol / l ) or sirna ( 0.2 0.4 mol / l ) complexed with lf according to the manufacturer 's instructions was added to the apical compartment . following the overnight incubation ( 15 hours ) , the transfection medium was exchanged with fresh dmem supplemented with 10% fbs in both compartments . for bcl-2 mrna expression analysis by qrt - pcr for bcl-2 protein expression analysis by western blot , cell monolayers were lysed 4 days after transfection in 25 l of lysis buffer ( 20 mmol / l tris hcl ph 7.7 , 150 mmol / l nacl , 5 mmol / l edta , 1% v / v triton x-100 , 25 mmol / l naf , 1 mmol / l pmsf , 1 mmol / l na3vo4 supplemented with complete protease inhibitors ) in line with previously described method . briefly , cell lysates were scraped from the transwells , centrifuged at 10,000 g for 15 minutes at 4c to remove cell debris , and protein concentration in the supernatants was determined by the micro bca assay according to the manufacturer 's instructions . 50 g of total protein per sample were resolved on 12% sds - page under reducing conditions and transferred to a pvdf membrane . the membrane was washed once with tbs - t buffer ( 20 mmol / l tris hcl ph 7.7 , 150 mmol / l nacl , 0.1% v / v polysorbate 20 ) and blocked with tbs - t containing 5% w / v skim milk ( blocking buffer ) for 1 hour . the membrane was cut in two at 35 kda ; the lower part containing bcl-2 ( 26 kda ) was incubated with anti - bcl-2 antibody diluted to 1:100 in blocking buffer , and the upper part containing -actin ( 42 kda , loading control ) was incubated with anti--actin antibody diluted to 1:4,000 overnight at 4c . membranes were washed 3 times for 5 minutes with pbs - t followed by 1.5-hour - incubation with the corresponding hrp - conjugated secondary antibodies diluted to 1:4,000 in blocking buffer . membranes were washed three times with tbs - t , and protein bands were detected with immunocruz luminol reagent and revealed on super rx x - ray films using an agfa curix 60 film processor ( agfa , mortsel , belgium ) . the relative intensities of the bands were analyzed using image j software ( national institutes of health , bethesda , md ) . statistical analysis . all treatment groups were compared pairwise using the one - way analysis of variance ( anova ) test combined with tukey 's ( holm - sidak ) post - hoc test assuming normal data distribution . the differences between treatment groups were considered statistically significant at p - values < 0.05 . supplementary material figure s1 . viability of hct-116 cells after transfection with various on derivatives at a dose of 1 mol / l in opti - mem medium overnight . bcl-2 mrna silencing and viability of hct-116 cells after transfection with various sirna derivatives complexed with lf and free ps sirna . mol / l of l - fana or 0.05 mol / l of sirna / lf in opti - mem medium . figure s4 . the influence of incubation time on knockdown efficacy in hct-116 cells transfected with l - fana or sirna / lf lipoplex . polyacrylamide gel electrophoresis images of l - fana conjugate and unconjugated fana incubated with oil emulsion . transfection of proliferating caco-2 cells with sirna / lf and negative control sirna ( sirnanc)/lf lipoplexes .

background . intertrochanteric fractures of the proximal femur are one of the most common fractures encountered , and dynamic hip screw with a side plate is the standard treatment . we compared a minimally invasive surgical technique with the conventional surgical technique used in the fixation of intertrochanteric fractures with the dynamic hip screw ( dhs ) device . methods . thirty patients with such fractures were treated with the conventional open technique and 30 with a new minimally invasive technique . patients in both groups were followed up for 1 year . results . there was less blood loss , minimal soft tissue destruction , shorter hospital stay , and early mobilization with the minimally invasive technique . conclusion . the present study finds minimally invasive technique superior to conventional ( open ) dhs . nucleic acid therapy can be beneficial for the local treatment of gastrointestinal diseases that currently lack appropriate treatments . indeed , several oligonucleotides ( ons ) are currently progressing through clinical trials as potential treatments for inflammatory bowel diseases . however , due to low uptake of carrier - free ons by mucosal cells , strategies aimed at increasing the potency of orally administered ons would be highly desirable . in this work , we explored the silencing properties of chemically modified and highly resistant ons derivatized with hydrophobic alkyl chain on intestinal epithelial cells . we screened a set of lipid - on conjugates for the silencing of model bcl-2 mrna and selected 2-deoxy-2-fluoro - arabinonucleic acid modified on bearing docosanoyl moiety ( l - fana ) as the most potent candidate with lowest toxicity . the efficacy of l - fana conjugate was preserved in simulated intestinal fluids and in the inverted transfection setup . importantly , l - fana conjugate was able to downregulate target gene expression at both mrna and protein levels in a difficult - to - transfect polarized epithelial cell monolayer in the absence of delivery devices and membrane disturbing agents . these findings indicate that lipid - on conjugates could be promising therapeutics for the treatment of intestinal diseases as well as a valuable tool for the discovery of new therapeutic targets .

increasing numbers of aesthetic procedures - especially cosmetic nonsurgical injection procedures - are performed every year ( 1 ) . these procedures are generally safe , although cosmetic facial filler injections could lead to devastating complications such as blindness . there are several reports ( most of which were case reports ) of cosmetic facial filler injection - associated retinal artery occlusion ( 234567891011 ) . a nation - wide survey was recently performed and a thorough analysis was performed on 44 patients regarding the clinical features of this condition with respect to its fluorescein angiographic findings . our group participated in that study and classified occlusion of the ophthalmic artery and its branches into 6 types on the basis of fluorescein angiographic findings and found that the more diffuse occlusion type was associated with poor visual outcomes ( 12 ) . several researchers proposed that the occlusion was caused by retrograde embolism by the injected material ( 378 ) . the supratrochlear and supraorbital arteries are thought to be the possible route for retrograde embolism in the glabellar region and the anastomosis of the dorsal nasal artery and angular artery seems to be the possible inlet of nasally injected embolic material ( 8) . however , the exact mechanism of retrograde embolism is still elusive and precise localization of emboli is difficult when it is evaluated only by fundus fluorescein angiography , especially when the presumed occlusion site is proximal to central retinal arteries . in the previous studies , the two most commonly injected materials , autologous fat and hyaluronic acid ( ha ) showed different clinical features . autologous fat injection was interestingly more frequently associated with diffuse occlusion , compared to ha injection ( 812 ) , while skin necrosis associated with facial filler injection was more common in ha injected cases ( 12 ) . the size difference between autologous fat and ha seems to be related in part to the differences in the clinical features of retinal artery occlusions associated with cosmetic facial injections of these materials ; however , the exact pathophysiology has never been thoroughly scrutinized . cerebral angiography provides more detailed information on blood flow of ophthalmic artery as well as surrounding orbital area which can not be covered by fundus fluorescein angiography . in this study , we aimed to evaluate cerebral angiographic features of cosmetic facial filler - related ophthalmic and retinal artery occlusion patients who had undergone intra - arterial thrombolysis ( iat ) , thus elucidate the pathophysiology of the disease . we retrospectively reviewed the medical records of patients who were diagnosed as having ophthalmic artery and/or retinal artery occlusion associated with cosmetic facial filler injections of autologous fat or ha and underwent iat along with transfemoral cerebral angiography at the seoul national university bundang hospital ( seongnam , korea ) between january 1 , 2008 and august 31 , 2014 . in particular , iat was considered for patients who presented less than 24 hr after symptom onset and for patients without any systemic conditions that restricted thrombolysis , such as uncontrolled hypertension , coagulation disorders , or a history of intracranial or extracranial hemorrhages within 6 months before presenting to the hospital . after a detailed ophthalmologic evaluation that included fundus fluorescein angiography , iat was performed with transfemoral cerebral angiography using a biplane angiographic unit ( integris allura ; philips medical systems , eindhoven , the netherlands ) . a microcatheter ( excelsior sl-10 ; stryker neurovascular , fremont , ca , usa ) was placed in the proximal part of the ophthalmic artery , and a thrombolytic agent - urokinase ( in patients with fat embolism , green cross , yongin , korea ) or hyaluronidase ( in patients with ha embolism , kuhnil pharm , seoul , korea ) or both- was slowly injected with mechanical disruption by microwire to dislodge the emboli distally . the hyaluronidase dosages ranged between 1,000 units and 9,000 units , and the urokinase dosage was up to 500,000 units . we evaluated cerebral angiography by localizing the embolic obstruction site on ophthalmic angiogram and characterizing the pattern of distal angiographic runoff on external carotid angiogram , which was corresponded to skin necrosis lesion . this study was approved by the institutional review board of seoul national university bundang hospital ( seongnam , korea , no . this study was approved by the institutional review board of seoul national university bundang hospital ( seongnam , korea , no . a total of 17 patients ( 10 ha - injected patients and 7 autologous fat - injected patients ) sustained ophthalmic artery and/or retinal artery occlusion associated with cosmetic facial filler injections during the period . of these patients , 7 patients ( 4 ha - injected patients and 3 autologous fat - injected patients ) underwent iat along with cerebral angiography , and were finally included in this study . the clinical findings for 4 of 7 patients had already been published in the literature ( 68912 ) . table 1 summarizes the demographics , clinical characteristics and cerebral angiographic findings of the study patients . all patients were women and most were young ( mean age of 40.612.7 yr ) . diffuse occlusions such as complete ophthalmic artery occlusion or occlusion of its branches were noted in all patients . three of 4 ha - injected patients showed skin necrosis , whereas all fat - injected patients and one ha - injected patient who were immediately treated with subcutaneous hyaluronidase injection by the physician who performed cosmetic filler injection showed none or mild skin lesion . one patient in fat - injected group showed concomitant middle cerebral artery territory infarct on brain magnetic resonance image . all patients arrived at the emergency room immediately ( i.e. , 4 hr or less ) after symptom onset and underwent iat on the same day . however , the result of thrombolysis was unfavorable because only 2 patients showed partial recanalization of the obstruction ( table 1 ) . the visual outcome was poor that all patients had final vision of no light perception . a case of autologous fat injection - associated retinal artery occlusion is shown in fig . 1 . on fundus photography , initially it appeared as occlusion of a branch of the retinal artery because one branch of the artery is occluded and retinal edema was present in the involved area ( fig . however , with fluorescein angiography , retinal perfusion and the choroidal perfusion were severely delayed ( fig . there is abrupt cut off of some of the arteriolar ends and showed focal hyperfluorescence in the late phase of fluorescein angiography , which suggests direct embolic obstruction of the arterioles ( fig . the selective ophthalmic angiogram revealed a large filling defect in the proximal ophthalmic artery , which explains the retinal and choroidal hypoperfusion in fluorescein angiography ( fig . the day after iat , retinal vessels were still segmented and the margin was blurred due to retinal edema secondary to ischemic injury ( fig . the choroidal perfusion was improved after iat , while retinal perfusion remained compromised ( fig . however , selective ophthalmic artery angiographic findings were different between ha - injected patients and fat - injected patients . a large filling defect was visible in the proximal part of the ophthalmic artery , and blood flow was compromised to the supratrochlear or supraorbital branch , and to the posterior ciliary branch in the fat - injected patients ( table 1 and fig . 3e - g ) . on the other hand , in the ha - injected patients , although there was flow stagnation in the distal branches of ophthalmic artery on initial angiogram , selective , pressurized infusion of contrast dye revealed grossly no mechanical obstruction in the supratrochlear branch or the supraorbital branch , while blood flow to the eyeball was compromised ( fig . however , the exact obstruction level was obscure . in two patients , obstruction was present at the level of the second segment of ophthalmic artery including the posterior ciliary branch ( table 1 , fig . 3a and d ) , while the other 2 patients did not show definite obstruction point in the second segment of ophthalmic artery ( table 1 , fig . the selective angiographic findings for the external carotid artery were also distinctly different between the ha - injected and fat - injected groups . three ha - injected patients showed diminished angiographic runoff in the distal branches of the internal maxillary and facial arteries , and decreased contrast staining in the periorbital area ( table 1 and fig . this finding was corresponded with skin lesion of the patients , as these patients revealed skin necrosis on injected area ( fig . on the other hand , all fat - injected patients and one ha - injected patient who was also treated with subcutaneous hyaluronidase injection showed preserved distal runoff in the distal branches of the internal maxillary and facial arteries and more prominent contrast staining in the periorbital area ( table 1 and fig . in this study , we carefully reviewed the angiographic findings of cosmetic facial filler injection - associated ophthalmic artery occlusions . although it is known that both ha and autologous fat injection could result in various spectrum of ophthalmic and retinal arterial occlusion ( 12 ) , all cases included in this study showed diffuse ophthalmic artery and its branches occlusion . both ha and autologous fat injection group showed severely compromised choroidal and retinal perfusion on fundus fluorescein angiography . despite these similar fundus findings , the cerebral angiographic findings were distinctively different between the ha - injected patients and the fat - injected patients . on selective ophthalmic angiography , all fat - injected patients had visible , large filling defects at the proximal part of the ophthalmic artery , whereas the ha - injected patients had obstruction at a more distal area than that observed in fat - injected patients . another interesting finding is that skin necrosis was mostly present in the ha - injected patients . in the previous study , our group also found the trend that 5 ( 23% ) of 22 fat - injected patients had skin lesions , whereas 9 ( 69% ) of 13 ha - injected patients had skin lesions ( p=0.007 ) ( 12 ) . however , by comparing the selective external carotid angiograms , we found that the angiographic runoff is diminished primarily in the distal branches of internal maxillary and facial arteries only in ha - injected group . this could have resulted from direct vascular obstruction by the injected filler material . on the other hand , the wide range of vascular runoff decrease around the filler - injected facial area also suggests another possibilities that the impediment of normal blood flow caused by elevation of distal intra - tissue pressure . some authors suggest that injected ha expands as it attracts water ; the facial artery and the angular artery or its branches become compressed , and skin necrosis ensues ( 1314 ) . in our cases , the skin necrosis lesion was most severe on a few days after cosmetic filler injection and this also supports the pressure necrosis mechanism secondary to local ischemic edema or to hydrophilic , volume - expansion properties of ha . interestingly , the patient who underwent immediate subcutaneous hyaluronidase injection showed relatively preserved angiographic runoff in the distal branches of internal maxillary and facial arteries and she sustained only mild erythema in filler injected area . hyaluronidase might have dissolved ha in injected area and reduced intra - tissue pressure , which improved vascular supply in the area and prevented skin necrosis . however , skin necrosis in ha - injected patients can not be fully explained only by pressure necrosis mechanism , as only small proportions of ha - injected patients suffer these complications . it would be more reasonable to explain as mechanical interruption of the vasculature of injected area are further compromised by increased tissue pressure secondary to both ischemic tissue edema and hydrophilic , volume - expansion properties of ha . in addition , increased distal tissue pressure could have worsened the blood flow into the periorbital area from the ophthalmic artery because the pressure gradient from ophthalmic artery to its peripheral branch is diminished . during the cerebral angiography in ha - injected patients , there was initial flow stagnation in the supratrochlear or supraorbital branches of ophthalmic artery , which was proven to be no mechanical obstruction after selective , pressurized infusion of contrast dye into ophthalmic artery . this finding also suggests that some proportion of flow impediment in ha - injected patients stems from decreased pressure gradient secondary to increased distal tissue pressure . previously thought as partial recanalization of frontal branches of the ophthalmic artery after iat might have been in reality , forced flow with pressure in the stagnated area ( 9 ) . based on all these findings , we summarized the different mechanisms of occlusion of the ophthalmic artery and its branches caused by ha and autologous fat in fig . fat particles in an aggregated form could completely block the proximal part of the ophthalmic artery . small fat particles could also obstruct smaller vessels , as proven by the presence of branch retinal artery occlusion cases in the previous literature ( 12 ) , and arteriolar obstruction found in late fundus fluorescein angiography in this study . on the other hand , ha seems to be more uniform in size and could obstruct the more distal part , compared to a fat embolism . this could be the reason for the less prevalent diffuse occlusion of ophthalmic and its branches in ha - injected patients compared to autologous fat - injected group ( 12 ) . furthermore , the exact obstruction level would be different between ha and autologous fat injected group with diffuse occlusion , although the clinical manifestation is similar as complete ophthalmic artery or central retinal artery occlusion . it is likely that diffuse ophthalmic artery occlusions caused by ha injection may arise from multiple obstructions of the retinal and posterior ciliary vessels rather than by a large bolus of filler material completely obstructing the proximal part of the ophthalmic artery , as in the fat - injected patients . another remarkable finding is that ha might increase distal intra - tissue pressure by attracting water , and thereby impede blood flow into these areas since the pressure gradient between the ophthalmic artery and its distal branches may be diminished ( fig . molecular characteristics of ha is determined by the amount of molecular cross - linking ( 15 ) . recently , by adding low molecular weight ha polymer chains into high molecular weight ha , more efficient cross - linking capability was achieved . the resultant material became more cohesive and viscous , and showed good clinical outcomes ( 16 ) . in addition to the size of the material , cohesiveness or viscosity might also affect material embolism . unfortunately , the information on the exact composition or brand name of ha injected in this study is lacking , thus the influence of cohesiveness or viscosity of the injected material on embolism is obscure . further study with regard to the cohesiveness or viscosity of injected material and its relationship with embolism is needed . only 2 patients showed partial recanalization of the obstructed vessel , but they had no visual gain . hyaluronidase is a soluble protein enzyme that breaks down and hydrolyzes ha by splitting the glucosaminidic bond of glucuronic acid ( 1718 ) . it is also used in cases of skin necrosis derived from ha filler injections , and results in a good clinical course ( 19 ) . the experimental study also suggested that hyaluronidase may reduce skin necrosis if injected within the first 4 hr ( 20 ) . in our study , in contrast to other three ha - injected patients , the patient who underwent subcutaneous hyaluronidase injection showed good vascular supply in periorbital area and no significant skin lesion . however , with regard to ophthalmic artery obstruction , the role of iat and hyaluronidase seems to be very limited . complete obstruction by a large fat aggregate may be associated with a poor outcome , and no current treatment method can be recommended . our study is limited by the small number of patients included in this study . however , considering that cosmetic facial filler related ophthalmic and retinal artery occlusion is very rare complication , this is by far the largest case series on cerebral angiographic findings of ophthalmic complication following cosmetic facial filler injection . furthermore , the distinctive angiographic characteristics between ha - injected and fat - injected patients noted even in this small case series provide valuable information on the pathophysiology of cosmetic facial filler injection - associated occlusion of the ophthalmic artery and its branches . in conclusion , the size difference between injected materials is associated with different angiographic findings . large , aggregated autologous fat particle is more prone to obstruct proximal part of ophthalmic artery , whereas small ha particle obstructs distal branches compared to fat embolism . in addition , hydrophilic and volume - expansion property of ha might exacerbate blood flow on injected area , either by compressing already disrupted vessels or by diminishing the pressure gradient between the ophthalmic artery and its distal end , which is also related to skin necrosis . considering the angiographic findings of the involved cases , iat has a limited role in reconstituting blood flow or regaining vision in cosmetic facial filler - associated ophthalmic artery occlusions . with the ever increasing importance of clinical effectiveness , audit and quality assurance ; it is becoming mandatory to grade one 's own treatment results . the idea of individuals grading their own performance is a self - teaching exercise and improves the quality of future treatment . the assessment of orthodontic treatment needs and the outcome has traditionally been accomplished using subjective opinion and the experiences of clinicians . however , the variations in criteria used by different orthodontists and lack of reproducibility are limitations of the subjective evaluation . visual analog scale ( vas ) has been the most widely used as a subjective assessment method for the validation of various indices . in early surveys , various classifications were used as determinants of treatment needs and outcome . however , various authors such as massler and frankel , drakers , bjoerk et al . , and summers devised indices to evaluate malocclusion on the basis of objective requirements of occlusal discrepancies . pickering and vig were the first to investigate the use of summers occlusal index as objective grading version to assess the outcome of treatment . devised peer assessment rating ( par ) indices to evaluate malocclusion on the basis of objective requirements of occlusal discrepancies . the american board of orthodontics ( abo ) model grading system is presently one of the most detailed scoring systems in use . comprehensive clinical evaluation as outcome assessment was developed which included facial form , dental esthetics , vertical control , arch form , periodontium management , root structure preservation , and treatment efficiency . to assess clinical performance , both objective and although there are many studies in the literature mentioning the orthodontic treatment outcome of various bracket systems , various extraction pattern , various bracket slot size , type of orthodontic practice , that is , private versus university practice , timing of treatment ; and general practitioner versus orthodontist but , to our knowledge , there are very few studies in the literature mentioning the effect of assessment of treatment outcome on the orthodontic treatment outcome . thus , the present study was conducted to find out the role of posttreatment critical evaluation ( ptce ) implementation on the effect of orthodontic treatment outcome . to eliminate any institutional bias , the study sample comprised of pretreatment and post treatment orthodontic records of 235 consecutively debonded cases in the department of orthodontics , oral health sciences centre , postgraduate institute of medical education and research , chandigarh . cases whose orthodontic treatment was completed during the period 1999 and 2004 were included in phase - i ( n = 109 ) evaluation . a total of 278 cases were debonded during this period , out of which 120 cases comprised of compromised debond and incomplete records and 49 cases comprised of cleft and surgical cases . cases whose orthodontic treatment was completed during the period 2006 and 2010 were included in phase - ii ( 126 ) evaluation . the sample was extracted from a total of 328 debonded cases , out of which 108 comprised of cleft and surgical cases , 94 had incomplete records . while selecting a case for the evaluation , no attempt was made to select a case on the basis of malocclusion type , gender or extraction pattern . orthodontic cases having good quality pretreatment and posttreatment records such as study models , photographs , and orthopantomograms ( opgs ) ; and orthodontic cases those had completed comprehensive orthodontic treatment in both the arches were included in the study . orthodontic cases having good quality pre - treatment and post - treatment records like study models , photographs and orthopantomograms and orthodontic cases those had completed comprehensive orthodontic treatment in both the arches were included in the study . in order to improve the quality of orthodontic care , ptce of each case was made mandatory as a part of the clinical protocol after phase - i study . the power point presentation contained detailed pre and post treatment records which included study models , intraoral and extra - oral photographs , intraoral periapical radiographs , opg , lateral cephalograms . detailed history , progress of treatment , number of missing appointments , data from study model , opg and lateral cephalometric analysis and abo model grading , superimpositions of pretreatment , stage , and post - treatment cephalograms ( to evaluate growth , change in facial profile and incisor molar positions ) were also included in the power point presentation . during the presentation , pre- and post - treatment records , data and treatment plan were critically evaluated by all the postgraduate residents and faculty . the presence of any lacunae in the treatment was discussed , and special emphasis was given to improve these lacunae subsequently . then , 6-years after implementation of compulsory ptce for each case , the orthodontic treatment outcome of all consecutively debonded cases was evaluated ( phase - ii ) . the treatment outcome was evaluated by american board of orthodontics model grading system ( abo mgs ) and subjective evaluation . the abo model grading system included eight criteria to measure the post - treatment finishing of the occlusion . post - treatment study models and panoramic radiographs were scored by one examiner for eight components of the abo mgs using the abo measuring gauge . the intraexaminer calibration was done by repeated abo scoring by the examiner at the interval of 4 weeks . as per the abo instruction , a case with < 20 points would pass , and the case with 30 points would fail . a case with points more than 20 but thus , the scores were thus divided into three categories , that is , grade - i ( score 020 points ) , grade - ii ( score 2030 points ) , and grade - iii ( score > 30 points ) . the subjective evaluation was carried out by three external examiners who were senior orthodontists with a minimum of 7 years of clinical experience and also involved in orthodontic teaching . the external examiners were chosen to avoid any bias in scoring cases from one 's own department . each of the three examiners judged debonded cases independently at different time periods for treatment outcome based on combined assessment of the pre- and post - treatment dental casts and photographs ( extraoral and intraoral , pre- and post - treatment photographs ) . the mean of the vas score allotted for each case by the three examiners was calculated for the purpose of analyzing the results . the scale used was 11-point ( 010 ; 0 = very poor , and 10 = excellent ) vas , which was anchored at its two ends with extreme limits of very poor and excellent . the total vas score was divided into three grades , that is , poor ( vas score 04.9 ) , fair ( vas score 56.9 ) , and good ( vas score 710 ) . the treatment outcome of cases in phase - i and phase - ii evaluation were noted on a standard performa . the mean values with standard deviation for the individual components and the total abo score of the abo model grading system for the total sample was recorded . the mean of the vas score allotted for each case by the three examiners was calculated for the purpose of analyzing the results . the statistical analysis was carried out using spss - software version-16.0 ( spss inc . released 2007 . the intra examiner reliability was determined among the various components of the abo mgs and the total score by using paired t - test and pearson 's correlation coefficient . the cronbach 's alpha test was used to determine the intraexaminer reliability for each of the three examiners . unpaired t - test was used to compare various components and a total score of abo model grading system and subjective evaluation between phase - i and phase - ii . the chi - square test was used for comparison between phase - i and phase - ii for different categories of abo model grading and subjective evaluation . the statistical analysis was carried out using spss - software version-16.0 ( spss inc . released 2007 . the intra examiner reliability was determined among the various components of the abo mgs and the total score by using paired t - test and pearson 's correlation coefficient . the cronbach 's alpha test was used to determine the intraexaminer reliability for each of the three examiners . unpaired t - test was used to compare various components and a total score of abo model grading system and subjective evaluation between phase - i and phase - ii . the chi - square test was used for comparison between phase - i and phase - ii for different categories of abo model grading and subjective evaluation . the intra examiner reliability among the various components of the abo mgs and the total score was found to be between 0.888 and 1 . the description of cases evaluated during the phase - i and phase - ii evaluation is summarized in table 1 . description of cases evaluated during phase - i and phase - ii evaluation the mean value for the individual components of the abo mgs and the total abo score for the total sample in phase - i and phase - ii evaluation is described in table 2 . the mean scores of alignment , marginal ridge relationship , occlusal contacts , and interproximal contacts were improved in phase - ii evaluation as compared to phase - i evaluation ; and the difference was comparable . the buccolingual inclination , occlusal relationship , and root angulation were improved significantly during phase - ii evaluation ( p < 0.001 ) . the score for overjet was improved from phase - i evaluation to the phase - ii evaluation ( p < 0.01 ) . the total abo score was improved significantly from 30.06 10.54 at phase - i evaluation to the 26.67 7.77 at phase - ii evaluation ( p < 0.01 ) . the mean values for the individual components of the abo mgs and the total abo score for the total sample in phase - i and phase - ii evaluation and their comparison the distribution of the sample according to abo score during phase - i and phase - ii evaluation is described in table 3 . the percentage of cases in the grade - i of abo category was similar during phase - i and phase - ii evaluation ( 20.10% vs. 20.63% ) . the percentage of cases in the grade - ii of abo category was improved significantly from phase - i evaluation ( 34.03% ) to phase - ii evaluation ( 50.79% ) ( p < 0.01 ) . the percentage of cases in the grade - iii of abo category was decreased significantly during phase - ii evaluation ( 45.87% ) as compared to the phase - i evaluation ( 28.57% ) ( p < 0.01 ) . comparison of total number of cases falling in each category of abo model grading system between phase - i and phase - ii evaluation the value of reliability for three examiners was found as 0.675 that indicated the good reliability between the examiners . the vas score for the total samples evaluated in phase - i and phase - ii is described in table 4 . the mean vas score for the total sample was improved from 5.66 0.77 at phase - i to 6.02 0.99 during phase - ii . the percentage of cases in the poor and fair grades was decreased from phase - i evaluation to the phase - ii evaluation . the percentage of cases scoring good score was increased significantly from 7.34% at phase - i evaluation to 19.05% at phase - ii evaluation indicating improvement in the orthodontic treatment outcome ( p < 0.01 ) . comparison of mean vas score and total number of cases falling in each category of vas grading between phase - i and phase - ii evaluation the excellence in finishing and the quality displayed at the end of orthodontic treatment constitutes a fundamental goal of orthodontic specialty . during all stages of treatment , one should keep the end results in mind , providing a protected occlusion , better aesthetics ( both dental and facial ) , good periodontal health and long - term stability , which are correlated to proper finishing . the sample for the study was collected from the records available in the department for a large sample in order to draw generalized conclusions . the gender distribution was not considered while including a case for the study because of uneven distribution of males and females . all the cases were evaluated by the same observer and the intraexaminer reliability value was found as 0.985 , which was > 0.85 recommended by abo members . the results of the present study showed a reduction in the mean total abo score from 30.06 10.54 to 26.67 7.77 . there was an improvement in the score of each component of abo model grading system . the cases falling in the grade - ii category had also been increased from 33.03% to 50.79% from the phase - i evaluation to phase - ii evaluation . the implementation of compulsory ptce was responsible for the improvement in the treatment outcome at phase - ii evaluation . the average total abo score ( 26.67 ) that we observed at the end of phase - ii evaluation was less than the average score of 45.54 18.33 for university group of illinois and 33.88 for abo group . the results of the present study correspond with the study done in indiana university school of dentistry . the better results in our study could be secondary to the implementation of compulsory ptce for orthodontic residents . we also considered a subjective assessment by three external orthodontists to compare the overall treatment outcome and treatment quality . in the subjective evaluation , not only study models were evaluated , but also intraoral and extraoral photographs were evaluated to identify the improvement in the profile . as the scoring on the vas was totally based on the personal opinion and judgment of the external examiners , this evaluation was purely subjective in nature and no standardization in terms of any set criteria or guidelines was carried out . therefore , an absolutely independent judgment was done by removing the confounding bias related to a discussion among the examiners . similar to abo model grading scores , the vas score was also improved at phase - ii evaluation , and the implementation of compulsory ptce was responsible for such improvement . however , unlike abo model grading system , the subjective evaluation scored the treatment results by looking at the change in occlusion , as well as facial appearance , thus it did not have the inherent limitation of objective indices . the lack of any objective measures to determine the results explained the low reliability of subjective criteria . similarly , to our study low reliability values were also reported by richmond et al . , where they used a 9-point scale for subjective assessment of dental casts with respect to deviation from normal occlusion in the validation study of the par index by 74 dentists . to reduce the effect of interexaminer variability the mean score of three examiners was used for the final score in the statistical comparison of results . thus , the present study showed that the implementation of ptce improved the quality of orthodontic treatment among orthodontic residents , and the ptce should be implemented as compulsory in a postgraduate orthodontic clinic . the quality of orthodontic care was improved significantly following the implementation of ptce in a postgraduate orthodontic clinic .

cosmetic facial filler - related ophthalmic artery occlusion is rare but is a devastating complication , while the exact pathophysiology is still elusive . cerebral angiography provides more detailed information on blood flow of ophthalmic artery as well as surrounding orbital area which can not be covered by fundus fluorescein angiography . this study aimed to evaluate cerebral angiographic features of cosmetic facial filler - related ophthalmic artery occlusion patients . we retrospectively reviewed cerebral angiography of 7 patients ( 4 hyaluronic acid [ ha ] and 3 autologous fat - injected cases ) showing ophthalmic artery and its branches occlusion after cosmetic facial filler injections , and underwent intra - arterial thrombolysis . on selective ophthalmic artery angiograms , all fat - injected patients showed a large filling defect on the proximal ophthalmic artery , whereas the ha - injected patients showed occlusion of the distal branches of the ophthalmic artery . three ha - injected patients revealed diminished distal runoff of the internal maxillary and facial arteries , which clinically corresponded with skin necrosis . however , all fat - injected patients and one ha - injected patient who were immediately treated with subcutaneous hyaluronidase injection showed preserved distal runoff of the internal maxillary and facial arteries and mild skin problems . the size difference between injected materials seems to be associated with different angiographic findings . autologous fat is more prone to obstruct proximal part of ophthalmic artery , whereas ha obstructs distal branches . in addition , hydrophilic and volume - expansion property of ha might exacerbate blood flow on injected area , which is also related to skin necrosis . intra - arterial thrombolysis has a limited role in reconstituting blood flow or regaining vision in cosmetic facial filler - associated ophthalmic artery occlusions . objective : the aim of the study was to evaluate the effect of post- treatment critical evaluation on the quality of orthodontic care in a postgraduate orthodontic clinic.materials and methods : orthodontic treatment outcome of 109 consecutively treated cases was evaluated in phase - i evaluation . following phase - i evaluation , ptce of each case was made mandatory . after 6-years of implementation of compulsory ptce for each case , orthodontic treatment outcome of all consecutively treated cases ( n = 126 ) was evaluated ( phase - ii ) . the treatment outcome was evaluated by american board of orthodontics model grading system ( abo mgs ) and subjective evaluation ( visual analogue scale , vas).results : based on the abo scores , the cases were divided into three grades , that is , grade - i , grade - ii , and grade - iii . the mean total abo score was improved significantly in phase - ii evaluation ( p < 0.01 ) . the total number of cases in abo grade - ii were increased significantly ( p < 0.01 ) whereas cases in abo grade - i remained comparable . the vas score was improved from 5.66 0.77 at phase - i to 6.02 0.99 at phase - ii evaluation ( p < 0.01).conclusion : the implementation of ptce significantly improved the quality of orthodontic care in a postgraduate orthodontic clinic.clinical significance : grading one 's own treatment improves the quality of future treatment .

concentrations of ambient pm2.5 ( particulate matter < 2.5 microm in aerodynamic diameter ) were associated with increased mortality in two prospective cohort studies . in this paper , i assess whether the weight of the evidence supports a causal association . i assumed the study population in each city to have the same exposure ; therefore , these are ecologic studies because exposure is at the group level . health outcome and confounding data are at the individual level . ambient pm concentrations are inadequate surrogates for personal exposure because they are at the group level and comprise only a small proportion of personal exposure , they change over time , and they constitute only a small proportion of a life span . the strength of association and exposure - response relationships can not be determined because the ecologic group - level risks of pm2.5 are overestimated 150- to 300-fold based on an analogy with individual - level exposure to inhaled cigarette smoke . risk estimates may also be high because of confounding from factors such as physical activity and lung function . the evidence is not coherent because the stronger associations are expected to be with morbidity , but instead are with mortality . for example , pm2.5 was associated with mortality but not with measurable reductions in lung function . biological plausibility is lacking because lifetime exposure of rats to combustion products at concentrations two to three orders of magnitude higher than air pollution levels cause lung overloading but no consistent reduction in survival . criteria for quantitative risk assessment are not met so the data are not useful for setting air quality standards . the weight of evidence suggests there is no substantive basis for concluding that a cause - effect relationship exists between long - term ambient pm2.5 and increased mortality.imagesfigure 1figure 2figure 3figure 4figure 5 beryllium was released into the air from routine operations and three accidental fires at the rocky flats plant ( rfp ) in colorado from 1958 to 1989 . we evaluated environmental monitoring data and developed estimates of airborne concentrations and their uncertainties and calculated lifetime cancer risks and risks of chronic beryllium disease to hypothetical receptors . this article discusses exposure - response relationships for lung cancer and chronic beryllium disease . we assigned a distribution to cancer slope factor values based on the relative risk estimates from an occupational epidemiologic study used by the u.s . environmental protection agency ( epa ) to determine the slope factors . we used the regional atmospheric transport code for hanford emission tracking atmospheric transport model for exposure calculations because it is particularly well suited for long - term annual - average dispersion estimates and it incorporates spatially varying meteorologic and environmental parameters . we accounted for model prediction uncertainty by using several multiplicative stochastic correction factors that accounted for uncertainty in the dispersion estimate , the meteorology , deposition , and plume depletion . we used monte carlo techniques to propagate model prediction uncertainty through to the final risk calculations . we developed nine exposure scenarios of hypothetical but typical residents of the rfp area to consider the lifestyle , time spent outdoors , location , age , and sex of people who may have been exposed . we determined geometric mean incremental lifetime cancer incidence risk estimates for beryllium inhalation for each scenario . the risk estimates were < 10(-6 ) . predicted air concentrations were well below the current reference concentration derived by the epa for beryllium sensitization.imagesfigure 1figure 2figure 3figure 4figure 5figure 6

the motility of nk cells in tissue or tumor microenvironment is likely driven by chemotactic signaling resulting in an approximation to key sites of interest , although nk cell motility in tumors is also driven by the expression of nkg2d ligands . therefore , similar to a t - cell sampling dendritic cells , nk cells undergo brief , exploratory interactions with multiple cell types , which may represent their initial encounter with a target ( figure 1a ) . it is not known what molecules specifically mediate these interactions , although there are a number of candidate receptors expressed on resting or primed nk cells , including a variety of tethering ' receptors as well as potentially more robust adhesion receptors . the former potentially include cd62l and psgl-1 ; the latter include cd2 , dnam-1 , nkg2d , the natural cytotoxicity receptors and lymphocyte function associated antigen-1 ( lfa-1 ) . unlike t cells , a subset ( 10% ) of freshly isolated human nk cells express the activated conformation of lfa-1 at rest , which presumably allows adhesion to a target before the engagement of other activating receptors . the transmission of a second activating signal through a receptor such as nkg2d , dnam-1 , 2b4 ( cd244 ) or cd2 strengthens these interactions by further activating lfa-1 , resulting in firm adhesion to the target and potentially providing minimal requirements for progression to mediating cytotoxicity . we are still learning of surprising interactions that nk cells engage in , likely in part due to the complex microenvironment that they patrol . one such unconventional form of communication is the formation of membrane nanotubes ( figure 1a ) . nanotubes appear to serve as intercellular tethers formed rapidly after contact with the target cell and can help guide the nk cell to the target cell or provide traction during its initial interaction . rather than hollow tubes that mediate free diffusion , nk cell nanotubes are heterogeneous structures that transmit signals over long distances and can even deliver lytic granules from an nk cell to a target . importantly , their formation enhances cytotoxicity . nkg2d and downstream activation molecules including dap10 and vav1 accumulate at the nanotube junction , resulting in the description of this junction as a nanoscale synapse . however , blocking either lfa-1 or 2b4 does not reduce the frequency of nanotube formation , suggesting that this is not simply a stretched conventional lytic synapse . cd2 expression specifically increases the frequency of nk target cell nanotubes , suggesting a role for specific receptor ligand interactions in their formation and partially explaining the observation that cd2 enhances nk cell cytotoxicity . the mechanism by which this occurs , and the prevalence of nanotubes during routine physiological nk surveillance , remains unclear . another example of the complex initial interaction may be the newly observed phenomenon of nk cell - derived exosomes : nk surface marker - expressing , fasl and perforin - containing nanovesicles ( 50100 nm ) that , upon release from nk cells , can mediate target cell killing independent of is formation . they may also potentially transfer key ligands to target cells to enable more robust interactions in environments in which exosome concentrations are high . this concept , in concert with nanotubes , harkens to a provocative early proposal of harpooning ' as a contributor to nk cell cytolytic function . despite their apparent constitutive secretion by human nk cells isolated from peripheral blood , the relative contribution of exosomes to an nk - mediated cytotoxic response following target cell contact , lfa-1 engagement initializes the first steps of is formation , including protein tyrosine kinase activation , pip(4,5)p2 generation and f - actin reorganization ( figure 1b ) . in synergy with activating receptor ligation , lfa-1 also induces an arrest signal to migrating cells , with a strong activating signal resulting in a stable radially symmetrical synapse and inhibitory signaling resulting in asymmetry and subsequent resumption of migration . whether nk cells kill through kinapses ' , or moving synapses , as has been described in t cells , remains to be determined . lfa-1-mediated outside - in signaling for f - actin polymerization brings together all the requisite components for f - actin polymerization and initiates this process . this initial signaling also includes the phosphorylation of vav1 by a src family kinase , its recruitment to lipid rafts and subsequent further activation as discussed in subsequent steps below . therefore , initial engagement of lfa-1 in concert with a second signal can help to ready an nk cell for target cell lysis . at this point the nk cell is likely still highly sensitive to a balance of activating and inhibitory signaling , and control of vav1 phosphorylation and subsequent actin accumulation is thought to be a critical axis in the regulation of cytotoxicity . at this stage , microclusters are likely present , may be functional and are also described further below . despite the tightly regulated steps leading to nk cell cytotoxicity , there is evidence that pre - docked granules are present in the cell cortex and can be accessed rapidly by nk cells . lytic granule secretion at the is before microtubule - organizing center ( mtoc ) polarization has been described in primary human cytotoxic t lymphocytes ( ctls ) . although it is conceivable that antigen specificity of t cells allows for the circumvention of further checkpoints to cytotoxicity , lytic granules are present in the cortex of resting nk cells and initial exocytosis bursts are detected ( figure 1b ) . despite these observations , imaging of both nk cell lines and primary nk cells by both live and fixed cell microscopy indicates that the majority of nk cells go through the multistep , highly regulated steps to cytotoxicity ( figure 2 ) . therefore , it is unclear as to what the significance of the presence and potential exocytosis of pre - docked granules is . in this light , it is also still unclear how many lytic granules are required to kill specific types of target cells . polarization of the mtoc and lytic granules to the is is required for nk cell cytotoxicity and occurs in the effector stage . however , preceding mtoc polarization , lytic granules rapidly move along microtubules and converge upon the mtoc ( figure 1c ) . this minus - ended lytic granule movement depends on dynein motor function but is independent of actin polymerization or microtubule dynamics . it also occurs whether the nk cell is conjugated to a susceptible or resistant target cell , as it is observed in the presence of an inhibitory synapse . this suggests that granule convergence is a step that precedes nk cell commitment to cytotoxicity , instead preparing the cell for it . accordingly , ligation of cd28 or lfa-1 alone is sufficient to induce lytic granule convergence , as is activation by soluble interleukin-2 . disruption of lamp-1 ( cd107a ) expression also results in impaired lytic granule motility , which corresponds with a decrease in association of the dynein component p150 with lytic granules . although this suggests that lamp-1 itself may mediate the interaction of granules with dynein , the mechanism of this is unknown . interestingly , mtoc polarization in lamp-1-deficient cells is intact , suggesting that convergence is not a prerequisite for polarization . lytic granule convergence before mtoc polarization likely represents the final juncture at which nk cells are susceptible to inhibitory signaling before a commitment to cytotoxicity . signaling through immunoreceptor tyrosine - based inhibitory motif ( itim)-containing inhibitory receptors induces formation of the inhibitory synapse , which is functionally dominated by phosphatases , particularly shp-1 , leading to exclusion of the downstream signaling molecules from the contact region ( figure 1d ) . inhibitory signaling acts swiftly to disrupt multiple points of activation , including conjugate formation and f - actin accumulation , lfa-1 activation , activating receptor clustering at the is and critically , ca flux . dephosphorylation of the activating phospho - tyrosine residues of vav1 appears to be a critical molecular switch for this control but this response is likely fine - tuned by other factors . these include the adaptor protein crk , which is also phosphorylated following inhibitory signaling and recruited to the inhibitory synapse . crk , through its association with c3 g and subsequent control of rap1 , likely has a role in the regulation of lfa-1 activation and subsequent target cell adhesion . interestingly , inhibitory receptors also serve to license ' nk cells during their developmental process , and nk cells that lack the recognition of mhc molecules during maturation are rendered hyporesponsive , at least partially by reduced signaling from activating receptors to lfa-1 . unlicensed cells form fewer conjugates , but those that do conjugate mediate lytic granule polarization normally . other mechanisms by which these unlicensed ' or anergic ' nk cells are restrained in their cytotoxic capacity represents an unknown cellular checkpoint that will be of great value in understanding full access to lytic capacity . at the beginning of the effector stage this stage is marked largely by significant cytoskeletal rearrangements , beginning with significant de novo f - actin polymerization and reorganization ( figure 1e ) . although the importance of the f - actin cytoskeleton in nk cell cytotoxicity has been appreciated for some time , recently many new contributors to the regulation of f - actin have been highlighted . one of these is the wiskott - aldrich syndrome protein ( wasp ) homolog wave2 . interestingly , while both wasp and wave2 are expressed in t and nk cells , wave2 may be critical for f - actin polymerization in ctls but is normally not accessed by nk cells . in nk cells , wasp appears to be the predominantly utilized family member . as an illustration , wiskott - aldrich syndrome patients who are deficient in wasp have severe nk cell functional impairment linked to an inability to rearrange f - actin . this can be overcome with interleukin-2 treatment , which activates wave2 in nk cells and restores f - actin rearrangement in was patient nk cells in vitro and in vivo . this not only offers therapeutic options for treatment of was but highlights an important difference between nk- and t - cell cytotoxicity . it also illustrates the potential cooperativity between adaptive and innate immunity via the ability of t cell - produced interleukin-2 to access distinct cell biology in nk cells . the rho gtpase cdc42 is also critical for f - actin polymerization by arp2/3 through direct binding and activation of wasp . recently , a role for the cdc42 effector dock8 has been described in nk cells . patients deficient in dock8 have decreased nk cell function with accompanying lytic synapse assembly and function defects that we now recognize as hallmarks of defective actin polymerization , including decreased lfa-1 recruitment , loss of granule polarization and accompanying decrease in cytotoxic function . importantly , dock8 deficiency does not affect overall nk cell f - actin content , but more specifically the targeted accumulation and polarization of f - actin at the synapse . this is in contrast to wasp deficiency in which nk cells have a reduced total f - actin content in addition to an inability to target actin accumulation . loss of dock2 function in mouse nk cells also results in impaired rac activation and subsequent loss of nk cell cytotoxic function , suggesting this family is an important regulator of nk cell cytotoxicity . in addition to the wasp and wave / scar family members , the arp2/3 complex can be activated by cortactin . the hematopoietic - specific homolog of cortactin , hs1 , is localized to the nk cell lytic synapse , where it is critical for signaling to and from lfa-1 . in nk cells , the formin homolog hdia is not required for lytic synapse branched actin , but instead is required for nk cell cytotoxicity by targeting microtubules to the synapse and subsequent lytic granule polarization , suggesting its function as the poorly understood link between the microtubule and actin cytoskeleton . once f - actin polymerization has been initiated , firm adhesion to the target cell is achieved and sustained ( figure 1e ) . as f - actin polymerization is required for lfa-1 recruitment and maintenance at the synapse , this synergy between actin and integrins provides firm adhesion throughout the period of nk cell activation . this transmembrane linkage results in the generation of significant force , which has been measured directly through the application of single - cell force spectroscopy . engagement of 2b4 leads to rapid , lfa-1- and actin - dependent increase in the force sufficient to disengage an nk cell from a susceptible target . therefore , the mechanical forces generated at the synapse also have an important role in adhesion activation signaling . this observation is supported by the increased lfa-1-mediated adhesion of nk cells bound to targets in which intercellular adhesion molecule-1 ( icam-1 ) is tethered and restrained , as opposed to freely diffusible . firm adhesion to the target cell and f - actin polymerization leads to the nk cell flattening and extending the diameter of the synapse ( figure 1f ) . this cell shape change ' is an actin - dependent process , as nk cells treated with actin inhibitors or from was patients fail to show this characteristic shape change . similarly , nk cells engaging in inhibitory synapses remain rounder , further underscoring the regulation of f - actin as a mechanism of nk cell inhibition . these can be defined as a critical mass of receptors that in concert can promote signal transduction . in t cells , f - actin - dependent microcluster movement occurs toward an actin - poor sink , with productive signaling occurring in the periphery of the synapse and terminating in the center . myosin iia also is required for centripetal microcluster movement and this sustained retrograde flow of microclusters is required for sustained ca release and activation signaling . although the kinetics may be different in nk cells , microclusters of both activating and inhibitory receptors are present and appear to have similar dynamics compared with those in t cells . studies using nk target cell conjugates show that phosphorylated kir2dl1-containing clusters originate in the periphery and coalesce in the center , thus reaffirming both the presence of microclusters and suggesting their utility in nk cells . furthermore , highly motile nkg2d - dap10-containing microclusters are also seen in the periphery of the synapse , and contain high levels of phospho - tyrosine , suggesting that , as in t cells , the periphery may also be the predominant site of activating signaling in nk cells . interestingly , kir2dl1 microclusters are found on resting nk cells , but become smaller and denser when nkg2d is ligated . this surprising observation was made possible using super - resolution microscopy , which allows for measurement of microcluster density and size ( 920 proteins / cluster and 120 nm , respectively ) . it is unclear what the significance of this microcluster compression actually is , but may reflect an interplay required for nk cell licensing , as speculated previously . the lipid microenvironment may also have a role in the spatial organization of receptors ( figure 1f ) . the role of higher - ordered microdomains , or lipid rafts , has been somewhat contentious . the use of multivalent labels , such as cholera toxin , always raises the possibility that aggregation of these probes may result in inaccurate detection . early studies , however , have implicated the recruitment of activating receptors to , and exclusion of inhibitory receptors from , lipid rafts . recent experiments in live jurkat t cells using a novel phase - sensitive membrane dye and high - resolution imaging have resolved higher - order membrane at the periphery of the synapse , suggesting that these domains may support microcluster or signaling platform formation . it remains to be seen whether this is also the case with nk cells , and what the effect of inhibitory signaling is on the membrane order at the synapse . two main axes of signaling are initiated and sustained through cytotoxicity once appropriate receptor ligand pairs are in place , namely , the pi3k and phospholipase c pathways . engagement of activating receptors leads to signaling through itam and yinm motifs and recruitment and activation of both membrane - proximal and distal proteins signaling and adaptor proteins . it is worth emphasizing the interdependence of activating signaling with the cytoskeleton , as disruption of one affects the other and prevents functional cytotoxicity . that said , the exact cellular mechanism of signaling protein localization and re - localization in nk cells remains unknown . one key outcome of activating receptor signaling is mobilization of ions , particularly calcium ( figure 1f ) . initial calcium flux occurs as a result of rapid release from endoplasmic reticulum ( er ) calcium stores mediated by ip3 channels downstream of plc signaling . depletion of er ca stores is sensed first by stim1 on the er surface . following activation , stim1 complexes reposition proximal to the plasma membrane ( visualized as puncta on the er surface ) and activate plasma membrane - associated ora1 . human genetic immunodeficiency has defined critical roles for both orai1 and stim1 in nk cell cytotoxicity . one manifestation of these patients ' disease is profound susceptibility to viral infection , a hallmark of reduced nk cell function , although they also present with many other signs and symptoms and have substantive defects in t - cell and b - cell function . importantly , while orai1-deficient nk cells form conjugates with target cells and polarize the mtoc and lytic granules , they fail to degranulate . although the two ca flux systems are the best characterized , there are other ion channels expressed and utilized by nk cells . recently , a critical role for intracellular mg has been described by a rare primary immune deficiency affecting the mg transporter magt1 . in these patients , intracellular levels of free mg are decreased , accompanied by defective nk cell and ctl killing and subsequent uncontrolled epstein interestingly , the role of mg in both ctl and nk cell control of epstein barr virus is through maintaining the stability of nkg2d at the cell surface , suggesting an unappreciated role for nkg2d in controlling epstein in addition , potassium and zinc channels are expressed and utilized by t cells ( reviewed in feske et al . ) , prompting the question of whether nk cells utilize similar mechanisms . presumably , in and around these steps a lytic cleft forms ( figure 1 g ) . once thought to be simply a gasket to prevent the leakage of lytic granules to surrounding tissues , it is now appreciated that the cleft is dynamic and complex . much of the activity that takes place in the cleft is critical to cytotoxic function , yet remains mysterious because of its inaccessibility . measurement of the lytic cleft by transmission electron microscopy in inhibitory and activating synapses shows that both have a similar range of distances between the nk cell and the apposing target cell membrane ( 1030 nm ) , which are proposed to account for distribution of proteins of different sizes at the synapse . these synaptic membrane features may also be a source of membrane transfer ( trogocytosis ) between an nk cell and a target ( figure 1 g ) , which has been defined for a number of receptors . this process is reciprocal , as transfer of both ligand from the target cell and receptor from the nk cell is observed . additional transmission electron microscopy experiments demonstrate that the target cell - derived receptors enter the nk cells in small , enclosed pits . both inhibitory and activating receptor exchange occurs across the nk cell synapse , although there is controversy as to whether cognate ligand engagement is required . the functional outcome of trogocytosis and intercellular receptor transfer is unclear , although transfer of nkg2d and micb at the synapse results in lower nk cell cytotoxic function . fascinatingly , nk cells that acquire nkg2d ligand from target cells become targets themselves , and are victims of fratricide by neighboring nk cells . this suggests that this ligand transfer may act to both enable and even control nk cell activity during infection , again suggesting the theory of harpooning . following firm adhesion and the formation of a cleft , f - actin continues to undergo substantial remodeling at the synapse . traditionally , this remodeling was thought to result in a large , central clearing of f - actin to enable centrosomal docking and lytic granule secretion . recent advances in imaging technology , namely , the adoption of super - resolution microscopy , have revealed that ligation of activating receptors creates granule - sized conduits through which granules are secreted ( figure 1h ) . interestingly , viral influenza particles can also result in conduit formation , presumably through the ligation of nkp46 , but this signal requires co - stimulation through lfa-1 . other activating receptors , such as nkg2d or cd16 , can signal solely for clearance formation . although not entirely understood , this seems to be an elegant mechanism by which an nk cell could discern a free viral particle from one bound to a target cell . whether proteins on the granules themselves form lytic granule conduits , they are a result of the inherent dynamism at the synapse , or occur in specific hotspots of signaling remains to be determined . it seems likely , based on the presence of actin nucleating factors at the synapse , that f - actin is dynamic and is likely undergoing continual remodeling . this may contribute to conduit kinetics , as has been suggested by analyses of synaptic plane actin dynamics using total internal reflection fluorescence imaging . the process of remodeling and specifically breaking down as opposed to building is of particular interest as conduits in f - actin are a requirement for degranulation . cofilin , which severs and depolymerizes f - actin , works in concert with arp2/3 to remodel actin to enable secretion in muscle cells . coronin 1a , which promotes the turnover of actin through binding to both arp2/3 and cofilin , regulates arp2/3 localization to the t - cell synapse , although a role for this important immune cell regulator in nk cells has yet to be defined . one appealing hypothesis is that hypodensity formation occurs at regions of high activating receptor density as a result of highly localized signaling events , such as is seen in mast cells . however , this is difficult to reconcile with what is likely the dynamic movement of microclusters of activating receptors themselves . an alternative explanation is that lytic granules create their own conduits , enabled by the presence of actin - remodeling proteins on their surface . this would be compatible with the observation that myosin iia is present on lytic granules and appears required for their penetrance through f - actin , but this hypothesis has not been directly tested . perhaps , in preparation for potential serial killing or nk cell recycling , lytic synapse formation also activates transcription factors and gene transcription ( figure 1i ) . ligation of nkp30 results in rapid translocation of nuclear factor b to the nucleus and de novo protein synthesis . perhaps , not surprisingly , perforin and granzyme is refilled in ctl lytic granules while they kill . the newly produced perforin then reaches the synapse independently of conventional lysosomal granules and mediates cytotoxicity . whether nk cells employ such a mechanism remains to be determined , as does the precise program of gene transcription that is activated during cytotoxicity . following the dynamic rearrangement of the actin cytoskeleton , microtubule dynamics result in a dramatic reorientation of the mtoc and associated lytic granules toward the synapse ( figure 1j ) . requirements for centrosome polarization include lfa-1 , pyk2 , erk2 , cip4 , the formin hdia and vav1 . however , it is important to note that f - actin polymerization is required for mtoc polarization ; therefore , any interference with f - actin dynamics will subsequently impair mtoc and granule polarization . one consideration in mtoc polarization is the significant amount of force likely needed to generate this reorientation . it is assumed from studies in other systems that microtubule insertion and anchoring in the cell cortex lead to either pushing ( microtubule growth ) or pulling ( microtubule shrinkage ) forces that can reposition the centrosome . dynein may again have a role , as a minus - ended motor it can generate significant pulling forces on shrinking microtubules when anchored in the cortex , and may contribute to the fine - tuning and positioning of microtubule asters . accordingly , it was recently shown that in t cells , mtoc repositioning occurs as a result of end - on capture shrinkage of microtubule focused at the center of the is and anchored to cortical dynein . interestingly , in nk cells , it appears that kinesin-1 has a role in the initial movement of the mtoc to the synapse , mediated through interactions with the small gtpase arl8b . iq motif containing gtpase - activating protein 1 ( iqgap1 ) may act as a linker between clip-170 on the plus ends of microtubule and specific regions of cortical actin . loss of iqgap1 results in a failure of nk cells to polarize the mtoc and degranulate . cip4 has also been implicated as a link between microtubules and f - actin at the cortex . although in t cells the mtoc docks in contact with the plasma membrane at the synapse , this has not been directly observed in nk cells . as the mtoc polarizes to the synapse , cellular organelles also reposition with some moving toward and others away from the synapse ( figure 1j ) . reorientation of the golgi along with microtubules toward the is presumably aids in directed secretion of granules toward the target cell . in t - helper cells , the mitochondria polarize toward the synapse to maintain ca flux across the plasma membrane for t - cell activation . in nk cells , the mitochondria reposition toward the nk cell is following nk stimulation with anti - nkgd2 antibodies but not with anti - kir2dl1 antibodies , suggesting that the mitochondrial dynamics are triggered as a result of nk cell activation . the polarization of these organelles is important for sufficient ca influx for signaling and granule exocytosis . it is conceivable that polarized mitochondria further serve as local sources of energy to power synaptic function , although this needs to be proven . following mtoc polarization to the is and anchoring at the plasma membrane , the delivery of the polarized lytic granules to the synapse occurs ( figure 1k ) . in t cells , this process requires plus - ended , kinesin-1-dependent movement of lytic granules upon microtubules to the membrane . in nk cells , other cellular machinery could also have a role in lytic granule movement . as mentioned above , whether this is through the facilitation of short runs across f - actin following mtoc polarization , the penetration of granules through the actin meshwork or the exocytosis of lytic granules is unclear , although obviously these scenarios are not mutually exclusive . following delivery to the plasma membrane , lytic granules have significant dynamic movement , which is followed by arrest , and subsequent degranulation in only a subset of granules ( figure 1l ) . this movement is not dependent upon f - actin dynamic rearrangement , but a role for microtubule dynamics has not been excluded . granule movement may be undirected rolling on synaptic actin , seeking regions of hypodensity . alternatively , it may represent the requirement for granules to find a primed fusion / tethering complex to mediate their arrest , as is suggested by high - resolution imaging of lytic granules in ctl utilizing munc 13 - 4 rab27a complexes for granule arrest and subsequent degranulation . at some point , lytic granules must traverse the pervasive actin network at the synapse ( figure 1 m ) . lytic granule may mechanistically identify areas of hypodense f - actin regions to pass through , or directed f - actin remodeling at the activating synapse may be required for clearing the way for lytic granules to transit through minimally permissive f - actin hypodensities . however , even in the presence of f - actin clearances , it is likely that lytic granule transit through the cortex requires the generation of force as was suggested by experiments evaluating degranulation in the absence of an f - actin meshwork . myosin iia may mechanically facilitate lytic granule motility through the f - actin - rich area of the is or may be involved in inducing local changes in the f - actin structure to allow polarized lytic granules access to the plasma membrane . after polarizing to the is and before degranulation , lytic granules dock at the is and fuse with the plasma membrane ( figure 1n ) . docking is likely mediated primarily by munc 13 - 4 and rab27a , both of which are recruited to lytic granules following activating receptor ligation . in ctl , lytic granules become fully mature only at late stages of the exocytic pathway , when munc13 - 4 and rab27a associate with perforin- and granzyme - containing lysosomal components . upon interacting with rab27a , munc13 - 4 may also regulate the interaction between vesicle and target soluble nsf attachment protein receptor ( snare ) required for lytic granule fusion with the plasma membrane . alternatively , this may be mediated by the interaction of syntaxin 11 and munc18 - 2 with r - snares ( such as vamp7 ) on the lytic granule membrane . priming then occurs as a result of munc13 - 4-mediated activation of syntaxin 11 or bridging of the plasma membrane with lytic granule . fusion occurs as a result of snare complex formation , and although this complex has not been defined in nk cells , mutations in the snare components syntaxin 11 , vamp4 and vamp7 prevent nk cell degranulation . in mast cells , coordinated oscillations of calcium in concert with n - wasp and pip(4,5)p2 whether such localized signaling dynamics contribute to nk cell exocytosis remains to be seen , although this hypothesis would complement the localized generation of actin hypodensities . in addition to the global calcium reserves such as the er and the store - operated reservoirs , local ca nanodomains created by channels on the lytic granule membrane in t cells are essential for their exocytosis . thus , local synaptic membrane nanoalterations may factor into a final step in allowing the release of lytic granule contents . nk cell lytic granules use at least two distinct modes of fusion : ( 1 ) complete fusion where granule content is completely discharged and the contents diffuse rapidly at the plasma membrane and ( 2 ) incomplete fusion , whereby formation of a transient fusion pore at the plasma membrane is accompanied by release of some but retention of most of the granule contents . this latter form of degranulation is suggestive of alternate forms of granule fusion seen in other secretory cells such as chromaffin cells and neurons . kiss and run ' , in which a granule pore is formed and resealed , preventing full release of granule contents , kiss and stay ' , in which granules remain at the membrane and are re - acidified , presumably for rapid recycling or reuse and crash fusion ' , in which granule contents are released without stable docking and priming . the true existence and significance of these alternate forms of granule fusion in nk cells is incompletely understood , although it is conceivable that it could be a means of facilitating serial killing through the rapid recycling of granule contents . as such , two - way vesicular trafficking occurs at the nk cell synapse , and rapid munc13 - 4-clathrin mediated endocytosis of granule membrane proteins exposed at the plasma membrane is necessary for the recycling of a pool of endocytic vesicles , allowing the serial killing event ( figure 1p ) . f - actin dynamics may have a role in the extrusion of granule contents and the persistence of lytic granules at the membrane following exocytosis . although this is presumably owing to force generation , the direct proof is presently unavailable . after degranulation , a period of relative inactivity begins and is presumably required as lytic granule contents cross the lytic cleft to act upon the target cell ( figures1o and p ) . the sustained presence of fused granules at the synaptic membrane , however , may serve additional functions as well . interestingly , the exposure of lamp-1 on the nk cell surface protects the nk cell from cytotoxicity - induced suicide , although this is likely not the only mechanism . subsequently , uptake of lytic granule contents by the target cell initiates apoptosis and marks the end of the effector stage of cytotoxicity , as the lytic hit has been administered . the termination stage of the synapse refers to the steps that immediately follow the release of lytic granules and up to either the reinitiating of a subsequent recognition or return to a resting state . initially , there is a longer time of apparent relative inactivity . during this time the target cell continues its death process , primarily by apoptosis ( figure 1q ) . phosphatidylserine on the surface of the cell may provide signaling to the nk cell to terminate the response , as nk cells express proteins that have been shown to bind to phosphatidylserine such as the itim - containing molecule cd300a . a further mechanism by which the nk cell could relax its activation status is through receptor downmodulation as has been documented for cd16 and nkg2d . ongoing is new lg biogenesis during this relatively quiet period as a likely requirement for nk cell recharging ' and subsequent serial kills . the final stage of cytotoxicity is defined by detachment from the target cell , a process that is not entirely arbitrary . kinetic studies show that the decision to end stable contacts with target cells is made faster when killing occurrs . interestingly , detachment was sometimes seen along with the formation of nanotubes connecting the nk and target cells ( figure 1r ) . the purpose of these connections in the context of a dying cell , however , is unclear and they may just represent the default behavior of an nk cell to tether to activating objects of interest . insight into the steps of the termination stage can be seen during the process of nk cell serial killing . serial killing can demonstrate burst kinetics , with a delayed first kill followed by more rapid subsequent kills . a proposed mechanism for burst kinetics is the idea of kinetic priming , which suggests that nk target interactions depend upon recent killing events . it is proposed that kinetic priming is a continuity of signaling , as interactions with old targets remain until a new target is found . as such , lytic granule may remain converged to the mtoc following target cell lysis in preparation for subsequent kills , as previously proposed . a consequence of serial killing is a near depletion of lytic granules and cytotoxic effector molecules . this depletion can leave the nk cells in an exhausted state until they detach from the target cell that has depleted their contents . after separation , exposure to locally available activating factors such as interleukin-2 can restore their cytotoxic function . interestingly , in cd8 cytotoxic t cells , it was found that rapid upregulation of nongranule - restricted perforin can be transported to the is and participate in cytotoxicity . as this form of perforin was identified in nk cells , perhaps this observation carries over to nk cells and contributes to the reacquisition of effector function after exhaustion . although seemingly the least is known about the specifics of the termination phase of the synapse , it can be interpreted as one of the most important parts , as recycling of nk cells in vivo in diseased tissue environments is likely to be of the essence in host defense . in recent years , explosive advances in technology have driven us deeper within the nk cell to better understand the mechanism of its critical function . in doing so , we have a much better understanding of the tightly regulated steps leading to cytotoxicity . there are critical and fundamental questions that remain to be answered , some of which require substantive technology development . these include , but are not limited to the following : ( 1 ) the exact number of , and temporospatial requirements for , degranulation events needed to kill particular target cells ; ( 2 ) how both inhibitory signaling and anergy ' restrain progression through the cell biological steps of synaptic formation ; ( 3 ) the mechanism by which conduits in the cell cortex are created to enable lytic granules to reach the nk cell synaptic membrane ; ( 4 ) the true purpose of lytic granule convergence to the mtoc , ( 5 ) the mechanism of delivery of lytic granules from mtoc to plasma membrane ; and ( 6 ) what specifically governs the release of an nk cell from its target cell . in addition , we still have much to learn about the fine - tuning of the cytotoxic response and the subversion of it by viruses and malignant cells . these represent only a sampling of the many questions begotten by the last 15 years of discovery , the answers to which will provide the tools to manipulate and control cytolytic host defense . better understanding of these facets of nk cells will enable more effective harnessing of nk cell functions and therapeutic intervention . the motility of nk cells in tissue or tumor microenvironment is likely driven by chemotactic signaling resulting in an approximation to key sites of interest , although nk cell motility in tumors is also driven by the expression of nkg2d ligands . therefore , similar to a t - cell sampling dendritic cells , nk cells undergo brief , exploratory interactions with multiple cell types , which may represent their initial encounter with a target ( figure 1a ) . it is not known what molecules specifically mediate these interactions , although there are a number of candidate receptors expressed on resting or primed nk cells , including a variety of tethering ' receptors as well as potentially more robust adhesion receptors . the former potentially include cd62l and psgl-1 ; the latter include cd2 , dnam-1 , nkg2d , the natural cytotoxicity receptors and lymphocyte function associated antigen-1 ( lfa-1 ) . unlike t cells , a subset ( 10% ) of freshly isolated human nk cells express the activated conformation of lfa-1 at rest , which presumably allows adhesion to a target before the engagement of other activating receptors . the transmission of a second activating signal through a receptor such as nkg2d , dnam-1 , 2b4 ( cd244 ) or cd2 strengthens these interactions by further activating lfa-1 , resulting in firm adhesion to the target and potentially providing minimal requirements for progression to mediating cytotoxicity . we are still learning of surprising interactions that nk cells engage in , likely in part due to the complex microenvironment that they patrol . one such unconventional form of communication is the formation of membrane nanotubes ( figure 1a ) . nanotubes appear to serve as intercellular tethers formed rapidly after contact with the target cell and can help guide the nk cell to the target cell or provide traction during its initial interaction . rather than hollow tubes that mediate free diffusion , nk cell nanotubes are heterogeneous structures that transmit signals over long distances and can even deliver lytic granules from an nk cell to a target . importantly , their formation enhances cytotoxicity . nkg2d and downstream activation molecules including dap10 and vav1 accumulate at the nanotube junction , resulting in the description of this junction as a nanoscale synapse . however , blocking either lfa-1 or 2b4 does not reduce the frequency of nanotube formation , suggesting that this is not simply a stretched conventional lytic synapse . cd2 expression specifically increases the frequency of nk target cell nanotubes , suggesting a role for specific receptor ligand interactions in their formation and partially explaining the observation that cd2 enhances nk cell cytotoxicity . the mechanism by which this occurs , and the prevalence of nanotubes during routine physiological nk surveillance , remains unclear . another example of the complex initial interaction may be the newly observed phenomenon of nk cell - derived exosomes : nk surface marker - expressing , fasl and perforin - containing nanovesicles ( 50100 nm ) that , upon release from nk cells , can mediate target cell killing independent of is formation . they may also potentially transfer key ligands to target cells to enable more robust interactions in environments in which exosome concentrations are high . this concept , in concert with nanotubes , harkens to a provocative early proposal of harpooning ' as a contributor to nk cell cytolytic function . despite their apparent constitutive secretion by human nk cells isolated from peripheral blood , the relative contribution of exosomes to an nk - mediated cytotoxic response following target cell contact , lfa-1 engagement initializes the first steps of is formation , including protein tyrosine kinase activation , pip(4,5)p2 generation and f - actin reorganization ( figure 1b ) . in synergy with activating receptor ligation , lfa-1 also induces an arrest signal to migrating cells , with a strong activating signal resulting in a stable radially symmetrical synapse and inhibitory signaling resulting in asymmetry and subsequent resumption of migration . whether nk cells kill through kinapses ' , or moving synapses , as has been described in t cells , remains to be determined . lfa-1-mediated outside - in signaling for f - actin polymerization brings together all the requisite components for f - actin polymerization and initiates this process . this initial signaling also includes the phosphorylation of vav1 by a src family kinase , its recruitment to lipid rafts and subsequent further activation as discussed in subsequent steps below . therefore , initial engagement of lfa-1 in concert with a second signal can help to ready an nk cell for target cell lysis . at this point the nk cell is likely still highly sensitive to a balance of activating and inhibitory signaling , and control of vav1 phosphorylation and subsequent actin accumulation is thought to be a critical axis in the regulation of cytotoxicity . at this stage , microclusters are likely present , may be functional and are also described further below . despite the tightly regulated steps leading to nk cell cytotoxicity , there is evidence that pre - docked granules are present in the cell cortex and can be accessed rapidly by nk cells . lytic granule secretion at the is before microtubule - organizing center ( mtoc ) polarization has been described in primary human cytotoxic t lymphocytes ( ctls ) . although it is conceivable that antigen specificity of t cells allows for the circumvention of further checkpoints to cytotoxicity , lytic granules are present in the cortex of resting nk cells and initial exocytosis bursts are detected ( figure 1b ) . despite these observations , imaging of both nk cell lines and primary nk cells by both live and fixed cell microscopy indicates that the majority of nk cells go through the multistep , highly regulated steps to cytotoxicity ( figure 2 ) . therefore , it is unclear as to what the significance of the presence and potential exocytosis of pre - docked granules is . in this light , it is also still unclear how many lytic granules are required to kill specific types of target cells . polarization of the mtoc and lytic granules to the is is required for nk cell cytotoxicity and occurs in the effector stage . however , preceding mtoc polarization , lytic granules rapidly move along microtubules and converge upon the mtoc ( figure 1c ) . this minus - ended lytic granule movement depends on dynein motor function but is independent of actin polymerization or microtubule dynamics . it also occurs whether the nk cell is conjugated to a susceptible or resistant target cell , as it is observed in the presence of an inhibitory synapse . this suggests that granule convergence is a step that precedes nk cell commitment to cytotoxicity , instead preparing the cell for it . accordingly , ligation of cd28 or lfa-1 alone is sufficient to induce lytic granule convergence , as is activation by soluble interleukin-2 . disruption of lamp-1 ( cd107a ) expression also results in impaired lytic granule motility , which corresponds with a decrease in association of the dynein component p150 with lytic granules . although this suggests that lamp-1 itself may mediate the interaction of granules with dynein , the mechanism of this is unknown . interestingly , mtoc polarization in lamp-1-deficient cells is intact , suggesting that convergence is not a prerequisite for polarization . lytic granule convergence before mtoc polarization likely represents the final juncture at which nk cells are susceptible to inhibitory signaling before a commitment to cytotoxicity . signaling through immunoreceptor tyrosine - based inhibitory motif ( itim)-containing inhibitory receptors induces formation of the inhibitory synapse , which is functionally dominated by phosphatases , particularly shp-1 , leading to exclusion of the downstream signaling molecules from the contact region ( figure 1d ) . inhibitory signaling acts swiftly to disrupt multiple points of activation , including conjugate formation and f - actin accumulation , lfa-1 activation , activating receptor clustering at the is and critically , ca flux . dephosphorylation of the activating phospho - tyrosine residues of vav1 appears to be a critical molecular switch for this control but this response is likely fine - tuned by other factors . these include the adaptor protein crk , which is also phosphorylated following inhibitory signaling and recruited to the inhibitory synapse . crk , through its association with c3 g and subsequent control of rap1 , likely has a role in the regulation of lfa-1 activation and subsequent target cell adhesion . interestingly , inhibitory receptors also serve to license ' nk cells during their developmental process , and nk cells that lack the recognition of mhc molecules during maturation are rendered hyporesponsive , at least partially by reduced signaling from activating receptors to lfa-1 . unlicensed cells form fewer conjugates , but those that do conjugate mediate lytic granule polarization normally . other mechanisms by which these unlicensed ' or anergic ' nk cells are restrained in their cytotoxic capacity represents an unknown cellular checkpoint that will be of great value in understanding full access to lytic capacity . at the beginning of the effector stage , the nk cell commits to cytotoxicity and proceeds toward degranulation . this stage is marked largely by significant cytoskeletal rearrangements , beginning with significant de novo f - actin polymerization and reorganization ( figure 1e ) . although the importance of the f - actin cytoskeleton in nk cell cytotoxicity has been appreciated for some time , recently many new contributors to the regulation of f - actin have been highlighted . one of these is the wiskott - aldrich syndrome protein ( wasp ) homolog wave2 . interestingly , while both wasp and wave2 are expressed in t and nk cells , wave2 may be critical for f - actin polymerization in ctls but is normally not accessed by nk cells . in nk cells , wasp appears to be the predominantly utilized family member . as an illustration , wiskott - aldrich syndrome patients who are deficient in wasp have severe nk cell functional impairment linked to an inability to rearrange f - actin . this can be overcome with interleukin-2 treatment , which activates wave2 in nk cells and restores f - actin rearrangement in was patient nk cells in vitro and in vivo . this not only offers therapeutic options for treatment of was but highlights an important difference between nk- and t - cell cytotoxicity . it also illustrates the potential cooperativity between adaptive and innate immunity via the ability of t cell - produced interleukin-2 to access distinct cell biology in nk cells . the rho gtpase cdc42 is also critical for f - actin polymerization by arp2/3 through direct binding and activation of wasp . recently , a role for the cdc42 effector dock8 has been described in nk cells . patients deficient in dock8 have decreased nk cell function with accompanying lytic synapse assembly and function defects that we now recognize as hallmarks of defective actin polymerization , including decreased lfa-1 recruitment , loss of granule polarization and accompanying decrease in cytotoxic function . importantly , dock8 deficiency does not affect overall nk cell f - actin content , but more specifically the targeted accumulation and polarization of f - actin at the synapse . this is in contrast to wasp deficiency in which nk cells have a reduced total f - actin content in addition to an inability to target actin accumulation . loss of dock2 function in mouse nk cells also results in impaired rac activation and subsequent loss of nk cell cytotoxic function , suggesting this family is an important regulator of nk cell cytotoxicity . in addition to the wasp and wave / scar family members , the arp2/3 complex can be activated by cortactin . the hematopoietic - specific homolog of cortactin , hs1 , is localized to the nk cell lytic synapse , where it is critical for signaling to and from lfa-1 . in nk cells , the formin homolog hdia is not required for lytic synapse branched actin , but instead is required for nk cell cytotoxicity by targeting microtubules to the synapse and subsequent lytic granule polarization , suggesting its function as the poorly understood link between the microtubule and actin cytoskeleton . once f - actin polymerization has been initiated , firm adhesion to the target cell is achieved and sustained ( figure 1e ) . as f - actin polymerization is required for lfa-1 recruitment and maintenance at the synapse , this synergy between actin and integrins provides firm adhesion throughout the period of nk cell activation . this transmembrane linkage results in the generation of significant force , which has been measured directly through the application of single - cell force spectroscopy . engagement of 2b4 leads to rapid , lfa-1- and actin - dependent increase in the force sufficient to disengage an nk cell from a susceptible target . therefore , the mechanical forces generated at the synapse also have an important role in adhesion activation signaling . this observation is supported by the increased lfa-1-mediated adhesion of nk cells bound to targets in which intercellular adhesion molecule-1 ( icam-1 ) is tethered and restrained , as opposed to freely diffusible . firm adhesion to the target cell and f - actin polymerization leads to the nk cell flattening and extending the diameter of the synapse ( figure 1f ) . this cell shape change ' is an actin - dependent process , as nk cells treated with actin inhibitors or from was patients fail to show this characteristic shape change . similarly , nk cells engaging in inhibitory synapses remain rounder , further underscoring the regulation of f - actin as a mechanism of nk cell inhibition . these can be defined as a critical mass of receptors that in concert can promote signal transduction . in t cells , f - actin - dependent microcluster movement occurs toward an actin - poor sink , with productive signaling occurring in the periphery of the synapse and terminating in the center . myosin iia also is required for centripetal microcluster movement and this sustained retrograde flow of microclusters is required for sustained ca release and activation signaling . although the kinetics may be different in nk cells , microclusters of both activating and inhibitory receptors are present and appear to have similar dynamics compared with those in t cells . studies using nk target cell conjugates show that phosphorylated kir2dl1-containing clusters originate in the periphery and coalesce in the center , thus reaffirming both the presence of microclusters and suggesting their utility in nk cells . furthermore , highly motile nkg2d - dap10-containing microclusters are also seen in the periphery of the synapse , and contain high levels of phospho - tyrosine , suggesting that , as in t cells , the periphery may also be the predominant site of activating signaling in nk cells . 2b4-containing interestingly , kir2dl1 microclusters are found on resting nk cells , but become smaller and denser when nkg2d is ligated . this surprising observation was made possible using super - resolution microscopy , which allows for measurement of microcluster density and size ( 920 proteins / cluster and 120 nm , respectively ) . it is unclear what the significance of this microcluster compression actually is , but may reflect an interplay required for nk cell licensing , as speculated previously . the lipid microenvironment may also have a role in the spatial organization of receptors ( figure 1f ) . the role of higher - ordered microdomains , or lipid rafts , has been somewhat contentious . the use of multivalent labels , such as cholera toxin , always raises the possibility that aggregation of these probes may result in inaccurate detection . early studies , however , have implicated the recruitment of activating receptors to , and exclusion of inhibitory receptors from , lipid rafts . recent experiments in live jurkat t cells using a novel phase - sensitive membrane dye and high - resolution imaging have resolved higher - order membrane at the periphery of the synapse , suggesting that these domains may support microcluster or signaling platform formation . it remains to be seen whether this is also the case with nk cells , and what the effect of inhibitory signaling is on the membrane order at the synapse . two main axes of signaling are initiated and sustained through cytotoxicity once appropriate receptor ligand pairs are in place , namely , the pi3k and phospholipase c pathways . engagement of activating receptors leads to signaling through itam and yinm motifs and recruitment and activation of both membrane - proximal and distal proteins signaling and adaptor proteins . it is worth emphasizing the interdependence of activating signaling with the cytoskeleton , as disruption of one affects the other and prevents functional cytotoxicity . that said , the exact cellular mechanism of signaling protein localization and re - localization in nk cells remains unknown . one key outcome of activating receptor signaling is mobilization of ions , particularly calcium ( figure 1f ) . initial calcium flux occurs as a result of rapid release from endoplasmic reticulum ( er ) calcium stores mediated by ip3 channels downstream of plc signaling . depletion of er ca stores is sensed first by stim1 on the er surface . following activation , stim1 complexes reposition proximal to the plasma membrane ( visualized as puncta on the er surface ) and activate plasma membrane - associated ora1 . human genetic immunodeficiency has defined critical roles for both orai1 and stim1 in nk cell cytotoxicity . one manifestation of these patients ' disease is profound susceptibility to viral infection , a hallmark of reduced nk cell function , although they also present with many other signs and symptoms and have substantive defects in t - cell and b - cell function . importantly , while orai1-deficient nk cells form conjugates with target cells and polarize the mtoc and lytic granules , they fail to degranulate . although the two ca flux systems are the best characterized , there are other ion channels expressed and utilized by nk cells . recently , a critical role for intracellular mg has been described by a rare primary immune deficiency affecting the mg transporter magt1 . in these patients , intracellular levels of free mg are decreased , accompanied by defective nk cell and ctl killing and subsequent uncontrolled epstein interestingly , the role of mg in both ctl and nk cell control of epstein barr virus is through maintaining the stability of nkg2d at the cell surface , suggesting an unappreciated role for nkg2d in controlling epstein in addition , potassium and zinc channels are expressed and utilized by t cells ( reviewed in feske et al . ) , prompting the question of whether nk cells utilize similar mechanisms . presumably , in and around these steps a lytic cleft forms ( figure 1 g ) . once thought to be simply a gasket to prevent the leakage of lytic granules to surrounding tissues , it is now appreciated that the cleft is dynamic and complex . much of the activity that takes place in the cleft is critical to cytotoxic function , yet remains mysterious because of its inaccessibility . measurement of the lytic cleft by transmission electron microscopy in inhibitory and activating synapses shows that both have a similar range of distances between the nk cell and the apposing target cell membrane ( 1030 nm ) , which are proposed to account for distribution of proteins of different sizes at the synapse . these synaptic membrane features may also be a source of membrane transfer ( trogocytosis ) between an nk cell and a target ( figure 1 g ) , which has been defined for a number of receptors . this process is reciprocal , as transfer of both ligand from the target cell and receptor from the nk cell is observed . additional transmission electron microscopy experiments demonstrate that the target cell - derived receptors enter the nk cells in small , enclosed pits . both inhibitory and activating receptor exchange occurs across the nk cell synapse , although there is controversy as to whether cognate ligand engagement is required . the functional outcome of trogocytosis and intercellular receptor transfer is unclear , although transfer of nkg2d and micb at the synapse results in lower nk cell cytotoxic function . fascinatingly , nk cells that acquire nkg2d ligand from target cells become targets themselves , and are victims of fratricide by neighboring nk cells . this suggests that this ligand transfer may act to both enable and even control nk cell activity during infection , again suggesting the theory of harpooning . following firm adhesion and the formation of a cleft , f - actin continues to undergo substantial remodeling at the synapse . traditionally , this remodeling was thought to result in a large , central clearing of f - actin to enable centrosomal docking and lytic granule secretion . recent advances in imaging technology , namely , the adoption of super - resolution microscopy , have revealed that ligation of activating receptors creates granule - sized conduits through which granules are secreted ( figure 1h ) . interestingly , viral influenza particles can also result in conduit formation , presumably through the ligation of nkp46 , but this signal requires co - stimulation through lfa-1 . other activating receptors , such as nkg2d or cd16 , can signal solely for clearance formation . although not entirely understood , this seems to be an elegant mechanism by which an nk cell could discern a free viral particle from one bound to a target cell . whether proteins on the granules themselves form lytic granule conduits , they are a result of the inherent dynamism at the synapse , or occur in specific hotspots of signaling remains to be determined . it seems likely , based on the presence of actin nucleating factors at the synapse , that f - actin is dynamic and is likely undergoing continual remodeling . this may contribute to conduit kinetics , as has been suggested by analyses of synaptic plane actin dynamics using total internal reflection fluorescence imaging . the process of remodeling and specifically breaking down as opposed to building is of particular interest as conduits in f - actin are a requirement for degranulation . cofilin , which severs and depolymerizes f - actin , works in concert with arp2/3 to remodel actin to enable secretion in muscle cells . coronin 1a , which promotes the turnover of actin through binding to both arp2/3 and cofilin , regulates arp2/3 localization to the t - cell synapse , although a role for this important immune cell regulator in nk cells has yet to be defined . one appealing hypothesis is that hypodensity formation occurs at regions of high activating receptor density as a result of highly localized signaling events , such as is seen in mast cells . however , this is difficult to reconcile with what is likely the dynamic movement of microclusters of activating receptors themselves . an alternative explanation is that lytic granules create their own conduits , enabled by the presence of actin - remodeling proteins on their surface . this would be compatible with the observation that myosin iia is present on lytic granules and appears required for their penetrance through f - actin , but this hypothesis has not been directly tested . perhaps , in preparation for potential serial killing or nk cell recycling , lytic synapse formation also activates transcription factors and gene transcription ( figure 1i ) . ligation of nkp30 results in rapid translocation of nuclear factor b to the nucleus and de novo protein synthesis . perhaps , not surprisingly , perforin and granzyme is refilled in ctl lytic granules while they kill . the newly produced perforin then reaches the synapse independently of conventional lysosomal granules and mediates cytotoxicity . whether nk cells employ such a mechanism remains to be determined , as does the precise program of gene transcription that is activated during cytotoxicity . following the dynamic rearrangement of the actin cytoskeleton , microtubule dynamics result in a dramatic reorientation of the mtoc and associated lytic granules toward the synapse ( figure 1j ) . requirements for centrosome polarization include lfa-1 , pyk2 , erk2 , cip4 , the formin hdia and vav1 . however , it is important to note that f - actin polymerization is required for mtoc polarization ; therefore , any interference with f - actin dynamics will subsequently impair mtoc and granule polarization . one consideration in mtoc polarization is the significant amount of force likely needed to generate this reorientation . it is assumed from studies in other systems that microtubule insertion and anchoring in the cell cortex lead to either pushing ( microtubule growth ) or pulling ( microtubule shrinkage ) forces that can reposition the centrosome . dynein may again have a role , as a minus - ended motor it can generate significant pulling forces on shrinking microtubules when anchored in the cortex , and may contribute to the fine - tuning and positioning of microtubule asters . accordingly , it was recently shown that in t cells , mtoc repositioning occurs as a result of end - on capture shrinkage of microtubule focused at the center of the is and anchored to cortical dynein . interestingly , in nk cells , it appears that kinesin-1 has a role in the initial movement of the mtoc to the synapse , mediated through interactions with the small gtpase arl8b . iq motif containing gtpase - activating protein 1 ( iqgap1 ) may act as a linker between clip-170 on the plus ends of microtubule and specific regions of cortical actin . loss of iqgap1 results in a failure of nk cells to polarize the mtoc and degranulate . cip4 has also been implicated as a link between microtubules and f - actin at the cortex . although in t cells the mtoc docks in contact with the plasma membrane at the synapse , this has not been directly observed in nk cells . as the mtoc polarizes to the synapse , cellular organelles also reposition with some moving toward and others away from the synapse ( figure 1j ) . reorientation of the golgi along with microtubules toward the is presumably aids in directed secretion of granules toward the target cell . in t - helper cells , the mitochondria polarize toward the synapse to maintain ca flux across the plasma membrane for t - cell activation . in nk cells , the mitochondria reposition toward the nk cell is following nk stimulation with anti - nkgd2 antibodies but not with anti - kir2dl1 antibodies , suggesting that the mitochondrial dynamics are triggered as a result of nk cell activation . the polarization of these organelles is important for sufficient ca influx for signaling and granule exocytosis . it is conceivable that polarized mitochondria further serve as local sources of energy to power synaptic function , although this needs to be proven . following mtoc polarization to the is and anchoring at the plasma membrane , the delivery of the polarized lytic granules to the synapse occurs ( figure 1k ) . in t cells , this process requires plus - ended , kinesin-1-dependent movement of lytic granules upon microtubules to the membrane . in nk cells , as mentioned above , myosin iia is associated with lytic granules and required for nk cell cytotoxicity . whether this is through the facilitation of short runs across f - actin following mtoc polarization , the penetration of granules through the actin meshwork or the exocytosis of lytic granules is unclear , although obviously these scenarios are not mutually exclusive . following delivery to the plasma membrane , lytic granules have significant dynamic movement , which is followed by arrest , and subsequent degranulation in only a subset of granules ( figure 1l ) . this movement is not dependent upon f - actin dynamic rearrangement , but a role for microtubule dynamics has not been excluded . granule movement may be undirected rolling on synaptic actin , seeking regions of hypodensity . alternatively , it may represent the requirement for granules to find a primed fusion / tethering complex to mediate their arrest , as is suggested by high - resolution imaging of lytic granules in ctl utilizing munc 13 - 4 rab27a complexes for granule arrest and subsequent degranulation . at some point , lytic granules must traverse the pervasive actin network at the synapse ( figure 1 m ) . lytic granule may mechanistically identify areas of hypodense f - actin regions to pass through , or directed f - actin remodeling at the activating synapse may be required for clearing the way for lytic granules to transit through minimally permissive f - actin hypodensities . however , even in the presence of f - actin clearances , it is likely that lytic granule transit through the cortex requires the generation of force as was suggested by experiments evaluating degranulation in the absence of an f - actin meshwork . myosin iia may mechanically facilitate lytic granule motility through the f - actin - rich area of the is or may be involved in inducing local changes in the f - actin structure to allow polarized lytic granules access to the plasma membrane . after polarizing to the is and before degranulation , lytic granules dock at the is and fuse with the plasma membrane ( figure 1n ) . docking is likely mediated primarily by munc 13 - 4 and rab27a , both of which are recruited to lytic granules following activating receptor ligation . in ctl , lytic granules become fully mature only at late stages of the exocytic pathway , when munc13 - 4 and rab27a associate with perforin- and granzyme - containing lysosomal components . upon interacting with rab27a , munc13 - 4 may also regulate the interaction between vesicle and target soluble nsf attachment protein receptor ( snare ) required for lytic granule fusion with the plasma membrane . alternatively , this may be mediated by the interaction of syntaxin 11 and munc18 - 2 with r - snares ( such as vamp7 ) on the lytic granule membrane . priming then occurs as a result of munc13 - 4-mediated activation of syntaxin 11 or bridging of the plasma membrane with lytic granule . fusion occurs as a result of snare complex formation , and although this complex has not been defined in nk cells , mutations in the snare components syntaxin 11 , vamp4 and vamp7 prevent nk cell degranulation . in mast cells , coordinated oscillations of calcium in concert with n - wasp and pip(4,5)p2 whether such localized signaling dynamics contribute to nk cell exocytosis remains to be seen , although this hypothesis would complement the localized generation of actin hypodensities . in addition to the global calcium reserves such as the er and the store - operated reservoirs , local ca nanodomains created by channels on the lytic granule membrane in t cells are essential for their exocytosis . thus , local synaptic membrane nanoalterations may factor into a final step in allowing the release of lytic granule contents . nk cell lytic granules use at least two distinct modes of fusion : ( 1 ) complete fusion where granule content is completely discharged and the contents diffuse rapidly at the plasma membrane and ( 2 ) incomplete fusion , whereby formation of a transient fusion pore at the plasma membrane is accompanied by release of some but retention of most of the granule contents . this latter form of degranulation is suggestive of alternate forms of granule fusion seen in other secretory cells such as chromaffin cells and neurons . kiss and run ' , in which a granule pore is formed and resealed , preventing full release of granule contents , kiss and stay ' , in which granules remain at the membrane and are re - acidified , presumably for rapid recycling or reuse and crash fusion ' , in which granule contents are released without stable docking and priming . the true existence and significance of these alternate forms of granule fusion in nk cells is incompletely understood , although it is conceivable that it could be a means of facilitating serial killing through the rapid recycling of granule contents . as such , two - way vesicular trafficking occurs at the nk cell synapse , and rapid munc13 - 4-clathrin mediated endocytosis of granule membrane proteins exposed at the plasma membrane is necessary for the recycling of a pool of endocytic vesicles , allowing the serial killing event ( figure 1p ) . f - actin dynamics may have a role in the extrusion of granule contents and the persistence of lytic granules at the membrane following exocytosis . although this is presumably owing to force generation , the direct proof is presently unavailable . after degranulation , a period of relative inactivity begins and is presumably required as lytic granule contents cross the lytic cleft to act upon the target cell ( figures1o and p ) . the sustained presence of fused granules at the synaptic membrane , however , may serve additional functions as well . interestingly , the exposure of lamp-1 on the nk cell surface protects the nk cell from cytotoxicity - induced suicide , although this is likely not the only mechanism . subsequently , uptake of lytic granule contents by the target cell initiates apoptosis and marks the end of the effector stage of cytotoxicity , as the lytic hit has been administered . the termination stage of the synapse refers to the steps that immediately follow the release of lytic granules and up to either the reinitiating of a subsequent recognition or return to a resting state . initially , there is a longer time of apparent relative inactivity . during this time the target cell continues its death process , primarily by apoptosis ( figure 1q ) . phosphatidylserine on the surface of the cell may provide signaling to the nk cell to terminate the response , as nk cells express proteins that have been shown to bind to phosphatidylserine such as the itim - containing molecule cd300a . a further mechanism by which the nk cell could relax its activation status is through receptor downmodulation as has been documented for cd16 and nkg2d . ongoing is new lg biogenesis during this relatively quiet period as a likely requirement for nk cell recharging ' and subsequent serial kills . the final stage of cytotoxicity is defined by detachment from the target cell , a process that is not entirely arbitrary . kinetic studies show that the decision to end stable contacts with target cells is made faster when killing occurrs . interestingly , detachment was sometimes seen along with the formation of nanotubes connecting the nk and target cells ( figure 1r ) . the purpose of these connections in the context of a dying cell , however , is unclear and they may just represent the default behavior of an nk cell to tether to activating objects of interest . insight into the steps of the termination stage can be seen during the process of nk cell serial killing . serial killing can demonstrate burst kinetics , with a delayed first kill followed by more rapid subsequent kills . a proposed mechanism for burst kinetics is the idea of kinetic priming , which suggests that nk target interactions depend upon recent killing events . it is proposed that kinetic priming is a continuity of signaling , as interactions with old targets remain until a new target is found . as such , lytic granule may remain converged to the mtoc following target cell lysis in preparation for subsequent kills , as previously proposed . a consequence of serial killing is a near depletion of lytic granules and cytotoxic effector molecules . this depletion can leave the nk cells in an exhausted state until they detach from the target cell that has depleted their contents . after separation , exposure to locally available activating factors such as interleukin-2 can restore their cytotoxic function . interestingly , in cd8 cytotoxic t cells , it was found that rapid upregulation of nongranule - restricted perforin can be transported to the is and participate in cytotoxicity . as this form of perforin was identified in nk cells , perhaps this observation carries over to nk cells and contributes to the reacquisition of effector function after exhaustion . although seemingly the least is known about the specifics of the termination phase of the synapse , it can be interpreted as one of the most important parts , as recycling of nk cells in vivo in diseased tissue environments is likely to be of the essence in host defense . in recent years , explosive advances in technology have driven us deeper within the nk cell to better understand the mechanism of its critical function . in doing so , we have a much better understanding of the tightly regulated steps leading to cytotoxicity . there are critical and fundamental questions that remain to be answered , some of which require substantive technology development . these include , but are not limited to the following : ( 1 ) the exact number of , and temporospatial requirements for , degranulation events needed to kill particular target cells ; ( 2 ) how both inhibitory signaling and anergy ' restrain progression through the cell biological steps of synaptic formation ; ( 3 ) the mechanism by which conduits in the cell cortex are created to enable lytic granules to reach the nk cell synaptic membrane ; ( 4 ) the true purpose of lytic granule convergence to the mtoc , ( 5 ) the mechanism of delivery of lytic granules from mtoc to plasma membrane ; and ( 6 ) what specifically governs the release of an nk cell from its target cell . in addition , we still have much to learn about the fine - tuning of the cytotoxic response and the subversion of it by viruses and malignant cells . these represent only a sampling of the many questions begotten by the last 15 years of discovery , the answers to which will provide the tools to manipulate and control cytolytic host defense . better understanding of these facets of nk cells will enable more effective harnessing of nk cell functions and therapeutic intervention . appropriate timing of treatment is the key to successful intensive care ; treatment that is delayed could be as detrimental as treatment that is premature . therefore , lange and colleagues , in this issue of critical care , reported on the time course of the expression of the different isoforms of the nitric oxide synthase ( nos ) - that is , neuronal nos [ nnos ] , inducible nos [ inos ] , and endothelial nos [ enos ] ( also known as nos1 , nos2 , and nos3 , respectively ) - of the tissue levels of nitrotyrosine and poly(adp - ribose ) , markers of nitrosative stress , and dna damage resulting from peroxynitrite formation as well as p65 , a mirror of the activation of the nuclear factor - kappa - b ( nf-b ) , in a well - established resuscitated ovine model of septic shock induced by pseudomonas aeruginosa pneumonia . ' downstream effects ' were evaluated by measuring nos activity , nitric oxide ( no ) production , and interleukin-8 concentrations . during the early phase ( that is , 4 to 12 hours after induction of pneumonia ) , enos expression was increased , and this coincided with increased tissue levels of nitrotyrosine , poly(adp - ribose ) , and nf-b activation , whereas in the later phase ( that is , until 24 hours of pneumonia ) , measurable nos activity and no production were related mainly to inos activation . the authors ' ' two - hit ' model of cotton smoke inhalation and subsequent instillation of live bacteria is characterized by a hyperdynamic circulation , hypoten - sion , tissue acidosis , and progressive impairment of gas exchange , lung mechanics , and morphological alterations typical of acute lung injury . furthermore , the model comprises resuscitation measures and thus allows the study of pathophysiological pathways in a clinically relevant , large - animal setting . numerous studies evaluated the no - related mediator orchestra and highlighted the friend - and - foe character of excess no formation : no not only is well established as a reactive nitrogen species ( rns ) , often referred to as a ' final mediator ' of sepsis - induced hypotension , but also acts as a scavenger of reactive oxygen species ( ros ) , such as the superoxide radical . this reaction , however , leads to the formation of the even more toxic peroxynitrite , which ultimately results in protein nitrosylation , dna damage , and activation of poly(adp - ribose ) polymerase ( parp ) . finally , the no production rate depends on the stimulus and the species , and therefore rodent data can not be transferred directly to the clinical scenario . endogenous no production in large animals is much closer to that of human beings , but although increased no production during sepsis is well established , these models yielded controversial results . the early and transient ( within minutes ) activation of parp would make parp inhibition an attractive approach , but unfortunately medical care is usually not available during this very early phase . moreover , the pathophysiological consequences of parp-1 activation are opposed to its vital role in the maintenance of genomic integrity through its function in base excision repair , and the effects of parp inhibition on dna damage and repair during shock are still a matter of debate . therefore , parp inhibitors are currently investigated in ischemia - reperfusion , oncology , and diabetes rather than in sepsis or acute lung injury , and peroxynitrite - neutralizing agents are a tempting alternative . several studies explored the potential of selective inhibition of nos isoforms under the assumptions that nnos and enos are constitutively producing homeostatic no and that inos responds to acute stimuli with excessive no production . in fact , the crucial role of enos expression seems to be unequivocal : enos activation improved microvascular perfusion and cardiac function in rodents , and enos polymorphism was associated with hypotension during human gram - negative sepsis . clearly , inos still seems to be a ' bad guy ' : several studies showed beneficial effects of various selective inos inhibitors on hemodynamics , lung function , deranged microcirculatory perfusion , coagulation disorders , and visceral organ injury [ 12 - 14 ] . recent data , however , suggest that nnos activation may also assume major importance , and a combined approach using selective nnos inhibition during the early phase ( 0 to 12 hours ) and inos inhibition during the later phase ( 12 to 24 hours ) yielded improved pulmonary function and attenuated nitrosative stress . finally , selective inos inhibition together with the ros scavenger tempol also afforded significant protection , further emphasizing the close interaction of no and oxidative / nitrosative stress . despite these encouraging results , nothing is simple or easy : in resuscitated murine septic shock , both genetic deletion and selective pharmacologic blockade of the inos were associated with markedly improved systolic contraction and catecholamine responsiveness but simultaneously deteriorated diastolic relaxation unfortunately , the authors did not report data on oxidative stress , so a complete overview of the whole rns - and ros - related mediator orchestra is only implicitly provided . nevertheless , the authors add an important piece to the complex puzzle of the no - related pathophysiological pathways : nitrosative stress ( that is , increased nitrotyrosine and poly[adp - ribose ] levels ) was aggravated only during the early phase up to 12 hours , whereas clear - cut increases in nos activity , no metabolites , and subsequently cytokines occurred only later on . interestingly , in this experiment , in contrast to previous reports from the same group , nnos synthesis was not increased . finally , despite its inherent protective properties , enos activation was also involved in the initiation of the septic response , and it remains to be elucidated whether this mirrors an adaptive or pathologic mechanism . how can we translate these data to daily care ? usually , there are few chances to intervene during the very early , evolving phase of sepsis and acute lung injury , and at the time of full - blown sepsis , all members of the mediator orchestra are already playing their ( un)coordinated , and unfortunately sometimes uncontrollable , concert . there are two ways to go from here , and both are worthy of being followed ! first , though extremely time- and resource - consuming , long - or longer - term large - animal models with a more prolonged observation period ( that is , days ) are needed and will probably yield valuable clues to the design of clinical studies . second , more studies on humans are warranted in order to better describe early and later phases of human sepsis in terms of nos , peroxynitrite formation , and parp activation . enos : endothelial nitric oxide synthase ; inos : inducible nitric oxide synthase ; nf-b : nuclear factor - kappa - b ; nnos : neuronal nitric oxide synthase ; no : nitric oxide ; nos : nitric oxide synthase ; parp : poly(adp - ribose ) polymerase ; rns : reactive nitrogen species ; ros : reactive oxygen species .

natural killer ( nk ) cell - mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps , which give rise to an extensive array of cellular checkpoints in accessing nk cell - mediated cytolytic defense . appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death . here we highlight recent discoveries in the formation of the nk cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process . appropriate timing of treatment assumes particular importance in critical care . lange and colleagues recently reported on the time course of the different nitric oxide synthase ( nos ) isoforms , nitrosative stress , and poly(adp - ribosylation ) during pseudomonas aeruginosa pneumonia - induced ovine septic shock . initially , endothelial nos expression was increased together with markers of peroxynitrite formation , dna damage , and nuclear factor - kappa - b activation . later on , measurable nos activity and nitric oxide production resulted mainly from inducible nos activation . these results emphasize the need for long - term , large - animal studies investigated over days so that future therapeutic interventions can be better tailored and matched to the exact time course of the activation of the mediator orchestra .

steroid hormones have been traditionally associated with regulation of peripheral organs , associated with stress ( corticosterone ) or with gonadal function ( estrogen and androgens ) . over the years , it became evident that these hormones also act within the hypothalamus , in a feedback regulatory loop , to affect the release of the neural factors that modulate production of the steroid hormones . more recently , several observations have elucidated new roles of steroid hormones in modulating higher cns functions . specifically , both stress and steroid hormones have been shown to affect synaptic receptors and ion channels and therefore regulate in several different ways synaptic transmission and neuronal plasticity . consequently , stress hormones have been implicated in processes ranging from homeostatic to cognitive functions . furthermore , in some disorders of the nervous system , hormones have been shown to play critical roles : favoring or halting the disease process . thus , the interaction between peripheral hormones and central networks seem to be more intense than ever imagined before . in the present study we review current knowledge on the effects of steroid hormones on synaptic plasticity and define their influence on hippocampal cognitive and emotional functions . following the exposure to stressful stimuli , the steroid hormone corticosterone ( cortisol in humans ) is released from the adrenal glands in order to set up the best response to the challenge by acting on steroid receptors ( de kloet et al . , 2005 ) . these are distributed throughout the body and have a particularly dense distribution in the cns ( de kloet et al . , 2005 ) . in the brain , the cellular and molecular targets for the action of corticosterone include , in addition to basic metabolic processes , an effect on excitatory ( karst and joels , 2005 ) and inhibitory ( maggio and segal , 2009a ) synaptic transmission , as well as an effect on voltage - gated calcium channels ( vgcc ; karst et al . , 2000 ; these effects are mediated by the activation of mineralocorticoid receptors ( mrs ) and glucocorticoid receptors ( grs ; joels , 1999 , 2008 ; de kloet et al . , 2005 ) . initially , it was suggested that both receptors act as nuclear transcription factors that modify protein synthesis and produce a slow , persistent change in the function of the cell ( de kloet et al . more recently , the existence of a new family of membrane - bound mr and gr ( mmr and mgr , respectively ) , which act through novel non - genomic pathways , has been reported ( karst et al . , 2005 ; de kloet et al . , 2008 ) . in this route , mmr and mgr can rapidly affect ionic conductances and thereby modify cell excitability and function ( karst et al . these membrane - bound receptors appear to differ from their intracellular cognates , not only in their location on the cell membrane , but also in their molecular structures ( joels et al . , 2008 ) , in their affinities for corticosterone , and in their downstream mechanisms of action which involve activation of g proteins ( joels et al . , 2008 ) . specifically , intracellular mr ( imr ) have a very high affinity for corticosterone and are highly expressed in all hippocampal subfields , as well as in cells of the central amygdala , lateral septum , and some motor nuclei in the brainstem ( joels , 2006 ) . intracellular gr ( igr ) have a relatively low affinity , are widely distributed throughout the brain , and are expressed both in neurons and in glia ( joels , 2006 ) . consequently , it has been proposed that imr hardly participate , if at all , in the fast response to stressful stimuli , due to their characteristic of being already saturated by the low ambient levels of corticosterone at rest ( joels , 2006 , 2008 ) . conversely , igr have been reported to become gradually activated by rising levels of corticosterone following a stressful event ( joels , 2006 , 2008 ; figure 1a ) . therefore , under physiological conditions , cells that coexpress both receptor types , such as principal cells in the ca1 region , the dentate gyrus ( dg ) , and the central amygdala , will shift between predominant imr activation and concurrent mmr and igr activation ( joels and krugers , 2007 ) . ( a ) time course of mr and gr activation following stressful stimuli . at a resting level , imr are already saturated by the baseline levels of corticosterone . rising concentration of corticosterone activates both mmr and igr , whereas an additional increase in corticosterone levels also activates mgr . mmr- and mgr - mediated effects appear in a faster time course than those mediated by the intracellular receptors . modified from maggio and segal ( 2010 ) . ( b ) proposed mechanism by which corticosteroid receptors differently regulate ltp and ltd in the hippocampus . imr are believed to be fully occupied at baseline level of corticosterone , therefore they might play a marginal role in synaptic plasticity . mmr might play a fundamental role in synaptic plasticity especially in vh : mmr activation reduces ipsc frequency . this determines an increase in the excitability of the pyramidal cells and raises the possibility of vgcc activation , thus enhancing ltp . in addition , a decrease in gabaergic inhibition can impair ltd through a group i mglur - mediated mechanism . igr are thought to both decrease nmda - mediated ltp and increase vgccs mediated ltp both in the hippocampus and amygdale . their effects may occur at longer time scale due to their lower affinity to corticosterone . mgr might be involved in the regulation of synaptic plasticity mainly in dh : mgr activation increases ipsc amplitude and following hyperpolarization of the pyramidal cell membrane and inactivation of nmda receptors , might impair ltp and enhance ltd . modified from maggio and segal ( 2010 ) . the identification of the molecular cascades linked to the effects of corticosteroids in the brain resulted in a series of studies examining the role of corticosterone in neuronal plasticity as well as in the cellular mechanisms underlying learning and memory such as long - term potentiation ( ltp ) and long - term depression ( ltd ; bliss and collingridge , 1993 ) . initial studies indicated that induction of ltp in the hippocampal area ca1 is impaired in a rat exposed to behavioral stress , such as inescapable shock ( foy et al . administration of high doses of corticosterone either in vivo ( diamond et al . , 1992 ) or in vitro ( pavlides et al . , 1996 ; alfarez et al . , 2002 ) produced the same effects , indicating that corticosterone is likely to mediate this action of stress . specifically , corticosterone - induced impairment of ltp seems to be due to the activation of igr , which depresses nmda receptor - dependent ltp ( krugers et al . conversely , it was also shown that ltp could be enhanced in the presence of low to moderate concentrations of corticosterone , while in absence of corticosterone ltp induction was impaired ( diamond et al . , 1992 ) . these studies show that the effects of corticosteroids on ltp induction are dose - dependent and follow an inverted u - shaped relationship ( diamond et al . , 1992 ; joels , 2006 ) . further studies , however , have presented a more complex picture of the effects of steroids on synaptic plasticity . specifically , it seems that the same dosage of corticosterone that impairs nmda - dependent ltp can indeed enhance vgcc - dependent ltp ( krugers et al . , 2005 ) . this species of ltp is found in the amygdale where it is believed to underlie the formation of fear memories ( blair et al . , 2001 ; bauer et al . , 2002 ) and can be evoked in the hippocampus as well ( borroni et al . , 2000 ; interestingly , in the hippocampus , corticosterone appears to enhance vgcc ltp through an igr - dependent mechanism ( krugers et al . , 2005 ) . it has been proposed that this effect requires a genomic pathway , as it occurs after a long delay between the exposure to stress and/or corticosterone and the recordings ( krugers et al . , 2005 ) , thus probably depending on the binding of gr homodimers to dna that causes an increase in calcium currents ( karst and joels , 2005 ; chameau et al . , 2007 ) . recent data from our group have shown that mrs are also able to enhance vgcc ltp ( maggio and segal , 2007b ) : either stress or physiological concentrations of corticosterone can enhance ltp in the ventral hippocampus ( vh ) , while inhibiting it in the dorsal hippocampus ( dh ; maggio and segal , 2007b ) . in particular , corticosterone enhances ltp through mrs since a selective mr agonist , aldosterone , shares the same effect in the vh ( maggio and segal , 2007b ) . the proposed mechanism excludes an interaction between mr and nmda receptors , as aldosterone by itself does not increase nmda - dependent synaptic potentials ( maggio and segal , 2007b ) . conversely , mr - induced ltp can be blocked by nifedipine , suggesting that vgccs are likely responsible for this effect ( maggio and segal , 2007b ; figure 1b ) . it is likely that mr activates vgcc by modulating ionic conductances or changing vgcc activation kinetics . in vivo experiments have shown that mr activation is able to increase ltp in the dh as well ( avital et al . , 2006 ) . specifically , animals which were injected with a gr antagonist prior to the stressful exposure , such that only mr could be activated by stress , show a much larger ltp than controls . in contrast , those animals previously injected with an mr antagonist and then exposed to stress , allowing only gr activation , show a much lower ltp than controls ( avital et al . , 2006 ) . these recordings were performed in the dg and even though there could be differences in the effects of stress and steroids between the dg and ca1 ( joels and krugers , 2007 ) , mrs were still shown to mediate an enhancement of ltp . it could be argued that the experiments in the vh were conducted using an in vitro preparation where ambient corticosterone maintained normally through the circulation is washed out . consequently , mrs are not occupied , and are ready to be activated by the superfused drug and produce ltp enhancement in the vh . this might not reflect the situation in the intact animal , where the brain is constantly exposed to fluctuating concentrations of corticosterone . in fact , mr should be already saturated by the resting concentration of corticosterone and should not respond to the stress - induced rise of corticosterone in the presence of a gr blockade . this , however , does not seem to be the case ( avital et al . , 2006 ) . furthermore , even though both mr and gr are expressed in the vh , corticosterone action is mediated by activation of mr rather than gr . this reflects the observation that in the vh , mr concentration is double that of gr ( robertson et al . , 2005 ) . if so , according to the u - shaped curve model of corticosterone effects , mr should be saturated rapidly by the rising concentration of corticosterone and their effect should fade away faster in favor of the slower gr activation . altogether , it seems that the simple , dose - dependent , inverted , u - shaped curve does not fully explain the modulatory functions of mr and gr on ltp in the different sectors of the hippocampus , therefore calling for the involvement of other factors . a possible mechanism that may clarify the mr - dependent enhancement of ltp should take into consideration the activation of mmr . these receptors act through a faster mechanism ( de kloet et al . , 2008 ) and have lower affinities for corticosterone compared to their intracellular cognates ( joels , 2008 ) and similar to that of the igr ( joels , 2008 ) . in addition , mr activation enhances ltp in the vh within 1 h , too short time window to be accounted for by activation of genomic mechanisms ( joels and krugers , 2007 ; joels , 2008 ) , but compatible with the faster time course of the non - genomic routes . thus , mmr could be the preferential target for rising concentrations of corticosterone in the vh if one takes into account the similar affinities for corticosterone between mmr and igr , and the denser distribution of the former over the latter ( robertson et al . this could most likely be due to the shorter time window of observation in our experiments compared to those done by others ( krugers et al . , both mr and gr were reported to increase vgcc ltp ( krugers et al . , 2005 ; maggio and segal , 2007b ) . this apparent contrast could probably be explained by considering the different time courses of mr and gr enhancement of vgcc ltp . specifically , mr has an earlier effect than gr and it could be that in the vh stress mediates a fast enhancement of ltp by mr followed by a second , slow increase in ltp due to gr activation . this proposal is compatible with the proposed role of the vh as a key player in the pathway that conveys stressful information to the hypothalamus and the amygdale so as to organize the stress response ( moser and moser , 1998 ; maggio and segal , 2010 ; segal et al . , 2010 ) . following stress , the quick mr - mediated increase in ltp facilitates the flow of the information related to stress from the vh to the ventral hypothalamus and other lower brain centers , so that the autonomic response to stress can be organized . later on , the mr - mediated response fades away and the effect of gr dominates . as previously mentioned , gr enhancement of vgcc ltp has been shown to have a role in the formation of fear memories in the amygdale ( blair et al . , 2001 ; bauer et al . , , gr could play the same function in the vh : the formation of the memory for the stressful event at the vh indeed , the evidence that mr and gr act on the same mechanism can have different purposes due to the time window of the respective outcomes that take place . considering this , it could be interesting to study the relationship between the mr and gr responses in the vh . in the dh , the reduction of ltp is mediated by gr ( maggio and segal , 2007b ) . this effect seems to occur in less than 1 h , a relatively quick response that is unlikely to be mediated by a genomic mechanism . gr could reduce nmda - mediated ltp either by a direct or an indirect mechanism . as far as it concerns the indirect mechanism hypothesis , we have demonstrated that a gr agonist , dexamethasone , increases ipscs and mipscs amplitude in the dh within 10 min ( maggio and segal , 2009a , 2012 ) , consistent with the possible activation of mgr . therefore , the increase in gabaa conductance could hyperpolarize the membrane , thus preventing the cell from reaching the threshold of depolarization that unlocks nmda receptors from the mg block ( figure 1b ) . all in all , our experiments indicate that gr affect ltp through a fast , probably non - genomic mechanism . even though this hypothesis needs to be explored further , the fast suppression of ltp in the dh can underlie the switch in the weight between the dh and vh ; by reducing dh ltp and simultaneously enhancing ltp in the vh , the stressful stimuli could temporarily suppress the cognitive route of the hippocampus to cortical structures and enable the transmission of the emotional information through the vh to the amygdala . conversely , ltd induction is facilitated by behavioral stress , through a mechanism that requires gr ( pavlides et al . we replicated previous experiments where both stress and corticosterone facilitate ltd through a gr - dependent mechanism in the dh , but we have also shown that ltd is impaired in the vh through a mr - dependent mechanism ( maggio and segal , 2009b ) . specifically in the latter case , ltd is transformed into a slow - onset ltp following the exposure to stressful stimulation ( maggio and segal , 2009b ) . as is the case for ltp , changes in ltd either in the dh or vh were observed at approximately 1 h after the exposure to the stress , a time window that could be compatible with non - genomic mechanisms . the mr - induced conversion of ltd to ltp in the vh could be due to the activation of vgcc , which will further facilitate the ventral route to the amygdale ( figure 1b ) . group i mglur have been shown to enhance ltd in ca1 ( fitzjohn et al.,2001 ; rammes et al . , 2003 ) , but , interestingly , they have been reported to induce a slow - onset potentiation in the dg ( manahan - vaughan and reymann , 1996 ) . in a previous study , we showed that , in the vh , application of dhpg , a group i mglur agonist , increases the population spike amplitude in response to a baseline stimulation ( maggio and segal , 2007a ) . taken together , these observations suggest that in the vh , a decrease in gabaergic inhibition can shift ltd to a slow - onset ltp through a group i mglur - mediated mechanism ( figure 1b ) . in conclusion , corticosteroid regulation of synaptic plasticity in the hippocampus is affected by several factors . an inverted u - shape effect of corticosterone partially explains the observed modulation of ltp . indeed , this hypothesis mainly refers to the activation of intracellular corticosteroid receptors and does not take into account the contribution of membrane - bound steroid receptors . in fact , mmr , which bears a similar corticosterone affinity to that of igr , will be activated at similar steroid concentrations . this implies that the effect of mmr appears earlier than that of igr , thus inducing an enhancement of ltp instead of ltd an additional factor to be considered is the distribution of mr and gr in specific brain areas , and the ratio of membrane - bound to intracellular receptors expressed therein . this is because at the same affinity value for corticosterone concentration , the receptor that is highly expressed will lead the effects on synaptic plasticity . mrs , for example , exist in different molecular configurations ( joels , 2008 ) , thus these receptors can be very diverse . this diversity in molecular structure could be linked to diverse intracellular pathways that differently influence neuronal functions . another issue that has to be considered is the clusters of brain areas that are involved in a particular stress situation . various brain regions have specific properties and are incorporated into unique networks , so that even if corticosterone evokes the same effect at the single cell level , this would not always result in the same effect on network functions such as ltp . for instance , both ca1 pyramidal neurons and granule cells in the dg highly express mr as well as gr ( joels , 2008 ) . in the dh , corticosterone and stress consistently suppress the induction of ca1 ltp in vivo and in vitro , unlike the case for the dg . tail shocks ( shors and dryver , 1994 ) can indeed suppress ltp ; however , in other situations , either no effect ( bramham et al . , 1998 ; gerges et al . , 2001 ; alfarez et al . , 2003 ) or enhancement of ltp has been reported ( kavushansky et al . , 2006 ) . this is because ltp in the dg seems to be more dependent on indirect inputs from the amygdale ( akirav and richter - levin , 2002 ; kavushansky and richter - levin , 2006 ) . finally , the response to a stressor is also determined by the history of the organism . for instance , the induction of ltp is impaired in animals that have been exposed to repetitive stress in the weeks prior to the experiment , even if corticosterone levels , at the time of ltp induction , are compatible with the expression of a normal ltp ( alfarez et al . , 2003 ) . studies on the effect of maternal care on synaptic plasticity report that animals that received very little maternal care have poor ltp when they are adult , as opposed to animals that received high maternal care ( champagne et al . , 2008 ) . interestingly , while ltp is suppressed by corticosterone in the latter group , it is enhanced in the former ( champagne et al . , 2008 ) . all in all , corticosteroid modulation of synaptic plasticity in the hippocampus seems to be more complex than previously thought and additional experiments are needed to address the role of membrane - bound as well as intracellular receptors on ltp / ltd regulation . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . primary amenorrhea is the absence of menstruation and secondary sexual characteristics in phenotypic women aged 14 years or older . common hormonal cause of primary amenorrhea includes pituitary dysfunction , chronic systemic disease , and absent ovarian function . gonadal failure in genetically xx individuals is ovarian failure ; when this occurs at any time before onset of sexual maturation , there will be primary amenorrhea and incomplete breast development . xy individuals with gonadal failure will have female genitalia because mullerian inhibiting factor and testosterone will not be produced . gonadal tumors occur in up to 25% of women with a y chromosome ; unlike complete androgen insensitivity , these gonads do not secrete hormones and should be removed at the time of diagnosis . the incidence of the chromosomal abnormality ( ca ) in live births is around 90 per 10,000 . included in the incidence are the numerical ( monosomy / trisomy / mosaicism ) as well as the structural ( translocation / isochromosome / deletion / duplication / ring ) ca . 233 women with primary amenorrhea who were referred to cytogenetic unit , children hospital , mansoura university , egypt from july 2008 to december 2010 . for all patients , steroidal hormonal assay were setup according to elisa technique,[810 ] and chromosomal cultures were setup according to g- banding , about 1 ml of blood was mixed with 5 ml of rpmi medium , 1 ml of fetal bovine serum , 0.1 g / ml of phytohemagglutinin ( pha ) and was incubated at 37c . after 72 hour of incubation , the colcimid ( 1 mg / ml ) was added and incubated for another 1.5 hour . the cells were then harvested by hypotonic treatment ( 1.5 hour with 0.075 m kcl at 37c ) , fixed and washed thrice with fixative solution ( methanol and acetic acid in a ratio of 3:1 ) , and then metaphases were spread and stained using standard g - banding technique . for each case , 50 spread metaphases were analyzed with cytovision system . fluorescence in situ hybridization ( fish ) technique was carried out in some of cases to more evaluation according to technique of . 10 l of the x , y centromeric probe were applied to the target area , and the cover slip was applied to the slide immediately . the cover slip was sealed with rubber cement , and the slide was placed on hot plate at 72c for 2 minutes . the slide was placed in a warmed humidified box , and the box was placed at 37c in an incubator overnight . the cover slip was removed from slide , and it was immersed in 70 ml of the 0.4 x ssc/0.3% np-40 in a coplin jar at a 731c water bath for 2 minutes . then , the slide was immersed in 70 ml of the 2 x ssc/0.1% np-40 in coplin jar at 25c for 2 minutes . the slide was air - dried in the dark , and 10 l of dapi ii counter stain were applied to the target area of slide , and the cover slip was applied to the each slide . 233 patients with primary amenorrhea were referred from different parts of egypt to cytogenetic laboratory of genetic unit , children hospital mansoura university , from july 2008 to december 2010 . ( 1 ) the numerical abnormalities of the x chromosome were detected in 23 ( 50% ) . ( 2 ) structural abnormalities of the x chromosome were detected in 11 ( 23.91% ) . ( 4 ) male karyotype 46 , xy was presented in 2 ( 4.35% ) , [ table 1 ] . the two patients with association of chromosome y was confirmed by fish [ figure 1 ] , and the presence of deletion of long arm of chromosome x in 3 patients was confirmed , using fish [ figure 2 ] . classification of chromosomal abnormalities of patients with primary amenorrhea interphase and metaphase from fish analysis showing one x chromosome and one y chromosome of female confirming the testicular feminization syndrome ( xy ) metaphase from fish analysis showing one x chromosome and short arm of another x of female confirming the form x , xq in table 2 , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) mean levels in sera of females with primary amenorrhea , whether they have numerical , structural , or mosaicism chromosomal abnormalities , were significantly higher than those of the control group ( p<0.05 ) . but , estradiol ( e2 ) mean levels in sera of them were significantly lower than those of the control group ( p<0.05 ) . 20.63% of chromosomal abnormalities in primary amenorrhea of present study compared with those in various study conducted between 1983 and 2010 [ table 3 ] . chromosome abnormalities in primary amenorrhea in various study and compared with present study genes essential for gonadal function are located on the proximal part of xp , the long arm of x proximal to xq13 and/or the long arm of x distal to xq26 . large deletion of xq with breakpoints at or proximal to q13 are expected to produce gonadal dysgenesis with primary amenorrhea , half of the patients with such deletions have turner`s syndrome . that explained the cause of our two patients with deletion of xq have primary amenorrhea , but have not feature of turner syndrome , whereas other three patients who have deletion of xp have primary amenorrhea and all the features of turner syndrome . ten et al . found one patient with primary amenorrhea and 47 , xxx had mental retardation and the cause of that , an extra x , can slow down embryonic cell development in a special way . similar to that , there were mental retardation in two of our patients with karyotype of 47 , xxx . xy individuals with gonadal failure will have female genitalia because mullerian inhibiting factor and testosterone will not be produced . gonadal tumors occur in up to 25% of women with a y chromosome ; unlike complete androgen insensitivity , these gonads do not secrete hormones and should be removed at the time of diagnosis . in our study , we diagnosed two patients of primary amenorrhea as testicular feminization syndrome ( xy ) using fish technique . the molecular cytogenetic technique , fish , accurately delineate the nature and origin of marker chromosomes in primary amenorrhea patients , which is difficult by conventional cytogenetics . as similar to that , in present study , the presence of deletion of long arm of chromosome x in 2 patients was confirmed , using fish , which is very difficult by conventional cytogenetic where short arm of the x chromosome is similar to the y chromosome . ten et al . found that , serum follicle stimulating hormones and luteinizing hormones were always increased in primary amenorrhea patients with chromosomal abnormalities . the present study showed that karyotype and fish are necessary to detect the causes of primary amenorrhea . this study also revealed the incidence of chromosomal abnormalities in women with primary amenorrhea in egypt is similar to that reported in previous literatures .

several new observations have shifted the view of the hippocampus from a structure in charge of cognitive processes to a brain area that participates in the formation of emotional memories , in addition to its role in cognition . specifically , while the dorsal hippocampus is involved in the processing of cognitive memories ; the ventral sector is mainly associated with the control of behavioral inhibition , stress , and emotional memory . stress is likely to cause this switch in control of hippocampal functions by modulating synaptic plasticity in the dorsal and ventral sectors of the hippocampus through the differential activation of mineralocorticosteroid or glucocorticosteroid receptors . herein , we will review the effects of stress hormones on synaptic plasticity in the hippocampus and outline the outcomes on stress - related global functions of this structure . we propose that steroid hormones act as molecular switches : by changing the strength of synaptic connectivity in the hippocampus following stress , they regulate the routes by which the hippocampus is functionally linked to the rest of the brain . this hypothesis has profound implications for the pathophysiology of psychiatric disorders . background : primary amenorrhea is defined as the absence of menstruation and secondary sexual characteristics in phenotypic women aged 14 years or older . hormonal disorders are main causes of primary amenorrhea . common hormonal cause of primary amenorrhea includes pituitary dysfunction and absent ovarian function . the aim of this study was to estimate the incidence and types of chromosomal abnormalities in patients with primary amenorrhea in egypt.materials and methods : chromosomal analysis and hormonal assay were carried out on 223 patients with primary amenorrhea that were referred from different parts of egypt to cytogenetic laboratory of genetic unit , children hospital mansoura university , from july 2008 to december 2010 . fish technique was carried out in some of cases to more evaluation.results:the frequency of chromosomal abnormalities was 46 ( 20.63% ) in primary amenorrhea patients . the chromosomal abnormalities can be classified into four main types . ( 1 ) the numerical abnormalities of the x chromosome were detected in 23 ( 50 % ) . ( 2 ) structural abnormalities of the x chromosome were detected in 11 ( 23.91% ) . ( 3 ) mosaicism of x chromosome was found in 10 ( 21.74% ) . ( 4 ) male karyotype 46 , xy was presented in 2 ( 4.35%).conclusion : the present study showed that karyotype and fish are necessary to detect the causes of primary amenorrhea . this study also revealed the incidence of chromosomal abnormalities in women with primary amenorrhea in egypt is similar to that reported in previous literatures .

we report the case of a 13yearold boy with neuromuscular proximal thoracic scoliosis who was found to have significant torsion of the left main bronchus during general anesthesia for posterior instrumented spinal fusion of t2 to l4 . this patient had a background history of multicore myopathy , which resulted in progressive scoliosis . endotracheal intubation was noted to be difficult on review of a previous anesthetic chart ( grade iii on the cormack and lehane scale ) . this patient reported increasing shortness of breath over the preceding few months , particularly on exertion . as part of his preoperative investigations , spirometry was performed , which showed that the forced vital capacity ( fvc ) was 36% of predicted ( 1.09 l ) and the forced expiratory volume in 1 sec ( fev1 ) to fvc ratio was 101% , which was in keeping with a restrictive lung defect . he underwent a level1 sleep study preoperatively which showed normal gas exchange with no significant desaturations or hypercapnoeic episodes . anesthesia was induced using a targetcontrolled infusion ( tci ) technique with propofol and remifentanil . the patient 's trachea was intubated using the airtraq videolaryngoscope ( prodol meditec s.a . , vizcaya , spain ) with the aid of a bougie . this resulted in rapid desaturation , a significant decrease in the end tidal carbon dioxide values and hypotension . there was a precipitous decrease in the expired tidal volume ( vte ) from 400 to 100 ml . a total of 100% oxygen , small aliquots of phenylephrine , and intravenous fluids was administered . the attending respiratory physician was called in for advice and flexible bronchoscopy was performed , which revealed a slitlike left main bronchus . following a multidisciplinary discussion involving the spinal surgeon , the anesthetic team and the respiratory physician , the decision was made to apply halo traction through the spine in order to reduce the magnitude of the scoliosis and , therefore , the extrinsic compression on the bronchus . this was achieved using gardner wells tongs to the skull and bilateral lower limb skin traction with 4 kg weight to the tongs and each lower limb . improved lung compliance and tidal volumes were achieved following application of traction , and the patient was able to tolerate the prone position without further ventilatory complication . neuromuscular scoliosis is caused by a heterogeneous group of conditions , in which the underlying pathology will have significant impact on the patient 's perioperative course 1 . he was noted to have restrictive lung disease on spirometry , which is well known to be the most common lung defect in young patients with progressive scoliosis 2 . extensive pulmonary testing including measurement of flowvolume loops and lung volumes were not carried out preoperatively . boyer et al . 3 in their review of lung function tests of 44 children with idiopathic scoliosis found that while only 7% met the criteria for airflow obstruction using standard spirometric techniques , 46% were found to have moderatetosevere gas trapping using by plethysmography and helium dilution methods . 2 demonstrated in a small case series of 18 pediatric patients with syndromic and congenital scoliosis that 33% of these patients had obstructive lung disease ( old ) as evidenced by pulmonary function tests . all patients with old demonstrated compression of the bronchial main stems as confirmed by preoperative flexible bronchoscopy and computed tomography ( ct ) . our patient had compression of his left main bronchus , which was associated with his primary leftsided thoracic curvature ( fig . it is also worth noting that this patient 's main curve was relatively proximal compared to the majority of patients with a main thoracic curve ; the apex was located at t6 compared to the usual t89 . furthermore , as the spirometric evidence of old in the above case series was supported by radiological imaging which showed airway compression , it is likely that we will consider performing preoperative thoracic ct imaging with threedimensional reconstruction on all our highrisk patients undergoing this type of surgery , particularly patients with significant or worsening respiratory symptoms . spinal radiograph taken preoperatively demonstrating a 79 left convex proximal thoracic scoliosis from t2t10 , apex at t6 , with compensatory thoracolumbar curve . we postulate that the degree of bronchial compression worsened significantly in prone position , resulting in gas trapping , high peak airway pressures , and the consequent circulatory disturbances . describe a similar case of a patient with thoracolumbar scoliosis whose ventilation deteriorated rapidly when moved into prone position . urgent flexible bronchoscopy performed in prone position revealed complete collapse of the trachea proximal to the carina . the principle anesthetist addressed the problem by inserting the endotracheal tube further , beyond the collapsed trachea , thus stenting the trachea open . this would not have possible in the case of our patient as the obstruction was at the level of the left main bronchus 4 . the application of modified halofemoral traction to this patient resulted in an immediate improvement in the ventilatory parameters , and the patient was moved to the prone position successfully . although studies have shown an improvement in pulmonary function with perioperative application of halogravity traction 5 , to the best of our knowledge , this is the first reported case of using halofemoral traction to negate the pressure effects of prone position in the context of bronchial torsion . portal hypertension , always accompanies by cirrhosis , can lead to lots of complications including varices hemorrhage , ascites , hypersplenism and hepatic encephalopathy . the risk of varices bleeding in patients with portal hypertension was approximately 30% over 2 years . nowadays , the treatment for patients with varices hemorrhage mainly depends on medical treatment , endoscopic therapy , and surgical shunting procedures . transjugular intrahepatic portosystemic shunts ( tipss ) are currently widely used to prevent or control variceal rebleeding for it could serve as a bridge to hepatic transplantation or fit for patients who are in need of portal decompression . tips was usually regarded as a first - line therapy and cost - effective solution to portal hypertension concomitant with variceal hemorrhage in recent years . therefore , tips has generally replaced surgical shunts , which require the involvement of surgeons and general anesthesia as the first treatment for patients with portal hypertension . besides advantages of cost - effective and less invasive , tips , however , was always accompanied by complications mainly including stenosis / occlusion , recurrent varices hemorrhage , and hepatic encephalopathy . its effectiveness beyond surgical shunting in bridging to hepatic transplantation , to some extent , was arguable . in addition , with issues of postoperative shunt stenosis , thrombosis and migration , almost half of the patients undergoing tips experienced shunt failure within 1 year of tips placement . however , surgical shunting that mostly refers to distal splenorenal shunts ( dsrss ) , portacaval shunts ( pcss ) or h - graft pcss ( hgpcss ) has generally disappeared for the widespread application of tips . recent article reported that patients receiving dsrs would have significantly lower rebleeding and encephalopathy rates than tips in the management of refractory variceal bleeding . also , pcs was reported significantly superior to endoscopic therapy in a longer survival rate , less morbidity and lower direct and indirect costs . with more randomized controlled trials ( rcts ) comparing tips with surgical shunting undertaken , the optimal management for patients with portal hypertension can be tested . there was still no all - round systemic evaluation comparing tips with surgical shunts for patients with portal hypertension . specifically , we focused on comparing postoperative complications ( including recurrent variceal hemorrhage , shunt stenosis , and encephalopathy ) , mortality and survival rate after surgery in patients undergoing tips versus surgical shunting . therefore , we performed a meta - analysis of all rcts concerning tips and surgical shunting ( including dsrs , pcs , and hgpcs ) to evaluate the optimal management for patients with portal hypertension . the aims of this study were to give more information concerning tips and surgical shunting with our proposed approach in evaluating the morbidity , mortality , and long - term survival . all databases including cbm , cnki , wfpd , pubmed , medline , embase and cochrane were searched using the mesh terms : hypertension , portal / complications , liver cirrhosis / complications , portosystemic shunt , transjugular intrahepatic / mortality , portosystemic shunt , surgical , esophageal and gastric varices / surgery , hypertension , portal / surgery independently or in combination for clinical trials comparing tips with surgical shunts . all literatures were screened to identify all suitable published rcts for eligibility until february 2014 . all eligible rcts were screened for their methods , characteristics and risk of bias strictly . in this meta - analysis , two different kinds of groups were generated from the included papers that met the criterion : the tips group and the surgical shunting group . the surgical shunting group referred to traditional portosystemic shunts that contained dsrs , hgpcs , and pcs . in sub - group analysis , hgpcs and pcs could be recognized as one group for their similar modus operandi and decompression principle . in our research , 246 patients underwent tips and 247 patients underwent surgical shunts . all included rcts were required to contain a direct comparison between portosystemic shunts ( including dsrs , hgpcs , and pcs ) and tips in a controlled manner . each trial was required to include postoperative complications , mortality and long - term survival rate in detail . primary outcomes to be assessed were mortality , 2- and 5-year survival and postoperative complications including recurrent variceal hemorrhage , shunt stenosis , and hepatic encephalopathy . secondary outcomes to be assessed were length of hospital stay , operating time and hospitalization charges . each of these papers was assessed using a standardized evaluation form by two investigators ( author # 1 , author # 5 ) independently for extraction in this meta - analysis . when consensus could nt be reached , discussion and joint collection of the paper could resolve . otherwise , a third reviewer ( author # 2 ) would take part in the discussion and determine the definite inclusion of papers as a referee . general information collected from abstracts , included the authors , publication year , characteristics of patients and outcomes . all papers were assessed for the risk of bias by two investigators ( author # 1 , author # 5 ) on the following areas : random sequence generation , allocation concealment , blinding of participants and personnel , blinding of outcome assessment , incomplete outcome data , selective reporting , other sources of bias . outcomes , including morbidity , mortality , 2-year survival rate , 5-year survival rate , hospital stay , operating time and hospitalization charge , were extracted from included papers . survival was defined as the time from the shunt procedure to the time of death from any cause . mortality in this meta - analysis was defined as postoperative death before discharge or within 30 postoperative days . transjugular intrahepatic portosystemic shunt placement referred to the technical procedure via interventional therapy that stents were placed between the portal vein and hepatic vein until a 510 mmhg gradient was achieved . the technique of hgpcs was described that small - diameter ( 810 mm ) externally ring - reinforced polytetrafluoroethylene graft was used with beveled ends oriented perpendicular ( 90 ) to each other to accommodate for the orientation of the portal vein to the inferior vena cava . pcs was defined as direct pcs , usually side to side , rarely end to side . dsrs was defined as distal splenic vein and renal vein shunted from end to side . the verified data were analyzed using review manager ( version 4.2 , oxford , england , the cochrane collaboration , 2003 ) . the odds ratio ( or ) , mean difference ( md ) and their corresponding 95% confidence intervals ( 95% cis ) were calculated for dichotomous or continuous outcome data respectively . statistical heterogeneity was assessed with the i test and q - test to justify the accuracy of the fixed or random effects model . a significant effect was assumed if the 95% ci did not include the value 1.0 for or or 0 for md . a fixed - effect model was used in case of no relevant statistical heterogeneity when i was < 50% and p > 0.1 . if the heterogeneity was high ( i > 50% and p < 0.1 ) , a sub - group analysis could be used to decrease the risk of bias . a fixed - effect model also could be used in sub - group analysis for decreased heterogeneity . a random - effect model was used when i > 50% and p < 0.1 . all databases including cbm , cnki , wfpd , pubmed , medline , embase and cochrane were searched using the mesh terms : hypertension , portal / complications , liver cirrhosis / complications , portosystemic shunt , transjugular intrahepatic / mortality , portosystemic shunt , surgical , esophageal and gastric varices / surgery , hypertension , portal / surgery independently or in combination for clinical trials comparing tips with surgical shunts . all literatures were screened to identify all suitable published rcts for eligibility until february 2014 . all eligible rcts were screened for their methods , characteristics and risk of bias strictly . in this meta - analysis , two different kinds of groups were generated from the included papers that met the criterion : the tips group and the surgical shunting group . the surgical shunting group referred to traditional portosystemic shunts that contained dsrs , hgpcs , and pcs . in sub - group analysis , hgpcs and pcs could be recognized as one group for their similar modus operandi and decompression principle . in our research , 246 patients underwent tips and 247 patients underwent surgical shunts . all included rcts were required to contain a direct comparison between portosystemic shunts ( including dsrs , hgpcs , and pcs ) and tips in a controlled manner . each trial was required to include postoperative complications , mortality and long - term survival rate in detail . primary outcomes to be assessed were mortality , 2- and 5-year survival and postoperative complications including recurrent variceal hemorrhage , shunt stenosis , and hepatic encephalopathy . secondary outcomes to be assessed were length of hospital stay , operating time and hospitalization charges . each of these papers was assessed using a standardized evaluation form by two investigators ( author # 1 , author # 5 ) independently for extraction in this meta - analysis . when consensus could nt be reached , discussion and joint collection of the paper could resolve . otherwise , a third reviewer ( author # 2 ) would take part in the discussion and determine the definite inclusion of papers as a referee . general information collected from abstracts , included the authors , publication year , characteristics of patients and outcomes . all papers were assessed for the risk of bias by two investigators ( author # 1 , author # 5 ) on the following areas : random sequence generation , allocation concealment , blinding of participants and personnel , blinding of outcome assessment , incomplete outcome data , selective reporting , other sources of bias . outcomes , including morbidity , mortality , 2-year survival rate , 5-year survival rate , hospital stay , operating time and hospitalization charge , were extracted from included papers . survival was defined as the time from the shunt procedure to the time of death from any cause . mortality in this meta - analysis was defined as postoperative death before discharge or within 30 postoperative days . transjugular intrahepatic portosystemic shunt placement referred to the technical procedure via interventional therapy that stents were placed between the portal vein and hepatic vein until a 510 mmhg gradient was achieved . the technique of hgpcs was described that small - diameter ( 810 mm ) externally ring - reinforced polytetrafluoroethylene graft was used with beveled ends oriented perpendicular ( 90 ) to each other to accommodate for the orientation of the portal vein to the inferior vena cava . pcs was defined as direct pcs , usually side to side , rarely end to side . dsrs was defined as distal splenic vein and renal vein shunted from end to side . the verified data were analyzed using review manager ( version 4.2 , oxford , england , the cochrane collaboration , 2003 ) . the odds ratio ( or ) , mean difference ( md ) and their corresponding 95% confidence intervals ( 95% cis ) were calculated for dichotomous or continuous outcome data respectively . statistical heterogeneity was assessed with the i test and q - test to justify the accuracy of the fixed or random effects model . a significant effect was assumed if the 95% ci did not include the value 1.0 for or or 0 for md . a fixed - effect model was used in case of no relevant statistical heterogeneity when i was < 50% and p > 0.1 . if the heterogeneity was high ( i > 50% and p < 0.1 ) , a sub - group analysis could be used to decrease the risk of bias . a fixed - effect model also could be used in sub - group analysis for decreased heterogeneity . a random - effect model was used when i > 50% and p < 0.1 . searching in medline , embase , pubmed and cochrane data of clinical trials was 376 trials . after checking for abstracts according to our predefined inclusion and exclusion criterion , 76 references remained for further evaluation . eventually , four rcts that documented adequate comparisons between tips and surgical shunting for portal hypertension were determined for this meta - analysis . no additional eligible studies were found after carefully examined the reference lists of all included papers [ figure 1 ] . the characteristics and risk of bias for included four studies that compared tips and surgical shunting for portal hypertension are performed in table 1 as suggested in the cochrane handbook . three included papers reported patients from america while the remaining one from india [ table 1 ] . characteristics of included rcts and risk of the bias summary tips : transjugular intrahepatic portosystemic shunting ; dsrs : distal splenorenal shunts ; pcs : portacaval shunts ; hgpcs : h - graft portacaval shunt ; rct : randomized clinical trial . from the four included trials , 493 patients of child - pugh a to c were included in our analysis . long - term results and postoperative complications mainly including recurrent variceal hemorrhage , shunt stenosis , and encephalopathy are summarized in table 2 . results and clinical characteristics comparing tips with surgical shunting tips : transjugular intrahepatic portasystemic shunting ; dsrs : distal splenorenal shunts ; pcs : portacaval shunts ; hgpcs : h - graft portacaval shunt ; sd : standard deviation ; na : not applicable . variceal rebleeding occurred in 27.6% of patients in tips group and in 4.5% of patients in surgical shunting group , shunt stenosis occurred in 66.1% of patients in tips group and in 9.9% of patients in surgical shunting group , hepatic encephalopathy occurred in 53.9% of patients in tips group and in 32.0% of patients in surgical shunting group . from our meta - analysis , morbidity in variceal rehemorrhage was significantly higher in tips than in surgical shunts ( or = 7.45 , 95% ci : ( 3.9314.15 ) , p < 0.00001 ) [ figure 2 ] . patients occurring postoperative shunt stenosis also was significantly higher in tips than in surgical shunts ( or = 20.01 , 95% ci : ( 6.6759.99 ) , p < 0.000001 ) [ figure 3 ] . besides , morbidity in hepatic encephalopathy was also significantly higher in tips than in surgical shunts ( or = 2.50 , 95% ci : ( 1.633.84 ) , p < 0.0001 ) , especially when compared with pcs in sub - group analysis ( or = 6.00 , 95% ci : ( 2.9312.27 ) , p < 0.00001 ) [ figure 4 ] . a sub - group analysis was conducted in the comparison of variceal rehemorrhage ( = 8.07 , p = 0.04 , i = 62.8% ) and hepatic encephalopathy ( = 12.41 , p = 0.002 , i = 83.9% ) to decrease high heterogeneity . mortality in this meta - analysis referred to the death rate at 30 days . in our research , mortality was equivalent among patients undergoing tips or surgical shunts ( or = 0.76 ; 95% ci : ( 0.451.30 ) , p = 0.32 ) [ figure 5 ] . no significant heterogeneity was found from the trials ( = 1.80 , p = 0.61 , i = 0% ) . survival at 2 years in sub - group analysis was also significantly higher among patients undergoing pcs than patients undergoing tips ( or = 0.48 ; 95% ci : ( 0.290.77 ) , p = 0.003 ) , but in sub - group analysis survival at 2 years was equivalent among patients undergoing tips and dsrs ( or = 1.38 , 95% ci : ( 0.662.88 ) , p = 0.39 ) . significant heterogeneity was found from the trials , and a sub - group analysis was conducted ( = 6.30 , p = 0.10 , i = 52.4% ) [ figure 6 ] . survival at 5 years was greater for patients undergoing surgical shunting than patients undergoing tips ( or = 0.44 ; 95% ci : ( 0.300.65 ) , p < 0.0001 ) , and survival in sub - group analysis was also higher among patients undergoing pcs than patients undergoing tips ( or = 0.29 ; 95% ci : ( 0.170.47 ) , p < 0.00001 ) . significant heterogeneity was found from the trials , and a sub - group analysis was conducted ( = 12.32 , p = 0.002 , i = 83.8% ) [ figure 7 ] . five - year survival comparing transjugular intrahepatic portosystemic shunt with surgical shunting . in the included rcts , only 1 paper reported the length of hospital stay in the form of the mean and standard deviation ( sd ) comparing tips with dsrs ( 3.1 [ 4.3 day ] vs. 10.5 [ 11.3 day ] ) , which suggested tips treatment had a shorter hospital stay [ table 2 ] . in this research , only 1 trial reported hospitalization charges comparing tips with dsrs ( 21,607 vs. 28,734 ) . however , cost analysis data of this article showed no overall significant difference in the cost of managing patients with either procedure [ table 2 ] . in our meta - analysis , only 1 paper reported operating time in the form of the mean and the range comparing tips with pcs ( 3 [ 1.2525 ] h vs. 3.8 [ 2.611 ] h ) [ table 2 ] . searching in medline , embase , pubmed and cochrane data of clinical trials was 376 trials . after checking for abstracts according to our predefined inclusion and exclusion criterion , 76 references remained for further evaluation . eventually , four rcts that documented adequate comparisons between tips and surgical shunting for portal hypertension were determined for this meta - analysis . no additional eligible studies were found after carefully examined the reference lists of all included papers [ figure 1 ] . the characteristics and risk of bias for included four studies that compared tips and surgical shunting for portal hypertension are performed in table 1 as suggested in the cochrane handbook . three included papers reported patients from america while the remaining one from india [ table 1 ] . characteristics of included rcts and risk of the bias summary tips : transjugular intrahepatic portosystemic shunting ; dsrs : distal splenorenal shunts ; pcs : portacaval shunts ; hgpcs : h - graft portacaval shunt ; rct : randomized clinical trial . from the four included trials , 493 patients of child - pugh a to c were included in our analysis . long - term results and postoperative complications mainly including recurrent variceal hemorrhage , shunt stenosis , and encephalopathy are summarized in table 2 . results and clinical characteristics comparing tips with surgical shunting tips : transjugular intrahepatic portasystemic shunting ; dsrs : distal splenorenal shunts ; pcs : portacaval shunts ; hgpcs : h - graft portacaval shunt ; sd : standard deviation ; na : not applicable . variceal rebleeding occurred in 27.6% of patients in tips group and in 4.5% of patients in surgical shunting group , shunt stenosis occurred in 66.1% of patients in tips group and in 9.9% of patients in surgical shunting group , hepatic encephalopathy occurred in 53.9% of patients in tips group and in 32.0% of patients in surgical shunting group . from our meta - analysis , morbidity in variceal rehemorrhage was significantly higher in tips than in surgical shunts ( or = 7.45 , 95% ci : ( 3.9314.15 ) , p < 0.00001 ) [ figure 2 ] . patients occurring postoperative shunt stenosis also was significantly higher in tips than in surgical shunts ( or = 20.01 , 95% ci : ( 6.6759.99 ) , p < 0.000001 ) [ figure 3 ] . besides , morbidity in hepatic encephalopathy was also significantly higher in tips than in surgical shunts ( or = 2.50 , 95% ci : ( 1.633.84 ) , p < 0.0001 ) , especially when compared with pcs in sub - group analysis ( or = 6.00 , 95% ci : ( 2.9312.27 ) , p < 0.00001 ) [ figure 4 ] . a sub - group analysis was conducted in the comparison of variceal rehemorrhage ( = 8.07 , p = 0.04 , i = 62.8% ) and hepatic encephalopathy ( = 12.41 , p = 0.002 , i = 83.9% ) to decrease high heterogeneity . mortality in this meta - analysis referred to the death rate at 30 days . in our research , mortality was equivalent among patients undergoing tips or surgical shunts ( or = 0.76 ; 95% ci : ( 0.451.30 ) , p = 0.32 ) [ figure 5 ] . no significant heterogeneity was found from the trials ( = 1.80 , p = 0.61 , i = 0% ) . survival at 2 years in sub - group analysis was also significantly higher among patients undergoing pcs than patients undergoing tips ( or = 0.48 ; 95% ci : ( 0.290.77 ) , p = 0.003 ) , but in sub - group analysis survival at 2 years was equivalent among patients undergoing tips and dsrs ( or = 1.38 , 95% ci : ( 0.662.88 ) , p = 0.39 ) . significant heterogeneity was found from the trials , and a sub - group analysis was conducted ( = 6.30 , p = 0.10 , i = 52.4% ) [ figure 6 ] . survival at 5 years was greater for patients undergoing surgical shunting than patients undergoing tips ( or = 0.44 ; 95% ci : ( 0.300.65 ) , p < 0.0001 ) , and survival in sub - group analysis was also higher among patients undergoing pcs than patients undergoing tips ( or = 0.29 ; 95% ci : ( 0.170.47 ) , p < 0.00001 ) . significant heterogeneity was found from the trials , and a sub - group analysis was conducted ( = 12.32 , p = 0.002 , i = 83.8% ) [ figure 7 ] . in the included rcts , only 1 paper reported the length of hospital stay in the form of the mean and standard deviation ( sd ) comparing tips with dsrs ( 3.1 [ 4.3 day ] vs. 10.5 [ 11.3 day ] ) , which suggested tips treatment had a shorter hospital stay [ table 2 ] . in this research , only 1 trial reported hospitalization charges comparing tips with dsrs ( 21,607 vs. 28,734 ) . however , cost analysis data of this article showed no overall significant difference in the cost of managing patients with either procedure [ table 2 ] . in our meta - analysis , only 1 paper reported operating time in the form of the mean and the range comparing tips with pcs ( 3 [ 1.2525 ] h vs. 3.8 [ 2.611 ] h ) [ table 2 ] . nowadays , tips placement has become the preferred first - line management for patients with portal hypertension . the present study is the first meta - analysis of studies comparing tips with surgical shunting in patients with portal hypertension focused on complications and survival . this meta - analysis included four rcts , the populations in three studies were from america and in the remaining study were from india . currently , there were few papers concerning tips versus surgical shunting in outcomes and survival rates , so the difference between tips and surgical shunting was unclear . in clinical practice , there was the lack of a definite guidance to determine the optimal treatment for patients with portal hypertension . this meta - analysis would help make a better decision in clinical work or develop a guideline . transjugular intrahepatic portosystemic shunt was generally considered as a first - line therapy in the definitive treatment of portal hypertension concomitant with variceal bleeding ; however , trials comparing tips with surgical shunts were few and drew little attention , so some disadvantages of tips were ignored . in this meta - analysis , we conducted a comparison between tips and surgical shunting in patients with cirrhosis , portal hypertension and bleeding varices from four trials , in order to get an all - around evaluation and give more information about how to decide the optimal management for patients with portal hypertension . as the measurement of patient 's survival and postoperative complications was representative , we could conclude that long - term survival and morbidity after shunting were promoted by surgical shunting relative to tips . but surgical shunts in this meta - analysis included dsrs and hgpcs , and the relations between the two types of surgeries and tips were unclear . as a result , the outcomes of this meta - analysis were proved to be helpful in answering questions that arose when comparing tips with surgical shunting . although each included trial did nt individually reveal the comparison between surgical shunting and tips in all respects , the outcomes of this meta - analysis proved the superiority of surgical shunting over tips . as a matter of fact , the outcomes were obvious that the results supported a role for surgical shunts in patients with portal hypertension over tips . in terms of morbidity , mortality , 2- and 5-year survival , in the included papers , three of four trials found significantly higher postoperative bleeding , stenosis and encephalopathy rates after tips . postoperative complications , including recurrent variceal hemorrhage , hepatic encephalopathy , shunt stenosis , severe infection and so on , were all deadly sign for patients after surgery that could lead to a series of disorders in metabolic systems . complications of tips placement proved to be critical when hepatic encephalopathy occurred , a limited number of procedures should be typically considered for the management . the postoperative rehemorrhage rate of nearly 11% after tips was the lowest reported in included trials while the rebleeding rate was reaching 5.5% after dsrs in two included papers and 7.5% after hgpcs in included paper . shunt stenosis could also cause recurrent variceal hemorrhage and result in a series of severe syndromes . the comparison of shunt stenosis between tips and surgical shunting was obvious as the rate was reaching almost 82% after tips and no more than 11% after surgical shunts . tips could effectively lower portal hypertension due to cirrhosis . according to past experience , it is significantly effective for hemorrhage of the digestive tract due to rupture of esophageal and fundic veins and for ascites and hydrothorax caused by portal hypertension . although shunt dysfunction has confused surgeons for the incidence of tips stenosis and occlusion , trials of successful treatment with tips of recurrent massive rectal bleeding due to portal hypertension has reinforced the confidence of surgeons in tips . recent article also reported bleeding from focal varices in the stoma could be treated with tips that spread the use of tips in other situations . in addition , a 25% incidence of hernia complications following tips creation in patients being treated for refractory ascites was higher than expected . the most common indications for tips placement were refractory ascites and variceal hemorrhage , also in properly selected candidate , tips placement could serve as a bridge to liver transplantation for tips was initially less expensive and led to an easier transplantation process . tips also performed as an effective way in preventing variceal rebleeding and may be more cost effective . in other words , it was equal to surgical procedures in preventing variceal rebleeding in patients who are medical failures . besides , when aimed at treating the budd chiari syndrome , a therapeutic strategy has been proposed that angioplasty for short length venous stenosis , tips , and ultimately liver transplantation could be done in proper order . although tips revealed superiority in some parts of treatment in patients with portal hypertension , the results of this meta - analysis performed significant deficiency in morbidity and survival . to some extent , people preferred shunting subjectively only when nonshunting modalities to control variceal bleeding failed or when these modalities were considered nonapplicable , thus strong points of this method were difficult to appear . this meta - analysis , which contained a number of patients in these trials , was able to give great credibility to the results and conclusions . pcs permanently stopped variceal bleeding and gained a longer time survival than tips , but nowadays the widespread practice of using pcs only served as a management for failure of endoscopic therapy or tips in unselected patients with cirrhosis . however , the 2-year survival was higher in pcs than in tips ( or = 0.48 ; 95% ci : ( 0.290.77 ) , p = 0.003 ) , and 5-year survival after pcs was longer than that after tips ( or = 0.29 ; 95% ci : ( 0.170.47 ) , p < 0.00001 ) . no matter what the surgical shunts include , the consecutive experiences of shunt operation are important for the improvement of surgical skills , both of the individual surgeon and the team . since 1988 , more and more patients have received hgpcs treatment for its effectiveness in reducing portal hypertension , and the outcome was favorable that the actual survival was better than predicted before . the actual 5- and 10-year survival after hgpcs by child class was : ( a ) 67% and 33% , ( b ) 49% and 16% , ( c ) 29% and 7% . hgpcs has been identified as an effective procedure for the treatment of variceal bleeding , as well as for the prevention of re - bleeding in patients , with a low rate of complications . some papers indicated that pcs with a small diameter interposition h - graft was an effective procedure for the treatment of variceal bleeding or as an elective or emergency procedure with a low rate of complications . papers published recently also indicated that the actual survival after hgpcs was better than other surgical procedures . central splenorenal shunt showed more negatively effect on hemodynamics of left kidney and symptoms of renal venous hypertension obviously due to shunting the large amounts of blood from a system of high pressure to a low . in contrast , dsrs or 8 mm h - graft shunt showed similar efficacy to tips , and it also served as the proper choice in preventing recurrent variceal bleeding . dsrs also revealed an effective and reliable procedure for children with portal hypertension and was still useful for selected pediatric patients . dsrs was suggested to be useful for the management of esophagogastric varices in patients with idiopathic portal hypertension . although a number of surgical procedures have been developed to manage esophageal varices , dsrs with splenopancreatic disconnection plus gastric transection was considered as an adequate treatment for patients with esophagogastric varices . the included studies regarding surgical shunting versus tips were rare , and surgical shunts in these papers included several kinds of operation methods . this could result in instability of the comparison between tips and surgical shunting as the different surgical types of shunts may lead to a slight bias of outcome . although sub - group analysis was taken , the bias of the result still existed . the only way to solve the problem was that more clinical trials comparing surgical shunting with tips should be undertaken . data such as cost and hospital stay from the included studies were rare , so further evidence concerning hospital charges and hospital stay was needed to fulfill this meta - analysis . although tips was performed most often with the use of the covered stent currently , there were no studies comparing covered stent with surgical shunting scientifically . the outcomes and survival rates between covered and uncovered tips varied , clark et al . concluded that there were no differences in rates of encephalopathy or survival between covered and uncovered tips , but bureau et al . revealed polytetrafluoroethylene - coated stents were superior to uncovered stents in morbidity , so more rcts concerning covered stents versus surgical shunting should be conducted to provide more evidence . limited rcts concerning covered tips was the restriction in this meta - analysis , so more clinical trials should be undertaken . finally , the risk of bias and heterogeneity of included rcts were still existed according to the cochrane handbook . allocation concealment was missed in some included rcts and high risk of bias was existed in some parts of our bias summary , these could lead to the bias of outcomes and influence the validity of result to some extent , so cochrane risk of bias should be examined exactly to reduce bias . in summary , surgical shunting would be recommended because of increased survival rate and fewer postoperative complications when compared with tips . as the patients with portal hypertension concomitant with variceal hemorrhage always remained in a large number and needed necessary treatment , they should be given more surgical advices for the superiority concerning complications and survival .

key clinical messagepulmonary function tests such as flowvolume loops and reconstructive radiological imaging may aid the detection of large airway obstruction prior to corrective surgery for severe scoliosis . intraoperative use of halogravity traction may help to reduce the severity of the scoliosis , and thus the extrinsic compression or torsion of the airways . background : the purpose of this article was to clarify the optimal management concerning transjugular intrahepatic portosystemic shunts ( tipss ) and surgical shunting in treating portal hypertension.methods:all databases , including cbm , cnki , wfpd , medline , embase , pubmed and cochrane up to february 2014 , were searched for randomized controlled trials ( rcts ) comparing tips with surgical shunting . four rcts , which were extracted by two independent investigators and were evaluated in postoperative complications , mortality , 2- and 5-year survival , hospital stay , operating time and hospitalization charges.results:the morbidity in variceal rehemorrhage was significantly higher in tips than in surgical shunts ( odds ratio [ or ] = 7.45 , 95% confidence interval[ci ] : ( 3.9314.15 ) , p < 0.00001 ) , the same outcomes were seen in shunt stenosis ( or = 20.01 , 95% ci : ( 6.6759.99 ) , p < 0.000001 ) and in hepatic encephalopathy ( or = 2.50 , 95% ci : ( 1.633.84 ) , p < 0.0001 ) . significantly better 2-year survival ( or = 0.66 ; 95% ci : ( 0.440.98 ) , p = 0.04 ) and 5-year survival ( or = 0.44 ; 95% ci : ( 0.300.66 ) , p < 0.00001 ) were seen in patients undergoing surgical shunting compared with tips.conclusions:compared with tips , postoperative complications and survival after surgical shunting were superior for patients with portal hypertension . application of surgical shunting was recommended for patients rather than tips .

the frequency of prenatal hydronephrosis diagnosed by ultrasound has been reported to be 0.65.4% of newborns [ 39 ] . among infants with hydronephrosis , hydronephrosis has been reported to be transient or physiological in nature ( 4188% ) or secondary to pelviureteric junction obstruction ( 1030% ) , vesicoureteric reflux ( 1020% ) , vesicoureteric junction obstruction ( 510% ) , multicystic dysplastic kidney ( 46% ) , duplex kidney with or without ureterocele ( 27% ) , and posterior urethral valves ( 12% ) . nevertheless , the detection of fetal hydronephrosis by ultrasound presents a dilemma in terms of diagnosis and treatment , and there is considerable debate and controversy regarding the optimal management of children with prenatally - diagnosed hydronephrosis . a recent report highlighted the substantial variations in the use of grading criteria for the diagnosis of fetal hydronephrosis and in the management of mild prenatal hydronephrosis . hence , antenatal hydronephrosis does not necessarily indicate the presence of an obstruction or poor renal function . indeed , most of the cases of antenatal hydronephrosis resolve spontaneously by the time of birth or during infancy period . the pediatric surgeon thus faces the challenge of distinguishing the affected infants who require long - term observation or surgical intervention from those who do not require observation or intervention . postnatal ultrasound is considered a critical investigation tool used to follow affected infants to monitor the progression of the disease , and can be complemented by other investigations such as micturating cystourethrography ( mcu ) and diuretic renography . therefore , in order to shed some light on the diagnosis and management of antenatal hydronephrosis , the present study aimed to examine the postnatal follow - up and treatment for hydronephrosis diagnosed prenatally in a pediatric hospital in china . this was a retrospective study of infants prenatally diagnosed with hydronephrosis and treated at the department of pediatric surgery of qilu hospital , shandong university ( china ) between january 2005 and december 2013 . the need for individual consent was waived by because of the retrospective nature of the study . exclusion criteria were : ( 1 ) patients with sfu grade 4 ; ( 2 ) ipsilateral or contralateral reflux ; ( 3 ) solitary kidney ; ( 4 ) pelvic stones ; ( 5 ) ureteral dilatation ; 6 ) anatomical or neurogenic abnormality of the lower urinary tract ; or ( 7 ) abnormality other than ureteropelvic junction obstruction of the contralateral normal kidney all patients underwent initial assessment of hydronephrosis by ultrasound one week after birth and underwent standardized dtpa renography one month after birth . the maximum anteroposterior diameter ( apd ) of the renal pelvis was measured on a transverse renal image . parenchymal thickness was measured at upper , middle , and lower poles , and the maximum parenchymal thickness was used as a variable . the society for fetal urology ( sfu ) grading system was used for hydronephrosis4[1921 ] as well as the method designed by grignon et al . infants who had an ultrasound within 72 hours of birth because of an emergency clinical status were re - evaluated at one week of age . for diuretic renogram , oral hydration was started two hours before the scan ; intravenous dn/2 was started at 15 ml / kg over 30 minutes , beginning 15 minutes before injection of the radiotracer , and then at 4 ml / kg / hour for the remaining of the study . furosemide 1 mg / kg was injected at the start of the study and renal function was measured , along with assessment of isotope washout after diuresis . during the diuresis phase , some patients were additionally investigated using emission computed tomography ( ect ) and magnetic resonance urography ( mru ) . urinary and blood urea nitrogen ( uun , bun ) and urinary and serum creatinine ( cr ) were measured routinely . patients with sfu grade 1 hydronephrosis ( equivalent to pyelectasis 1.5 cm in grignon s approach ; table 1 ) had follow - up with b - mode ultrasound . a regular follow - up examination to clarify the pathological obstruction was deemed necessary in patients with sfu grade 2 hydronephrosis ( expanded renal pelvis of > 1.5 cm ) . patients diagnosed with sfu grade 3 hydronephrosis were closely followed due to the possibility of obstructive pathological factors and permanent renal damage . the ect and mru investigations were carried out at one month of age , and ultrasound examinations were undertaken every two to four weeks . surgery was performed at the time of presentation if the initial differential renal function ( drf)was < 35% , with an obstructed curve on renogram and/or parenchymal thickness below 3.5 mm . post - surgery , the patients were closely monitored by ultrasound and followed at one , three , six , and 12 months . the other patients were followed using a standard protocol , with ultrasound and radionuclide studies being performed every three months during the first two years . if renal function and sfu grade remained stable , the patients were followed by ultrasound every six months for another two years . all patients were followed for at least two years after birth . normally distributed continuous variables are presented as mean standard deviation ( sd ) and were analyzed using the student s t test or anova with the tukey s post hoc test , as appropriate . non - normally distributed continuous variables are presented as median ( range ) and were analyzed using the mann - whitney test . the occurrence of surgery during follow - up was analyzed using kaplan - meier curves , which were analyzed by the log - rank test . a multivariate analysis using cox regression analysis was performed to identify the independent factors involved in the occurrence of surgery . the receiver operating characteristic ( roc ) curve approach was used to determine the accuracy of factors associated with the occurrence of surgery . spss 16.0 ( ibm , armonk , ny , usa ) was used for analysis . this was a retrospective study of infants prenatally diagnosed with hydronephrosis and treated at the department of pediatric surgery of qilu hospital , shandong university ( china ) between january 2005 and december 2013 . the need for individual consent was waived by because of the retrospective nature of the study . exclusion criteria were : ( 1 ) patients with sfu grade 4 ; ( 2 ) ipsilateral or contralateral reflux ; ( 3 ) solitary kidney ; ( 4 ) pelvic stones ; ( 5 ) ureteral dilatation ; 6 ) anatomical or neurogenic abnormality of the lower urinary tract ; or ( 7 ) abnormality other than ureteropelvic junction obstruction of the contralateral normal kidney all patients underwent initial assessment of hydronephrosis by ultrasound one week after birth and underwent standardized dtpa renography one month after birth . the maximum anteroposterior diameter ( apd ) of the renal pelvis was measured on a transverse renal image . parenchymal thickness was measured at upper , middle , and lower poles , and the maximum parenchymal thickness was used as a variable . the society for fetal urology ( sfu ) grading system was used for hydronephrosis4[1921 ] as well as the method designed by grignon et al . ( table 1 ) . infants who had an ultrasound within 72 hours of birth because of an emergency clinical status were re - evaluated at one week of age . for diuretic renogram , oral hydration was started two hours before the scan ; intravenous dn/2 was started at 15 ml / kg over 30 minutes , beginning 15 minutes before injection of the radiotracer , and then at 4 ml / kg / hour for the remaining of the study . furosemide 1 mg / kg was injected at the start of the study and renal function was measured , along with assessment of isotope washout after diuresis . during the diuresis phase , some patients were additionally investigated using emission computed tomography ( ect ) and magnetic resonance urography ( mru ) . urinary and blood urea nitrogen ( uun , bun ) and urinary and serum creatinine ( cr ) were measured routinely . patients with sfu grade 1 hydronephrosis ( equivalent to pyelectasis 1.5 cm in grignon s approach ; table 1 ) had follow - up with b - mode ultrasound . a regular follow - up examination to clarify the pathological obstruction was deemed necessary in patients with sfu grade 2 hydronephrosis ( expanded renal pelvis of > 1.5 cm ) . patients diagnosed with sfu grade 3 hydronephrosis were closely followed due to the possibility of obstructive pathological factors and permanent renal damage . the ect and mru investigations were carried out at one month of age , and ultrasound examinations were undertaken every two to four weeks . surgery was performed at the time of presentation if the initial differential renal function ( drf)was < 35% , with an obstructed curve on renogram and/or parenchymal thickness below 3.5 mm . post - surgery , the patients were closely monitored by ultrasound and followed at one , three , six , and 12 months . the other patients were followed using a standard protocol , with ultrasound and radionuclide studies being performed every three months during the first two years . if renal function and sfu grade remained stable , the patients were followed by ultrasound every six months for another two years . normally distributed continuous variables are presented as mean standard deviation ( sd ) and were analyzed using the student s t test or anova with the tukey s post hoc test , as appropriate . non - normally distributed continuous variables are presented as median ( range ) and were analyzed using the mann - whitney test . the occurrence of surgery during follow - up was analyzed using kaplan - meier curves , which were analyzed by the log - rank test . a multivariate analysis using cox regression analysis the receiver operating characteristic ( roc ) curve approach was used to determine the accuracy of factors associated with the occurrence of surgery . spss 16.0 ( ibm , armonk , ny , usa ) was used for analysis . most patients ( 59.5% ) were sfu grade 1 . at the last follow - up figure 1 presents the kaplan - meier curves of the occurrence of surgery according to the sfu grade determined one week after birth by ultrasound . only a small proportion of patients who were originally grade 1 underwent surgery ( n=3 ) , while most grade 23 patients underwent surgery . patients who underwent surgery were more often sfu grade 2 or 3 ( p<0.001 ) , had a larger apd ( p<0.001 ) , and had a smaller parenchymal thickness ( p<0.001 ) . the variables that were significant in univariate analyses were included in a multivariate model ( table 4 ) . results showed that sfu grade 3 ( hr=9.23 , 95% ci : 1.4359.74 , p=0.02 ) , apd ( hr=2.81 , 95% ci : 1.117.10 , p=0.03 ) , and parenchymal thickness ( hr=0.42 , 95% ci : 0.240.71 , p=0.001 ) were independently associated with the occurrence of surgery . figure 2 presents the roc curve analysis for the accuracy of apd and parenchymal thickness for predicting the occurrence of surgery . for apd , using a cut - off point of 1.1 , the area under the curve ( auc ) was 0.86 , youden index was 0.556 , sensitivity was 70.4% , and specificity was 85.3% . for parenchymal thickness , using a cut - off point of 5 , auc was 0.79 , youden index was 0.478 , sensitivity was 74.1% , and specificity was 73.8% . most patients ( 59.5% ) were sfu grade 1 . at the last follow - up figure 1 presents the kaplan - meier curves of the occurrence of surgery according to the sfu grade determined one week after birth by ultrasound . only a small proportion of patients who were originally grade 1 underwent surgery ( n=3 ) , while most grade 23 patients underwent surgery . patients who underwent surgery were more often sfu grade 2 or 3 ( p<0.001 ) , had a larger apd ( p<0.001 ) , and had a smaller parenchymal thickness ( p<0.001 ) . the variables that were significant in univariate analyses were included in a multivariate model ( table 4 ) . results showed that sfu grade 3 ( hr=9.23 , 95% ci : 1.4359.74 , p=0.02 ) , apd ( hr=2.81 , 95% ci : 1.117.10 , p=0.03 ) , and parenchymal thickness ( hr=0.42 , 95% ci : 0.240.71 , p=0.001 ) were independently associated with the occurrence of surgery . figure 2 presents the roc curve analysis for the accuracy of apd and parenchymal thickness for predicting the occurrence of surgery . for apd , using a cut - off point of 1.1 , the area under the curve ( auc ) was 0.86 , youden index was 0.556 , sensitivity was 70.4% , and specificity was 85.3% . for parenchymal thickness , using a cut - off point of 5 , auc was 0.79 , youden index was 0.478 , sensitivity was 74.1% , and specificity was 73.8% . hydronephrosis is a common congenital condition that can be detected by prenatal ultrasound , but there is considerable debate and controversy regarding the optimal management of children with prenatally - diagnosed hydronephrosis . therefore , this study aimed to examine postnatal follow - up and treatment for hydronephrosis diagnosed prenatally , as well as the characteristics associated with the occurrence of surgery . results showed that 2.4% , 18.9% , and 90.9% of patients with sfu grade 1 , 2 , and 3 hydronephrosis , respectively , underwent surgery . sfu grade 3 , apd , and parenchymal thickness were independently associated with the occurrence of surgery . for anterioposterior diameter , using a cut - off point of 1.1 , sensitivity was 70.4% , and specificity was 85.3% . for parenchymal thickness , using a cut - off point of 5 , sensitivity was 74.1% , and specificity was 73.8% . . however , the recent developments in ultrasound and prenatal medical technologies have allowed for the diagnosis of hydronephrosis during the fetal period , and its monitoring after birth . studies are now available concerning the management of hydronephrosis in children , and guide clinicians to make better decisions regarding the selection of suitable follow - up strategies and surgical interventions . indeed , compared with a newborn , a fetus has a relatively low renal vascular resistance and a relatively high renal blood flow , and the combination of a higher glomerular filtration rate and a lower renal concentrating capacity may thus lead to a relatively high urine volume . in addition , as the compliance of the ureter increases , it becomes easier to expand . since the fetal and neonatal kidneys are not fully developed , the renal medullary cone may be transparent under ultrasound examination , which leads to a wrong diagnosis of hydronephrosis . in contrast , pathological hydronephrosis is due to renal obstruction , resulting in the separation of the collection system . since the fetal and neonatal renal tubules are relatively short and straight , the consequences of obstruction are more serious than in the adult . hence , it is very important to distinguish between physiological and pathological hydronephrosis , since their management is different . ultrasound investigations that ate carried out during prenatal screening and subsequent follow - up after birth , are the primary and preferred means of monitoring patients diagnosed with hydronephrosis . although ultrasound has a limited role in predicting the degree of renal impairment and identifying the cause of the restriction / obstruction , it is highly useful for the diagnosis of hydronephrosis and evaluation of disease progression . early studies have shown that the patients with renal parenchyma thickness > 4 mm had normal glomerular , tubular , and cell structure ; at 3 mm thickness of renal parenchyma , some surviving glomeruli and tubules were evident but there was cell deformation with the presence of abnormal nuclei , and removal of the obstruction resulted in partial restoration of the cell morphology ; at 2 mm , glomerular and tubular structures were not apparent , and the normal cell structure was not evident through electron microscopy . after a gestational age of 24 weeks , ultrasound is able to demonstrate clearly the structure of the fetal kidney , enabling direct measurement of the collection system , the expansion of the renal pelvis , and the thickness of the renal cortex . since it is a non - invasive and straightforward technique , it has an advantage over other methods such as ect , mru , and intravenous pyelography , particularly in young children . an apd of 4 mm or greater at 20 weeks of gestation or larger threshold of 7 mm in the third trimester has been used as an indicator for the diagnosis of prenatal hydronephrosis . in addition to diagnosis of the condition , antenatal ultrasound has also been reported to have some prognostic value . interestingly , the second and third trimester apd cut - offs of 5 mm and 8 mm to10 mm , respectively , have been reported to have 100% sensitivity for predicting the requirement for postnatal surgery . similarly , in another study , apd thresholds of 10 mm at 20 weeks and 12 mm at 30 weeks of gestation were found to have 100% sensitivity , and 86% and 67% specificity ( respectively ) for the diagnosis of hydronephrosis postnatally . the risk of postnatal pathology increases with the severity of prenatal hydronephrosis , while antenatal apd of < 12 mm is associated with stabilization or resolution in 98% of the infant population with hydronephrosis . in this study , using an apd cut - off of 1.1 had 70.4% sensitivity and 85.3% specificity for the occurrence of surgery . using parenchymal thickness of 5 mm had 74.1% sensitivity and 73.8% specificity . the sfu guidelines recommend that all children with a prenatal diagnosis of hydronephrosis should be investigated with an ultrasound within one week of birth , and the results should be compared with the last prenatal ultrasound , in order to determine the classification of the hydronephrosis . have reported that ultrasound examination 48 hours after birth showed either no hydronephrosis or a mild hydronephrosis with no obvious significant obstructive renal injury at one year . however , wiener et al . have reported that the grading of hydronephrosis using an ultrasound examination two days after birth may differ from the ultrasound examination 710 days after birth , possibly due to the presence of oliguria in the first few days . the present study suggests that an initial b - mode screening 710 days after birth with a second investigation 42 days after birth could be a good approach , but severe cases may be investigated earlier . the hydronephrosis may then be graded based on these investigations , and an appropriate management approach can be selected . some authors believe that the majority of young infants with mild - to - moderate hydronephrosis may be managed by observation rather than surgery . a previous study showed that there was a recovery rate of 98% in infants with sfu grade 1 or 2 hydronephrosis or apd < 12 mm , compared with 51% recovery with grade 3 or 4 hydronephrosis or apd > 12 mm . in addition , the sfu reported that surgery was eventually needed in 25% of children aged < 6 months with grade 34 hydronephrosis and a relative renal function > 40% . furthermore , surgery was found to reduce the degree of hydronephrosis and improve the renal pelvis evacuation , and was superior to the conservative approach of close observation . reported that almost all children with grade 34 hydronephrosis required surgery : in a review of 464 medical records , the vast majority of patients receiving surgery had grade 3 or 4 hydronephrosis with an evidence of obstruction ( renography ) , whereas those managed by observation tended to have grade 2 or 1 disease without obstruction . consistent with these data , a recent retrospective review of 629 infants found that the proportion of patients requiring surgery for grade 1 , 2 , 3 , and 4 hydronephrosis was 0,% 13% , 70% , and 100% , respectively . in clinical practice , the decision to go ahead with surgical intervention , and its timing , are based on the following main considerations : ( 1 ) sfu grade 34 ; ( 2 ) continued expansion of the renal pelvis collection system ; ( 3 ) a renal cortex < 5 mm ; and ( 4 ) a single - kidney decrease in gfr , as reflected by ect and mru examinations . future research should be directed at improving the understanding of areas where knowledge remains limited such as the renal processes underlying physiological and pathological hydronephrosis and their progression or resolution , and the optimization of the point at which surgical intervention is deemed necessary . it is important to standardize the guidelines based on up - to - date research , to help clinicians to make better decisions regarding the management of infants with a prenatal diagnosis of hydronephrosis . in conclusion , patients with sfu grade 2 hydronephrosis require long - term follow - up . surgery and close postsurgical observation may be necessary for patients with sfu grade 3 and 4 hydronephrosis . an initial b - mode ultrasound screening at 710 days after birth may help making an optimal diagnosis and treatment selection . d - penicillamine has been known as one of the agents that are able to induce pemphigus ( 1,2 ) . n-(2-mercapto-2-methylpropionyl)-l - cysteine ] is a thiol compound contained two free sulfhydryl groups , whereas d - penicillamine has only one sulfhydryl radical ( 3 ) . while drug - induced pemphigus was not infrequently demonstrated in association with d - penicillamine , it has rarely been reported with bucillamine and has never been reported in rheumatoid arthritis ( ra ) and polymyositis ( pm ) overlap syndrome . hereby , we report a case of bucillamine - induced pemphigus vulgaris in a patient with ra and pm overlap syndrome . a 46-yr - old woman was referred to our rheumatology department due to polyarthralgia , and myalgia in may 2001 . soft tissue swelling and tenderness were bilaterally noted at the proximal interphalangeal and metacarpophalangeal joints with morning stiffness lasting at least 1 hr . proximal motor weakness on upper arms and legs has insidiously developed over three months and findings of magnetic resonance image on both thighs were compatible with myositis . in addition , elevated level of muscle enzymes including creatine kinase ( ck ) , aldolase , alanine aminotransferase ( ast and alt ) , and lactate dehydrogenase was shown . antinuclear antibody was positive at 1:80 of speckled pattern , and 1:2,560 of cytoplasmic pattern . auto - antibodies including anti - jo-1 , anti - rnp , anti - double stranded dna , anti - sm , and anti - ss - a / ss - b were absent . level of rheumatoid factor was 3,420 iu / ml , but level of c3 and c4 was 97.9 mg / dl ( normal range 79 - 152 mg / dl ) and 30.8 mg / dl ( normal range 16 - 38 mg / dl ) respectively . chest radiograph showed pattern of interstitial lung disease and pulmonary function test revealed significantly decreased dlco . she was diagnosed as ra and pm overlap , and had been treated with prednisolone , azathioprine and non - steroidal anti - inflammatory drug since may 2001 . myositis became silent over time with above regimen , however she had continuous synovitis and joint pain on right knee and small joints on both hands . blisters consisted of erythematous patches and superficial erosions with turbid bullae suggesting pemphigus vulgaris ( fig . laboratory tests showed c - reactive protein 0.831 mg / dl ( normal range < 0.8 mg / dl ) , and erythrocyte sedimentation rate 66 mm / hr . a skin biopsy revealed intraepidermal vesicle formation with acantholysis and the vesicle formation is suprabasal ( fig . indirect immunofluorescence , performed by using serial dilutions of the patient 's serum , demonstrated no igg , iga and c3 staining along epidermis and papillary dermis . bucillamine was discontin - ued and the patient was placed on oral prednisolone 20 mg daily . d - penicillamine and bucillamine have been used for patients with rheumatoid arthritis as a disease modifying anti - rheumatic drug . d - penicillamine can cause various autoimmune diseases including pemphigus , pm , and myasthenia gravis ( 4 - 6 ) . although bucillamine has a chemical structure similar to d - penicillamine , autoimmune diseases are rarely associated with bucillamine . some cases of bucillamine - induced interstitial lung disease , nephropathy , and myasthenia gravis were described ( 7 - 9 ) . it was reported that the incidence of d - penicillamine - induced pemphigus was about 7% ( 5,10 ) and mean duration until the onset of pemphigus was 11 to 13 months ( 11,12 ) . it has been suggested that the epidermal cell surface proteins play a role as an antigen to the host through a sulfhydryl radical and other unknown factors ( 1 ) . in d - penicillamine- induced pemphigus , the prevalence of igg and iga antibodies directed against the intercellular region is lower than that in spontaneously occurring pemphigus ( 13 ) . it has been shown that these antibodies might not be detected in some cases of drug - induced pemphigus like our case ( 13,14 ) . to date , only 2 cases of bucillamine - induced pemphigus were reported in english literature . one was drug - induced pemphigus foliaceus with features of pemphigus vulgaris and the other was subcorneal pustular dermatosis - type iga pemphigus induced by thiol drugs ( 12,14 ) . the discontinuation of d - penicillamine may be inadequate in treatment because it frequently fails to stop disease progression.initial dose of corticosteroid is typically about 0.75 - 1 mg / kg / day . it has been suggested that high - dose or pulse therapy with steroids usually clears blisters promptly . prednisolone at a lower dose of 20 - 30 mg / day can maintain these patients without blistering but other adjuvant immunosuppressive drugs is need to achieve steroid - sparing effect or prevention of relapse ( 15,16 ) . bucillamine may cause pemphigus even at a low rate therefore we have to keep in mind that skin lesions with blisters should be carefully investigated in patients with bucillamine treatment .

backgroundhydronephrosis is a common congenital condition . the detection of fetal hydronephrosis by ultrasound presents a treatment dilemma . this study aims to examine postnatal follow - up and treatment for hydronephrosis diagnosed prenatally.material/methodsthis was a retrospective study of 210 infants with hydronephrosis diagnosed at the qilu hospital ( shangdong , china ) between january 2005 and january 2013 . the patient cohort was divided into four groups based on prenatal ultrasound examinations using the society for fetal urology ( sfu ) classification system . data on follow - up investigations and treatment methods were extracted from the charts and analyzed.resultspatients with sfu grade 1 , 2 , and 3 hydronephrosis ( n=125 , n=74 , and n=11 , respectively ) were followed for two years . in all , 2.4% , 18.9% , and 90.9% of patients with sfu grade 1 , 2 , and 3 hydronephrosis , respectively , underwent surgery . sfu grade 3 ( hr=9.23 , 95% ci : 1.4359.74 , p=0.02 ) , apd ( hr=2.81 , 95% ci : 1.117.10 , p=0.03 ) , and parenchymal thickness ( hr=0.42 , 95% ci : 0.240.71 , p=0.001 ) were independently associated with the occurrence of surgery . for anterioposterior diameter , using a cut - off point of 1.1 , the area under the curve was 0.86 , youden index was 0.556 , sensitivity was 70.4% , and specificity was 85.3% . for parenchymal thickness , using a cut - off point of 5 , auc was 0.79 , youden index was 0.478 , sensitivity was 74.1% , and specificity was 73.8%.conclusionspatients with sfu grade 2 hydronephrosis require long - term follow - up . surgery and close postsurgical observation may be necessary for patients with sfu grade 3 and 4 hydronephrosis . an initial b - mode ultrasound screening at 710 days after birth may help make an optimal diagnosis and treatment selection . bucillamine is a disease modifying anti - rheumatic drug , structurally similar to d - penicillamine . although d - penicillamine - induced pemphigus has been not infrequently demonstrated , pemphigus associated with bucillamine was rarely reported . we describe a patient complicating pemphigus vulgaris after bucillamine treatment in rheumatoid arthritis ( ra ) and polymyositis ( pm ) overlap syndrome . pm and ra overlap syndrome was diagnosed three years ago and bucillamine was administrated for 20 months . skin lesions including erythematous flaccid blisters on her chest , axillae , and back were occurred and were compatible with pemphigus vulgaris by typical pathology . withdrawal from bucillamine and prednisolone treatment made rapid improvement of pemphigus lesions .

the - and -lactam scaffolds are found in various biologically relevant compounds . additionally , these core structures are crucial intermediates in the synthesis of various natural products as well as pharmaceuticals , prompting the development of new methods for the synthesis of these valuable structures . our group has explored the synthesis of -lactams by the reactions of aryl- , thioaryl- , and cyano - substituted anhydrides . furthermore , we have demonstrated that one - pot four - component reactions ( 4crs ) are possible in the case of thioaryl substitution . recent computational studies of the cyanosuccinic anhydride reactions demonstrate that they proceed by a mannich - like mechanism via the enol tautomer of the anhydride . related reactions reported by castagnoli , cushman , and haimova likely proceed by this mechanism as well . consistent with the proposed mechanism , those anhydrides are poorly enolizable and require forcing conditions . two decades later , cushman and haimova reported analogous reactions with homophthalic anhydride , which is highly enolizable . the high reactivity of this anhydride at lower temperatures is consistent with the mannich - like mechanism . these results prompted us to explore mannich - type reactions and related 4crs of variously substituted anhydrides . although aryl - substituted succinic anhydrides showed some variation in reactivity based on the substituents attached to the aromatic ring , direct attachment of an electron - withdrawing group resulted in higher reactivity . furthermore , in the case of cyanosuccinic anhydrides , the diastereoselectivity was reversed relative to the aryl and thioaryl cases and could be controlled by resident stereogenic centers . we envisioned that the electron - withdrawing capability of a sulfone would impart the anhydride with similar reactivity as the nitrile - substituted anhydride , albeit with drastically different steric requirements ( scheme 1 ) . herein we report the development of anhydride mannich reactions ( amrs ) using sulfone - substituted anhydrides for the synthesis of - and -lactams as well as a six - step formal synthesis of ( )-isoretronecanol . multigram - scale syntheses of anhydrides 3a d were achieved from readily available starting materials . anhydride 3a was synthesized by conjugate addition of benzenesulfinic acid sodium salt ( 7 ) to maleic anhydride to yield a diacid , which was cyclized in one step ( scheme 2a ) . the syntheses of anhydrides 3b d were achieved by michael addition of sulfone acetate 8 to various tert - butyl acrylates followed by ester cleavage and cyclization ( scheme 2b ) . anhydride 3a readily reacted with imines to form -lactams . as with previous amrs , the carboxylic acid 5 ( table 1 ) is the first - formed product . unlike all previous related intermediates , these carboxylic acids were prone to facile decarboxylation . mild heating drove the decarboxylation to completion , allowing isolation of the sulfone - substituted lactams in high yields with only the anti diastereomer detectable by h nmr spectroscopy . in all cases , non - enolizable substrates the lack of reactivity of ketones and enolizable aldehydes in the amr probably stems from the rapid equilibration to the enamine tautomer , which is unreactive in the mannich addition step . the stereochemical configuration of the products was assigned by x - ray crystallography . lactam 10e has an anti configuration between the aromatic ring and the sulfonyl group . the other lactams were assigned by comparison of the coupling constants of the adjacent protons ( e.g. , 10f ; figure 1a ) . furthermore , acid intermediate 5f , which leads to 10f by decarboxylation , was also crystalline and was found to have an anti configuration between the sulfone and aromatic substituents ( figure 1b ) . our previous mechanistic work on the cyano - substituted anhydrides suggests that the anti - configured carboxylic acids are the kinetic products of the amrs . subsequent decarboxylation leads to the anti - sulfones , which would also be expected to be thermodynamically preferred . x - ray structures of 5f , 10f , and 10 g and j values for 10e and 10j . the stereochemical outcome of the reactions of imines with 3a is adequately explained by a transition state that is analogous to what is operative for cyanosuccinic anhydrides the iminium ion is attacked by the enolate of the anhydride , and the cyclic transition state is stabilized by hydrogen bonding to the sulfone . after the mannich reaction , subsequent rapid transannular acylation forms the lactam product.1 the carboxylic acid intermediates could be trapped with trimethylsilyldiazomethane ( tmschn2 ) . after many esterification conditions were tried , treatment of crude mixtures of 5 with a commercially available solution of tmschn2 resulted in reasonable yields of methyl esters 11a c ( table 2 ) . these intermediates could also be smoothly desulfonylated with magnesium in methanol to produce the corresponding esters 12 in good yield . although this reaction was successful for 11a and 11b , no ester product was observed in the attempted desulfonylation of 11c . unlike the analogous -lactams , these intermediates were impossible to isolate , thus preventing assignment of the configuration of the predominant diastereomer . upon decarboxylation , sulfone lactams 13 were formed as single isomers in high yields ( table 3 ) . determined by h this change in diastereoselectivity upon decarboxylation suggests the formation of a planar anion followed by selective protonation from one face of the anion . this result is in accordance with experimental data and models of sulfone anions proposed by corey . similar to our results with the succinic anhydride , the reaction tolerated a variety of non - enolizable aldehydes and a variety of alkylamines and anilines . while some of our initial reactions gave low yields ( 4050% ) , the use of commercially available imines or those purified by distillation significantly increased the yields of the lactam products . methyl - substituted anhydrides 3c and 3d provided trisubstituted -lactams in high yields with good diastereomeric ratios ( scheme 3 ) . these results suggest that substitution on the anhydride provides excellent facial selectivity , resulting in the formation of only two diastereomers of the intermediate carboxylic acids . studies are ongoing in our laboratory to develop an enantioselective michael addition with sulfone acetate 8 to provide the anhydrides as single enantiomers , which would presumably provide enantiomerically pure lactam products . this new amr was applied to a formal synthesis of ( )-isoretronecanol ( scheme 4 ) . the reaction of imine 16 with anhydride 3a followed by ring - closing metathesis provides the pyrrolizidine core of the natural product from cinnamaldehyde , allylamine , and 3a in two steps . the elimination of 18 yields 21 , which has been converted to ( )-isoretronecanol in two steps in low yield . further reduction yields lactam ester 22 , which has been converted to ( )-isoretronecanol in one step . while this second route is redox - inefficient , it provides material in a much higher yield and avoids the high - pressure conditions necessary to reduce pyrrole 21 . this synthetic route showcases the versatility of the anhydride michael reaction of the sulfone - substituted anhydride , exemplifying its usefulness in the assembly of heterocyclic targets . this synthesis demonstrates that the amr of sulfone - substituted anhydrides will be useful for the assembly of many heterocyclic targets , natural and otherwise . in summary , we have successfully synthesized various sulfone - substituted - and -lactams in good yields with high diastereoselectivities ( > 95:5 ) by an anhydride mannich reaction . we have previously studied the reactivity of several different substituted succinic anhydrides , but these are the first new results employing glutaric anhydyrides since the original work of castagnoli . unlike previous reactions of this type to date , the carboxylic acid products were prone to decarboxylation . although the -lactam acids derived from the substituted succinic anhydride could be trapped as esters , the analogous -lactam products could not be intercepted . another important difference was observed in the diastereoselectivity of the initial mannich - type reaction . although the reactions of succinic anhydyrides were very selective , the acid products derived from the glutaric anhydrides were generally formed as diastereomeric mixtures . in both cases , we have shown that this methodology is highly robust and have showcased it in the formal synthesis of a biologically active natural product . unless otherwise specified , all commercially available reagents were used as received . all reactions using dried solvents were carried out under an atmosphere of argon in flame - dried glassware with magnetic stirring . dry solvent was dispensed from a solvent purification system that passes solvent through two columns of dry neutral alumina . h nmr spectra and proton - decoupled c nmr spectra were obtained on a 400 or 600 mhz nmr spectrometer . chemical shifts ( ) are reported in parts per million ( ppm ) relative to residual solvent ( chcl3 , s , 7.26 ) . multiplicities are given as s ( singlet ) , d ( doublet ) , t ( triplet ) , dd ( doublet of doublets ) , m ( multiplet ) , br m ( broad multiplet ) , or br s ( broad singlet ) ; c nmr chemical shifts are reported relative to cdcl3 ( t , 77.23 ) unless otherwise noted . high - resolution mass spectra were recorded in positive esi mode in methanol or acetonitrile . silica gel chromatographic purifications were performed by flash chromatography with silica gel packed in glass columns . the eluting solvent for each purification was determined by thin - layer chromatography ( tlc ) on glass plates coated with silica gel and visualized by uv light or by staining with ceric ammonium molybdate ( cam ) followed by gentle heating . the following abbreviations are used throughout : ethyl acetate ( etoac ) , hexanes ( hex ) , dichloromethane ( dcm ) , triethylamine ( tea ) , diisopropylethylamine ( dipea ) . benzenesulfinic acid sodium salt ( 10.0 g , 60.9 mmol ) was dissolved in 100 ml of h2o at 0 c . then 10% hcl was added , and the mixture was extracted two times with etoac . the combined organic layers were dried over na2so4 , and the solvent was removed in vacuo to yield a white solid , which was dissolved in 50 ml of toluene . ac2o ( 70 ml , 20 mmol ) was added , and the reaction mixture was heated at reflux for 3 h. the solvent was evaporated in vacuo , and the resulting brown solid was washed several times with cold dcm and collected in a fritted funnel , yielding a white to light - brown solid ( 6.8 g , 46% yield over the two steps ) . mp 132.4133.1 c ( dec . ) ; h nmr ( 400 mhz , cdcl3 ) 7.96 ( dd , j = 8.4 , 1.3 hz , 2h ) , 7.867.76 ( m , 1h ) , 7.727.63 ( m , 2h ) , 4.52 ( dd , j = 10.3 , 4.4 hz , 1h ) , 3.68 ( dd , j = 19.9 ( gem ) , 4.4 hz , 1h ) , 3.39 ( dd , j = 19.7 ( gem ) , 10.6 hz , 1h ) ; c nmr ( 100 mhz , cdcl3 ) 166.0 , 162.9 , 135.8 , 135.4 , 129.9 , 129.7 , 64.4 , 30.1 ; ir ( neat ) 1782 , 1739 ( c = o ) , 1169 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + h ] calcd for c10h9o5s 241.0171 , found 241.0165 . the amine and the aldehyde were dissolved in thf ( 0.10 m ) , and 3 equiv of triethyl orthoformate was added . the anhydride was added , and then the reaction mixture was allowed to stir for 3 h. water was added to the mixture ( final concentration 0.05 mmol ) , followed by the addition of 3 equiv of k2co3 and heating at reflux for 4 h , unless otherwise stated . the reaction mixture was allowed to cool to rt and then extracted two times with etoac . the solvent was evaporated in vacuo , and the crude mixture was purified by flash chromatography using an etoac/3050% gradient , unless otherwise stated . general procedure a was used with sulfone anhydride 3a ( 0.24 g , 0.99 mmol ) , benzylamine ( 109 l , 0.99 mmol ) , and benzaldehyde ( 102 l , 1.0 mmol ) to yield 10a ( 0.32 g , 83% ) as a foam ( 50:50 etoac / hex , rf = 0.59 ) . h nmr ( 600 mhz , cdcl3 ) 7.687.64 ( m , 2h ) , 7.62 ( tt , j = 7.5 , 1.3 hz , 1h ) , 7.487.41 ( m , 2h ) , 7.327.23 ( m , 6h ) , 7.097.06 ( m , 2h ) , 6.836.79 ( m , 2h ) , 5.09 ( d , j = 14.9 hz , 1h ) , 4.70 ( d , j = 3.3 hz , 1h ) , 3.66 ( ddd , j = 9.9 , 4.4 , 3.3 hz , 1h ) , 3.44 ( d , j = 14.9 hz , 1h ) , 3.03 ( dd , j = 18.2 ( gem ) , 4.3 hz , 1h ) , 2.95 ( ddd , j = 18.2 ( gem ) , 9.9 , 1.2 hz , 1h ) ; c nmr ( 101 mhz , cdcl3 ) 170.5 , 137.7 , 136.4 , 134.7 , 134.4 , 129.5 , 129.3 , 128.9 , 128.7 , 128.7 , 128.4 , 127.8 , 126.1 , 63.5 , 60.6 , 44.5 , 30.7 ; ir ( thin film ) 1692 ( c = o ) , 1322 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h21no3sna 414.1140 , found 414.1120 . general procedure a was used with sulfone anhydride 3a ( 0.30 g , 1.26 mmol ) and ( e)-n - methyl-1-phenylmethanimine ( 156 l , 1.26 mmol ) . because the commercially available imine was used in this experiment , the anhydride was added immediately after the imine was dissolved instead of waiting for 3 h or overnight . the reaction yielded 10b ( 0.332 g , 83% ) as a foam ( 50:50 etoac / hex , rf = 0.35 ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( d , j = 7.5 hz , 2h ) , 7.69 ( t , j = 7.5 hz , 1h ) , 7.58 ( t , j = 7.8 hz , 2h ) , 7.367.31 ( m , 3h ) , 7.067.02 ( m , 2h ) , 4.97 ( d , j = 3.5 hz , 1h ) , 3.66 ( dt , j = 9.8 , 4.1 hz , 1h ) , 2.90 ( dd , j = 18.1 ( gem ) , 4.7 hz , 1h ) , 2.82 ( dd , j = 18.1 ( gem ) , 9.8 hz , 1h ) , 2.64 ( s , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.6 , 138.2 , 136.8 , 134.6 , 129.7 , 129.5 , 129.0 , 128.9 , 126.1 , 64.3 , 63.6 , 31.0 , 28.4 ; ir ( thin film ) 1690 ( c = o ) , 1308 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h17no3sna 338.0827 , found 338.0818 . general procedure a was used with sulfone anhydride 3a ( 0.240 g , 1.00 mmol ) , prop-2-yn-1-amine ( 64.0 l , 1.00 mmol ) , and benzaldehyde ( 102 l , 1.00 mmol ) to yield 10c ( 0.182 g , 54% ) as a foam . the residue was purified by flash column chromatography ( 50:50 etoac / hex , rf = 0.53 ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( m , 2h ) , 7.68 ( t , j = 7.4 hz , 1h ) , 7.607.53 ( t , 2h ) , 7.357.29 ( m , 3h ) , 7.077.02 ( m , 2h ) , 5.23 ( d , j = 4.0 hz , 1h ) , 4.51 ( dd , j = 17.6 , 2.6 hz , 1h ) , 3.75 ( ddd , j = 9.4 , 5.1 , 4.0 hz , 1h ) , 3.253.20 ( m , 1h ) , 2.96 ( dd , j = 18.2 ( gem ) , 5.1 hz , 1h ) , 2.89 ( ddd , j = 18.2 ( gem ) , 9.8 , 1.1 hz , 1h ) , 2.25 ( t , j = 2.5 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 169.9 , 137.3 , 136.6 , 134.6 , 129.6 , 129.4 , 129.0 , 128.9 , 126.4 , 76.2 , 73.3 , 63.9 , 60.9 , 31.1 , 30.5 ; ir ( thin film ) 1699 ( c = o ) , 1414 , 1322 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h17no3sna 362.0827 , found 362.0831 . general procedure a was used with sulfone anhydride 3a ( 0.24 g , 0.99 mmol ) , propylamine ( 82 l , 0.99 mmol ) , and furan-2-carbaldehyde ( 83 l , 1.00 mmol ) to yield 10d ( 0.167 g , 50% ) as a foam . the residue was purified by flash column chromatography ( 50:50 etoac / hex , rf = 0.47 ) . h nmr ( 600 mhz , cdcl3 ) 7.907.86 ( m , 2h ) , 7.707.65 ( m , 1h ) , 7.57 ( dd , j = 8.3 , 7.2 hz , 2h ) , 7.32 ( d , j = 1.8 hz , 1h ) , 6.27 ( dd , j = 3.3 , 1.9 hz , 1h ) , 6.19 ( d , j = 3.3 hz , 1h ) , 5.07 ( d , j = 4.1 hz , 1h ) , 3.96 ( ddd , j = 8.9 , 6.1 , 4.1 hz , 1h ) , 3.40 ( ddd , j = 13.9 , 8.8 , 7.1 hz , 1h ) , 2.952.85 ( m , 2h ) , 2.65 ( ddd , j = 13.9 , 8.6 , 5.2 hz , 1h ) , 1.471.37 ( m , 1h ) , 1.331.24 ( m , 1h ) , 0.82 ( t , j = 7.4 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 169.9 , 149.4 , 143.5 , 136.7 , 134.5 , 129.6 , 128.6 , 110.6 , 109.8 , 60.8 , 55.2 , 42.7 , 31.2 , 20.1 , 11.1 ; ir ( thin film ) 1690 ( c = o ) , 1416 , 1308 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h19no4sna 356.0932 , found 356.0929 . general procedure a was used with sulfone anhydride 3a ( 0.3 g , 1.2 mmol ) , propylamine ( 100 l , 1.2 mmol ) , and benzaldehyde ( 126 l , 1.2 mmol ) to yield 10e ( 0.36 g , 87% ) as a solid ( 50:50 etoac / hex , rf = 0.36 ) . mp 149.8150.4 c ; h nmr ( 400 mhz , cdcl3 ) 7.89 ( dt , j = 8.5 , 1.4 hz , 2h ) , 7.767.54 ( m , 3h ) , 7.437.24 ( m , 3h ) , 7.136.98 ( m , 2h ) , 5.09 ( d , j = 3.5 hz , 1h ) , 3.703.56 ( m , 2h ) , 2.972.76 ( m , 2h ) , 2.54 ( dddd , j = 13.7 , 8.4 , 5.1 , 1.1 hz , 1h ) , 1.551.31 ( m , 2h ) , 0.85 ( td , j = 7.3 , 1.2 hz , 3h ) ; c nmr ( 100 mhz , cdcl3 ) 170.6 , 138.4 , 136.7 , 134.5 , 129.6 , 129.3 , 128.8 , 128.7 , 126.0 , 63.8 , 61.1 , 42.6 , 30.9 , 19.9 , 11.1 ; ir ( thin film ) 1688 ( c = o ) cm ; hrms ( esi - tof ) m / z [ m h ] calcd for c19h20no3s 342.1164 , found 342.1155 . general procedure a ( except that the decarboxylation was performed at room temperature overnight ) was used with sulfone anhydride 3a ( 0.3 g , 1.2 mmol ) , propylamine ( 102 l , 1.2 mmol ) , and 4-cyanobenzaldehyde ( 0.164 g , 1.3 mmol ) to yield 10f ( 0.36 g , 80% ) as a foam ( 50:50 etoac / hex , rf = 0.35 ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( dt , j = 8.5 , 1.8 hz , 2h ) , 7.777.54 ( m , 5h ) , 7.26 ( m , 2h ) , 5.20 ( d , j = 3.6 hz , 1h ) , 3.67 ( dt , j = 13.7 , 8.0 hz , 1h ) , 3.57 ( dt , j = 10.0 , 4.3 hz , 1h ) , 2.88 ( dd , j = 18.2 ( gem ) , 4.5 hz , 1h ) , 2.76 ( dd , j = 18.1 ( gem ) , 10.0 hz , 1h ) , 2.52 ( ddd , j = 13.8 , 8.6 , 5.0 hz , 1h ) , 1.44 ( m , 2h ) , 0.87 ( t , j = 7.3 hz , 3h ) ; c nmr ( 600 mhz , cdcl3 ) 170.7 , 144.1 , 136.6 , 134.9 , 133.3 , 129.9 , 128.9 , 127.2 , 118.1 , 113.1 , 63.8 , 60.6 , 43.1 , 31.3 , 20.1 , 11.2 ; ir ( thin film ) 1692 ( c = o ) , 1445 , 1412 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h20n2o3sna 391.1092 , found 391.1098 . general procedure a was used with sulfone anhydride 3a ( 88 mg , 0.36 mmol ) , propylamine ( 30 l , 0.36 mmol ) , and aldehyde ( 50 mg , 0.37 mmol ) to yield 10 g ( 87 mg , 65% ) as a solid ( 50:50 etoac / hex , rf = 0.17 ) . mp 125.3126.1 c ; h nmr ( 600 mhz , cdcl3 ) 7.927.86 ( m , 2h ) , 7.697.62 ( m , 1h ) , 7.55 ( t , j = 7.9 hz , 2h ) , 7.28 ( m , 1h ) , 6.936.82 ( m , 3h ) , 5.16 ( s , 1h ) , 3.80 ( d , j = 9.8 hz , 1h ) , 3.69 ( s , 3h ) , 3.49 ( dt , j = 13.5 , 8.1 hz , 1h ) , 2.96 ( dd , j = 17.9 ( gem ) , 4.0 hz , 1h ) , 2.85 ( dd , j = 17.9 ( gem ) , 9.9 hz , 1h ) , 2.48 ( ddd , j = 13.7 , 8.5 , 5.2 hz , 1h ) , 1.461.28 ( m , 2h ) , 0.82 ( t , j = 7.3 hz , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 171.2 , 157.5 , 137.7 , 134.2 , 130.5 , 129.5 ( 2c ) , 129.0 ( 2c ) , 125.7 , 121.1 , 111.5 , 62.8 , 55.6 , 42.8 , 31.7 , 20.3 , 11.3 ; ir ( thin film ) 1681 ( c = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no4sna 396.1245 , found 396.1252 . general procedure a was used with sulfone anhydride 3a ( 0.18 g , 0.73 mmol ) , benzylamine ( 76 l , 0.69 mmol ) , and aldehyde ( 0.1 ml , 0.79 mmol ) to yield 10h ( 0.25 g , 82% ) as a foam ( 50:50 etoac / hex , rf = 0.36 ) . h nmr ( 600 mhz , cdcl3 ) 7.817.74 ( m , 2h ) , 7.657.14 ( m , 13h ) , 6.12 ( d , j = 15.8 hz , 1h ) , 5.74 ( dd , j = 15.6 , 8.5 hz , 1h ) , 4.93 ( d , j = 15.0 hz , 1h ) , 4.38 ( dd , j = 8.8 , 4.3 hz , 1h ) , 3.90 ( d , j = 15.0 hz , 1h ) , 3.65 ( dt , j = 9.9 , 5.1 hz , 1h ) , 3.01 ( dd , j = 18.0 ( gem ) , 5.5 hz , 1h ) , 2.86 ( dd , j = 18.1 ( gem ) , 10.0 hz , 1h ) ; c nmr ( 151 mhz , cdcl3 ) 170.1 , 136.8 , 135.3 , 135.2 , 135.0 , 134.4 , 129.5 , 128.8 , 128.7 , 128.7 , 128.4 , 127.7 , 126.8 , 126.7 , 125.3 , 61.7 , 59.9 , 44.7 , 30.8 ; ir ( thin film ) 1690 ( c = o ) , 1146 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c25h23no3sna 440.1296 , found 440.1282 . general procedure a was used with sulfone anhydride 3a ( 0.3 g , 1.26 mmol ) , isopropylamine ( 100 l , 1.22 mmol ) , and benzaldehyde ( 128 l , 1.26 mmol ) to yield 10i ( 0.297 g , 70% ) as a foam ( 50:50 etoac / hex , rf = 0.42 ) . h nmr ( 600 mhz , cdcl3 ) 7.987.86 ( m , 2h ) , 7.72 ( tt , j = 7.2 , 1.5 hz , 1h ) , 7.687.56 ( m , 2h ) , 7.407.26 ( m , 3h ) , 7.176.97 ( m , 2h ) , 5.10 ( d , j = 2.3 hz , 1h ) , 4.203.98 ( m , 1h ) , 3.57 ( ddd , j = 9.6 , 2.6 , 2.1 hz , 1h ) , 2.93 ( ddd , j = 18.2 ( gem ) , 9.6 , 1.9 hz , 1h ) , 2.882.75 ( m , 1h ) , 1.21 ( dd , j = 6.8 , 2.0 hz , 3h ) , 0.85 ( dd , j = 6.9 , 1.9 hz , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 170.7 , 140.7 , 136.6 , 134.5 , 129.6 , 129.2 , 128.8 , 128.7 , 125.9 , 64.5 , 60.1 , 45.5 , 31.2 , 20.8 , 19.5 ; ir ( thin film ) 1693 ( c = o ) , 1145 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h21no3sna 366.1140 , found 366.1147 . general procedure a was used with sulfone anhydride 3a ( 0.30 g , 1.26 mmol ) , allylamine ( 93 l , 1.24 mmol ) , and benzaldehyde ( 128 l , 1.26 mmol ) to yield 10j ( 0.33 g , 78% ) as a foam ( 50:50 etoac / hex , rf = 0.42 ) . h nmr ( 600 mhz , cdcl3 ) 7.88 ( dt , j = 8.5 , 2.0 hz , 2h ) , 7.777.67 ( m , 1h ) , 7.59 ( m , 2h ) , 7.437.27 ( m , 3h ) , 6.99 ( m , 2h ) , 5.55 ( dddd , j = 17.6 , 10.0 , 7.8 , 4.3 hz , 1h ) , 5.235.15 ( m , 1h ) , 5.145.02 ( m , 2h ) , 4.40 ( ddt , j = 15.4 , 3.9 , 1.9 hz , 1h ) , 3.753.65 ( m , 1h ) , 2.95 ( m , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 170.42 , 138.2 , 136.8 , 134.5 , 131.0 , 129.7 , 129.5 , 129.4 , 128.9 , 126.2 , 119.0 , 63.9 , 60.7 , 43.4 , 31.0 ; ir ( thin film ) 1691 ( c = o ) , 1145 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h19no3sna 364.0983 , found 364.0974 . general procedure a was used with sulfone anhydride 3a ( 0.30 g , 1.2 mmol ) , propylamine ( 100 l , 1.2 mmol ) , and 4-methoxybenzaldehyde ( 151 l , 1.2 mmol ) to yield 10k ( 0.38 g , 84% ) as a foam ( 50:50 etoac / hex , rf = 0.27 ) . h nmr ( 600 mhz , cdcl3 ) 7.927.86 ( d , j = 7.8 hz , 2h ) , 7.69 ( m , 1h ) , 7.58 ( t , j = 7.8 hz , 2h ) , 6.96 ( dd , j = 9.2 , 2.5 hz , 2h ) , 6.84 ( dd , j = 9.0 , 2.5 hz , 2h ) , 5.04 ( d , j = 3.5 hz , 1h ) , 3.80 ( sharp s , 3h ) , 3.663.53 ( m , 2h ) , 2.90 ( dd , j = 18.2 ( gem ) , 4.7 hz , 1h ) , 2.852.76 ( dd , j = 18.2 ( gem ) , 10.2 hz , 1h ) , 2.52 ( m , 1h ) , 1.511.32 ( m , 2h ) , 0.880.79 ( t , j = 7.8 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.4 , 159.9 , 136.9 , 134.4 , 130.3 , 129.6 , 128.8 , 127.4 , 114.7 , 64.1 , 60.7 , 55.3 , 42.5 , 31.1 , 20.0 , 11.1 ; ir ( thin film ) 1687 ( c = o ) , 1354 , 1144 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no4sna 396.1245 , found 396.1252 . n - benzylidenebenzylamine ( 0.1 ml , 0.53 mmol ) was added to a round - bottom flask containing na2so4 ( 0.37 g , 2.6 mmol ) and thf ( 6 ml ) , and then sulfone anhydride 3a ( 0.13 g , 0.54 mmol ) was added . the reaction mixture was allowed to stir for 1.5 h , after which the na2so4 was removed by filtration and the solvent was removed in vacuo . the residue was redissolved in etoac , transferred to a separatory funnel , and washed with hcl ( 1.2 m or 10% ) and brine . the combined organic layers were dried over na2so4 , and the solvent removed in vacuo . the resulting acid was redissolved in a mixture of toluene and methanol ( 12 ml:7 ml ) and cooled to 0 c . to the cold solution , tmschn2 ( 0.53 ml , 1.1 mmol , 2 m solution in hexanes ) after 2 h the solvent was removed in vacuo , and the resulting material was purified by column chromatography ( 3060% etoac : hex ) to yield 11a ( 0.157 g , 66% over the two steps ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.47 ) . h nmr ( 600 mhz , cdcl3 ) 7.647.18 ( m , 15h ) , 5.21 ( d , j = 14.5 hz , 1h ) , 4.89 ( s , 1h ) , 3.70 ( d , j = 18.7 ( gem ) hz , 1h ) , 3.383.30 ( m , 2h ) , 3.13 ( s , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.0 , 164.9 , 134.9 , 134.8 , 134.8 , 134.4 , 131.1 , 129.6 , 129.5 , 129.5 , 129.0 , 128.9 , 128.9 , 128.2 , 76.2 , 63.9 , 52.7 , 44.9 , 35.0 ; ir ( thin film ) 1783 , 1784 ( c = o ) , 1124 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c25h23no5sna 472.1195 , found 472.1176 . propylamine ( 68 l , 0.83 mmol ) and 3,5-dimethoxybenzaldehyde ( 0.14 g , 0.84 mmol ) were dissolved in thf ( 8 ml ) . na2so4 ( 0.59 g , 4.2 mmol ) was added , and the mixture was allowed to stir for 1.5 h. sulfone anhydride 3a ( 0.20 g , 0.83 mmol ) was added , and the reaction was allowed to stir for an additional 1 h. the na2so4 was filtered off , and the solvent was removed in vacuo . the residue was redissolved in etoac , transferred to a separatory funnel , and washed with hcl ( 1.2 m or 10% ) and brine . the combined organic layers were dried over na2so4 , and the solvent was removed in vacuo . the resulting crude acid was dissolved in a mixture of toluene and methanol ( 13 ml:7 ml ) and cooled to 0 c . to the cold solution , tmschn2 ( 0.83 ml , 1.7 mmol , 2 m solution in hexanes ) was added dropwise over a period of 5 min , and the reaction was monitored by tlc for the disappearance of starting material . after 2 h the solvent was removed in vacuo , and the resulting material was purified by column chromatography ( 3060% etoac : hex ) to yield 11b ( 0.22 g , 57% over the two steps ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.31 ) . h nmr ( 600 mhz , cdcl3 ) 8.017.83 ( m , 2h ) , 7.817.68 ( m , 1h ) , 7.687.52 ( m , 2h ) , 6.39 ( d , j = 2.3 hz , 1h ) , 6.22 ( s , 2h ) , 5.38 ( s , 1h ) , 3.74 ( s , 6h ) , 3.663.59 ( m , 1h ) , 3.55 ( d , j = 18.4 ( gem ) hz , 1h ) , 3.26 ( m , 3h ) , 3.14 ( d , j = 18.5 ( gem ) hz , 1h ) , 2.58 ( ddd , j = 13.8 , 8.7 , 5.2 hz , 1h ) , 1.651.32 ( m , 2h ) , 0.92 ( t , j = 7.3 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.9 , 164.7 , 160.9 , 137.3 , 135.3 , 134.9 , 130.4 , 129.2 , 100.8 , 76.7 , 64.3 , 55.4 , 52.8 , 43.2 , 35.8 , 20.2 ( 2c ) , 11.3 ; ir ( thin film ) 1748 , 1702 ( c = o ) , 1144 , 1457 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h27no7sna 484.1406 , found 484.1404 . n - benzylidenemethylamine ( 0.1 ml , 0.81 mmol ) was added to a round - bottom flask containing na2so4 ( 0.57 g , 4.0 mmol ) and thf ( 8 ml ) , and then sulfone anhydride 3a ( 0.19 g , 0.80 mmol ) was added . the reaction mixture was allowed to stir for 1.5 h , after which the na2so4 was removed by filtration and the solvent removed in vacuo . the residue was redissolved in etoac , transferred to a separatory funnel , and washed with hcl ( 1.2 m or 10% ) and brine . the combined organic layers were dried over na2so4 , and the solvent was removed in vacuo . the crude acid was redissolved in a mixture of toluene and methanol ( 13 ml:7 ml ) and cooled to 0 c . to the cold solution , tmschn2 ( 0.81 ml , 1.6 mmol , 2 m solution in hexanes ) was added dropwise over a period of 5 min , and the reaction was monitored by tlc for the disappearance of starting material . after 2 h the solvent was removed in vacuo , and the resulting material was purified by column chromatography ( 3060% etoac : hex ) to yield 11c ( 0.20 g , 67% over the two steps ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.28 ) . h nmr ( 400 mhz , cdcl3 ) 7.88 ( dt , j = 8.6 , 2.2 hz , 2h ) , 7.727.63 ( m , 1h ) , 7.647.50 ( m , 2h ) , 7.367.23 ( m , 4h ) , 7.05 ( d , j = 7.2 hz , 2h ) , 5.31 ( s , 1h ) , 3.49 ( d , j = 18.4 ( gem ) hz , 1h ) , 3.15 ( d , j = 2.3 hz , 3h ) , 3.01 ( d , j = 18.5 ( gem ) hz , 1h ) , 2.58 ( s , 2h ) ; c nmr ( 100 mhz , cdcl3 ) 169.75 , 164.51 , 135.32 , 135.1 , 134.3 , 130.2 , 129.4 , 129.3 , 128.7 , 127.6 , 76.7 , 66.5 , 52.9 , 35.5 , 28.3 ; ir ( thin film ) 1736 , 1690 ( c = o ) , 1177 , 1360 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h19no5sna 396.0882 , found 396.0887 . ester 11a ( 0.104 g , 0.23 mmol ) was dissolved in anhydrous meoh ( 12 ml ) , and magnesium turnings ( 169 mg , 6.9 mmol , flame - dried under argon ) were added to the reaction mixture . the mixture was heated to 50 c for 15 min and then to 64 c for 1.5 h , cooled to rt , poured into an erlenmeyer flask containing 10% hcl ( 20 ml ) , and extracted with diethyl ether . the organic layer was dried over na2so4 , and the solvent was removed in vacuo . purification by flash chromatography ( 3050% etoac / hex ) yielded 12a as an oil ( 0.54 g , 76% ) . h nmr data for the major diastereomer ( 600 mhz , cdcl3 ) : 7.417.33 ( m , 4h ) , 7.307.23 ( m , 3h ) , 7.187.13 ( m , 1h ) , 7.057.01 ( m , 2h ) , 5.11 ( d , j = 14.7 hz , 1h ) , 4.634.56 ( d , j = 5.4 hz , 1h ) , 3.64 ( s , 3h ) , 3.48 ( d , j = 14.9 hz , 1h ) , 3.07 ( m , 1h ) , 2.942.75 ( m , 2h ) . the h nmr data are in accordance with the literature . ester 11b ( 75.4 mg , 0.163 mmol ) was dissolved in anhydrous meoh ( 12 ml ) , and magnesium turnings ( 162 mg , 6.67 mmol , flame - dried under argon ) were added to the reaction mixture . the mixture was heated to 50 c for 15 min and then to 64 c for 1.5 h , cooled to rt , poured into an erlenmeyer flask containing 10% hcl ( 30 ml ) , and extracted with diethyl ether . the organic layer was dried over na2so4 , and the solvent was removed in vacuo . purification by flash chromatography ( 3050% etoac / hex ) yielded 12b as an oil ( 0.35 g , 67% ) . h nmr ( 400 mhz , cdcl3 ) 6.42 ( t , j = 2.2 hz , 1h ) , 6.35 ( d , j = 2.3 hz , 2h ) , 4.83 ( d , j = 5.3 hz , 1h ) , 3.79 ( s , 6h ) , 3.74 ( s , 3h ) , 3.66 ( dt , j = 13.6 , 8.1 hz , 1h ) , 3.04 ( ddd , j = 9.6 , 7.0 , 5.2 hz , 1h ) , 2.84 ( dd , j = 17.2 , 9.8 hz , 1h ) , 2.76 ( d , j = 7.0 hz , 1h ) , 2.61 ( ddd , j = 13.6 , 8.3 , 5.2 hz , 1h ) , 1.541.37 ( m , 2h ) , 0.84 ( t , j = 7.4 hz , 3h ) ; c nmr ( 100 mhz , cdcl3 ) 173.1 , 172.7 , 161.5 , 142.0 , 104.5 , 100.1 , 64.2 , 55.5 , 52.6 , 45.9 , 42.5 , 33.8 , 20.2 , 11.3 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h23no5na 344.1474 , found 344.1465 . to a 500 ml round - bottom flask were added benzenesulfinic acid sodium salt ( 5.1 g , 30.8 mmol ) , tert - butyl bromoacetate ( 3.8 ml , 25.6 mmol ) , and 170 ml of ethanol . the crude mixture was suspended in et2o ( 150 ml ) and washed with water ( 2 150 ml ) and brine ( 150 ml ) . the organic layer was dried over sodium sulfate and concentrated in vacuo to yield 8 ( 6.31 g , 96% ) as a clear oil . h nmr ( 300 mhz , cdcl3 ) 7.90 ( m , 2h ) , 7.68 ( m , 1h ) , 7.55 ( m , 2h ) , 4.03 ( s , 2h ) , 1.33 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 ) 161.2 , 138.8 , 134.1 , 129.1 , 128.5 , 83.6 , 62.0 , 27.6 . these data are in accordance with the literature . to a dry 250 ml round - bottom flask were added 8 ( 6.31 g , 24.6 mmol ) , cesium carbonate ( 400 mg , 1.23 mmol ) , tert - butyl acrylate ( 3.6 ml , 24.6 mmol ) , and 80 ml of acetonitrile . the reaction mixture was heated to 50 c overnight and then diluted with water and extracted with ethyl acetate ( 3 100 ml ) . the crude product was purified on silica gel ( 20:80 etoac / hex ) to yield 3ba ( 4.91 g , 52% ) as an amorphous solid . h nmr ( 300 mhz , cdcl3 ) 7.89 ( d , j = 7.3 hz , 2h ) , 7.68 ( m , 1h ) , 7.57 ( dd , j = 8.4 , 6.9 hz , 2h ) , 3.99 ( m , 1h ) , 2.24 ( m , 4h ) , 1.42 ( s , 9h ) , 1.35 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 ) 170.9 , 164.4 , 137.4 , 134.1 , 129.4 , 129.0 , 83.5 , 81.0 , 70.1 , 32.1 , 28.0 , 27.7 , 22.3 ; ir ( thin film ) 2979 , 1730 , 1309 , 1138 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h28o6sna 407.1504 , found 407.1502 . to a dry 250 ml round - bottom flask were added 8 ( 3.00 g , 11.7 mmol ) , cesium carbonate ( 382 mg , 1.17 mmol ) , tert - butyl crotonate ( 1.90 ml , 11.7 mmol ) , and 40 ml of acetonitrile . the reaction mixture was heated to reflux overnight and then diluted with water and extracted with ethyl acetate ( 3 100 ml ) . the crude product was purified on silica gel ( 20:80 etoac / hex ) to yield 3ca ( 3.75 g , 80% ) as an amorphous solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 600 mhz , cdcl3 ) 7.90 ( m , 3h ) , 7.64 ( m , 2h ) , 7.53 ( m , 3h ) , 4.10 ( d , j = 8.0 hz , 1h ) , 2.71 ( m , 2h ) , 2.43 ( m , 1h ) , 1.42 ( s , 16h , overlap with minor diastereomer ) , 1.27 ( s , 14h , overlap with minor diastereomer ) , 1.12 ( d , j = 6.7 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.7 , 164.4 , 138.4 , 134.0 , 129.2 , 128.9 , 83.3 , 80.8 , 73.7 , 39.9 , 29.6 , 28.1 , 27.6 , 17.1 ; ir ( thin film ) 2980 , 1729 , 1325 , 1135 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h30o6sna 421.1661 , found 421.1653 . to a dry 250 ml round - bottom flask were added 8 ( 3.00 g , 11.7 mmol ) , cesium carbonate ( 382 mg , 1.17 mmol ) , tert - butyl methacrylate ( 1.90 ml , 11.7 mmol ) , and 40 ml of acetonitrile . the reaction mixture was heated to reflux for 48 h and then diluted with water and extracted with ethyl acetate ( 3 100 ml ) . the crude product was purified on silica gel ( 20:80 etoac / hex ) to yield 3da ( 4.20 g , 90% ) as an amorphous solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 400 mhz , cdcl3 ) 7.89 ( m , 2h ) , 7.69 ( m , 1h ) , 7.58 ( m , 2h ) , 4.03 ( dd , j = 11.9 , 3.3 hz , 1h ) , 2.28 ( m , 2h ) , 1.99 ( m , 1h ) , 1.43 ( s , 9h ) , 1.37 ( s , 9h ) , 1.16 ( d , j = 6.6 hz , 3h ) ; c nmr ( 101 mhz , cdcl3 ) 174.2 , 164.7 , 137.6 , 134.3 , 129.5 , 129.2 , 83.6 , 81.1 , 69.6 , 38.2 , 30.6 , 28.2 , 27.8 , 18.4 ; ir ( thin film ) 2978 , 2936 , 1727 , 1325 , 1138 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h30o6sna 421.1661 , found 421.1662 . to a dry 250 ml flask were added 3ba ( 4.91 g , 12.7 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . the reaction mixture was stirred for 1 h and then concentrated in vacuo and azeotropically distilled with ch2cl2 ( 3 10 ml ) . the crude mixture was taken up in 45 ml of trifluoroacetic anhydride and stirred overnight . the reaction mixture was concentrated in vacuo and azeotroped with toluene ( 3 10 ml ) . filtering the off - white solid with diethyl ether yielded 3b ( 3.01 g , 93% ) as a white solid . mp 122.2122.8 c ; h nmr ( 600 mhz , cd3cn ) 7.97 ( m , 2h ) , 7.83 ( m , 1h ) , 7.70 ( m , 2h ) , 4.55 ( m , 1h ) , 3.06 ( m , 1h ) , 2.87 ( m , 1h ) , 2.50 ( m , 2h ) ; c nmr ( 150 mhz , cd3cn ) 165.3 , 160.7 , 137.3 , 135.0 , 129.5 , 129.0 , 63.4 , 27.1 , 17.1 ; ir ( thin film ) 3098 , 2929 , 1816 , 1746 1322 , 1152 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c11h10o5sna 277.0147 , found 277.0145 . to a dry 250 ml flask were added 3ca ( 3.73 g , 9.30 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . the reaction mixture was stirred for 1 h and then concentrated in vacuo and azeotropically distilled with ch2cl2 ( 3 10 ml ) . to the crude mixture was added acetic anhydride ( 33 ml ) , and the mixture was taken up in 100 ml of toluene and stirred overnight . the mixture was concentrated in vacuo and azeotroped with benzene ( 3 10 ml ) . trituration with diethyl ether afforded 3c ( 2.31 g , 92% ) as a solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 600 mhz , cd3cn ) 7.93 ( m , 2h ) , 7.84 ( m , 1h ) , 7.70 ( m , 2h ) , 4.40 ( m , 1h ) , 3.29 ( m , 1h ) , 3.03 ( m , 1h ) , 2.75 ( m , 1h ) , 1.18 ( m , 3h ) ; c nmr ( 151 mhz , cd3cn ) 164.8 , 160.4 , 135.2 , 129.6 , 129.3 , 128.9 , 69.6 , 33.9 , 24.4 , 19.4 ; ir ( thin film ) 2991 , 2957 , 1814 , 1758 , 1311 , 1146 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c12h12o5sna 291.0293 , found 291.0303 . to a dry 250 ml flask were added 3da ( 4.00 g , 10.0 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . the reaction mixture was stirred for 1 h and then concentrated in vacuo and azeotropically distilled with ch2cl2 ( 3 10 ml ) . to the crude mixture were added acetic anhydride ( 33 ml ) and 100 ml of toluene , and the resulting mixture was stirred overnight , concentrated in vacuo , and azeotroped with benzene ( 3 10 ml ) . trituration with diethyl ether afforded 3d ( 2.53 g , 94% ) as a solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 400 mhz , cdcl3 ) 7.90 ( m , 2h ) , 7.74 ( m , 1h ) , 7.62 ( m , 3h , overlap with minor diasteromer ) , 4.22 ( dd , j = 3.0 , 9.0 hz , 1h ) , 2.95 ( ddd , j = 2.1 , 6.1 , 15.2 hz , 1h ) , 2.762.58 ( m , 1h ) , 2.13 ( ddd , j = 6.2 , 13.2 , 15.2 hz , 1h ) , 1.44 ( s , 3h ) ; c nmr ( 100 mhz , cdcl3 ) 167.8 , 160.3 , 137.1 , 135.2 , 129.8 , 129.4 , 64.8 , 32.6 , 25.5 , 16.4 ; ir ( thin film ) 3066 , 2970 , 1803 , 1755 , 1310 , 1154 cm ; hrms ( esi - tof ) m / z [ m + h2o + na ] calcd for c12h14o6sna 309.0409 , found 309.0402 . to a dry 10 ml flask were added 2 ml of acetonitrile , triethyl orthoformate ( 0.11 ml , 0.63 mmol ) , amine ( 0.39 mmol ) , and aldehyde ( 0.39 mmol ) . anhydride 3b ( 0.39 mmol ) in 2 ml of acetonitrile was added , and the reaction mixture was stirred for 30 min at room temperature and then refluxed for 4 h. the mixture was cooled to room temperature and concentrated in vacuo . the product was purified by flash column chromatography ( conditions given for each compound ) . to a dry 10 ml flask was added 4 ml of acetonitrile , triethyl orthoformate ( 0.11 ml , 0.63 mmol ) , the prepared imine ( 0.39 mmol ) , and anhydride 3b d ( 0.39 mmol ) . the reaction mixture was stirred for 30 min at room temperature and then refluxed for 4 h. the mixture was cooled to room temperature and concentrated in vacuo . the residue was purified by flash column chromatography ( conditions given for each compound ) . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 13a as a solid ( 105 mg , 80% ) . mp 152.6153.1 c ; h nmr ( 300 mhz , cdcl3 ) 7.91 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.32 ( m , 3h ) , 7.04 ( m , 2h ) , 5.10 ( s , 1h ) , 3.36 ( ddd , j = 5.0 , 4.9 , 2.3 hz , 1h ) , 2.84 ( s , 3h ) , 2.73 ( m , 1h ) , 2.45 ( ddd , j = 17.9 ( gem ) , 6.6 , 4.6 hz , 1h ) , 2.08 ( m , 2h ) ; c nmr ( 75 mhz , cdcl3 ) 169.3 , 139.0 , 137.3 , 134.4 , 129.6 , 129.3 , 128.6 , 128.4 , 126.0 , 65.0 , 60.7 , 34.4 , 27.9 , 17.8 ; ir ( thin film ) 3057 , 2930 , 1635 , 1302 , 1142 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c18h19no3sna 352.0983 , found 352.0973 . general procedure b. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13b as a foam ( 117 mg , 84% ) . h nmr ( 300 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.747.64 ( m , 1h ) , 7.60 ( m , 2h ) , 7.30 ( m , 3h ) , 7.15 ( m , 2h ) , 5.40 ( s , 1h ) , 4.68 ( m , 1h ) , 3.28 ( m , 1h ) , 2.78 ( m , 1h ) , 2.49 ( ddd , j = 12.3 , 11.9 , 9.8 hz , 1h ) , 1.92 ( m , 2h ) , 1.31 ( d , j = 6.8 hz , 3h ) , 0.89 ( d , j = 7.0 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 169.7 , 141.6 , 137.5 , 134.3 , 129.6 , 129.0 , 128.6 , 128.0 , 126.0 , 65.0 , 53.8 , 47.6 , 28.5 , 20.3 , 20.1 , 17.5 ; ir ( thin film ) 2967 , 2945 , 1641 , 1303 , 1143 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no3sna 380.1296 , found 380.1306 . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.48 ) to yield 13c as a foam ( 130 mg , 85% ) . h nmr ( 600 mhz , cdcl3 ) 7.63 ( t , j = 7.4 hz , 1h ) , 7.39 ( m , 7h ) , 7.30 ( m , 3h ) , 7.25 ( t , j = 6.1 hz , 2h ) , 6.79 ( m , 2h ) , 5.76 ( d , j = 14.6 hz , 1h ) , 4.69 ( s , 1h ) , 3.22 ( d , j = 14.6 hz , 1h ) , 3.17 ( m , 1h ) , 3.07 ( m , 1h ) , 2.67 ( dd , j = 18.1 ( gem ) , 6.8 hz , 1h ) , 2.43 ( m , 1h ) , 2.18 ( m , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 169.0 , 138.8 , 137.0 , 136.1 , 134.0 , 129.5 , 129.4 , 129.3 , 128.6 , 128.6 , 128.5 , 127.7 , 125.9 , 63.9 , 57.2 , 47.6 , 28.3 , 16.1 ; ir ( thin film ) 3059 , 2936 , 1642 , 1307 , 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c24h23no3sna 428.1296 , found 428.1303 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13d as a foam ( 103 mg , 75% ) . h nmr ( 600 mhz , cdcl3 ) 7.99 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.30 ( m , 3h ) , 6.98 ( dd , j = 7.3 , 2.0 hz , 2h ) , 5.40 ( d , j = 2.4 hz , 1h ) , 4.95 ( dd , j = 17.5 , 2.6 hz , 1h ) , 3.38 ( td , j = 5.0 , 2.5 hz , 1h ) , 3.30 ( dd , j = 17.5 , 2.5 hz , 1h ) , 2.81 ( ddd , j = 17.7 , 10.5 , 6.9 hz , 1h ) , 2.52 ( ddd , j = 18.0 ( gem ) , 6.7 , 4.3 hz , 1h ) , 2.28 ( m , 1h ) , 2.12 ( m , 2h ) ; c nmr ( 150 mhz , cdcl3 ) 168.7 , 138.4 , 137.3 , 134.4 , 129.5 , 129.3 , 128.9 , 128.6 , 126.2 , 77.7 , 73.1 , 64.6 , 57.9 , 34.3 , 28.3 , 17.3 ; ir ( thin film ) 3303 , 3055 , 1650 , 1267 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h19no3sna 376.0983 , found 376.0977 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13e as a foam ( 124 mg , 89% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.71 ( m , 1h ) , 7.60 ( m hz , 2h ) , 7.29 ( m , 3h ) , 6.96 ( dd , j = 7.5 , 1.9 hz , 2h ) , 5.83 ( dddd , j = 17.7 , 10.5 , 8.2 , 4.3 hz , 1h ) , 5.21 ( m , 2h ) , 5.11 ( s , 1h ) , 4.86 ( ddd , j = 15.0 , 4.3 , 2.0 hz , 1h ) , 3.31 ( td , j = 4.7 , 1.8 hz , 1h ) , 3.002.82 ( m , 2h ) , 2.54 ( ddd , j = 18.0 ( gem ) , 6.8 , 3.7 hz , 1h ) , 2.19 ( m , 2h ) ; c nmr ( 151 mhz , cdcl3 ) 169.1 , 139.1 , 137.3 , 134.4 , 132.4 , 129.5 , 129.3 , 128.7 , 128.4 , 126.0 , 118.8 , 64.4 , 57.2 , 47.9 , 28.0 , 17.1 ; ir ( thin film ) 3054 , 2988 , 1641 , 1307 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h21no3sna 378.1140 , found 378.1136 . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 13f as a foam ( 110 mg , 72% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.67 ( t , j = 7.5 hz , 1h ) , 7.56 ( m , 2h ) , 7.32 ( m , 2h ) , 7.27 ( m , 3h ) , 7.19 ( m , 5h ) , 5.54 ( s , 1h ) , 3.48 ( m , 1h ) , 2.95 ( m , 1h ) , 2.66 ( dt , j = 18.1 ( gem ) , 6.2 hz , 1h ) , 2.20 ( m , 2h ) ; c nmr ( 150 mhz , cdcl3 ) 169.8 , 142.3 , 139.7 , 137.2 , 134.4 , 129.6 , 129.2 , 129.1 , 128.6 , 128.3 , 127.3 , 127.0 , 126.2 , 64.9 , 62.2 , 28.8 , 18.2 ; ir ( thin film ) 3066 , 3008 , 2946 , 1654 , 1304 , 1143 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h21no3sna 414.1140 , found 414.1131 . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.46 ) to yield 13 g as a foam ( 126 mg , 91% ) . h nmr ( 600 mhz , cdcl3 ) 7.91 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( t , j = 7.8 hz , 2h ) , 7.31 ( dd , j = 8.4 , 6.6 hz , 2h ) , 7.25 ( m , 1h ) , 7.05 ( d , j = 7.4 hz , 2h ) , 5.22 ( s , 1h ) , 3.95 ( dtd , j = 13.6 , 6.8 , 3.3 hz , 1h ) , 3.32 ( td , j = 5.0 , 2.0 hz , 1h ) , 2.81 ( m , 1h ) , 2.43 ( m , 2h ) , 1.99 ( m , 2h ) , 1.61 ( m , 2h ) , 0.87 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.2 , 139.7 , 137.4 , 134.3 , 129.6 , 129.2 , 128.5 , 128.3 , 126.1 , 64.7 , 57.9 , 48.0 , 28.0 , 20.3 , 17.6 , 11.3 ; ir ( thin film ) 2967 , 2929 , 2871 , 1639 , 1308 , 1147 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no3sna 380.1296 , found 380.1290 . general procedure b. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.50 ) to yield 13h as an orange amorphous solid ( 101 mg , 68% ) . h nmr ( 300 mhz , cdcl3 ) 7.93 ( dd , j = 7.2 , 1.6 hz , 2h ) , 7.70 ( m , 1h ) , 7.60 ( dd , j = 8.2 , 6.6 hz , 2h ) , 7.32 ( m , 5h ) , 6.37 ( dd , j = 16.0 , 1.2 hz , 1h ) , 6.07 ( dd , j = 15.8 , 6.4 hz , 1h ) , 4.94 ( dt , j = 6.6 , 1.6 hz , 1h ) , 4.66 ( m , 1h ) , 3.35 ( td , j = 6.4 , 2.0 hz , 1h ) , 2.68 ( dt , j = 17.2 , 7.0 hz , 1h ) , 2.40 ( dt , j = 17.2 , 7.1 hz , 1h ) , 2.09 ( dt , j = 14.4 , 7.2 hz , 1h ) , 1.99 ( dt , j = 14.4 , 7.2 hz , 1h ) , 1.33 ( d , j = 6.8 hz , 3h ) , 1.18 ( d , j = 6.9 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 169.7 , 137.6 , 135.4 , 134.2 , 132.0 , 129.8 , 129.6 , 128.8 , 128.6 , 128.4 , 126.5 , 63.5 , 51.8 , 47.3 , 29.1 , 20.7 , 20.2 , 18.78 ; ir ( thin film ) 3059 , 2975 , 1639 , 1446 , 1305 , 1145 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h25no3sna 406.1453 , found 406.1447 . general procedure b. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.42 ) to yield 13i as a foam ( 96 mg , 69% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( d , j = 8.4 hz , 2h ) , 7.69 ( t , j = 7.4 hz , 1h ) , 7.60 ( t , j = 7.8 hz , 2h ) , 6.97 ( d , j = 8.6 hz , 2h ) , 6.84 ( m , 2h ) , 5.18 ( m , 1h ) , 3.93 ( m , 1h ) , 3.77 ( s , 3h ) , 3.30 ( m , 1h ) , 2.81 ( m , 1h ) , 2.45 ( m , 2h ) , 2.01 ( m , 2h ) , 1.61 ( m , 2h ) , 0.88 ( t , j = 7.5 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.2 , 159.5 , 134.2 , 131.5 , 129.5 , 128.6 , 127.3 , 114.5 , 105.0 , 64.9 , 57.5 , 55.3 , 48.0 , 28.1 , 20.3 , 17.6 , 11.3 ; ir ( thin film ) 2964 , 2920 , 1633 , 1250 , 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c21h25no4sna 410.1402 , found 410.1400 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13j as a foam ( 133 mg , 87% ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.28 ( m , 2h ) , 7.01 ( m , 2h ) , 5.21 ( s , 1h ) , 3.92 ( ddd , j = 13.6 , 9.0 , 7.4 hz , 1h ) , 3.27 ( td , j = 5.1 , 2.2 hz , 1h ) , 2.78 ( ddd , j = 17.4 , 10.1 , 6.8 hz , 1h ) , 2.41 ( m , 2h ) , 1.96 ( m , 2h ) , 1.59 ( m , 2h ) , 0.86 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.2 , 138.3 , 137.2 , 134.4 , 134.2 , 129.6 , 129.4 , 128.5 , 127.5 , 64.6 , 57.5 , 48.0 , 28.1 , 20.3 , 17.9 , 11.3 ; ir ( thin film ) 2964 , 2937 , 1641 , 1306 , 1146 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h22clno3sna 414.0907 , found 414.0921 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.42 ) to yield 13k as a foam ( 92 g , 61% ) . h nmr ( 600 mhz , cdcl3 ) 7.95 ( d , j = 8.4 hz , 2h ) , 7.70 ( t , j = 7.4 hz , 1h ) , 7.61 ( t , j = 7.9 hz , 2h ) , 7.24 ( m , 1h ) , 7.17 ( m , 2h ) , 7.05 ( d , j = 7.8 hz , 1h ) , 5.51 ( s , 1h ) , 3.97 ( m , 1h ) , 3.14 ( m , 1h ) , 2.94 ( m , 1h ) , 2.53 ( dd , j = 18.3 ( gem ) , 7.4 hz , 1h ) , 2.42 ( m , 1h ) , 2.34 ( m , 2h ) , 2.07 ( m , 1h ) , 1.98 ( m , 1h ) , 1.69 ( m , 2h ) , 1.14 ( t , j = 7.5 hz , 3h ) , 0.92 ( t , j = 7.4 hz , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 168.9 , 141.1 , 137.6 , 136.1 , 134.3 , 129.63 , 129.60 , 128.63 , 128.60 , 126.5 , 126.1 , 62.5 , 54.2 , 48.2 , 27.3 , 24.3 , 20.4 , 16.3 , 15.4 , 11.5 ; ir ( thin film ) 3006 , 2966 , 1639 , 1300 , 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h27no3sna 408.1609 , found 408.1620 . general procedure b. the residue was purified by flash column chromatography ( 80:20 etoac / hex , rf = 0.50 ) to yield 13l as a foam ( 104 mg , 64% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.69 ( t , j = 7.1 hz , 1h ) , 7.60 ( m , 2h ) , 6.32 ( s , 1h ) , 6.16 ( d , j = 2.1 hz , 2h ) , 5.14 ( d , j = 2.1 hz , 1h ) , 3.95 ( ddd , j = 13.4 , 9.6 , 6.5 hz , 1h ) , 3.72 ( s , 6h ) , 3.34 ( td , j = 4.8 , 1.9 hz , 1h ) , 2.79 ( m , 1h ) , 2.46 ( m , 2h ) , 2.03 ( m , 2h ) , 1.63 ( m , 2h ) , 0.89 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.1 , 161.5 , 142.3 , 137.4 , 134.2 , 129.6 , 128.6 , 104.2 , 99.5 , 64.6 , 58.0 , 55.4 , 48.2 , 28.0 , 20.3 , 17.7 , 11.3 ; ir ( thin film ) 2962 , 2937 , 1641 , 1594 , 1306 , 1145 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h27no5sna 440.1508 , found 440.1517 . general procedure b. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.35 ) to yield 13 m as a foam ( 101 mg , 75% ) . h nmr ( 600 mhz , cdcl3 ) 7.88 ( d , j = 7.2 hz , 2h ) , 7.65 ( m , 1h ) , 7.56 ( t , j = 7.7 hz , 2h ) , 7.27 ( dd , j = 1.9 , 1.0 hz , 1h ) , 6.24 ( m , 1h ) , 6.10 ( m , 1h ) , 5.16 ( d , j = 2.9 hz , 1h ) , 3.78 ( ddd , j = 13.6 , 9.6 , 6.4 hz , 1h ) , 3.59 ( td , j = 6.1 , 3.1 hz , 1h ) , 2.71 ( m , 2h ) , 2.43 ( dt , j = 17.5 , 6.6 hz , 1h ) , 2.09 ( m , 2h ) , 1.58 ( m , 1h ) , 1.49 ( m , 1h ) , 0.85 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.4 , 151.3 , 143.0 , 137.3 , 134.2 , 129.5 , 128.5 , 110.6 , 108.4 , 61.8 , 52.9 , 47.8 , 28.5 , 20.4 , 19.0 , 11.3 ; ir ( thin film ) 2965 , 2933 , 2875 , 1647 , 1503 , 1306 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c18h21no4sna 370.1089 , found 370.1076 . the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.35 ) to yield 14 as a solid ( 111 mg , 87% ) . mp 127.4128.1 c ; h nmr ( 600 mhz , cdcl3 ) 7.99 ( m , 2h ) , 7.74 ( m , 1h ) , 7.65 ( m , 2h ) , 7.35 ( dd , j = 8.4 , 7.1 hz , 2h ) , 7.28 ( m , 1h ) , 7.05 ( d , j = 6.8 hz , 2h ) , 5.21 ( s , 1h ) , 3.45 ( d , j = 6.9 hz , 1h ) , 2.91 ( s , 3h ) , 2.53 ( m , 1h ) , 2.45 ( dd , j = 15.7 , 5.3 hz , 1h ) , 2.11 ( dd , j = 15.8 , 11.6 hz , 1h ) , 0.91 ( d , j = 6.7 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 171.1 , 139.3 , 137.0 , 134.5 , 129.6 , 129.4 , 129.1 , 127.9 , 125.3 , 73.7 , 60.3 , 38.1 , 34.4 , 29.1 , 22.4 ; ir ( thin film ) 2912 , 1658 , 1305 , 1130 cm ; hrms ( esi - tof ) m / z the residue was purified by flash column chromatography ( 80:20 etoac / hex , rf = 0.35 ) to yield 15 as a white solid ( 169 mg , 79% ) . mp 143.7144.3 c ; h nmr ( 600 mhz , cdcl3 ) 7.70 ( d , j = 7.5 hz , 2h ) , 7.50 ( t , j = 7.4 hz , 1h ) , 7.37 ( t , j = 7.7 hz , 2h ) , 5.89 ( s , 2h ) , 5.31 ( d , j = 9.5 hz , 1h ) , 4.22 ( ddd , j = 13.4 , 9.5 , 3.6 hz , 1h ) , 3.72 ( s , 3h ) , 3.71 ( s , 6h ) , 2.48 ( m , 4h , nch3 and 1 methylene h ) , 2.40 ( m , 1h ) , 1.84 ( m , 1h ) , 1.27 ( d , j = 6.9 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 173.1 , 161.4 , 158.6 , 137.4 , 133.4 , 128.50 , 128.48 , 106.8 , 90.6 , 61.9 , 55.6 , 55.2 , 51.9 , 35.0 , 31.8 , 29.3 , 17.1 ; ir ( thin film ) 3006 , 2939 , 1614 , 1286 , 1134 cm ; hrms ( esi - tof ) m / z [ m + h ] calcd for c22h28no6s 434.1637 , found 434.1649 . allylamine ( 0.29 ml , 3.8 mmol ) and trans - cinammaldehyde ( 0.53 ml , 4.2 mmol ) were added to a round - bottom flask containing thf ( 38 ml ) and na2so4 ( 3.3 g , 23.0 mmol ) , and the mixture was allowed to stir for 1 h. the anhydride ( 0.92 g , 3.8 mmol ) was added , and the resulting mixture was allowed to stir for 2 h. the na2so4 was filtered off , and the solvent was evaporated in vacuo . the residue was triturated with hexanes , transferred to a fritted funnel , and washed in portions with hexanes five times ( 5 ml ) and two times with a 9:1 mixture of cold hexanes and chcl3 ( 10 ml total ) , yielding 1.2 g of an off - white solid , which was 85% pure by nmr . a portion of the resulting acid ( 0.885 g , 2.2 mmol ) was redissolved in anhydrous toluene and methanol ( 60 ml:30 ml ) , and tmschn2 ( 2.2 ml , 4.4 mmol , 2 m in hexanes ) was added at 0 c dropwise over a period of 5 min . the mixture was allowed to stir for 1.5 h and monitored by tlc for disappearance of the starting material . the solvent was removed in vacuo , and the crude mixture was purified by flash column chromatography ( etoac / hex 3060% ) , yielding 17 ( 0.85 g , 92% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.44 ) . h nmr ( 600 mhz , cdcl3 ) 7.987.89 ( m , 2h ) , 7.817.56 ( m , 3h ) , 7.417.24 ( m , 5h ) , 6.54 ( d , j = 15.8 hz , 1h ) , 5.845.72 ( m , 2h ) , 5.345.25 ( m , 2h ) , 5.02 ( d , j = 8.4 hz , 1h ) , 4.40 ( ddt , j = 15.3 , 4.7 , 1.7 hz , 1h ) , 3.713.42 ( m , 3h ) , 3.443.27 ( m , 2h ) , 3.13 ( d , j = 18.2 ( gem ) hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 169.4 , 165.3 , 136.7 , 135.8 , 135.2 , 135.0 , 131.7 , 130.6 , 129.3 , 128.9 , 128.9 , 126.8 , 121.9 , 119.4 , 76.1 , 62.0 , 53.4 , 43.8 , 35.6 . ir ( thin film ) 1736 , 1715 , 1694 ( c = o ) , 1309 , 1148 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h23no5sna 448.1195 , found 448.1195 . ester 17 ( 0.602 g , 1.41 mmol ) was dissolved in anhydrous benzene ( 114 ml ) , which was degassed by bubbling with argon for approximately 5 min . then grubbs second - generation catalyst ( 35 mg , 3 mol % ) was added , and the mixture was heated to reflux for 9 h. the solvent was evaporated in vacuo , and the residue was redissolved in etoac ( 30 ml ) . saturated nh4cl ( 10 ml ) was added , and the mixture was allowed to stir for 30 min . the mixture was transferred to a separtory funnel , and the organic layer was washed with brine , dried over na2so4 , and then purified ( etoac / hex 3060% ) , yielding 18 ( 0.39 g , 86% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.16 ) . h nmr ( 600 mhz , cdcl3 ) 8.027.93 ( m , 2h ) , 7.74 ( td , j = 7.4 , 1.3 hz , 1h ) , 7.61 ( t , j = 7.9 hz , 2h ) , 5.90 ( dq , j = 6.0 , 2.1 hz , 1h ) , 5.55 ( dq , j = 6.2 , 2.2 hz , 1h ) , 5.38 ( tt , j = 4.3 , 1.8 hz , 1h ) , 4.33 ( ddt , j = 15.7 , 4.2 , 2.1 hz , 1h ) , 3.783.67 ( m , 2h ) , 3.62 ( s , 3h ) , 3.00 ( d , j = 15.7 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 172.8 , 165.7 , 136.9 , 134.9 , 130.6 , 130.3 , 129.3 , 129.2 , 79.0 , 70.1 , 53.3 , 50.7 , 39.5 ; ir ( thin film ) 1735 , 1716 , 1687 ( c = o ) , 1311 , 1148 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c15h15no5sna 344.0569 , found 344.0568 . alkene 18 ( 0.25 g , 0.77 mmol ) was dissolved in anhydrous meoh ( 30 ml ) , and the reaction mixture was degassed by bubbling with argon for 5 min . then pd / c ( 45 mg ) was added and h2 was bubbled into the reaction flask , and the mixture was allowed to stir for 3 h ( monitored by tlc ) . the pd / c was filtered off and washed with etoac ( 3 10 ml ) . the solvent was removed in vacuo , yielding 19 ( 0.246 g , 99% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.1 ) . h nmr ( 600 mhz , cdcl3 ) 7.987.90 ( m , 2h ) , 7.797.70 ( m , 1h ) , 7.60 ( dd , j = 8.5 , 7.3 hz , 2h ) , 4.61 ( dd , j = 9.7 , 5.9 hz , 1h ) , 3.72 ( s , 3h ) , 3.623.49 ( m , 2h ) , 3.17 ( d , j = 17.1 hz , 1h ) , 3.153.05 ( m , 1h ) , 2.071.93 ( m , 2h ) , 1.91 ( dtd , j = 12.2 , 6.0 , 3.9 hz , 1h ) , 1.25 ( dq , j = 12.6 , 9.3 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 170.6 , 166.0 , 134.9 , 130.3 , 130.2 , 129.2 , 75.6 , 63.9 , 53.4 , 42.1 , 39.4 , 28.5 , 25.8 ; ir ( thin film ) 1734 , 1686 ( c = o ) , 1146 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c15h17no5sna 346.0725 , found 346.0730 . to sulfone 19 ( 0.10 g , 0.31 mmol ) dissolved in 3 ml of chcl3 was added dbu ( 70 l , 0.46 mmol ) , and the reaction mixture was stirred overnight . the mixture was concentrated in vacuo and purified by flash chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 20 as a colorless oil ( 0.047 g , 84% ) . h nmr ( 600 mhz , cdcl3 ) 3.72 ( s , 3h ) , 3.58 ( t , j = 7.1 hz , 2h ) , 3.52 ( m , 2h ) , 2.93 ( m , 2h ) , 2.40 ( p , j = 7.4 hz , 2h ) ; c nmr ( 150 mhz , cdcl3 ) 172.8 , 164.0 , 161.6 , 98.6 , 51.0 , 41.5 , 41.2 , 26.6 , 25.6 . these data ester 18 ( 50 mg , 0.16 mmol ) was dissolved in 2 ml of chcl3 , and dbu ( 36 l , 0.24 mmol ) was added . after 90 min , the solvent evaporated in vacuo , and the crude mixture was purified by flash chromatography ( 20:80 etoac / hex , rf = 0.45 ) , yielding 21 as an oil ( 12 mg , 41% ) . h nmr ( 600 mhz , cdcl3 ) 7.04 ( d , j = 3.4 hz , 1h ) , 6.47 ( t , j = 3.5 hz , 1h ) , 6.17 ( d , j = 3.1 hz , 1h ) , 4.20 ( dd , j = 9 , 3 hz , 1h ) , 3.78 ( s , 3h ) , 3.48 ( dd , j = 19.8 ( gem ) , 3.6 hz , 1h ) , 3.19 ( dd , j = 18.6 ( gem ) , 9 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 170.6 , 169.6 , 136.1 , 119.2 , 112.2 , 106.6 , 52.9 , 38.1 , 38.0 . alkene 20 ( 47 mg , 0.25 mmol ) was dissolved in 2.2 ml of methanol under an atmosphere of argon , and 10% pd / c ( 14 mg ) was added to the vial . the flask was purged with h2 gas via a balloon , and the reaction mixture was stirred under an atmosphere of h2 overnight . the reaction vessel was purged with argon , and then the mixture was filtered through celite and concentrated to yield 22 as an oil ( 45 mg , 99% ) . h nmr ( 600 mhz , cdcl3 ) 4.09 ( ddd , j = 9.7 , 9.1 , 3.8 hz , 1h ) , 3.69 ( s , 3h ) , 3.57 ( q , j = 9.3 hz , 1h ) , 3.38 ( td , j = 8.5 , 4.8 hz , 1h ) , 3.03 ( t , j = 11.7 hz , 1h ) , 2.78 ( d , j = 8.9 hz , 2h ) , 2.05 ( q , j = 9.5 hz , 1h ) , 1.95 ( m , 1h ) , 1.82 ( m , 1h ) , 1.24 ( m , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 174.0 , 172.2 , 62.7 , 51.9 , 41.8 , 39.9 , 36.0 , 27.4 , 26.0 . these data almost 40%60% of patients with colorectal cancer ( crc ) develop metastasis , predominantly in the liver . tumor angiogenesis has been implicated as a major factor in the development and spread of these metastases . the recent discovery of vascular endothelial growth factor - c ( vegf - c ) and vegf receptor-3 ( vegfr-3 ) involvement in lymphatic vessel development and specific lymphatic markers has provided new insights into the field of lymphangiogenesis . using these markers , studies have suggested that lymphangiogenesis plays an active role in the formation and spread of colorectal liver metastases ( clm ) [ 4 , 5 ] . the patterns of intratumoral lymphatics may have potential clinical significance as a predictive marker of disease recurrence and patient survival . in this study , we investigated the patterns of tumor lymphangiogenesis using monoclonal d2 - 40 and lyve-1 antibody , as a predictive marker for disease - free survival in patients with clm . blood vessels were examined using cd34 antibody to differentiate the blood vessels from the lymphatic vessels . tissue was obtained from the victorian cancer biobank for 49 patients who underwent hepatic resection for clm . informed consent had been obtained from these patients at the time of surgery for long - term storage of specimen samples and subsequent research according to well - established protocols . formal ethics approval was obtained from the human research ethics committee ( hrec ) of the austin hospital for the study ( hrec submission number : h2012/04618 ) . the tumor mass was divided into four regions for each parameter measured ; tumor periphery , tumor centre , liver immediately adjacent to tumor , and host liver distal to tumor . previously , we demonstrated that , following the treatment with a novel vascular destructive agent ( vda ) , oxi4503 , the bulk of the tumor died leaving a viable rim of tumor cells at the periphery extending one hundred microns from the tumor - host interface towards the tumor centre . based on findings from our previous study , the tumor periphery is defined as the area covering the tumor - host interface and extending one hundred microns towards the tumor centre . the tumor centre is the remaining bulk of the tumor without considering the periphery . the liver immediately adjacent to the tumor is defined as the area at the tumor - host interface and extending one hundred microns away from the tumor . the host liver distal to the tumor is host liver which is farther than one hundred microns from the tumor . tissue samples from the specimen were fixed in 10% buffered formalin , embedded in paraffin , and cut into four m thick sections . d2 - 40 is a new selective monoclonal antibody for lymphatic endothelium which does not cross react with blood vessel endothelium . for d2 - 40 and lyve-1 staining , heat antigen retrieval was performed using tris buffer ( 50 mm ) ph 9.5 for 15 minutes at 99c . the sections were immunostained with mouse monoclonal antibody d2 - 40 used at 0.03475 mg / ml ( d2 - 40 , dako , victoria , australia ) and lyve-1 used at 0.0067 mg / ml ( abcam , cambridge , usa ) . cd34 is an established endothelial vessel marker normally expressed on tumor vessels and host vessels undergoing neovasculature . for cd34 , no antigen retrieval was required . normal goat serum ( 20% ) cd34 was used at 0.005 mg / ml ( abcam , cambridge , usa ) . a polymer - based detection kit containing goat anti - mouse immunoglobulins ( igg ) coupled with horseradish peroxidase ( hrp ) ( envision plus , dakocytomation pty , ltd , botany , nsw , australia ) was used . images of positively stained vessels were captured using a digital light microscope ( nikon coolscope , nikon corporation , japan ) between 10x and 400x magnification . the images of tumor fields were captured to be representative of the entire tumor , using a raster pattern which allowed for captured fields to be random and avoid overlap . the images were analyzed using image - pro plus ( version 5 , media cybernetics , perth australia ) . vessels were assessed as the number of positively stained vessels per tumor area to provide a microvascular density index . the lymphatic vessel density ( lvd ) assessments were performed by researchers blinded to the patient outcome data . pairwise comparisons of group means for parametric data were performed using analysis of variance ( anova ) with post hoc analysis as appropriate . nonparametric data were performed using mann - whitney u or kruskal - wallis tests , as appropriate . disease - free survival was calculated from the date of surgery to the date of progression or the date of last follow up . overall survival was not used in this study as a result of a relatively small number of deaths ( 7/49 ) within the follow up period . receiver operating characteristic ( roc ) analysis was conducted to assess the discriminative performance and the predictive capability of lvd in each region ( tumor periphery , tumor centre , and adjacent liver ) with tumor recurrence as the end point . an area under the curve ( auc ) of 1 represents a perfect test ; an area of 0.5 represents a worthless test . cut - off points that maximized sensitivity and specificity were established by analyzing roc curve coordinate points . using optimal cut - off thresholds determined from roc analysis , kaplan - meier survival curves were generated to compare survival between groups of high and low lvd in the three regions . all statistical analyses were performed using spss ( statistics package for social sciences , spss , chicago , il ) . median follow - up time for all patients was 27 months ( range 495 months ) . all patients had been assessed by a multidisciplinary team consisting of radiologists , hpb surgeons , oncologists , and nuclear physicians prior to commencement of treatment . standard indications for liver resection of clm were followed after excluding extrahepatic metastases by multidetector computed tomography ( mdct ) of chest and triple phase mdct of abdomen and pelvis in addition to whole body positron emission tomography with fluorodeoxyglucose integrated with computed tomography ( fdg pet / ct ) scans . patients received a combination chemotherapy regimen including oxaliplatin ( folfox or capecitabine / oxaliplatin ) or irinotecan ( folfiri ) based regimen . cd34 appears to only stain blood vessel endothelial cells , leaving lymphatic endothelial cells negative . serial immunohistochemistry staining using cd34 and d2 - 40 antibodies demonstrates the specificity of the markers . no overlapping of vessels was observed between cd34 and d2 - 40 ( figure 1 ) . quantification of d2 - 40 staining ( figure 2 ) revealed greater density at the tumor periphery compared to the tumor centre ( 49.22 24.3 positive lvd / mm versus 22.1 11.5 positive lvd / mm , p < 0.001 ) , adjacent liver ( 49.22 24.3 positive lvd / mm versus 4.3 4.9 positive lvd / mm , p < 0.001 ) , normal liver ( distal to the tumor ) ( 49.22 24.3 positive lvd / mm versus 6.7 3.1 positive lvd / mm , p < 0.001 ) , and benign liver ( 49.22 24.3 positive lvd / mm versus 8.7 6.2 positive lvd / mm , p < 0.001 ) . lyve-1 was found to be expressed in the liver sinusoids but absent from the tumor ( figure 3 ) . the roc curve in figures 4(a ) , 5(a ) and 6(a ) shows the ability of lvd in the tumor periphery , centre , and adjacent liver to be used as a prognostic marker to predict the likelihood of disease recurrence following hepatic resection . the roc graph shows a statistically significant ability of peripheral ( p < 0.01 ) , central ( p < 0.05 ) , and adjacent liver ( p = 0.01 ) lvd to predict disease recurrence . peripheral lvd was the most discriminative , with an area under the curve ( auc ) of 0.713 , followed by lvd in the adjacent liver with an auc equal to 0.708 and central lvd with an auc equal to 0.692 . according to the optimal cut - off values provided by roc analysis , patients were categorized into two groups : low lvd and high lvd in different regions . low d2 - 40 stained lvd in the tumor periphery ( p < 0.01 ) , centre ( p = 0.01 ) , and liver adjacent to the tumor ( p = 0.018 ) ( figures 4(b ) , 5(b ) and 6(b ) , resp . ) correlated significantly with disease - free survival . table 2 shows that patients , with high lvd in the periphery , centre , and liver adjacent to the tumor , appear to demonstrate close correlation to disease recurrence within the first and second year ( p < 0.05 ) after resection . however , only patients with high lvd in the tumor periphery significantly correlated with disease - free survival within the third year ( p = 0.009 ) after resection . in cox multivariate analysis , taking into consideration the other variables including sex , age , total tumor volume ( ttv ) , largest tumor volume ( ltv ) , and number of lesions , only high lvd in the normal liver adjacent to the tumor showed significant correlation ( p = 0.046 ) to disease recurrence following resection ( table 3 ) . the potential role of lymphangiogenesis in the process of tumor metastasis has been largely overshadowed by the role of angiogenesis . for decades , angiogenesis , the formation of new blood vessels , has been the main focus of research in the pathogenesis of tumor metastases . over the decades , much knowledge has accumulated linking angiogenesis as an essential step in tumor growth and development . it has been reported that , in the absence of active angiogenesis , the growing tumor may undergo necrosis and apoptosis beyond 1 - 2 mm in diameter due to limitation imposed by diffusion . several studies have focused on the microvessel density ( mvd ) as a prognostic tool ; hasan et al . observed elevated levels of mvd in crc to be associated with poor prognosis . our study is in concordance with those findings and demonstrates significant differences in mvd between different regions of the tumor and adjacent liver . this highlights the spatial differences within the tumor microenvironment and the heterogeneity of the tumor . in contrast to the extensive characterization of molecular mechanisms involved in angiogenesis , research into the role and mechanisms of lymphangiogenesis in cancer has been hampered by singular lack of specific markers . however , recent discovery of lymphangiogenic factor , vascular endothelial growth factor - c ( vegf - c ) , and specific lymphatic markers ( lyve-1 and d2 - 40 ) now allow researchers to identify and focus on the role of lymphangiogenesis in tumor metastases and to determine whether they have prognostic implications . the recent introduction of lyve-1 and d2 - 40 has paved the way for exciting research into the field of lymphangiogenesis : its mechanisms and the possible role these vessels play in the spread and dissemination of tumor cells [ 7 , 13 ] . despite the recent focus on lymphangiogenesis research , literature on the role of lymphatic vessels in tumor metastasis early studies reported the absence of lymphangiogenesis within the tumor , initially believed to be due to the high interstitial pressure created by rapidly proliferating tumor cells . due to the absence of intratumoral lymphatic vessels , it was suggested that lymphatic vessels at the periphery were responsible for the spread of tumors . however , more recent studies have reported the presence of intratumoral lymphatics in several different cancers such as breast cancer and colon cancer . dadras et al . investigated the possibility of using lvd as a marker for prognosis ; the study reported high intratumoral lvd in metastatic lesions compared to primary lesions significantly correlated with poor disease - free survival in cutaneous melanoma . in agreement , saad et al . also reported the presence of intratumoral lymphatics in 46% of cases with stage 1 endocervical adenocarcinoma ; however , they observed small and flattened vessels within the tumor , in contrast to the wide open lymphatic vessels found in the tumor periphery , casting a doubt of functionality of intratumoral lymphatic vessels . in contrast to dadras ' findings , saad et al . showed a significant correlation with peritumoral d2 - 40 lvd and depth of invasion . due to many conflicting results and the absence of a general consensus on the prognostic value of lvd , different types of cancers need to be investigated separately to identify the prognostic value of lymphangiogenesis . few studies have investigated lvd as a prognostic marker in clm and the results have been contradictory [ 1921 ] . despite recent progress in this field , the potential use of lvd as a prognostic marker in clm remains unclear . investigating the expression of vegf - c , a marker associated with lymphangiogenesis , matsumoto et al . reported that vegf - c overexpression significantly correlated with tumor invasion , lymphatic invasion , and lymph node metastases . in contrast , using lyve-1 as a marker for lymphatic vessels , brundler et al . observed no significant correlation between lvd and clinical outcome and concluded that lvd had no prognostic value in esophageal adenocarcinoma . one possible explanation for the results reported by brundler et al . maybe due to the antibody used to identify lymphatic vessels . lyve-1 does not appear to be a sensitive and reliable marker for lymphatic vessels in all solid tumors . in agreement with previous studies lyve-1 stained liver sinusoidal endothelial cells and was not able to identify lymphatic vessels in the tumor . lyve-1 is a known hyaluronan ( ha ) receptor found on lymphatic endothelial cells ; however , the liver sinusoidal endothelial cells play a major role in ha catabolism and as a result also display ha receptors . in addition , ichida et al . reported elevated ha levels associated with liver injury including cirrhosis and hepatocellular carcinoma ( hcc ) . it is believed that reduced expression of the scavenging lyve-1 ha receptors during liver injury and hcc leads to increased ha serum levels . indeed , in contrast to brundler et al . , saad et al . reported a significant correlation between lvd and lymph node metastasis and lymphovascular invasion and tumor stage in esophageal adenocarcinoma using d2 - 40 as a marker for lymphatic vessels . due to these few and conflicting results , clear consensus on the potential use of lvd as a prognostic marker remains to be elucidated . using d2 - 40 as a specific marker for lymphatic vessels , our data demonstrated that lvd in the tumor periphery and centre and adjacent liver significantly correlated with disease - free survival . lvd in the tumor periphery and centre and adjacent liver correlated with recurrence within the first two years following resection , while lvd in the tumor periphery continued to correlate with disease recurrence three years after resection . however , in the multivariate analysis , only the lvd in the adjacent liver was significantly correlated with disease - free survival . the contradictory results regarding the prognostic significance of lvd in tumor metastases may be due to different patient cohorts , specific tumors included in the analysis , or the method / markers used to detect lymphatic vessels . one of the limitations of this study is that samples were obtained only from a single tumor from each patient . this , therefore , may not necessarily reflect the lvd in other tumors in the same patient and , hence , be a confounding factor . this study has demonstrated that d2 - 40 is effective in identifying lymphatic vessels in human clm . the monoclonal antibody strongly labeled lymphatic vessels without staining blood vessels as observed in the serial staining of d2 - 40 and cd34 . d2 - 40 was , therefore , found to be an appropriate and selective marker for lymphatic vessels in clm . our results further demonstrate the potential predictive value of lvd detected by d2 - 40 , as a prognostic marker clm . despite the limitations imposed by the retrospective nature of the study , relatively short follow - up period , and sampling of single lesions from each patient , the results have established a foundation for investigating what appears to be a potentially significant predictive factor in the long - term survival of patients with clm . determining the lymphatic development within tumors may further play a significant role in the selective use of biological agents with the ability to target lymphatics being currently under development .

sulfone - substituted - and -lactams have been prepared in a single step with high diastereoselectivity . sulfonylglutaric anhydrides produce intermediates that readily decarboxylate to provide -lactams with high diastereoselectivity . substituents at the 3- or 4-position of the glutaric anhydride induce high levels of stereocontrol . sulfonylsuccinic anhydrides produce intermediate carboxylic acids that can be trapped as methyl esters or allowed to decarboxylate under mild conditions . this method has been applied to a short synthesis of the pyrrolizidine alkaloid ( )-isoretronecanol . background . colorectal cancer ( crc ) is the most common form of cancer diagnosed in australia across both genders . approximately , 40%60% of patients with crc develop metastasis , the liver being the most common site . almost 70% of crc mortality can be attributed to the development of liver metastasis . this study examines the pattern and density of lymphatics in colorectal liver metastases ( clm ) as predictors of survival following hepatic resection for clm . methods . patient tissue samples were obtained from the victorian cancer biobank . immunohistochemistry was used to examine the spatial differences in blood and lymphatic vessel densities between different regions within the tumor ( clm ) and surrounding host tissue . lymphatic vessel density ( lvd ) was assessed as a potential prognostic marker . results . patients with low lymphatic vessel density in the tumor centre , tumor periphery , and adjacent normal liver demonstrated a significant disease - free survival advantage compared to patients with high lymphatic vessel density ( p = 0.01 , p > 0.01 , and p = 0.05 , resp . ) . lymphatic vessel density in the tumor centre and periphery and adjacent normal liver was an accurate predictive marker of disease - free survival ( p = 0.05 ) . conclusion . lymphatic vessel density in clm appears to be an accurate predictor of recurrence and disease - free survival .

carotid blow - out syndrome is the most feared complication in head and neck surgery . the syndrome occurs in 3% to 4% of all head and neck cancer patients and is easy to happen following fistula formation , bad wound healing and tumor recurrence . carotid blow - out syndrome presents an average of 2.7 years after the initial diagnosis of cancer , usually occurs proximal to the carotid bifurcation , and is bilateral in 2% of cases . carotid blow - out syndrome occurred in a patient undergoing reoperation for recurrent thyroid cancer , and was repaired using the remaining carotid sheath , sterno - thyroid muscle , sterno - hyoid muscle and the harvested external jugular vein . local recurrence occurred on both sides 5 years and 6 months later , and the re - operation was done by an otolaryngologist ( fig . 1 ) . the sterno - hyoid muscle and sterno - thyroid muscle was severed and the tumor around the cricoid cartilage was removed . the tumor extended into the space between the right common carotid artery and internal jugular vein . the recurrent tumor was also located under the right common carotid artery and vagus nerve on the lateral side . enhanced cervical ct scan showed the recurred thyroid cancer in juxta - carotid regions of both sides . the tumor on the right side ( white arrowhead ) attached to the right common carotid artery ( white arrow ) . the carotid sheath of the right common carotid artery was hard and had no elasticity . the right common carotid artery had two layers , which were very fragile , so the direct suture or repair with a graft was impossible . the carotid artery could not be trapped with ligation because the cerebral vascular supply was not examined preoperatively . the carotid sheath was sutured to the carotid artery on the distal and proximal end of the lacerated portion ( fig . thereafter , the lacerated portion was covered with the remaining carotid sheath surrounding the carotid artery ( fig . fibrin glue was used to seal the space between the carotid artery and carotid sheath . however , re - bleeding occurred spontaneously at the proximal portion of the previous laceration during the dissection of the opposite side . fibrin glue with oxidized cellulose was initially used to seal the second small hole in this lesion ( fig . the ruptured section was then covered with the remaining sterno - thyroid muscle as the first layer and the sterno - hyoid muscle as the second layer ( fig . the proximal portion of the right common carotid artery was covered with the harvested external jugular vein because there were no vascularized tissues surrounding this artery ( fig . finally , the repaired right common carotid artery was widely covered with the sternomastoid muscle . blood pressure was strictly controlled after the operation , and no anti - platelet drugs were administered . the carotid sheath ( arrows ) was sutured to the right common carotid artery at the distal end of the lacerated portion ( arrowhead ) . the lacerated portion was covered with the remaining carotid sheath surrounding the right common carotid artery . re - bleeding occurred at the proximal portion ( black arrow ) of the previous laceration . fibrin glue with oxidized cellulose ( arrow head ) was initially used to seal the second small hole ( black arrow ) in this lesion . the ruptured section was then covered with the remaining sterno - thyroid muscle ( white arrow ) as the first layer . the ruptured section was then covered with the remaining sterno - hyoid muscle ( white arrow ) as the second layer . the proximal portion of right common carotid artery ( black arrow ) was covered with the harvested external jugular vein ( white arrow ) . three - dimensional ct angiography showed irregular stenosis in the right common carotid artery just after the re - operation ( fig . 3 ) . digital subtraction angiography also showed irregular stenosis in the right common carotid artery 5 days after the re - operation ( fig . however , the stenosis was found to recover on magnetic resonance angiography 11 days later . no cerebral infarction occurred after the operation and the patient s general condition was good . the pathological specimen revealed the papillary carcinoma which showed prominent invasion of the nervous system . the patient is in good condition without neurological complication , and recurrence of the tumor 22 months after the operation . the carotid sheath used for the repairment was partially pushed into the arterial cavity ( white arrow ) . thyroid cancer frequently invades the recurrent laryngeal nerve , strap muscles and trachea , and it is followed by invasion of the esophagus , internal jugular vein and carotid artery . surgical resection is the primary treatment for locally advanced thyroid cancer , and the carotid sheath invaded by a tumor must be excised with the thyroid tumor . during this procedure , carotid blow - out syndrome patients present with sentinel bleeding , but 60% of patients will develop a life - threatening hemorrhage requiring emergent intervention . the incidence is higher due to the lack of supporting healthy tissues if there is a history of radical neck dissection . other risk factors for carotid blow - out syndrome include radiotherapy , flap necrosis , mucocutaneous fistula , wound infection , and recurrent tumor invasion . injury to the vasa vasorum and necrotizing vasculitis in all three layers is an important factor in the development of radiation - induced vasculopathy of large arteries . the carotid rupture in the current case was related to the previous radical neck dissection followed by the recurrent tumor invasion . over 90% of patients with carotid blow - out syndrome are treated with endovascular therapy ( embolization 56% , stenting 36% ) , and surgical ligation ( 7% ) is rarely indicated . carotid artery embolization has a 15% to 20% neurologic morbidity rate and a lower incidence of associated mortality . endovascular embolization can also help predict cerebrovascular complications with temporary balloon occlusion and collateral cerebral blood flow analysis . stent - grafts may be useful in the treatment of carotid blow - out syndrome and show a high procedural success rate in selected cases . however , stent placement has a higher risk of recurrent carotid blow - out syndrome ( 44% ) than embolization therapy ( 10% ) . other studies of carotid blow - out syndrome treated with surgical ligation report an average mortality rate of 40% and an average morbidity rate of 60% . although trapping with ligation was considered in the current case , trapping of the right common carotid artery was not done , because the cerebral vascular supply was not assessed preoperatively . instead fibrin glue was used to seal the space between the carotid artery and the surrounding covering flap . we thought that it was important to seal the ruptured portion in multiple layers using vital , vascularized tissue . thus , the reconstruction was reinforced by covering the common carotid artery with the harvested external jugular vein and sternocleidomastoid muscle . this surgical technique was an emergency measure following the sudden rupture of the common carotid artery . given the large diameter of the common carotid artery , obstruction could be avoided even after the thrombus was formed around the repaired sites . this procedure can be one option when an unexpected rupture of the carotid artery occurs . errors in the health care system are due to a diverse interaction of human behavior , socio - cultural aspects , technical aspects of the system , as well as a range of system weaknesses . various categories of errors present as an overlap between human and system causes . when working conditions lead to circumstances in which it is easy to commit an error , this is known as a latent error or system failure. lack of experienced staff on duty , leading to staff fatigue and poor administration verdicts for example , may lead to latent errors or system failure . this , in turn , can lead to violation - producing conditions , where an individual has little choice but to violate protocol . this includes knowledge - based errors , rule - based errors , skill - based errors , technical errors and violations . tackling only the active failures will lead to an accretion of latent conditions , and an inevitable error will ultimately occur , completing the cascade and resulting in a tragic outcome . a holistic approach to incident reporting would allow for the possibility that an error or adverse event suffered by a patient in one part of the world would be a transmitted source of learning that benefits future patients in many other countries . learning from both adverse events and near misses is essential for improving the quality of care however one of the greatest frustrations for patients and professionals alike is the apparent failure of the health care systems to learn from their mistakes . commonly , neither health care professionals nor health care organizations counsel others when a mishap occurs , nor do they share what they have learned when an investigation has been carried out . consequently , the same mistakes occur repeatedly in many settings and patients continue to be harmed by preventable errors . health - care organizations and individuals benefit from incident reporting if they receive back useful information , gained by analysis of similar cases at other institutions . if the event and the results of the analysis are not reported to an external authority , the lessons learned are trapped within the walls of that hospital . the opportunity to analyze the problem although the importance of incident reporting has been established , under - reporting remains a significant problem occurring for example , at a rate of 50%96% annually in the united states . one solution to this dilemma is reporting by the primary care providers within the hospital or health - care organization , and by the organization to a broader audience through a system - wide , regional , or national reporting system . researchers in the field of quality in health care believe that an effective reporting system is the foundation of safe practice and , within a hospital or other health - care organization , a corner stone towards achieving a culture of safety . at a minimum , reporting can help identify hazards and risks , and provide information as to where the system is breaking down . this can help target improvement efforts and systems changes to reduce the likelihood of injury to future patients . extensive work has been done in the west regarding the role of incident reporting systems in preventing harm to patients thus improving the quality and safety of health care . a report prepared for the department of health in the uk indicated that an adverse event was associated with 10% of hospital admissions . with over 850,000 events per year , costing more than 2 billion per year in direct health care costs . in one year , errors involving medical devices led to death or serious injury in 400 people . the cost of hospital - acquired infection was over 1 billion in direct health care costs alone , of which 15% were considered to be preventable . clinical negligence claims currently amount to 400 million annually , with an estimated potential liability of 2.4 billion in existing and expected claims . in the united states , analysis of the harvard medical practice study of 1984 medical records and the colorado / utah study of 1992 records showed adverse events to have been associated with 3.7% and 2.9% of admissions , 13.6% and 8.8% deaths respectively . peer review indicated that 55% of these events were preventable , and almost 28% were due to negligence . medication errors , technical errors , diagnostic errors and failure to prevent injury were the most common type of incidences reported . this report estimated that the total cost of preventable adverse events is between $ 17 billion and $ 29 billion , with direct health care costs accounting for over half . adverse events were associated with 16.6% of hospital admissions ( with approximately half leading to the admission , and half occurring during the admission ) , 4.9% mortality and permanent disability in 13.7% . of all adverse events , the preventable cost of adverse events may be as much as $ 2 billion annually , or 5% of the $ 40 billion spent each year on health care . in addition , costs arising from legal expenses and compensation for medical error currently total $ 400 million per year , which consumes a further 1% of the health budget . since the publication of the us institute of medicine report to err is human , and the uk department of health report an organization with a memory , there has been increasing recognition of the need for healthcare organizations to monitor and learn from patient safety incidents . over the last few years , several countries have established national or system - wide reporting systems to facilitate large scale monitoring and analysis of incident data[1517 ] . the national reporting and learning system ( nrls ) for england and in wales , established by the national patient safety agency , was rolled out in late 2003 and has now received over one million reports , mainly from acute hospitals . limited framework for incident reporting system exists in most of the health care system in pakistan and therefore poses risk to the patients and results in compromised quality of care . development of a nationwide incident reporting system is inevitable in pakistan . recognizing the attitudes and perceptions of health professionals who will implement this system is mandatory for its success . this study aims assess the attitudes and perceptions of doctors and nurses towards incident / error reporting in tertiary level health care of pakistan and to identify potential barriers at the grass root level to the implementation of an error reporting system . to the best of our knowledge the study was conducted in shifa international hospital ( sih ) , a 600 bed tertiary care facility , employing 520 registered health professionals . fifty percent reporting of error was taken as identified factor . for 95% confidence interval and precision of 5% , the questionnaire was designed by modifying those currently used by agency of health related quality ( ahrq ) and other researchers . a small description of key terminology such as incidence , error , adverse events , near misses or close calls , and medication errors was attached to each copy of the questionnaire . the questionnaire consisted of 3 sections , which encompasses determination of : the support or lack thereof provided by the working environment to affirm incident reporting ; health care professional 's perception regarding attitudes of managers and most important barriers to incident reporting ; the motivators to incident reporting ; and patient outcomes that influence reporting behavior of health professionals . variables explored were : working environment ( supportive , culture of blame and shame ) ; attitudes of managers ( " we are informed about the errors that happen in this unit " ) ; reasons to report the incident ( to get immediate help for patient , system development so that repetition of incidents can be minimized ) ; to whom incident reporting would be easy ( administration , head of the department ) ; and perceived barriers to incident reporting ( lack of feedback , legal and financial penalties and administrative sanctions ) . mean standard deviation ( sd ) of age and working hours per week were reported . frequency ( percentage % ) were presented for gender , staff position , primary area of employment , patient 's outcome influencing reporting behavior , and individual reporting of an un - witnessed incident . chi square test was used to test the significance of association of professional groups ( doctors and nurses ) with reasons to report , to whom incident reporting would be easy , perceived barriers to incident reporting and patient outcomes that influence reporting behavior . the only exclusion criteria used in the study was medical and nursing students . the ethical approval of the study was obtained from the institutional review board ( irb ) of shifa college of medicine . written informed consent was obtained from all participants . one hundred and fourteen doctors ( 52.5% ) and 103 nurses ( 47.5% ) completed and returned the questionnaire . of these participants , 116 ( 53.5% ) were men and 101 ( 46.5% ) were women . background details of the sample ( n=217 ) considerable homogeneity is found in the incident reporting attitude among different health professionals : 100% among consultants and registrars , 94% among medical officers and 97% among nurses are ready to report the incident happened through them . house officers are reluctant to report the incident happened through them , that is , 75% responded impartially ( neither likely / unlikely ) to report the incident . ( n=217 ) only 19.3% ( n=42 ) doctors and nurses believe that tertiary health care centers have enough staff to handle the workload . this result matches up with the findings that 70% percent ( n= 151 ) health professional believe that their working hours are too long and 60.4% ( n=131 ) health professional are working more than 80 hours per week . some other characteristics of the working environment ( such as mutual respect among workers ) and attitudes of management towards patient safety ( working fast by taking shortcuts ) are depicted in table 2 . working environment and attitudes of management ( agreed ) frequency - percentage ( n=217 ) table 3 shows the main motivator for incident reporting ; to whom reporting is easy ; perceived barriers to incident reporting and patient outcome that influence the reporting behavior of doctors and nurses . a statistically significant difference ( p<0.001 , or 5.035 , 95%ci 2.52 , 10.04 ) was found between doctors ( 42% ) and nurses ( 13% ) in learning for self and others from your mistake as the main reason for incident reporting . eighty percent doctors and 84% nurses think that system development to minimize the repetition of particular incidents is the main reason for incident reporting , although this association is not significant ( or 0.727 . sixty percent doctors ( n=69 ) and 80% nurses ( n= 83 ) think that incidents should be reported to the head of the department ( or 0.37 , 95% ci 0.19 , 0.68 . eighty eight percent of doctors ( n=101 ) and 84% of nurses ( n=87 ) share a common barrier to incident reporting as lack of feedback generation while the significance of association is low ( or 1.42 , 95% ci 0.65 , 3.13 . reasons ( motivators ) to report , feasible to report to and barriers to incident reporting ( n=217 ) we presented three hypothetical situations , in which different outcomes of patients could influence the reporting behavior of health professionals . in first situation , an incident occurred but was corrected before affecting the patient . in the second , the incident happened but has no potential harm to the patient and lastly , an incident happened that can harm the patient but does not . in all three situations nurses tend to report more than doctors and the associations were statically significant ( p<0.001 ) . only 37% doctors will report the incident that could harm the patient contrary to their counterparts nurses ( 79% ) who reported significantly more in this situation ( or 0.13 , 95% ci 0.07 , 0.24 . overall results of health professional 's incident reporting behaviors in different situations are shown in table 4 . any program that aims to improve patient safety must contain all - inclusive information on incidents , near misses , adverse events or errors so that , as a source , it can be used for learning and grounds for precautionary action in the future . some systems focus on specific types of incidents / errors concerning technologies or on areas where incidents / errors occur frequently ( i.e. beeping equipment , infusion pumps , and blood transfusion ) . some systems are open ended taking into account all incidents/ errors along with the entire spectrum of quality of care provided . the rationale for any reporting system is learning . reporting can lead to learning and patient safety in several ways . first , through generating alerts regarding new hazards ( e.g. complications or adverse effects of new drugs ) . finally , report analysis can provide insight into recognizing hazard trends and system failures to aid in the establishment of best practices guidelines . our study shows that incident reporting for the purpose of learning is not well avowed by health professionals , particularly nurses . significant differences exist between doctors ( 42% ) and nurses ( 12% ) for learning as the main reason for incident reporting ( or 5.035 , 95% ci 2.52 , 10.04 . p<0.001 ) . whereas the majority of health professionals ( doctors 80% and nurses 84% ) will report an incident in order to minimize its repetition in the future . incident - reporting behavior differs between doctors and nursing professional groups , with nurses reporting significantly more often than doctors . a study in the uk indicated that health professionals are reluctant to report an incident in which there was a negative outcome for the patient . our study showed similar findings in that nurses are more willing to report than doctors . an incident which harmed the patient negatively influenced the reporting behavior of both doctors and nurses . this may be because health professionals feel insecure about their job and are afraid that they will have to face administrative fury after committing and reporting an error . this is supported by the finding in our study that 69% of doctors ( n=79 ) and 68% of nurses ( n=70 ) believe that administrative sanction is the most important barrier to incident reporting . it is vital to note that a reporting system itself does not bring about or improve patient safety . it is the action or response to the reporting that brings the change . within an organization , reporting of incidents/ adverse events should lead to an in - depth investigation to assess the etiological factors ( active or latent ) so the system can be changed and recurrence can be prevented . at a national level , report analysis by experts and dissemination of information is required to improve patient safety through incident reporting . in this study more than 88% of doctors and 84% of nurses believe that the lack of feedback generation is the most influential barrier to incident reporting . a similar study conducted in south australia ( 2006 ) also found that almost two thirds of the health professionals ( doctors and nurses ) believed lack of feedback was the greatest deterrent to reporting . a non - supportive environment , a culture of blame and shame and the culture of medicine , with its emphasis on professional autonomy , collegiality , and self - regulation , is unlikely to foster incident reporting . our study identified that only 54% of health professionals believe that their hospital environment is supportive . moreover 57.1% of health professionals perceive lack of value in incident reporting because when an event is reported , it feels like the person is written up , not the problem. some other barriers to incident reporting identified from peer reviewed literature is the lack of knowledge about how , what and whom to report . the evidence suggests that an autonomous body to collect and analyze incident reports should be established within the hospitals and that it should not work under the influence of manager / supervisors , head of the department or senior faculty members . our study shows that a significant proportion of doctors ( 60% ) and nurses ( 80% ) are in favor of reporting an incident to the head of the department , while , only 19% of doctors and 9% of nurses prefer reporting to the hospital administration . this preference may be because department heads are more accessible , offer a certain level of confidentiality and feedback may be pursued easily . our research confirms the previous finding that , in the presence of written protocols and guidelines , an incident is more likely to be reported . this finding may provide an initiative to introduce protocols and guidelines in writing , as these are less likely to be violated and violations are more likely to be reported . the willingness of health professionals to report incidents in order to improve patient safety indicates that fertile grounds are available for development of an incident reporting system in pakistan . the core and theme of any incident / error reporting system is to learn from mistakes . this fact however , is not well acknowledged by health professionals in pakistan . more work is needed to raise the awareness among health professionals pertaining to incident reporting . furthermore , any system of incident reporting that might be implemented in the future would need to consider providing : a supportive working environment ; prompt feedback ; and immunity from penalties ( administrative and financial ) .

abstractthe patient had thyroid cancer and underwent subtotal thyroidectomy . local recurrence occurred on both sides 5 years and 6 months later . the sterno - hyoid muscle and sterno - thyroid muscle were severed and the tumor around the cricoid cartilage was removed . the tumor extended into the space between the right common carotid artery and internal jugular vein and was located under the right common carotid artery and vagus nerve on the lateral side . the carotid sheath was peeled off of the carotid artery quite easily . the right common carotid artery ruptured abruptly at the distal side during this procedure.the right common carotid artery had two layers , which were very fragile , so the direct suture or repair with a graft was impossible . the carotid artery could not be trapped with ligation because the cerebral vascular supply was not examined preoperatively . this portion was repaired using the remaining carotid sheath . however , re - bleeding occurred at the proximal portion of the previous laceration spontaneously . fibrin glue with oxidized cellulose was initially used to seal the second small hole in this lesion . the second ruptured section was repaired using the remaining sterno - thyroid and sterno - hyoid muscles . the proximal portion of the right common carotid artery was reinforced with the harvested external jugular vein.these procedures resulted in hemostasis . three - dimensional ct angiography showed irregular stenosis just after the operation , but it recovered 11 days later . no cerebral infarction occurred after the operation and the patient s general condition was good . background : a limited framework of incident reporting exists in most of the health care system in pakistan . this poses a risk to the patient population and therefore there is a need to find the causes behind the lack of such a system in healthcare settings in pakistan.aims:to determine the attitudes and perceived barriers towards incident reporting among tertiary care health professionals in pakistanmaterials and methods : the study was done in shifa international hospitals and consisted of a questionnaire given to 217 randomly selected doctors and nurses . mean sd of continuous variables and frequency ( percentage % ) of categorical variables are presented . chi square statistical analysis was used to test the significance of association among doctors and nurses with various outcome variables ( motivators to report , perceived barriers , preferred person to report and patient 's outcome that influence reporting behaviors ) . p value of < 0.05 was considered significant . student doctors and student nurses were not included in the study.results:unlike consultant , registrars , medical officers and nurses ( more than 95% are willing to report ) , only 20% of house officers will report the incident happened through them . sixty nine percent of doctors and 67% of nurses perceive administration sanction as a common barrier to incident reporting . sixty percent of doctors and 80% of nurses would prefer reporting to the head of the department.conclusions:by giving immunity from administrative sanction , providing prompt feedback and assurance that the incident reporting will be used to make changes in the system , there is considerable willingness of doctors and nurses to take time out of their busy schedules to submit reports .

sulfonamides and sulfones serve both as useful intermediates in organic synthesis and as important target molecules in their own right . for example , enantioenriched secondary benzylic sulfonamides and sulfones display a range of biological activity ( e.g. , protein tyrosine phosphatase inhibitor , antisepsis agent , and -secretase inhibitor ) . however , to the best of our knowledge , there are no methods for the direct catalytic asymmetric synthesis of such sulfonamides , and just a few for such sulfones . one potential route to these compounds is the stereoconvergent coupling of a racemic -halosulfonamide / sulfone with an appropriate nucleophile ( eq 1 ) . during the past several years , we have described an array of nickel - catalyzed enantioselective cross - couplings of secondary alkyl electrophiles . whereas couplings of activated electrophiles ( a leaving group to a carbonyl , aryl , alkenyl , alkynyl , or cyano group ) proceed in good ee with a variety of nucleophiles ( alkyl- , aryl- , and alkenylmetal ) , reactions of unactivated electrophiles have been limited , with three exceptions , to alkylmetals , specifically alkyl-(9-bbn ) reagents.1 because -halosulfonyl compounds are generally poor substrates for sn2 reactions , and the sulfonyl group does not effectively stabilize an adjacent radical ( bond dissociation energy for a c h bond of dimethylsulfone : 99 kcal / mol ) , we view them as unactivated alkyl electrophiles . herein , we report the first examples of the stereoconvergent cross - coupling of unactivated alkyl electrophiles with nucleophiles other than organoboranes , as well as the first general method for their coupling with aryl- or alkenylmetal reagents ; in particular , we describe nickel / bis(oxazoline)-catalyzed cross - couplings of racemic -bromosulfonamides and -sulfones with organozinc and organozirconium reagents , thereby furnishing secondary benzylic and allylic sulfonamides and sulfones in good enantiomeric excess ( eq 2).2 upon investigating a variety of reaction parameters , we determined that a nickel / bis(oxazoline ) catalyst can achieve the cross - coupling of an -bromosulfonamide with an arylzinc reagent in very good ee and yield at 20 c ( table 1 , entry 1 ) ; nicl2glyme and bis(oxazoline ) l1 ( ( r , r ) and ( s , s ) ) are commercially available . all previous reports of stereoconvergent negishi reactions of racemic electrophiles have employed activated coupling partners . the yield was determined through gc analysis with the aid of a calibrated internal standard . essentially no cross - coupling is observed in the absence of nicl2glyme ( table 1 , entry 2 ) , and a low yield of the desired product is obtained if ligand l1 is omitted ( entry 3 ) . at room temperature , the coupling proceeds in good ee , but hydrodebromination predominates over c c bond formation . the corresponding valine - derived bis(oxazoline ) ( l2 ) is nearly as effective as phenylglycine - derived l1 ( entries 1 and 5 ) , whereas removal of the gem - dimethyl substituents on the linker between the oxazolines of l1 leads to a substantial drop in efficiency ( entry 6 ) . although pybox ligands are often the ligand of choice for nickel - catalyzed stereoconvergent negishi reactions , pybox l4 is not effective for this particular cross - coupling , nor is a 1,2-diamine ligand ( entries 7 and 8) . the use of phmgbr in place of phzni leads to a small loss in ee and a substantial loss in yield ( entry 9 ) . when the catalyst loading is reduced in half , no erosion in enantioselectivity is observed , although the yield falls by a modest amount ( entry 10 ) . the process is somewhat oxygen - sensitive ( entry 11 ; 66% conversion ) but not particularly moisture - sensitive ( entry 12 ) . under these conditions , we can achieve stereoconvergent negishi cross - couplings of a variety of racemic -bromosulfonamides with phzni to generate highly enantioenriched benzylic sulfonamides ( table 2 ) . the nitrogen of the sulfonamide can bear either an alkyl or an aryl substituent , with little impact on ee or yield ( entries 15 ) . furthermore , the r side chain can include functional groups and can be either primary ( entries 68 ) or secondary ( entry 9 ) , although in the case of the latter a diminished yield is observed . on a gram scale , the cross - coupling illustrated in entry 2 proceeds in 92% ee and 98% yield . the conditions that we developed for stereoconvergent negishi reactions of -bromosulfonamides ( table 2 ) can be applied without modification to the corresponding sulfones ( table 3 ) . thus , the r substituent can range in steric demand from methyl to t - butyl ( entries 14 ) , and it can be aromatic ( entry 5 ) . with respect to the r group , it may be linear or branched ( entries 15 ) . we have examined the scope of arylzinc nucleophiles that participate in this method for the catalytic asymmetric synthesis of benzylic sulfonamides and sulfones ( table 4 ) . electron - rich and electron - poor arylzinc reagents are suitable partners , as is a heteroarylzinc reagent , furnishing the desired product in very good ee . especially noteworthy is our observation that o - substituted phenylzinc reagents can be employed with both sulfonamide- and sulfone - based electrophiles ( entries 57 and 911 ) ; in our previous studies of nickel - catalyzed enantioselective arylations , o - substituted nucleophiles have generally been poor coupling partners . all data are the average of two experiments . amount of arzni : 2.0 equiv ; amount of catalyst : 20% nicl2glyme , 26% ( r , r)-l1 . next , we sought to expand the scope of stereoconvergent cross - couplings of -bromosulfonamides and -sulfones to include a second family of nucleophiles , specifically , alkenylmetal reagents . we determined that , whereas reactions of alkenylzinc reagents proceed in poor yield using the method described above , alkenylzirconium reagents serve as suitable nucleophiles under modified conditions ( table 5 ) . thus , an array of allylic sulfonamides and sulfones can be synthesized with good enantioselectivity in the presence of functional groups such as a thiophene and a primary alkyl chloride . we have suggested that , for at least some nickel - catalyzed cross - couplings of alkyl electrophiles , an alkyl radical may be generated during the oxidative - addition step of the catalytic cycle . in the case of electrophiles that bear an appropriately positioned pendant olefin , we have observed complete cyclization of the putative radical onto the olefin in some instances , and no cyclization in another . in order to gain insight into the mechanism of the stereoconvergent arylations described herein , we investigated the negishi reaction of an -bromosulfonamide bearing a pendant olefin ( eq 3 ) . at 70% conversion of the electrophile , we observe a 38% yield of the direct ( uncyclized ) cross - coupling product ( 8) and a 17% combined yield of cyclized cross - coupling products ( 9).3 cyclopentane derivatives cis-9 and trans-9 are each an 1:1 mixture of diastereomers ( differing in the relative stereochemistry at the deuterium - bearing carbon ) , consistent with the expectation for cyclization of radical intermediate 10 ( but not for a simple organometallic pathway involving only -migratory insertion and then reductive elimination ) . our observation that uncyclized cross - coupling product 8 has deuterium only in the trans position suggests that , when intermediate 10 does cyclize , the process is irreversible . simple 5-hexenyl radical cyclizations proceed with first - order rate constants of 10 s. although , to the best of our knowledge , the rate of cyclization of sulfonamide - substituted radical 10 has not been determined , it is very likely to be significantly slower than diffusion ( 10 s ) . our observation of cyclized , cross - coupled products ( 9 ) therefore represents evidence that a nickel - catalyzed asymmetric cross - coupling process may include a noncage radical pathway . consistent with this suggestion , the ratio of uncyclized ( d)/cyclized ( c ) product changes as the concentration of catalyst changes ( figure 1 ) . dependence of the ratio of uncyclized ( d)/cyclized ( c ) product on the concentration of nickel . we have developed methods for the enantioselective synthesis of secondary sulfonamides and sulfones , specifically , nickel - catalyzed negishi arylations and alkenylations of racemic -bromosulfonamides and -sulfones with readily available organozinc and organozirconium reagents ; with regard to stereoconvergent couplings of unactivated alkyl electrophiles , previous examples had been limited to organoboron nucleophiles and , with the exception of three isolated examples , to alkylation reactions . in terms of mechanism , a cyclization / stereochemical probe has provided evidence for a radical intermediate that has a sufficient lifetime to escape the solvent cage and cyclize irreversibly under the coupling conditions . additional studies directed at elucidating the mechanisms of cross - coupling reactions of alkyl electrophiles , as well as expanding the scope of such processes , are underway . gold nanorods ( gnrs ) are anisotropic particles whose surface plasmon modes can be tuned as a function of aspect ratio , with optical resonances ranging from visible to near - infrared wavelengths . gnrs have been investigated as contrast agents for optical biomedical imaging modalities such as optical coherence tomography and photoacoustic tomography ; they are also capable of producing linear and two - photon excited luminescence , with detection limits at the single - particle level . the large absorption cross sections of gnrs can also generate localized photothermal effects , with application toward the release of molecular cargo and hyperthermic effects on diseased cells and tissues . these attributes have sparked a global effort to develop gnrs into theranostic agents for nanomedicine . one hurdle in the scalable manufacturing of gnr - based materials is the efficient exchange and removal of cetyltrimethylammonium bromide ( ctab ) , a micellar surfactant used in the batch synthesis of gnrs . ctab is cationic and moderately cytotoxic ( although not insupportably so ) , most of which can be removed by multiple washes and exchanges with chemisorptive surfactants ( e.g. , pegylated thiols or dithiocarbamates ) , phospholipids , or other surface - active agents . however , ctab - coated gnr dispersions are frequently destabilized during surfactant exchange , resulting in partial aggregation and low recovery yields . furthermore , ligand - modified gnrs are often contaminated with residual ctab , which can induce nonspecific protein adsorption and cell uptake under physiological conditions , or produce surface charge defects in materials applications . we have previously shown that ctab - depleted gnr dispersions can be prepared when using sodium polystyrenesulfonate ( na - pss ) as a mild detergent ; nevertheless , the stability of such suspensions remains capricious in subsequent manipulations . we thus sought to develop a practical method for producing ctab - free gnr dispersions that would be universally compatible with surface conjugation protocols . in this letter we describe an efficient method for converting ctab - stabilized gnrs into citrate - stabilized gnrs ( cit - gnrs ) via intermediate treatment with pss . the surface exchange process was monitored in stages by x - ray photoelectron spectroscopy ( xps ) , an invaluable tool for quantitative elemental analysis of surface adsorbates , as well as by attenuated total reflectance infrared ( atr - ir ) spectroscopy and surface - enhanced raman scattering ( sers ) . our analyses indicate that ctab removal by pss treatment is highly efficient , as is the subsequent displacement of pss by citrate . the cit - gnrs are fully dispersible in low - salt solutions , and are easily functionalized by further ligand exchange ( discussed in a companion article ) . a six - stage protocol was developed to convert ctab - gnrs ( 68 26 nm , lpr = 713 nm ) into cit - gnrs , using standard ultrafiltration and centrifugation equipment . ctab - gnrs were prepared on a gram scale by the method described by khanal and zubarev , and diluted 4-fold to prevent premature flocculation of ctab - gnrs during the initial purification stages . in a typical process , a suspension of gnrs ( optical density ( o.d . ) = 3.2 ) stabilized in 25 mm ctab was concentrated by stirred ultrafiltration to remove excess surfactant , then diluted with deionized water ( stage 1 ; figure 1 ) . the gnrs were then subjected to three cycles of centrifugation and redispersion ( c / r ) to deplete ctab to trace levels , using 0.15 wt % na - pss ( mw = 70 kda ; stages 24 ) . we note that the residual ctab in the pss - stabilized gnr suspension after stage 4 is < 1 m based on simple dilution factors ; however , the free ctab concentration is likely even lower due to its favorable adsorption to pss . the pss - gnrs were then subjected to two additional c / r cycles using 5 mm sodium citrate ( stages 5 and 6 ) for exchange with pss , yielding stable dispersions of cit - gnrs . a complete procedure with exact volumes and concentrations is provided in the supporting information . process flow diagram for converting ctab - gnrs into cit - gnrs by ultrafiltration ( stage 1 ) with successive c / r cycles using 0.15 wt % na - pss ( stages 24 ) and 5 mm na3-cit ( stages 5 and 6 ) , with estimated surfactant concentrations after each stage . the conversion of ctab - gnrs into cit - gnrs was achieved with an overall efficiency of 7585% , based on initial and final o.d . the main source of loss is due to incomplete recovery of gnrs during the first two c / r cycles . absorption spectroscopy and transmission electron microscopy ( tem ) analysis of the citrate - stabilized gnrs indicated essentially no changes in optical resonance ( max = 710 nm ; figure 2 ) or size distribution ( figure s1 , supporting information ) . on the other hand , the exchange of pss to citrate was readily discernible by electrokinetic measurements : the zeta potential of gnrs in dilute na - pss solution ( 0.25 mg / ml , ph 5 ) was 55.6 8.3 mv , while that of cit - gnrs in dilute phosphate buffered solution ( pbs ) adjusted to ph 9.5 ( i 8.2 mm ) was less negative ( mean 26.9 13.2 mv ; mode 21.1 7.1 mv ) . the cit - gnr suspensions are stable for months at low ionic strength but aggregate if dispersed in highly saline solutions ( also see below ) , and thus behave similarly as other citrate - stabilized au nanoparticles . normalized absorption spectra of gnr dispersions after ultrafiltration ( stage 1 , red ) , c / r cycles with na - pss ( stage 3 , green ) , and c / r cycles with na3-cit ( stages 5 and 6 , blue ) . the incubation of gnrs with 70-kda na - pss ( minimum 1 h , prior to each c / r step ) is essential for producing well - dispersed cit - gnrs . previous studies in our laboratory have established that pss is useful as a detergent for removing residual ctab from gnr dispersions , below the level of observable cytotoxicity . however , na - pss by itself is not suitable as a peptizing agent : it adsorbs weakly onto gnr surfaces in the absence of ctab , resulting in metastable dispersions whose relative stability depends on polymer concentration , suggestive of depletion effects . gnrs cleansed with pss can be stabilized afterward by introducing surface - active agents that do not associate strongly with the polyelectrolyte . in this regard , polyanions with moderate binding activity such as citrate are ideal for surface exchange ; the pss is gently displaced from the gnrs while maintaining a negative zeta potential during the exchange process . nonionic , hydrophilic surfactants can also be used to displace pss from gnr surfaces , but the efficiency of exchange depends on the specific qualities of the surface - active agent . attempts to treat ctab - gnrs directly with citrate without na - pss cleansing invariably led to rapid aggregation , regardless of surfactant concentrations ( figure s2 , supporting information ) . this is not surprising , as the citrate anions neutralize the positive surface charge supported by the resident ctab layer , resulting in particle destabilization prior to complete ligand exchange . it has been reported that citrate preferentially adsorbs onto the ends of ctab - gnrs , resulting in their end - to - end aggregation . the ligand exchange process from stage 3 ( pss wash # 2 ) to stage 6 ( citrate wash # 2 ) was evaluated by xps , based on the binding energies ( b.e . ) of various atomic species ( table 1 and figure 3 ) . the xps signal corresponding to bromine ( br 3d ; 68.5 ev ) could not be detected even at stage 3 , whereas that of nitrogen ( n 1s ; 401 ev ) was reduced below the limit of detection by stage 5 , establishing the essentially complete displacement of ctab from the gnr surfaces . the xps signals for carbon ( c 1s ) , oxygen ( o 1s ) , sodium ( na 1s ) , and sulfur ( s 2p ) were also greatly reduced upon washing with 5 mm citrate ( stage 5 ) , indicating the gross displacement of na - pss from the gnr surfaces ( figure 3a ) . the s 2p signal was almost completely gone by the second citrate wash ( stage 6 ) , and below trace levels in the supernatant . high - resolution analysis of the c 1s region further revealed that citrate exchange was accompanied by a change in the population of carbon subtypes , with a reduction in hydrocarbon species ( c h / c c ; b.e . = 284.8 ev ) and an increase in the density of carboxyl groups ( c(=o)o ; b.e . = 288.2 ev ) , corresponding with the adsorption of citrate ( figure 3b ) . binding energies from survey xps spectra . at or below limit of detection . ( a ) survey xps spectra acquired from pelleted gnr samples after stage 4 ( pss wash # 3 ) and stage 5 ( citrate wash # 1 ; 5 magnification ) , indicating the disappearance of the n 1s signal . ( b ) high - resolution ( regional ) xps spectra of the c 1s region , indicating relative changes in carbon subtype after treatment with citrate . it is worth noting that the xps spectra also indicated the significant presence of silver ( ag 3d ; ca . 25% versus au 4f ) , which remained approximately constant throughout the ligand exchange process . agno3 is used as an additive in the seeded growth of gnrs , and has been postulated to contribute toward shape control by its selective deposition onto longitudinal facets . this notion has recently been brought into question by high - resolution tem imaging using energy dispersive x - ray spectroscopy , which indicated the distribution of ag over the entire gnr surface . regardless of mechanism , the intensity of the ag 3d xps signal in our study also suggests that the deposited ag is mostly localized near the gnr surface . the ligand exchange process was also characterized by atr - ir spectroscopy and sers , to identify the molecular species associated with the gnr samples ( figures s3 and s4 , supporting information ) . ir analysis of pelleted gnr samples from stages 1 , 3 , and 4 confirmed the presence ( and depletion ) of ctab and pss , with the former producing strong bands associated with sp c h stretching ( 28002900 cm ) , ch3 deformation ( 14501480 cm ) , and c n stretching ( 900950 cm ) , and the latter producing peaks associated with the so3 group ( 10201200 cm ) . pss displacement by citrate anions produced additional spectral changes , dominated by co2 stretching ( 1575 cm ) and ch2 deformation ( 1390 cm ) . sers analysis also confirmed that pss was adsorbed onto gnrs by the enhanced so3 raman signal , but was displaced after two washes with na3-cit . overall , these vibrational analyses further support the conclusions drawn from the xps data . citrate - stabilized au nanoparticles are widely appreciated for their versatility in surface modification protocols , with minimal interference by the displaced electrolyte . we conducted a preliminary survey of cit - gnrs dispersed in different aqueous solutions , to determine their relative stability during surface functionalization . dispersions of cit - gnrs are stable in 10 mm sodium citrate ( i = 60 mm ) , but slowly aggregate in standard pbs ( i 160 mm ) or 1 m nacl . however , cit - gnrs treated with nonionic surfactants such as tween-20 , chemisorptive surfactants such as thiolated polyethylene glycol , or serum proteins such as albumin all form stable dispersions in pbs . such coated gnrs have been characterized and are further described in our companion paper . the protocol described above is a mild and general approach for depleting ctab and other cationic surfactants from aqueous nanoparticle suspensions , allowing their clean exchange with citrate ions . the citrate - stabilized gnrs are compatible with surface conjugation chemistries commonly applied to metal colloids , and provide a reliable platform for making rational changes in chemical and biological properties as a function of surface modification .

the development of efficient methods for the generation of enantioenriched sulfonamides and sulfones is an important objective for fields such as organic synthesis and medicinal chemistry ; however , there have been relatively few reports of direct catalytic asymmetric approaches to controlling the stereochemistry of the sulfur - bearing carbon of such targets . in this report , we describe nickel - catalyzed stereoconvergent negishi arylations and alkenylations of racemic -bromosulfonamides and -sulfones that furnish the desired cross - coupling product in very good ee and yield for an array of reaction partners . mechanistic studies are consistent with the generation of a radical intermediate that has a sufficient lifetime to diffuse out of the solvent cage and to cyclize onto a pendant olefin . stable aqueous dispersions of citrate - stabilized gold nanorods ( cit - gnrs ) have been prepared in scalable fashion by surfactant exchange from cetyltrimethylammonium bromide ( ctab)-stabilized gnrs , using polystyrenesulfonate ( pss ) as a detergent . the surfactant exchange process was monitored by infrared spectroscopy , surface - enhanced raman scattering ( sers ) , and x - ray photoelectron spectroscopy ( xps ) . the latter established the quantitative displacement of ctab ( by pss ) and of pss ( by citrate ) . the cit - gnrs are indefinitely stable at low ionic strength , and are conducive to further ligand exchange without loss of dispersion stability . the reliability of the surface exchange process supports the systematic analysis of ligand structure on the hydrodynamic size of gnrs , as described in a companion paper .

many psychoactive substances have therapeutic function as analgesics or anesthetics and high addiction potential ( 1 ) . are commonly used on the other hand an opioid consumption is traditionally common in iran . the effects of different addictives on body systems have been reported ; however , there is not enough information about different blood parameters ( 2 ) . the effect of some psychoactive substances on commonly observed diseases like coronary artery disease , diabetes mellitus and some psychiatric disorders has been investigated , but effects of long - term use of these substances on endocrine system have not been studied enough ( 3 ) . studies have demonstrated controversial results about effects of psychoactive substances on hypothalamic - pituitary axis and thyroid function ( 4 ) . psychoactive substances have many different effects on physiologic and neuroendocrine functions in humans ( 5 ) . it has been demonstrated that endogenous opioids like -endorphin , enkephalins and opiate receptors influence neuroendocrine regulation ( 6 ) . previous studies showed that some psychoactive substances modulate immune function and affect resistance to bacterial , viral and protozoan infections ( 7 , 8) . monitorization of the psychoactive substance use can be done objectively by urine analysis . since substance concentrations in urine are higher than blood , urine is the preferred material for determination of substance use . testing schedules and determination of cut - offs are important for confirmation of the psychoactive substance use ( 9 ) . since polydrug abuse is common worldwide , determination of the effects of only one drug is difficult ( 10 ) . recent studies demonstrated controversial results about the effects of opium on serum electrolytes , lipid markers and glucose metabolism . defining the real side effects of these psychoactive substances may increase awareness about disadvantages of consumption ( 11 ) . studies about the effects of psychoactive substances on different biochemical and hematological parameters were limited in number ( 2 , 12 ) . this study was conducted to determine the effects of different psychoactive substances on serum biochemical parameters . this study was conducted on 324 patients and 69 controls admitted to erenkoy mental health and neurology training and research hospital between january 2013 and january 2014 . patients were determined according to the diagnostic and statistical manual of mental disorders , fourth edition dependent group involves subjects determined as at least one or more than one type of psychoactive substance users . in the control group urine toxicology results were negative . levels of toxicology parameters were determined using a hitachi model 902 automatic analyzer ( hitachi high - technologies corporation , roche diagnostics ) with an enzyme immunoassay ( microgenics cedia fremont , california , usa , for urine toxicology ) . after hospitalization , each patient s first urine and blood sample before treatment were taken into account for comparison . cases and controls with accompanying diseases like diabetes , cancer , metabolic disorders etc . are excluded from the study . urine samples were tested simultaneously for heroin , cannabinoids , cocain , benzodiazepins , opiates , buprenorfine , amphetamines , extacy and ethyl glucuronide . routine biochemistry parameters were measured using the architect ci 4100 ( abbott diagnostics products ) automatic biochemistry analyzer . this study was approved by the ethics committee of erenkoy mental health and neurology training and research hospital . spss ibm 20.0 software ( chicago , illinois , usa ) was used for statistical analysis . spss ibm 20.0 software ( chicago , illinois , usa ) was used for statistical analysis . a total of 324 patients ( 316 males and 8 females ) and 69 controls ( 64 males and 5 females ) were involved in this study . the median ages of psychoactive substance users and controls were 26.5 ( 23 - 34 ) and 27 ( 20.5 - 34.5 ) years , respectively ( table 1 ) . the concentration of biochemical parameters in the serum of the substance - dependent group was compared to the control group ( table 2 ) . we found a statistically significant difference in gamma - glutamyl transferase ( ggt ) , uric acid , creatinin , urea , albumin , aspartate aminotransferase ( ast ) medians between the patient and control groups ( p < 0.05 ) . abbreviations : alp , alkaline phosphatase ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; crp , c - reactive protein ; ggt , gamma - glutamyl transferase ; hdl , high density lipoprotein ; ldh , lactate dehydrogenase ; and ldl , low density lipoprotein . forty - six out of 424 addicts tested positive for only opium ( cut - off , 300 ng / ml ) . several biochemical parameters in the opium - dependent group were compared to the control group ( table 3 ) . there was a statistically significant difference in the serum concentrations of sodium and albumin between the opium - dependent and the control groups ( p < 0.05 ) . moreover , sixty - nine patients were tested positive for only cannabinoids ( cut - off , 50 ng / ml ) . biochemical parameters did not show any significant difference in the cannabinoid - dependent group compared to the control group ( p > 0.05 ) . thirteen out of all addicts were tested positive for only benzodiazepine ( cut - off > 300 ng / ml ) . there was a statistically significant difference in ggt , urea , glucose , sodium , total protein , and ast levels between the benzodiazepine - dependent group and control group ( p < 0.05 ) . also , 12 patients were tested positive for only ethyl glucuronide ( cut - off > 500 ng / ml ) . a statistically significant difference was found in triglyceride and ggt levels between the ethyl glucuronide and control groups ( p < 0.05 ) . clinical observations demonstrated that adverse effects of psychoactive substances were associated with the addiction period and route of administration . psychoactive substance users commonly have nutritional problems since they spend their money largely for drugs ( 13 ) . coexistence of psychiatric disorders in drug addicts is also important since some psychiatric disorders may cause loss of appetite and poor nutritional status and changes in biochemical parameters ( 14 ) . clinical studies showed that long - term use of opium and heroin can affect serum glucose , potassium , calcium , uric acid and cholesterol levels ( 12 ) . in our study , we found a significant difference in ggt , uric acid , creatinine , urea , albumin , and ast medians between the dependent and control groups ( p < 0.05 ) . studies about the effects of opium addiction on blood glucose showed that the opium - dependence decreased fasting blood glucose temporarily without any effect on hemoglobin a1c ( 15 ) . we found a statistically significant decrease in glucose levels only in patients tested positive for benzodiazepine ( p < 0.05 ) . some psychoactive substances may affect gluconeogenesis so that blood glucose levels may decrease , especially in cases with poor nutrition . some people believe that psychoactive substances like opium can have beneficial effects on blood lipid profile and cardiovascular disease , but clinical observations demonstrated that opium can increase risk of atherosclerotic plaque formation and adversely affect lipid profile in animal models ( 16 ) . we found a statistically significant increase in triglyceride levels in ethylglucuronide group ( p < 0.05 ) . in our study , we did not found statistically significant difference in total cholesterol , low density lipoprotein ( ldl ) , and high density lipoprotein ( hdl ) levels between dependent and control groups ( p > 0.05 ) . cardiovascular side effects of some psychoactive substances may not be attributed to changes in lipid profile . there is a need to conduct a prospective study with the larger sample size for the prevalence of the cardiovascular disease . another study , which was performed on opium addicts showed that serum adiponectin levels were decreased but no significant change was observed in serum leptin levels . it was also indicated in previous studies that a decrease in adiponectin levels can be associated with an increased risk of metabolic disorders like insulin resistance and cardiovascular disease since it has known antidiabetic and antiatherogenic effects ( 17 ) . changes in nutritional patterns may also be related with changes in albumin levels in these patients ( 18 ) . we found a statistically significant difference in albumin levels between the drug - dependant and control groups . in our study , the amount of total protein showed a significant difference only in the benzodiazepine dependent group ( p < 0.05 ) . since in our study , patients with significant derangements were referred to specialized medical centers for further assessment and excluded from the study , changes in their albumin levels may not be due to viral infections or metabolic disorders . in a previous study , it has been demonstrated that morphine consumption for long period in animal models increases uric acid and creatinine levels ( 19 ) . we found a statistically significant decrease in uric acid and creatinine levels in the dependant group compared to the control group ( p < 0.05 ) . poor living conditions and nutritional factors like low protein intake may likely to contribute to changes in biochemical parameters in psychoactive substance users . these effects on renal system include changes in urinary output and urinary sodium excretion ( 20 ) . divsalar et al . showed that a sodium level in the heroin - dependent group did not change ; however , it was significantly high in the ex - heroin dependent group compared to the control group . however , no significance difference was found in potassium and calcium levels between the ex - heroin dependent and control groups ( 14 ) . we found that the concentration of sodium in the serum of the opium - dependent and benzodiazepine dependent groups was significantly increased compared to the control group ( p < 0.05 ) . no statistically significant difference was observed in calcium levels between the dependent and control groups ( p > 0.05 ) . routine biochemistry , and hematology parameters , and vitamin , and mineral levels are affected by nutritional factors . determining the differences in biochemical parameters , vitamin and mineral levels between addicts and healthy subjects may help to define individuals at nutritional risk and provide these patients with the corrective nutritional programs ( 18 ) . in this study we tried to describe the extent of changes in various blood parameters due to psychoactive drug use . our study involves a range of psychoactive substances like heroin , cannabinoids , cocaine , benzodiazepine , opiate , buprenorphine , amphetamine , extacy , ethyl glucuronide and their potential effects on many biochemical parameters . raising awareness about potential adverse effects of psychoactive substances health problems associated with physiological side effects of these substances may increase economic burden on health care resources . therefore , in psychoactive substance users defining the real need for intensive monitoring and treatment programs are extremely important . research interest is increasing in biomaterials with the potential to stimulate bone healing have gained interest to improve treatment outcome for patients with osteoporotic fractures . calcium phosphate cements ( cpc ) has been used in bone surgery for many years . the bio - inspired combination of the inorganic component silica and the organic component collagen has lead to a novel class of innovative biomaterials because silica and collagen turned out to be ideal partners for composite formation , resulting in advantageous synergistic effects in material properties suitable for soft - tissue or hard - tissue substitution applications . the chemical and technological flexibility of the silica / collagen system has generated a remarkably wide range of biomaterials individually adapted for a large number of biomedical applications , including soft hydrogels , macroposous scaffolds , and solid compact xerogels . in addition , metal foams open new opportunities for design of bone implants due to their mechanically stability . in cardiovascular applications , iron has been used successfully in animal implantation studies and in clinical cases . the use of pet scanning as a diagnostic tool for fracture healing is attractive because it can provide a direct quantitative assessment of metabolic activity in a region of interest . we recently showed that the fixation of fluoride in the bone matrix may be enhanced using cpc , especially cpc with strontium carbonate as biomaterial for bone replacement and defect healing in osteoporotic bone . the present study was designed to examine the efficacy of multifunctional bioabsorbable implants , including cpc - based , silica / collagen - based , and iron - based composites for the treatment of experimental osteoporotic fractures . the treatment response was assessed by f-18-sodium fluoride ( naf ) dynamic pet - ct ( dpet - ct ) . all animal procedures were carried out in accordance with the guide for care and use of laboratory animals of the national institutes of health . the animal studies were approved by the corresponding authority ( regierungsprsidium gieen , gi 20/28 nr . the study included 65 3-month - old female sprague - dawley rats ( charles rivers wiga , sulzbach , germany ) with a body weight between 250 and 400 g. after an acclimatization of 4 weeks , an osteoporotic bone status was induced by bilateral ovariectomy and a calcium- , phosphorous- , vitamin d3- , soy- , and phytoestrogen - free diet , as previously described by heiss et al . . after 3 months , the animals underwent femur surgery under general anaesthesia with xylazine ( 4 mg / kg body weight , rompun , bayer ) and ketamine ( 100 mg / kg body weight , hostaket , hoechst ) by intraperitoneal injection . the left femur was desinfected with povidone iodine ( braunol , braun , melsungen , germany ) and draped in a sterile manner . after making a 4-cm skin incision at the lateral aspect of the distal thigh , the lateral vastus muscle and the lateral head of the femoral biceps muscle were dissected in their septum intermusculare and the femur from shaft to the condyle massif was exposed . subsequently , a 7-hole t - shaped miniplate ( leibinger xs - miniplate , stryker , schnkirchen , germany ) was slightly bent and fixed to the lateral femur with 1.2-mm screws . afterwards , a wedge - shaped defect with a lateral height of 4 mm was then created at the metaphyseal area of the femur . the cutting lines were marked with a custom - made triangular sawing guide with a lateral length of 4 mm . the distal transverse osteotomy was made just above the border of the femoropatellar joint , without bruising the articular cartilage . calcium phosphate cement ( cpc ) and cpc enriched with strontium ( srcpc ) were prepared as described previously . in brief , cpc powder was composed of -tricalcium phosphate ( -ca3(po4)2 ) , calcium hydrogen phosphate ( cahpo4 ) , calcium carbonate ( caco3 ) , and hydroxyapatite ( ca10(po4)6(oh)2 ) , and was supplied by innotere gmbh ( radebeul , germany ) . srcpc powder was obtained by substitution of caco3 by srco3 ( alfa aesar ) in the cpc cement precursor powder . a mouldable paste was obtained by mixing the cement powder with na2hpo4 solution using a liquid - to - powder ratio of 500 and 350 l / g for cpc and srcpc , respectively , immediately prior to implantation . srcpc was previously shown to release significant amounts of sr upon immersion in aqueous media ( e.g. , cell culture medium ) and to positively influence both in vitro osteoblast proliferation and differentiation and in vivo bone formation ( 15 ) . composites of silica and fibrillar bovine collagen were used for implantation as monolithic xerogels ( b30 , 70 wt% silica , 30 wt% collagen ) or as porous scaffold ( sc - b30 , xerogel particles b30 , size < 250 m , embedded in a collagen matrix with xerogel / matrix weight ratio of 1.0 ) . for preparation of compact silica / collagen xerogels , bovine tropocollagen type i ( gfn , germany ) was dialysed ( nominal molecular weight cut - off 1214 kda , roth , germany ) against deionized water followed by fibrillation in 30-mm neutral sodium phosphate buffer solution , lyophilisation ( christ alpha 14 laboratory freeze - dryer , osterode , germany ) , and resuspension in 0.1 m trishcl ph 7.4 ( roth ) to obtain homogeneous 30 mg / ml suspensions . the silica component was prepared by hydrolysing tetraethoxysilane ( teos , 99% , sigma , germany ; molar ratio teos / water=1/4 ) under acidic conditions ( 0.01 m hcl ) to obtain silicic acid . vigorous stirring of calculated volumes of silicic acid and collagen suspension to obtain a final composition of 70% silica and 30% collagen ( b30 ) resulted in the formation of 800-l hydrogels . a modification of the scaffolds was prepared by using xerogel particles consisting of 50 wt% silica , 30 wt% fibrillar bovine collagen , and 20 wt% strontium carbonate ( sc - b30sr20 ) . strontium carbonate was introduced as a third phase by being previously added to the collagen suspension . b30 or b30sr20 gels were transferred to molds , stabilized , and dryed for 7 days in an espec sh-221 climate chamber ( japan ) at 37c and 95% relative humidity . the resulting xerogel samples were ground and classified according to the particle size . on the one hand , b30 xerogel particles < 0.250 mm were compacted using a custom - made pressing tool to obtain monolithic b30 xerogel samples exhibiting exactly the above - described bone defect shape . on the other hand , b30 or b30sr20 xerogel particles < 0.120 mm were added to a 30-mg / ml collagen suspension adjusting a xerogel / collagen weight ratio of 1/1 . these xerogel particle / collagen suspensions were transferred to custom - made silicon molds to again obtain the above - described bone defect shape , cooled at 0.5 k / min to 20c ( espec sh-221 climate chamber , osaka , japan ) followed by lyophilisation ( christ alpha 14 laboratory freeze - dryer , osterode , germany ) . the scaffolds were chemically cross - linked by immersing in 1 wt% n-(3-dimethylaminopropyl)-n0-ethylcarbodiimide ( edc)/n - hydroxysuccinimide ( nhs ) ( sigma ) in 40% ethanol for 24 h. finally , the scaffolds were rinsed in deionized water and freeze - dried again . according to the xerogel particle composition used , scaffold sample labels were scb30 or scb30sr20 , respectively . all xerogel or scaffold samples were gamma - sterilized at 25 kgy before used for implantation . moreover , iron foam with interconnected pores ( fe ) was applied . for preparation of strontium coating of iron foams , 153 ml of ultrapure water and 17 ml of phosphoric acid ( 85% h3po4 ) were added in a 250-ml glass bottle . the supernatant was diluted 20-fold using ultrapure water , which yielded the solution of sr(h2po4)2 . ten fe foams eroded and cleaned by ifam ( fraunhofer institute for manufacturing technology and advanced materials , dresden ) with an average weight of 170 mg were coated by suspension in 100 ml of sr(h2po4)2 solution for 4 h under vacuum . sr - coated foams were settled in fresh coating solution for another 3 days at room temperature and rinsed 3 times using absolute ethanol with subsequent drying at 40c in a drying cabinet . sr - coating resulted in an average weight gain of 37 mg or approximately 22% . in addition , the alloy element phosphor is added with fe3p - powder ( atmix , japan ) with a particle size of 3.8 m . to achieve a phosphate ratio of 0.6% the iron foam was coated with zolendronic acid ( fe - bp ) that is a member of the bisphosphonate family . . then the complex was carefully washed , dried , ground , and finally led to a coating of 35-g zolendronic acid , the basic iron foam . these 8 different formulations of bone substitution materials were implanted in the current animal model ( n=78 ) ( table 1 ) . eighteen weeks after induction of osteoporosis and 6 weeks after femoral surgery , dpet - ct with f - sodium fluoride ( naf ) scans were performed . during scanning , rats were anesthetized using a mixture of nitrous oxide ( 1 l / min ) , oxygen ( 0.5 l / min ) and isoflurane ( 1.5 vol.% ) . dpet - ct studies were performed for 60 min after the intravenous application of 20 to 40 mbq f - sodium fluoride , using a 28-frame protocol ( 10 frames of 30 s , 5 frames of 60 s , 5 frames of 120 s , and 8 frames of 300 s ) . a dedicated pet - ct system ( biograph mct , 128 s siemens co , erlangen , germany ) with an axial field of view of 21.6 cm with truev and truepoint , operated in a 3-dimensional mode , was used for all animal studies . the system provides the simultaneous acquisition of 369 transverse pet slices with a slice thickness of 0.6 mm . two rats were examined in parallel and were positioned in the axial plane of the system to maintain the optimum resolution in the center of the system . a high - resolution ct scan was performed prior the pet scanning with 160 ma , 80 kv , pitch of 0.85 cm in addition to the low - dose attenuation ct ( 80 ma , 80 kv ) for attenuation correction of the acquired dynamic emission pet data . all pet images were attenuation - corrected and an image matrix of 400400 pixels was used for iterative image reconstruction ( voxel size 1.5651.5650.6 mm ) based on the syngo mi pet / ct 2009c software version . the reconstructed images were converted to standardized uptake value ( suv ) images based on the formula ( 18 ) : suv = tissue concentration ( bq / g)/[injected dose ( bq)/body weight ( g ) ] . volumes of interest were placed over the created defect area in the femur , and rotated pet images according to the ct images . irregular regions of interest were drawn manually according to the fused pet - ct images , as mentioned before . to precisely select the region to draw the voi of the created defect , the fusion of pet images and ct scans by co - registration preprocessing was performed after rotation of each rat individually . first , we rotated the ct series to determine the optimum angle for the visualisation of the complete defect and the adjacent metal plates in the longitudinal direction . the next step was to fuse ct and pet and to use these fused images for the quantitative pet evaluation . the spatial resolution was 2 mm for pet image and 0.33 mm for ct image . the voi for the defect was placed between the 2 screws located at the left and the right side of the 4-mm created defect . for input , we used 10 contiguous pet slices in the middle and lower third of the aorta . we avoided using the upper part of the thoracic aorta due to spillover from the heart . herein , a 2-tissue compartment model was used to evaluate the dynamic studies and to gain quantitative data about the kinetics . in the current study , the learning - machine 2-tissue compartment model was used for the fitting , and provided 5 parameters : the plasma clearance to the bone extracellular fluid ( ecf ) compartment and the rate constant for return of tracer to plasma ; k1 and k2 ; the rate constants describing movement of tracer into and out of the bound bone compartment ; k3 and k4 ; and the fractional blood volume , also called vessel density ( vb ) , which reflects the amount of blood in the voi . following compartment analysis , we calculated the plasma clearance of tracer to bone mineral from the compartment data using the formula : influx ( ki)=(k1*k3)/(k2+k3 ) . compared to the standard iterative method , the machine learning method has the advantage of a fast convergence and avoidance of over - fitting . the model parameters were accepted when k1k4 was less than 1 and vb exceeded 0 . the fractal dimension is a parameter of heterogeneity and was calculated for the time - activity data of each individual volume of interest . the values for fractal dimension vary from 0 to 2 , showing the deterministic or chaotic distribution of tracer activity . we used a subdivision of 77 and a maximal suv of 20 for the calculation of fractal dimension . statistical evaluation was performed with stata / se 11.1 ( statacorp , college station , tx , usa ) using the descriptive statistics and box plots . linear discriminant analysis was performed for calculating the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and highest accuracy . the 2-sided wilcoxon rank sum test was applied for all pet parameters , which also included suv , using a single parameter analysis to assess groups . all animal procedures were carried out in accordance with the guide for care and use of laboratory animals of the national institutes of health . the animal studies were approved by the corresponding authority ( regierungsprsidium gieen , gi 20/28 nr . the study included 65 3-month - old female sprague - dawley rats ( charles rivers wiga , sulzbach , germany ) with a body weight between 250 and 400 g. after an acclimatization of 4 weeks , an osteoporotic bone status was induced by bilateral ovariectomy and a calcium- , phosphorous- , vitamin d3- , soy- , and phytoestrogen - free diet , as previously described by heiss et al . . after 3 months , the animals underwent femur surgery under general anaesthesia with xylazine ( 4 mg / kg body weight , rompun , bayer ) and ketamine ( 100 mg / kg body weight , hostaket , hoechst ) by intraperitoneal injection . the left femur was desinfected with povidone iodine ( braunol , braun , melsungen , germany ) and draped in a sterile manner . after making a 4-cm skin incision at the lateral aspect of the distal thigh , the lateral vastus muscle and the lateral head of the femoral biceps muscle were dissected in their septum intermusculare and the femur from shaft to the condyle massif was exposed . subsequently , a 7-hole t - shaped miniplate ( leibinger xs - miniplate , stryker , schnkirchen , germany ) was slightly bent and fixed to the lateral femur with 1.2-mm screws . afterwards , a wedge - shaped defect with a lateral height of 4 mm was then created at the metaphyseal area of the femur . the cutting lines were marked with a custom - made triangular sawing guide with a lateral length of 4 mm . the distal transverse osteotomy was made just above the border of the femoropatellar joint , without bruising the articular cartilage . calcium phosphate cement ( cpc ) and cpc enriched with strontium ( srcpc ) were prepared as described previously . in brief , cpc powder was composed of -tricalcium phosphate ( -ca3(po4)2 ) , calcium hydrogen phosphate ( cahpo4 ) , calcium carbonate ( caco3 ) , and hydroxyapatite ( ca10(po4)6(oh)2 ) , and was supplied by innotere gmbh ( radebeul , germany ) . srcpc powder was obtained by substitution of caco3 by srco3 ( alfa aesar ) in the cpc cement precursor powder . a mouldable paste was obtained by mixing the cement powder with na2hpo4 solution using a liquid - to - powder ratio of 500 and 350 l / g for cpc and srcpc , respectively , immediately prior to implantation . srcpc was previously shown to release significant amounts of sr upon immersion in aqueous media ( e.g. , cell culture medium ) and to positively influence both in vitro osteoblast proliferation and differentiation and in vivo bone formation ( 15 ) . composites of silica and fibrillar bovine collagen were used for implantation as monolithic xerogels ( b30 , 70 wt% silica , 30 wt% collagen ) or as porous scaffold ( sc - b30 , xerogel particles b30 , size < 250 m , embedded in a collagen matrix with xerogel / matrix weight ratio of 1.0 ) . for preparation of compact silica / collagen xerogels , bovine tropocollagen type i ( gfn , germany ) was dialysed ( nominal molecular weight cut - off 1214 kda , roth , germany ) against deionized water followed by fibrillation in 30-mm neutral sodium phosphate buffer solution , lyophilisation ( christ alpha 14 laboratory freeze - dryer , osterode , germany ) , and resuspension in 0.1 m trishcl ph 7.4 ( roth ) to obtain homogeneous 30 mg / ml suspensions . the silica component was prepared by hydrolysing tetraethoxysilane ( teos , 99% , sigma , germany ; molar ratio teos / water=1/4 ) under acidic conditions ( 0.01 m hcl ) to obtain silicic acid . vigorous stirring of calculated volumes of silicic acid and collagen suspension to obtain a final composition of 70% silica and 30% collagen ( b30 ) resulted in the formation of 800-l hydrogels . a modification of the scaffolds was prepared by using xerogel particles consisting of 50 wt% silica , 30 wt% fibrillar bovine collagen , and 20 wt% strontium carbonate ( sc - b30sr20 ) . strontium carbonate was introduced as a third phase by being previously added to the collagen suspension . b30 or b30sr20 gels were transferred to molds , stabilized , and dryed for 7 days in an espec sh-221 climate chamber ( japan ) at 37c and 95% relative humidity . the resulting xerogel samples were ground and classified according to the particle size . on the one hand , b30 xerogel particles < 0.250 mm were compacted using a custom - made pressing tool to obtain monolithic b30 xerogel samples exhibiting exactly the above - described bone defect shape . on the other hand , b30 or b30sr20 xerogel particles < 0.120 mm were added to a 30-mg / ml collagen suspension adjusting a xerogel / collagen weight ratio of 1/1 . these xerogel particle / collagen suspensions were transferred to custom - made silicon molds to again obtain the above - described bone defect shape , cooled at 0.5 k / min to 20c ( espec sh-221 climate chamber , osaka , japan ) followed by lyophilisation ( christ alpha 14 laboratory freeze - dryer , osterode , germany ) . the scaffolds were chemically cross - linked by immersing in 1 wt% n-(3-dimethylaminopropyl)-n0-ethylcarbodiimide ( edc)/n - hydroxysuccinimide ( nhs ) ( sigma ) in 40% ethanol for 24 h. finally , the scaffolds were rinsed in deionized water and freeze - dried again . according to the xerogel particle composition used , all xerogel or scaffold samples were gamma - sterilized at 25 kgy before used for implantation . moreover , iron foam with interconnected pores ( fe ) was applied . for preparation of strontium coating of iron foams , 153 ml of ultrapure water and 17 ml of phosphoric acid ( 85% h3po4 ) were added in a 250-ml glass bottle . the supernatant was diluted 20-fold using ultrapure water , which yielded the solution of sr(h2po4)2 . ten fe foams eroded and cleaned by ifam ( fraunhofer institute for manufacturing technology and advanced materials , dresden ) with an average weight of 170 mg were coated by suspension in 100 ml of sr(h2po4)2 solution for 4 h under vacuum . sr - coated foams were settled in fresh coating solution for another 3 days at room temperature and rinsed 3 times using absolute ethanol with subsequent drying at 40c in a drying cabinet . sr - coating resulted in an average weight gain of 37 mg or approximately 22% . in addition , the alloy element phosphor is added with fe3p - powder ( atmix , japan ) with a particle size of 3.8 m . to achieve a phosphate ratio of 0.6% the iron foam was coated with zolendronic acid ( fe - bp ) that is a member of the bisphosphonate family . . then the complex was carefully washed , dried , ground , and finally led to a coating of 35-g zolendronic acid , the basic iron foam . these 8 different formulations of bone substitution materials were implanted in the current animal model ( n=78 ) ( table 1 ) . eighteen weeks after induction of osteoporosis and 6 weeks after femoral surgery , dpet - ct with f - sodium fluoride ( naf ) scans were performed . during scanning , rats were anesthetized using a mixture of nitrous oxide ( 1 l / min ) , oxygen ( 0.5 l / min ) and isoflurane ( 1.5 vol.% ) . dpet - ct studies were performed for 60 min after the intravenous application of 20 to 40 mbq f - sodium fluoride , using a 28-frame protocol ( 10 frames of 30 s , 5 frames of 60 s , 5 frames of 120 s , and 8 frames of 300 s ) . a dedicated pet - ct system ( biograph mct , 128 s siemens co , erlangen , germany ) with an axial field of view of 21.6 cm with truev and truepoint , operated in a 3-dimensional mode , was used for all animal studies . the system provides the simultaneous acquisition of 369 transverse pet slices with a slice thickness of 0.6 mm . two rats were examined in parallel and were positioned in the axial plane of the system to maintain the optimum resolution in the center of the system . a high - resolution ct scan was performed prior the pet scanning with 160 ma , 80 kv , pitch of 0.85 cm in addition to the low - dose attenuation ct ( 80 ma , 80 kv ) for attenuation correction of the acquired dynamic emission pet data . all pet images were attenuation - corrected and an image matrix of 400400 pixels was used for iterative image reconstruction ( voxel size 1.5651.5650.6 mm ) based on the syngo mi pet / ct 2009c software version . the reconstructed images were converted to standardized uptake value ( suv ) images based on the formula ( 18 ) : suv = tissue concentration ( bq / g)/[injected dose ( bq)/body weight ( g ) ] . volumes of interest were placed over the created defect area in the femur , and rotated pet images according to the ct images . irregular regions of interest were drawn manually according to the fused pet - ct images , as mentioned before . to precisely select the region to draw the voi of the created defect , the fusion of pet images and ct scans by co - registration preprocessing was performed after rotation of each rat individually . first , we rotated the ct series to determine the optimum angle for the visualisation of the complete defect and the adjacent metal plates in the longitudinal direction . the next step was to fuse ct and pet and to use these fused images for the quantitative pet evaluation . the spatial resolution was 2 mm for pet image and 0.33 mm for ct image . the voi for the defect was placed between the 2 screws located at the left and the right side of the 4-mm created defect . for input , we used 10 contiguous pet slices in the middle and lower third of the aorta . we avoided using the upper part of the thoracic aorta due to spillover from the heart . herein , a 2-tissue compartment model was used to evaluate the dynamic studies and to gain quantitative data about the kinetics . in the current study , the learning - machine 2-tissue compartment model was used for the fitting , and provided 5 parameters : the plasma clearance to the bone extracellular fluid ( ecf ) compartment and the rate constant for return of tracer to plasma ; k1 and k2 ; the rate constants describing movement of tracer into and out of the bound bone compartment ; k3 and k4 ; and the fractional blood volume , also called vessel density ( vb ) , which reflects the amount of blood in the voi . following compartment analysis , we calculated the plasma clearance of tracer to bone mineral from the compartment data using the formula : influx ( ki)=(k1*k3)/(k2+k3 ) . compared to the standard iterative method , the machine learning method has the advantage of a fast convergence and avoidance of over - fitting . the model parameters were accepted when k1k4 was less than 1 and vb exceeded 0 . the unit for the rate constants k1k4 was 1/min . besides the compartmental analysis , a noncompartmental model based on the fractal dimension was used . the fractal dimension is a parameter of heterogeneity and was calculated for the time - activity data of each individual volume of interest . the values for fractal dimension vary from 0 to 2 , showing the deterministic or chaotic distribution of tracer activity . we used a subdivision of 77 and a maximal suv of 20 for the calculation of fractal dimension . statistical evaluation was performed with stata / se 11.1 ( statacorp , college station , tx , usa ) using the descriptive statistics and box plots . linear discriminant analysis was performed for calculating the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and highest accuracy . the 2-sided wilcoxon rank sum test was applied for all pet parameters , which also included suv , using a single parameter analysis to assess groups . we have reported that k3 was the most sensitive pet parameter for the characterization of healing process and revealed the best differentiation for the empty defect and the cpc group , as well as the srcpc , for the defect region . however , no pet parameter demonstrated a significant difference between the cpc and srcpc group in the defect region . in this study , the volume of interest ( voi ) of the biomaterial - bone interface was selected for evaluation ( figure 1 ) . k3 showed a statistically significant increase in the bone formation for the srcpc group compared to the cpc group ( p=0.018 ) in the biomaterial - bone interface ( figure 2 ) . three types of the composites were used : monolithic xerogels ( b30 ) and porous scaffolds ( sc - b30 and sc - b30sr20 ) . comparing the pet parameters , we could not detect significant changes between the 3 composites and the empty defect for both rois . only k3 in the sc - b30 group exhibited a significant increase in comparison to the b30 group in the defect region ( figure 3 , p=0.022 ) . three different formulations of the iron foam were used as implants in the different rat groups : a ) plain iron foam ( fe ) , b ) strontium functionalized iron foam ( fe - s ) , c ) iron foam with coating of the bisphosphonate zoledronic acid ( fe - bp ) . no significant differences were found in the pet parameters between the 3 implants and the empty defect for both rois . however , there was a significant increase of ki in the fe - bp group as compared with the fe group for the defect region ( figure 4 , p=0.037 ) . a multiparameter analysis was performed to find differences between the 2 different groups based on the dpet - ct data . the aim was to identify a combination of variables that best discriminate each group of rats with different biomaterials in the defect region . an overall accuracy of > 75% was achieved for the combination of some kinetic parameters between the 2 different groups . furthermore , all sensitivity and specificity values from the multiparameter analysis were high ( > 71% ) ( table 2 ) . the highest overall accuracy of 78.6% was achieved for k3 between the cpc and srcpc group based on the biomaterial - bone interface . the positive predictive value ( npv ) and the negative predictive value ( ppv ) were 85.7% and 71.4% , respectively . therefore , 6/7 rats with cpc in the defect and as 5/7 rats with srcpc in the defect could be correctly classified based on the k3 as measured . between the sc - b30 and sc - b30sr group , the highest overall accuracy of 86.7% was achieved for a combination of k1 , k3 , ki , and suv . we could correctly classify 7/8 rats with sc - b30 in the defect ( npv of 86.3% ) and 6/7 rats with sc - b30sr ( ppv of 85.7% ) . between the sc - b30 and b30 group , the highest overall accuracy of 92.3% was achieved for a combination of k3 and ki . we could correctly classify 5/6 rats with b30 in the defect ( npv of 83.3% ) and 7/7 rats with sc - b30 ( ppv of 100% ) . between the sc - b30sr20 and b30 group , the highest overall accuracy of 78.6% was achieved for a combination of k3 and vb . we could correctly classify 3/6 rats with b30 in the defect ( npv of 50.0% ) and 8/8 rats with sc - b30sr20 ( ppv of 100% ) . between the fe and fe - s group , we could correctly classify 6/8 rats with fe - s in the defect ( npv of 75.0% ) and 5/7 rats with fe ( ppv of 71.4% ) . between the fe and fe - bp groups , the highest overall accuracy of 80.0% we could correctly classify 7/8 rats with fe - bp in the defect ( npv of 86.3% ) and 5/7 rats with fe ( ppv of 71.4% ) . between the fe - s and fe - bp , the highest overall accuracy of 75.0% was achieved for a combination of vb , k1 , k3 , ki , and suv . we could correctly classify 6/8 rats with fe - bp in the defect ( npv of 75.0% ) as well as 6/8 rats with fe ( ppv of 75.0% ) . we have reported that k3 was the most sensitive pet parameter for the characterization of healing process and revealed the best differentiation for the empty defect and the cpc group , as well as the srcpc , for the defect region . however , no pet parameter demonstrated a significant difference between the cpc and srcpc group in the defect region . in this study , the volume of interest ( voi ) of the biomaterial - bone interface was selected for evaluation ( figure 1 ) . k3 showed a statistically significant increase in the bone formation for the srcpc group compared to the cpc group ( p=0.018 ) in the biomaterial - bone interface ( figure 2 ) . three types of the composites were used : monolithic xerogels ( b30 ) and porous scaffolds ( sc - b30 and sc - b30sr20 ) . comparing the pet parameters , we could not detect significant changes between the 3 composites and the empty defect for both rois . only k3 in the sc - b30 group exhibited a significant increase in comparison to the b30 group in the defect region ( figure 3 , p=0.022 ) . three different formulations of the iron foam were used as implants in the different rat groups : a ) plain iron foam ( fe ) , b ) strontium functionalized iron foam ( fe - s ) , c ) iron foam with coating of the bisphosphonate zoledronic acid ( fe - bp ) . no significant differences were found in the pet parameters between the 3 implants and the empty defect for both rois . however , there was a significant increase of ki in the fe - bp group as compared with the fe group for the defect region ( figure 4 , p=0.037 ) . a multiparameter analysis was performed to find differences between the 2 different groups based on the dpet - ct data . the aim was to identify a combination of variables that best discriminate each group of rats with different biomaterials in the defect region . an overall accuracy of > 75% was achieved for the combination of some kinetic parameters between the 2 different groups . furthermore , all sensitivity and specificity values from the multiparameter analysis were high ( > 71% ) ( table 2 ) . the highest overall accuracy of 78.6% was achieved for k3 between the cpc and srcpc group based on the biomaterial - bone interface . the positive predictive value ( npv ) and the negative predictive value ( ppv ) were 85.7% and 71.4% , respectively . therefore , 6/7 rats with cpc in the defect and as 5/7 rats with srcpc in the defect could be correctly classified based on the k3 as measured . between the sc - b30 and sc - b30sr group , the highest overall accuracy of 86.7% was achieved for a combination of k1 , k3 , ki , and suv . we could correctly classify 7/8 rats with sc - b30 in the defect ( npv of 86.3% ) and 6/7 rats with sc - b30sr ( ppv of 85.7% ) . between the sc - b30 and b30 group , the highest overall accuracy of 92.3% was achieved for a combination of k3 and ki . we could correctly classify 5/6 rats with b30 in the defect ( npv of 83.3% ) and 7/7 rats with sc - b30 ( ppv of 100% ) . between the sc - b30sr20 and b30 group , the highest overall accuracy of 78.6% was achieved for a combination of k3 and vb . we could correctly classify 3/6 rats with b30 in the defect ( npv of 50.0% ) and 8/8 rats with sc - b30sr20 ( ppv of 100% ) . between the fe and fe - s group , we could correctly classify 6/8 rats with fe - s in the defect ( npv of 75.0% ) and 5/7 rats with fe ( ppv of 71.4% ) . between the fe and fe - bp groups , the highest overall accuracy of 80.0% we could correctly classify 7/8 rats with fe - bp in the defect ( npv of 86.3% ) and 5/7 rats with fe ( ppv of 71.4% ) . between the fe - s and fe - bp , the highest overall accuracy of 75.0% was achieved for a combination of vb , k1 , k3 , ki , and suv . we could correctly classify 6/8 rats with fe - bp in the defect ( npv of 75.0% ) as well as 6/8 rats with fe ( ppv of 75.0% ) . in this study , 3 different classes of biomaterials , including cpc - based implants , composites of silica , and collagen and iron foam implants , were investigated using dpet - ct after they were implanted in 4-mm created rat femoral defects . cpc is a well - defined , hydroxyapatite - forming cement with a formulation based on -tricalcium phosphate as initially described by driessens et al . . srcoc is a strontium - enriched modification thereof , in which caco3 was substituted with srco3 during cement precursor fabrication . this srcpc was recently shown to enhance osteoblast precursor proliferation and osteogenic differentiation in vitro as well as to support new bone formation in vivo . the 4-mm defect represented a critical - size defect with a fracture gap , which has been proven to be a good model to investigate potential bone enhancement effects of biomaterials . our data demonstrate differences in the tracer kinetics within the region of the 4-mm created defect , which includes new bone formation in the defects filled with biomaterials . for f - sodium fluoride , pet parameter k3 describes the formation of fluoroapatite , which is indicative of new bone formation . both cpc and srcpc revealed statistically significant enhanced new bone formation as compared to the empty defect in the defect region . however , no significant difference between the 2 different biomaterials in the defect region was detected . recently , ventura et al . reported pet - suv only indicated the speed of new bone formation but not the absolute amount by correlation of pet - ct and histology . therefore , it might be possible that pet in our study also describes the speed of new bone formation . bone healing is a dynamic process over time , and the use of a single time point is a limitation of this study . we are considering a series of measurements in further related studies , as part of an ongoing project . in addition , in this study , srcpc - treated animals exhibited a statistically significant higher rate of new bone formation at the biomaterial - bone interface , suggesting that a considerable amount of strontium is released from the srcpc into the interface region and into the surrounding tissue , which are most likely related to enhanced bone formation . recently , andersen et al . revealed that local delivery of strontium from surface - functionalized titanium implants could increase bone - to - implant interaction for implants in the femoral shaft of healthy female wister rats . demonstrated a high sr concentration in the interface region of the srcpc implant by tof - sims , which is in line with our findings . it may be concluded that strontium is released into the local milieu of osseointegrating implants , enhancing bone ingrowth into the implant surface . bone tissue has a highly nano - hierarchical structure , consisting mainly of collagen type i fibres and nano hydroxyapatite crystals as the matrix . scaffolds based on polymeric materials act as temporary templates for bone regeneration and actively stimulate vascularized bone growth by improving material - tissue interaction due to the porous form of the biomaterial structure . our results demonstrate significantly higher k3 values of f - sodium fluoride in the defect of rats filled with porous silicate / collagen ( sc - b30 ) than with silicate / collagen ( b30 ) ( figure 3 ) , which allows us to conclude that the formation of fluoroapatite , and therefore the new bone formation , is enhanced in the porous form of the biomaterial structure rather than in the solid form . however , a non - significant increase of k3 in the sc - b30sr20 group was detected as compared with the b30 group . it is uncertain if this is caused by a lack of statistical power or by a greater degree of tissue heterogeneity than can be explained by a 2-tissue compartmental model . the efficacy of bisphosphonates in reducing bone resorption has been clinically estimated using biochemical indices of bone turnover . reported a highly significant correlation ( r=0.84 ) between ki and bone formation rate assessed by levels of serum alkaline phosphatase , as well as histomorphometry in patients with renal osteodystrophy . our results demonstrate that ki , which reflects bone remodeling , was significantly higher in the defect with fe - bp than in the defect with fe only ( figure 4 ) . the increase of ki is related to the enhanced bone formation , confirming the reduced rate of bone resorption . however , a non - significant increase of ki in the fe - s group was detected as compared with the fe group . although we did not find further significant differences for any pet parameters between the other 2 different material group in the defect or in the biomaterial - tissue interface region , each group could be still well classified by the combination of some parameters as measured in the defect ( table 2 ) . it is unclear whether this is because pet is not sensitive enough for discriminating differences of bone metabolism due to the small changes of bone remodeling using different biomaterials and their enrichments , or is due to lack of statistical power . with f - sodium fluoride , k3 was the most significant parameter for the characterization of healing processes and showed the best differentiation between the 2 different biomaterials . pet scanning using f - sodium fluoride seems to be a sensitive and useful method for evaluation of bone healing after replacement with these biomaterials .

background : psychoactive substances affect mainly central nervous system and brain function causing changes in behavior.objectives:the purpose of this study was to determine the effects of different psychoactive substances on serum biochemical parameters.patients and methods : the study included 324 drug dependents , and 69 controls . the patient group was determined according to dsm - iv ( the diagnostic and statistical manual of mental disorders , fourth edition ) criteria . all patients and control subjects were tested for routine biochemical parameters and urine toxicology parameters for psychoactive substance use . cases and controls with accompanying diseases like diabetes , cancer , metabolic disorders etc . are excluded from the study . moreover , an association between urine toxicology results and changes in biochemical parameters was evaluated for statistical significance.results:there was a statistically significant difference in the gamma - glutamyl transferase ( ggt ) , uric acid , creatinine , urea , albumin , aspartate aminotransferase ( ast ) medians between the dependent and control groups ( p < 0.05 ) . we found a statistically significant difference in sodium and albumin levels between the opium - dependent and control groups ( p < 0.05 ) . in the benzodiazepin dependent group , we found a significant difference in ggt , urea , glucose , sodium , t protein , and ast levels ( p < 0.05 ) . moreover , a statistically significant difference was observed in triglyceride and ggt levels between the ethyl glucuronide and control groups ( p < 0.05).conclusions : in psychoactive substance dependents , serum routine biochemistry parameters can be used to predict the need for intensive monitoring and treatment programs . backgroundthe aim of the current study was to measure and compare the effect of various biomaterials for the healing of osteoporotic bone defects in the rat femur using 18f - sodium fluoride dpet-ct.material/methodsosteoporosis was induced by ovariectomy and a calcium - restricted diet . after 3 months , rats were operated on to create a 4-mm wedge - shaped defect in the distal metaphyseal femur . bone substitution materials of calcium phosphate cement ( cpc ) , composites of collagen and silica , and iron foams with interconnecting pores were inserted . strontium or bisphosphonate , which are well known for having positive effects in osteoporosis treatment , were added into the materials . eighteen weeks after osteoporosis induction and 6 weeks following femoral surgery , dpet - ct studies scan were performed with 18f - sodium fluoride . standardized uptake values ( suvs ) and a 2-tissue compartmental learning - machine model ( k1-k4 , vessel density [ vb ] , influx [ ki ] ) were used for quantitative analysis.resultsk3 , reflecting the formation of fluoroapatite , revealed a statistically significant increase at the biomaterial - bone interface due to the sr release from strontium - modified calcium phosphate cement ( srcpc ) compared to cpc , which demonstrated enhanced new bone formation . in addition , k3 as measured in the porous scaffold silica / collagen xerogel ( sc - b30 ) , showed a significant increase based on wilcoxon rank - sum test ( p<0.05 ) as compared with monolithic silica / collagen xerogel ( b30 ) in the defect region . furthermore , ki , reflecting the net plasma clearance of tracer to bone mineral measured in the iron foam with coating of the bisphosphonate zoledronic acid ( fe - bp ) , was enhanced as compared with plain iron foam ( fe ) in the defect region.conclusionsk3 was the most significant parameter for the characterization of healing processes and revealed the best differentiation between the 2 different biomaterials . pet scanning using 18f - sodium fluoride seems to be a sensitive and useful method for evaluation of bone healing after replacement with these biomaterials .

m. tuberculosis isolates were obtained from the laboratory of mycobacteriology of the institut pasteur de tunis as part of the national tuberculosis surveillance program . all samples ( 884 specimens ) from patients with suspected tb residing in northern tunisia ( bizerte ) from august 2001 to october 2003 were forwarded to us by the referral regional hospital . the incidence of tb in this area from 2001 to 2002 was 29/100,000 male patients and 11/100,000 female patients . all patients received the standard chemotherapy regimen of the tunisian national tuberculosis program , i.e. , 2 months of rifampicin , isoniazid , pyrazinamide , and streptomycin , followed by 4 months of rifampicin and isoniazid ( 2rhzs/4rh ) . the corresponding patients were interviewed , and detailed epidemiologic investigations were conducted according to described protocols ( 6 ) . in april 2004 , while the study was in progress , a new mdr case was diagnosed . analyses by is6110 restriction fragment length polymorphism ( is6110 rflp ) , ligation - mediated polymerase chain reaction ( pcr ) , and spoligotyping were carried out by using standard protocols ( 79 ) . typing of the polymorphic gc - rich repetitive sequence ( pgrs ) with probe mtb484 ( 1 ) was conducted according to a previously reported protocol ( 10 ) , with the exception that dna was digested with alui . isolates were assigned to principal genetic groups according to the polymorphisms in the katg and gyra genes ( 11 ) . the following primer pairs were used to sequence rpob , katg , and pnca gene mutations that confer resistance to rifampicin , isoniazid , and pyrazinamide , respectively : rpob ( 5-atcacaccgcagacgttg-3 , 5-tgcatcacagtgatgtagtcg-3 ) ; katg ( 5- cgtcgaaacagcggcgctga-3 , 5-caagcgccagcagggctctt-3 ) ; and pnca ( 5-ggcgcacacaatgatcggtg-3 , 5-gctttgcggcgagcgctcca-3 ) . the recently described single nucleotide polymorphisms in putative m. tuberculosis mutator genes mutt1 , mutt2 , mutt3 , and ogt were investigated with the same protocol reported by rad et al . ( 12 ) . dna sequencing was conducted directly on the purified pcr products by using the prism ready reaction dye deoxy terminator cycle sequencing kit on an abi prism 377 dna sequencer ( applied biosystems , foster city , ca , usa ) . epidemiologic and clinical data indicated that all patients with mdr - tb were male with a mean age of 31 years at diagnosis ( table 1 ) . all were tunisians and permanently resided in the northern part of the country ( bizerte ) . all patients were seronegative for hiv with no documented history of travel abroad , and none had a history of immunosuppression , diabetes , or respiratory diseases other than tb . mapping of the 21 patients with mdr - tb according to their residence sites showed that they were mostly scattered over the northeastern part of the region ( surface area 1,000 km ) with no concentration in a particular locality ( data not shown ) . resistance to 5 first - line drugs was observed for most isolates ( table 2 ) . * pt , previously treated ; nc , new case ; nd , not determined . * rflp , restriction fragment length polymorphism ; pgg , principal genetic grouping ; wt , wild type ( identical to strain h37rv ) ; nd , not determined . h , isoniazid ; s , streptomycin ; r , rifampicin ; e , ethambutol ; z , pyrazinamide . number of is6110 bands . principal genetic grouping according to gyra and katg polymorphisms ( 11 ) . # is6110 typing was determined by ligation - mediated polymerase chain reaction and the profile was identical to the other outbreak - associated strains . * * absence of spacers 15 , 2124 , and 3336 . as indicated in table 1 , with the exception of patient p20 , the dna samples subjected to molecular typing were obtained from the initial isolate of all new patients . rflp showed that 18 patients had nearly identical is6110 profiles ( figures 1 and 2 ) . the predominant profile ( occurring in 13 patients ) showed 11 bands , while the remaining 5 patients had an additional is6110 band . the presence or absence of the additional is6110 band the rflp pattern of patient p11 , a new patient , clearly showed a mixture of the 12-band profile and some additional is6110 bands ( figure 2a ) . typing of his follow - up culture , which was obtained after 6 months of directly observed short - course therapy , as recommended by the world health organization , yielded only the 12-band profile ( figure 2a ) . is6110 restriction fragment length polymorphism ( rflp ) analysis of mycobacterium tuberculosis isolates from 16 patients associated with the multidrug - resistant tuberculosis outbreak , bizerte , tunisia , 20012004 . a ) is6110 restriction fragment length polymorphism ( rflp ) analysis ( left ) and polymorphic gc - rich repetitive sequence ( pgrs ) typing ( right ) of patient p11 . lane 1 , initial isolate ; lane 2 , follow - up isolate . b ) is6110 rflp ( left ) and pgrs typing ( right ) of patient p20 ( lane 1 ) compared with patient p3 ( lane 2 ) , a typical outbreak - associated patient . effective epidemiologic links were identified for 9 ( 47% ) patients ( table 1 ) . it shows 10 is6110 bands ( figure 2b ) , 9 of which are common to the 12-band rflp pattern described for the other isolates . the isolate from patient p19 displayed a 9-band is6110 profile that was clearly distinct from all the other patients with mdr - tb ( data not shown ) . with the exception of patient p19 , the mdr isolates were identical in their pgrs profile ( figure 2 ) and spoligotype patterns ( table 2 ) , which is characteristic of the haarlem3 type ( 4 ) . sequence analysis of mutator and drug resistance genes conclusively confirmed that the 19 mdr isolates with nearly identical is6110 ( both 12- and 11-band profiles ) are genetically closely related . they all harbor the l209l , t15s , s531l , and s315 t mutations in mutt3 , ogt , rpob , and katg genes , respectively ( table 2 ) , whereas mutt1 and mutt2 showed a wild type genotype ( data not shown ) . the occurrence of an additional uncommon mutation in the rpob gene ( v610 m ) confirmed the clonality of this mdr haarlem strain since it was present only in 19 patients with mdr - tb . the variability of resistance to pyrazinamide and the mutational profile within the pnca gene ( table 2 ) strongly suggest that primary transmission from person to person occurred mainly with a strain that was simultaneously resistant to isoniazid and rifampicin . to extend our analysis of the situation that prevailed in this region , samples from 143 ( 83% ) of 172 patients without mdr strains were spoligotyped . of these 143 patients , 41 ( 29% ) in contrast to the mdr - tb outbreak that involved only men , 6 women had a non - mdr haarlem3 strain . aside from the absence of clustering , ligation - mediated pcr typing showed that none of these non - mdr haarlem3 isolates displayed a profile similar to the 19 mdr isolates involved in the transmission chain . sequencing of the rpob gene of 10 isolates randomly selected from the 31 non - mdr haarlem isolates showed the absence of the outbreak - associated mutation v610 m . this finding is strongly indicative of a true clonal expansion and a typical mdr - tb outbreak . the results indicate that an mdr strain of m. tuberculosis has been actively transmitted among 19 hiv - negative male patients in tunisia . several observations indicate that this particular haarlem strain displays increased transmissibility , virulence , or both . first , the outbreak peaked suddenly within a relatively short period of 21 months ; 17 new cases ( 89% ) were reported from september 2001 to june 2003 . inspection of the hospital register for 2000 showed only 3 new patients with mdr isolates , including outbreak - associated patients p5 and p7 ( table 1 ) . second , no epidemiologic links or contact points could be traced for several patients , which suggests that brief exposure would have been sufficient for effective transmission . because patients with mdr - tb do not respond to treatment such a situation is likely to have occurred for the patients with established epidemiologic links . third , the incidence of tb in the region in which the outbreak occurred is not particularly high . fifth , no aids - associated tb outbreak that might have increased the adaptability of the strain within the indigenous population had occurred in the region . sixth , although the haarlem strain was mdr , it was able to cause an outbreak in those vaccinated with bacille calmette - gurin and in persons who were not hospitalized . among the identified m. tuberculosis strain families ( 4,5 ) , the w / the haarlem strain family appears to be widespread ( 4 ) , but its ability to cause outbreaks has been reported only twice , once in argentina ( 13 ) and once in the czech republic ( 14 ) . the distinctive feature of the present haarlem mdr - tb outbreak is its accelerated transmission compared with the first 2 mdr - tb outbreaks . alterations within dna repair genes ( mutator genes ) are thought to favor the emergence of mdr strains with an increased adaptability ( 12 ) . in this respect , both widespread mdr strains might also benefit from their intrinsic adaptability ( 15 ) . from an epidemiologic point of view , tb programs must conduct extensive surveillance of mdr strains of m. tuberculosis strain families because they might cause serious outbreaks . the goals of surgical management include correction and maintenance of sagittal and coronal alignment , establishment of length and rotation , and early functional range of movements of knee and ankle . interlocking nailing of tibial fractures is desirable because this technique allows some load sharing , spares extra osseous blood supply , avoids extensive soft tissue dissection , and is familiar to most surgeons . nailing of metaphyseal fractures with short distal fragment is associated with an increase in malalignment particularly in coronal plane , nonunion , and need for secondary procedures to achieve union . as there is a mismatch between the diameters of the nail and the medullary canal , with no nail - cortex contact , the nail may translate laterally along coronally placed locking screws and increased stress is placed on the locking holes to maintain fracture alignment after surgery . various techniques have been recommended to improve nailing the metaphyseal fractures including blocking screws ( poller screw ) , temporary unicortical plating , percutaneous reduction clamps , and fibular plating . blocking screws placed adjacent to the nail and perpendicular to the screw holes usually in an anteroposterior direction had been suggested as one possible method of improving the stability of metaphyseal fractures and had been described as a reduction tool used to overcome the displacing forces at the time of introduction of intramedullary nail . the blocking screws functionally decrease the width of the metaphyseal medulla and are particularly useful with nails of smaller diameter . in 1994 krettek et al . described the clinical application of blocking screws , termed poller screws , as a tool for the prevention of axial deformities of proximal and distal third fractures of tibia during intramedullary nailing [ 2 , 3 ] . this was a prospective study of 20 cases of distal tibial metaphyseal fractures treated with statically locked intramedullary nailing with supplementary blocking screws between august 2006 and september 2007 at our institute with a minimum followup of 5 years . displaced distal tibial metaphyseal fractures in adults treated with intramedullary nailing were included in the study . the fractures included were acute fractures and delayed union . both open and closed fractures were included in the study . metaphyseal fractures treated with statically locked intramedullary nails but with additional procedures like fibular plating were also excluded from the study . among there were 16 males and 4 female patients with a mean age of 37.75 years ( 95% lower confidence limit of ( lcl ) 33.13 years and 95% upper confidence limit of ( ucl ) 42.36 ) . the mechanism of injury was road traffic accident in all except three in whom it was fall from height in two and fall of a heavy object over the leg in one . according to ao guidelines there were five type 43 a1 , eleven type 43 a2 , and four type 43 a3 fractures . injury was closed in 15 fractures and gustilo anderson grade i in 2 and grade ii in 3 patients . the mean distance from the articular surface was 4.6 cm ( 95% lcl 2.8 cm and 95% ucl 5.5 cm ) and the mean length of the fracture was 3.4 cm ( 95% lcl 2.69 cm and 95% ucl 4.10 cm ) . the mean delay between the injury and the surgery was 3.75 weeks ( 95% lcl 1.23 weeks and 95% ucl 6.26 weeks ) . among the 20 cases two the mean diameter of the medullary canal at the level of isthmus was 11.9 mm and at the fracture site was 22.9 mm ( table 1 ) . blocking screws were selected for use for one or more of the following reasons : to correct alignment after insertion of nail ( 8 fractures),to maintain alignment or to improve the stability of bone implant complex ( 20 fractures),to control the nail during insertion ( 5 fractures ) . to correct alignment after insertion of nail ( 8 fractures ) , to maintain alignment or to improve the stability of bone implant complex ( 20 fractures ) , to control the nail during insertion ( 5 fractures ) . in 7 cases single blocking screw was used on the concave side of the deformity , close to the fracture site in the short fragment . in 2 cases single blocking screw was used on the convex side of the deformity , near the end of the nail . and in the rest of cases 2 blocking screws were placed , the first one on the concave side of the deformity close to the fracture site and the second screw on the convex side of deformity near the end of the nail in the distal fragment . depending on the amount of correction needed , the screws used for blocking were 4 mm locking screws or 4.5 mm cortical screws . x - ray of the injured leg in ap and lateral views was taken . the fracture tendency for valgus or varus and antecurvatum or recurvatum malalignment the diameters of medullary canal at isthmus and at the level of fracture were also measured . appropriate length of the nail was measured in the contralateral leg , from the tibial tuberosity to medial malleolus . metaphyseal fractures were stabilized with statically locked intramedullary nails on a standard radio lucent table with manual traction . tourniquet was not used in any case . through patellar tendon splitting approach , entry point was made in the midline . closed reduction was attempted and we had to do open reduction as there was a marked overriding of the fragments due to a delay of 18 weeks before surgery . the nails used were unreamed cannulated stainless nails , with 2 proximal ( mediolateral ) and 3 distal ( 2 mediolateral and 1 anteroposterior ) locking options , of diameter 8 or 9 mm . in one case the tibia was too narrow and too short where we have used a nail of 7 mm diameter . the blocking screw was used on the concave side of the deformity close to the fracture in the short fragment when single screw was used between the cortex and the nail under image intensification . when 2 blocking screws were placed , the second screw was on the convex side of deformity near the end of the nail in the short fragment . but in 2 cases the distal segments were too short and there was significant comminution on the concave side of the deformity . hence we used single poller screw on the convex side of deformity near the tip of the nail . in cases of malalignment and instability the screw holes 2.5 mm or 3 mm k wire was used to drill the pilot hole for poller screw as the drill bit may damage the nail while drilling with the nail in situ . for fractures which were stable but malaligned , the nail was temporarily removed , the blocking screws were placed , and the nail was reinserted ( figures 1 and 2 ) . distal and proximal locking was done after achieving the alignment using blocking screws ( figure 3 ) . partial weight bearing was started in the second postoperative week in all except two cases . in one where we have used 7 size nail , we recommended non - weight - bearing till radiological evidence of union and in the other where tibialis anterior tendon was found cut and the patient had both bones fractures in the contralateral leg , partial weight bearing could not be started . partial weight bearing continued up from 4 to 8 weeks ; thereafter full weight bearing started depending on clinical and radiological evidence of union . all the fractures were followed through till union of fracture with clinical and radiological examination at intervals of 4 to 6 weeks . valgus and antecurvatum were expressed as positive values and varus and recurvatum were expressed as negative values . radiographs were analyzed for correction , maintenance of position , or loss of reduction . shortening and rotational malalignment were not measured . fracture was defined as united when patient was able to bear full weight on the injured limb without pain and without support and when radiographs showing bridging call us in at least 3 cortices . it is an independent measurement , not influenced by other comorbid conditions and associated injuries . all the fractures eventually united in a mean period of 11.5 weeks ( 95% lcl 10.11 weeks and 95% ucl 12.88 weeks ) . karlstrom - olerud score was excellent in 14 fractures ( 70% ) , good in 4 patients ( 20% ) , and fair in 2 patients ( 10% ) . radiologically the mean postoperative varus / valgus alignment was 1.7 degrees ( 95% lcl 0.5 degrees and 95% ucl 2.9 degrees ) when compared to the mean preoperative varus / valgus alignment of 10.3 degrees ( 95% lcl 8.2 degrees and 95% ucl 12.4 degrees ) . the alignment was maintained till union with the mean remaining the same in the coronal plane . repeated measures anova test showed the f - test value of 45.29 which is significant as the p value is 0.00000 ( p < 0.05 ) . the mean postoperative antecurvatum / recurvatum alignment was 0.2 degrees ( 95% lcl 0.1 degrees and 95% ucl 0.5 degrees ) when compared to the mean preoperative antecurvatum / recurvatum alignment of 8.0 degrees ( 95% lcl 4.6 degrees and 95% ucl 11.3 degrees ) . f - test value in repeated measures anova is 22.845 with a p value of 0.0000 ( < 0.05 ) which is statistically significant . the mean ratio of fracture segment to the nail length ( i.e. , the length of tibia ) was 14% . the poller screw related complication was encountered in one case where we had new fracture lines while introducing the nail after placement of poller screw ( figures 4 and 5 ) . but the alignment was achieved and maintained and the fracture united within 8 weeks ( figure 6 ) . complications which were not related to poller screw were encountered in two cases . both of them had developed deep infection and went in for delayed union of which one required dynamisation to achieve union . there were no incidences of breakage of nail , locking screw , or blocking screw . we can not overemphasize the potential advantages of intramedullary nailing more than any other form of fixation like external fixator or plating in tibial fractures . but the problems in extending the indications to metaphyseal fractures have to be analyzed and resolved . trafton 's recommendation is generally agreed on by many authors . as per trafton 's recommendation the acceptable malalignment is less than 5 degrees of varus - valgus angulation , 10 degrees of anteroposterior angulation , 10 degrees of rotation , and 15 mm of shortening . in our study merchant and dietz in 1989 suggested that for tibial fractures deformity of > 5 was associated with radiographic changes in the ankle . van der schoot reported a 15-year followup of 88 patients with fractures of lower leg . more arthritis was found in the knee and ankle adjacent to fracture than in comparable joints of the uninjured leg . recorded the long - term effects of tibial angular malunion on knee and ankle joints in his 28 tibial fractures with an average followup of 8.2 years . kyro a in his series of 64 tibial shaft fractures concluded that malunion of tibial shaft fractures seems to be especially harmful in distal fractures , in fractures with marked previous displacement , in fractures caused by high - energy injury , and among patients less than 45 years of age . ahlers and von issendorf analyzed 386 fractures of tibia treated by intramedullary nailing of which 32 were proximal and 138 were distal third fractures . in both the groups one - quarter to one - third had varus - valgus deformities greater than 4 degrees . in another study , mosheiff in 1999 found that 42% of distal third fractures treated with interlocking nailing required secondary procedures to achieve union . there has been discrepancy in the literature regarding the locking bolt orientation and its effect on fracture nail construct stability . chen compared the intrinsic stability in tibial intramedullary nail constructs in distal third diaphyseal fractures without isthmal support , between two mediolateral distal locking screws and two perpendicular ( one mediolateral and one anteroposterior ) distal locking screws . he concluded that fixation stability of intramedullary nail is not significantly influenced by distal locking screw orientation in response to sagittal , coronal , or rotational forces . on the contrary , smucker et al . found two parallel locking bolts being a better construct than perpendicular locking bolts in their study . we have analyzed the mismatch between the diameters of medullary canal at the level of isthmus ( i.e. , maximum possible nail size ) and at the fracture site in all cases . the mean diameter of medullary canal at the level of isthmus was 11.9 mm compared to 22.9 mm at the level of fracture site . this mismatch explained the cause of instability in metaphyseal fractures when treated with intramedullary nailing . to overcome the issue of malalignment various techniques have been developed . in distal third fractures , fibular plating , one cross screw across fracture site as lag screw , use of large reduction forceps , temporary unicortical plating , percutaneous manipulation with shanz pins , femoral distracter and cutting the distal few millimeters of nail distal to the distal screw hole to allow two cross locking screws in the distal fragment , have been the supplementary procedures used to achieve the alignment [ 1424 ] . the primary aim of the study was to analyze the effectiveness of achieving and maintaining reduction in metaphyseal fractures of tibia treated with intramedullary nailing using supplementary blocking screws . we have also measured the maximum diameter of the metaphysis distal tibia , thereby the length of the distal metaphyseal segment in our population was reached . as described in various literatures the malalignment in these circumstances was significantly high when done without any supplementary procedures . published the mechanical effect of blocking screw in stabilizing tibial fractures with short proximal or distal fragments after insertion of small diameter intramedullary nails . created bone implant constructs ( bic ) in fresh cadaveric tibiae and demonstrated in distal bics the addition of blocking screws decreasing the average deformation of the bics by 57% ( p < 0.0001 ) [ 25 , 26 ] . explored the effect of blocking screws on the breakage of interlocking intramedullary nails and concluded that blocking screws improve the stability of fracture area distinctively and hence reduce the breakage of intramedullary nailing . according to james kellam , meticulous intramedullary techniques combined with the use of fibular plate fixation or blocking screws will achieve the best results in maintaining the reduction of distal tibial fractures till union . the use of blocking screw as reduction tool was established in our study by the repeated measures anova test and was comparable to the study by krettek . blocking screws improved the stability of the metaphyseal fractures after nailing and promoted union in our study . the fracture was originally a grade ii compound fracture treated with external fixator which was removed once the wound healed . no cases required bone grafting , bone marrow injection , or exchange of nailing . the ratio of short metaphyseal fragment length to the total tibial length was analyzed . the total length of the tibia was approximately derived from the length of the nail used . this indicates that even such short metaphyseal fragments had been effectively stabilized till union with intramedullary nailing when supplemented with blocking screw . blocking screws functionally reduce the width of the metaphyseal medulla , and usually blocking screw is applied in anteroposterior direction as the coronal plane malalignment is more prone to occur than the sagittal plane . moreover deformities in the sagittal plane are better tolerated and are less common if the fracture is reduced at the time of initial locking . but when the fracture pattern suggests instability in sagittal plane , blocking screw should be used in the mediolateral direction . paige whittle a and george w wood ii in their analyses of the influence of fibular fractures on maintaining alignment in 40 distal tibial fractures treated with locked intramedullary nailing concluded that 60% of unfixed fibular fractures occurring at the same level as the tibial fracture were malaligned . in our study , it was at the same level of tibial fracture in 18 cases of which 2 were segmental and distal to tibial fracture in two cases . only 11% ( 2/18 ) of unfixed fibular fractures occurring at the same level as the tibial fractures were malaligned , which is statistically not significant . we found that in interlocking nailing of distal third tibial fractures , when supplemented with blocking screw , level of fibula fracture did not influence the stability or the functional outcome . when compared to other techniques described for preventing metaphyseal malalignment during nailing , blocking screws are technically easy and reproducible , do not require any special instrumentation , and do not need any special design modifications in the nail . there is no need for excessive soft tissue dissection or additional hardware like unicortical plating or fibular plating . we had excellent to satisfactory outcome in 90% by karlstrom - olerud scoring which is comparable to the results of krettek et al . we conclude that blocking screw by acting as a reduction tool , functionally reducing the medullary width and preventing the loss of initial reduction , definitely extends the indication of intramedullary nailing to distal tibial metaphyseal fractures .

multidrug - resistant tuberculosis was diagnosed in 21 hiv - negative , nonhospitalized male patients residing in northern tunisia . a detailed investigation showed accelerated transmission of a mycobacterium tuberculosis clone of the haarlem type in 90% of all patients . this finding highlights the epidemic potential of this prevalent genotype . aim . to evaluate the clinical use of blocking screws as a supplement to stability in distal tibial metaphyseal fractures treated with statically locked intramedullary nail . main outcome measurement . alignment and reduction preoperatively , postoperatively , and at healing were the main outcome measured with an emphasis on maintenance of initial reduction on followup . patients and methods . this was a prospective study of 20 consecutive cases of distal tibial metaphyseal fractures treated with statically locked intramedullary nailing with supplementary blocking screw between august 2006 and september 2007 with a maximum followup of 3 years . medullary canal diameter was measured at the levels of fracture and isthmus . results . the mean diameter of tibia at the level of isthmus was 11.9 mm and at the fracture site was 22.9 mm . mean length of distal fracture segment was 4.6 cm . mean varus / valgus alignment was 10.3 degrees preoperatively and 1.7 degrees immediatly postoperatively and was maintained till union . using karlstrom - olerud score the outcome was excellent to good in 90% . conclusion . we conclude that the use of blocking screw as a supplement will aid in achieving and maintaining the reduction of distal tibial metaphyseal fractures when treated with intramedullary nailing thereby extending the indication of intramedullary nailing .

this was a retrospective study that included consecutive cases undergoing surgery for primary rhegmatogenous rd in patients with pseudophakia after phacoemulsification between the dates of january 2000 and june 2008 that had a follow - up duration of at least 12 months . eyes were excluded from the study if they had non - primary rhegmatogenous rds , such as from trauma , rds associated with coloboma , uveitis , or glaucoma , rds with a macular hole , or had an axial length > 26 mm . patients were excluded from the study if they had atopic dermatitis or if they had a pre - operative proliferative vitreoretinopathy grade c-1 or higher according to the retina society classification . the baseline data ( gender , age , laterality , intraocular lens [ iol ] status , best - corrected visual acuity , and intraocular pressure [ iop ] ) and the intra - operative findings ( operative procedure , macular status , lens capsule status , extension of the rd , number of retinal breaks , and location of retinal breaks ) were recorded . the visual acuity was recorded in snellen units and converted to logarithm of the minimum angle of resolution ( logmar ) units for statistical analysis . each patient underwent a slit lamp biomicroscopy , fundus photography , and goldmann 's three mirror lens in order to identify retinal breaks at baseline . the duration of time from when the cataract surgery took place to the time at which the rd was diagnosed was recorded as the time of rd evolution . scleral buckling ( sb ) or 20-gauge pars plana vitrectomy was performed according to the surgeon 's preference . vitrectomy combined with sb was considered for total rd , and multiple or unidentified retinal breaks . univariate and multivariate linear regression analyses were undertaken for the dependent variable in each model . univariate categorical analysis was performed using the two paired t - test , chi - square test , mann - whitney u - test , or the fisher exact test , as appropriate . data were analyzed via a repeated - measures analysis of variance with a bonferroni correction . statistical significance was set at 0.05 ( two - sided ) for all of the tests . this study comprised 106 eyes of 104 patients ( 70 males and 34 females ) , with a mean age of 55.4 16.7 years ( range , 20 to 85 years ) . the time between onset of rd ( defined by the onset of early signs and symptoms , including flashes and floaters ) and surgery was 15.4 34.7 days ( range , 4 to 75 days ) . the time of rd evolution was 35.8 26.9 months ( range , 1 to 120 months ) . the mean duration of follow - up was 18.0 8.7 months ( range , 12 to 60 months ) . ten of the eyes ( 9.4% ) have had previous ocular surgery ( an anterior vitrectomy in six eyes , a penetrating keratoplasty in one eye and refractive surgery in two of the eyes ) . cataract surgery was performed on 16 eyes in our hospital and surgery was performed on the remaining 90 eyes in another hospital . there were no sclera tunnel incisions and no iris - fixated or anterior chamber lenses . a superior incision was made in four of the eyes ( 3.8% ) and 60 eyes ( 56.5% ) had a temporal incision . the incisional approach for 42 of these eyes ( 39.6% ) was ill - defined . the location of the iol was in the ciliary sulcus in six eyes , scleral fixation in four eyes , and in the bag in the remaining eyes . univariate analysis did not show any statistically significant differences between the baseline visual acuity and the following variables : gender ; age ; laterality ; the time of rd evolution ; the duration of symptoms ; lower iop ; iol location ; lens capsule status ; identification of a retinal break ; number of retinal breaks ; location of retinal breaks ; and type of surgery . a smaller extension of the rd and a macula - on rd had better baseline visual acuity ( p = 0.005 , p = 0.002 ) . in the sb group ( 14 eyes ) , ten of the eyes had segmental sb and four of the eyes had encircling with segmental sb . a primary vitrectomy was performed in 56 eyes ( 52.8% ) and a vitrectomy with sb was performed in 36 eyes ( 33.9% ) ( table 1 ) . intraocular gas tamponade was used for all 92 eyes that underwent vitrectomy ( 20% sf6 [ 40 eyes ] and 14% c3f8 [ 52 eyes ] ) . there were no cases in which air or silicone oil tamponade was used during the primary surgery . post - operatively , 24 eyes ( 22.6% ) underwent re - operation due to recurrence of rd . ten eyes had repeat vitrectomies , eight eyes had vitrectomies with silicone oil tamponade , and six of the eyes had fluid - gas exchange . silicone oil was used for the long - standing tamponade according to the surgeon 's preference and the removal of the silicone oil was performed in four of the eyes one year from the date of the last surgery . three of the four eyes that underwent silicone oil removal achieved final and complete retinal attachment . the remaining eye that had the silicone oil removal received a silicone - oil re - injection due to a recurrence of the rd . at the time of the last follow - up , silicone oil tamponade was continued in eight eyes including the cases that received further silicone oil injections . there were no significant silicone oil - related complications in the silicone oil - filled eyes throughout the follow - up period except for controlled iop elevation . in addition , one eye with permanent silicone tamponade and one eye receiving further fluid - gas exchange presented complete retinal attachment at the time of the patient 's last follow - up . thus , among the 24 eyes that were re - operated on , five of them ( 20.8% ) achieved retinal reattachment . the final visual acuities of the recurrent rd cases were 1.80 0.25 ( range , 0 to 3 ) , which were worse than the 0.63 0.55 of the eyes without recurrence ( p = 0.045 ) ( table 2 ) . the major complications after surgery were macular pucker , which occurred in four of the eyes , and iol dislocation , which occurred in another four eyes . visually significant macular pucker was diagnosed approximately 3 to 6 months after surgery in three of the eyes and all of these eyes underwent a subsequent removal of the epiretinal membrane . four eyes began to develop iol dislocation , and thus two of these eyes underwent one or two point iol fixation and the other two eyes underwent iol repositioning . intra - operatively , the macula was already detached in 58 of the eyes ( 54.7% ) . twenty - eight eyes ( 26.4% ) had a defect in the posterior lens capsule and the remaining 78 eyes had an intact posterior lens capsule . during surgery , a retinal break was not identified in 30 of the eyes ( 28.3% ) , even though the surgeon performed a careful examination with 360 indentations . in regards to the number of retinal breaks that occurred in 76 eyes , 50 of the eyes had on break , 14 eyes had two breaks , eight of the eyes had more than three breaks present , and four eyes presented with retinal dialysis < 1 quadrant . retinal breaks were predominately small horse - shoe tears that were often located anterior to the equator , near the ora serrata . breaks were found in the superotemporal quadrant in 32 of the eyes , the superonasal quadrant in 16 of the eyes , the inferotemporal quadrant in 14 of the eyes , the inferonasal quadrant in 12 of the eyes , and the perimacular area in two of the eyes ( table 1 ) . the retina was attached in 82 of the eyes ( 77.3% ) after primary surgery and in 87 of the eyes ( 82.1% ) at the time of the last follow - up . multivariate analysis did not show any statistically significant differences between anatomic success and the following pre - operative variables : baseline visual acuity ; gender ; age ; laterality ; presence of systemic complications ; the time of rd evolution ; the time between the onset of rd and surgery ; an intact lens capsule ; the location of the intraocular lens ; and a lower iop . an inability to indentify retinal breaks was associated with a significantly lower anatomic success rate ( p = 0.002 ) . the other analyzed intra - operative factors and surgical factors were not associated with different reattachment rates ( table 3 ) . in eyes with unidentified retinal breaks , the retinal attachment rate after the primary surgery was performed was 15 ( 50% ) out of 30 eyes . fifteen ( 50% ) of 30 eyes had a re - operation and tiny retinal breaks were identified in three eyes during the re - operation . the final retinal attachment rate was 20 ( 66.6% ) of 30 eyes , which is significantly lower than the eyes with identified breaks ( p = 0.02 ) ( table 4 ) . the final visual acuities ( logmar ) were improved from the baseline of 1.51 1.14 ( range , 0.1 to 3 ) to 0.65 0.49 ( range , 0 to 3 ) ( p < 0.001 ) . there was no correlation between the baseline and the final visual acuity ( r = 0.128 , p = 0.463 ) . at the last follow - up , 50 of the eyes ( 47.1% ) had a visual acuity of 20/50 or better and 42 of the 106 eyes ( 39.6% ) achieved a visual acuity of 6 / 18 or better . based on multivariate analysis , the characteristics associated with a worse final visual acuity were an inability to identify retinal breaks , a lower iop , and recurrence after the primary surgery ( table 3 ) . the operative procedure , including the use of perfluorocarbon liquid , gas for intraocular tamponade , retinotomy for drainage , and type of implant used for encircling bands and/or scleral sponges , did not influence the final visual acuity . eyes with unidentified retinal breaks presented larger extension of the rd and a higher rate of pre - operative macula - off rd and the final visual acuity in the eyes with unidentified retinal breaks was worse than in the eyes with identified breaks ( 1.40 1.32 and 0.73 0.75 , respectively ; p = 0.02 ) ( table 4 ) . in addition , a lower iop was observed in 10 of the eyes ( 9.4% ) pre - operatively and a lower iop resulted in worse final visual acuity when compared to the eyes with an iop in the normal range ( 2.50 0.83 and 1.39 1.12 , respectively ; p = 0.02 ) . in the sb group ( 14 eyes ) , ten of the eyes had segmental sb and four of the eyes had encircling with segmental sb . a primary vitrectomy was performed in 56 eyes ( 52.8% ) and a vitrectomy with sb was performed in 36 eyes ( 33.9% ) ( table 1 ) . intraocular gas tamponade was used for all 92 eyes that underwent vitrectomy ( 20% sf6 [ 40 eyes ] and 14% c3f8 [ 52 eyes ] ) . there were no cases in which air or silicone oil tamponade was used during the primary surgery . post - operatively , 24 eyes ( 22.6% ) underwent re - operation due to recurrence of rd . ten eyes had repeat vitrectomies , eight eyes had vitrectomies with silicone oil tamponade , and six of the eyes had fluid - gas exchange . silicone oil was used for the long - standing tamponade according to the surgeon 's preference and the removal of the silicone oil was performed in four of the eyes one year from the date of the last surgery . three of the four eyes that underwent silicone oil removal achieved final and complete retinal attachment . the remaining eye that had the silicone oil removal received a silicone - oil re - injection due to a recurrence of the rd . at the time of the last follow - up , silicone oil tamponade was continued in eight eyes including the cases that received further silicone oil injections . there were no significant silicone oil - related complications in the silicone oil - filled eyes throughout the follow - up period except for controlled iop elevation . in addition , one eye with permanent silicone tamponade and one eye receiving further fluid - gas exchange presented complete retinal attachment at the time of the patient 's last follow - up . thus , among the 24 eyes that were re - operated on , five of them ( 20.8% ) achieved retinal reattachment . the final visual acuities of the recurrent rd cases were 1.80 0.25 ( range , 0 to 3 ) , which were worse than the 0.63 0.55 of the eyes without recurrence ( p = 0.045 ) ( table 2 ) . the major complications after surgery were macular pucker , which occurred in four of the eyes , and iol dislocation , which occurred in another four eyes . visually significant macular pucker was diagnosed approximately 3 to 6 months after surgery in three of the eyes and all of these eyes underwent a subsequent removal of the epiretinal membrane . four eyes began to develop iol dislocation , and thus two of these eyes underwent one or two point iol fixation and the other two eyes underwent iol repositioning . intra - operatively , the macula was already detached in 58 of the eyes ( 54.7% ) . twenty - eight eyes ( 26.4% ) had a defect in the posterior lens capsule and the remaining 78 eyes had an intact posterior lens capsule . during surgery , a retinal break was not identified in 30 of the eyes ( 28.3% ) , even though the surgeon performed a careful examination with 360 indentations . in regards to the number of retinal breaks that occurred in 76 eyes , 50 of the eyes had on break , 14 eyes had two breaks , eight of the eyes had more than three breaks present , and four eyes presented with retinal dialysis < 1 quadrant . retinal breaks were predominately small horse - shoe tears that were often located anterior to the equator , near the ora serrata . breaks were found in the superotemporal quadrant in 32 of the eyes , the superonasal quadrant in 16 of the eyes , the inferotemporal quadrant in 14 of the eyes , the inferonasal quadrant in 12 of the eyes , and the perimacular area in two of the eyes ( table 1 ) . the retina was attached in 82 of the eyes ( 77.3% ) after primary surgery and in 87 of the eyes ( 82.1% ) at the time of the last follow - up . multivariate analysis did not show any statistically significant differences between anatomic success and the following pre - operative variables : baseline visual acuity ; gender ; age ; laterality ; presence of systemic complications ; the time of rd evolution ; the time between the onset of rd and surgery ; an intact lens capsule ; the location of the intraocular lens ; and a lower iop . an inability to indentify retinal breaks was associated with a significantly lower anatomic success rate ( p = 0.002 ) . the other analyzed intra - operative factors and surgical factors were not associated with different reattachment rates ( table 3 ) . in eyes with unidentified retinal breaks , the retinal attachment rate after the primary surgery was performed was 15 ( 50% ) out of 30 eyes . fifteen ( 50% ) of 30 eyes had a re - operation and tiny retinal breaks were identified in three eyes during the re - operation . the final retinal attachment rate was 20 ( 66.6% ) of 30 eyes , which is significantly lower than the eyes with identified breaks ( p = 0.02 ) ( table 4 ) . the final visual acuities ( logmar ) were improved from the baseline of 1.51 1.14 ( range , 0.1 to 3 ) to 0.65 0.49 ( range , 0 to 3 ) ( p < 0.001 ) . there was no correlation between the baseline and the final visual acuity ( r = 0.128 , p = 0.463 ) . at the last follow - up , 50 of the eyes ( 47.1% ) had a visual acuity of 20/50 or better and 42 of the 106 eyes ( 39.6% ) achieved a visual acuity of 6 / 18 or better . based on multivariate analysis , the characteristics associated with a worse final visual acuity were an inability to identify retinal breaks , a lower iop , and recurrence after the primary surgery ( table 3 ) . the operative procedure , including the use of perfluorocarbon liquid , gas for intraocular tamponade , retinotomy for drainage , and type of implant used for encircling bands and/or scleral sponges , did not influence the final visual acuity . eyes with unidentified retinal breaks presented larger extension of the rd and a higher rate of pre - operative macula - off rd and the final visual acuity in the eyes with unidentified retinal breaks was worse than in the eyes with identified breaks ( 1.40 1.32 and 0.73 0.75 , respectively ; p = 0.02 ) ( table 4 ) . in addition , a lower iop was observed in 10 of the eyes ( 9.4% ) pre - operatively and a lower iop resulted in worse final visual acuity when compared to the eyes with an iop in the normal range ( 2.50 0.83 and 1.39 1.12 , respectively ; p = 0.02 ) . surgical techniques in patients with pseudophakia after phacoemulsification have rapidly advanced in parallel with the development of better equipment . previous studies involving patients with pseudophakic rds have been reported before the era of phacoemulsification with micro - corneal incisions . thus , the reported surgical outcomes of patients with pseudophakic rds may be improved upon . this retrospective study involved 106 patients with a primary of idiopathic pseudophakic rhegmatogenous rds who had surgery by one surgeon over an 8-year period . data relating to 21 pre- , intra- , and post - operative variables were studied . the final reattachment rate was 82% , which is slightly greater than what previous studies have reported [ 6 - 8 ] . previous reports have identified the following negative prognostic factors for reattachment after surgery : age > 65 years ; poor pre - operative vision ; rd involving > 3 quadrants ; macula - off rds ; a lower iop ; inability to identify a retinal break ; longer duration of symptoms ; and grades c or d of proliferative vitreoretinopathy [ 6 - 13 ] . in the current study , only an inability to identify a retinal break was significantly associated with lower reattachment success . proliferative vitreoretinopathy grade c or d was not analyzed due to the exclusion criteria used in this study . a critical step in successful rd surgery is to locate and seal all of the causative retinal breaks . however , in pseudophakia , tiny breaks , poor mydriasis , cortical remnants , capsular opacification , glare or pitting from the intraocular lens implant , and corneal or vitreous opacities may make identification difficult , especially since anterior breaks commonly occur in pseudophakic and aphakic rds . thus , a retinal break is not identified in 9% to 20% of pseudophakic rds , which would be a risk factor for surgical failure [ 11 - 17 ] . cases with unidentified retinal breaks have traditionally been managed using sb and broad application cryopexy , which has a reported primary success rate of 53% to 70% . we think an internal approach with vitrectomy offers some advantages that might result in the detection of these breaks . scleral external indentation together with endoillumination and magnification during the vitrectomy may help locate previously undetected breaks . also during vitrectomy , breaks can be searched for with an aspirating instrument or by using perfluorocarbon liquids that may identify a stream of fluid that is exiting the subretinal space via the hole . we performed vitrectomy combined with either segmental or encircled sb in all 30 of the eyes that had unidentified breaks with a final anatomical success rate of 66% . previous case series have reported that the anatomical success rate of vitrectomy when combined with sb was superior to sb alone [ 19,22 - 24 ] . in contrast , some authors have found that vitrectomy was not of additional value in terms of a better success rate . a larger prospective randomized trial is needed in order to determine an accurate surgical method for rds with unidentified breaks . as mentioned above , we indicated that identifying a retinal break is an important factor for both anatomic and functional outcomes for pseudophakic rd repair . one of the most important changes in the posterior segment after cataract surgery is anomalous posterior vitreous detachment , which would induce a retinal tear [ 27 - 29 ] . although the reasons for an increased incidence of posterior vitreous detachment after cataract surgery remain unknown , it is thought that the anterior movements of the vitreous during surgery might play a role in the development of posterior vitreous detachment . we believe that it is possible that the insertion of an instrument during surgery via the main incision could produce vitreous movement and the subsequent vitreous traction may then result . in the current cases , the main incision for the cataract surgery was via a temporal approach and the eyes with an undetermined incisional approach may have undergone a temporal corneal approach . although it is not clear why retinal breaks occur more frequently in the superiotemporal quadrant , meticulous examinations of the superiotemporal area during surgery are important to identify these retinal breaks . it has been reported that the previously mentioned negative prognostic variables for anatomical outcomes are also associated with worse visual outcomes [ 5 - 13 ] . among these variables , a lower iop , unidentified retinal breaks , and the recurrence after primary surgery were associated with poorer final visual acuity in the current study . a lower iop was related to a poor final visual acuity and this relationship has previously been reported [ 32 - 35 ] . this finding might reflect a more bullous rd , a pre - operative choroidal detachment or surgical difficulty . these factors are related with retinal glial cell up - regulation and retinal pigment epithelium dispersion , which resulted in a delayed morphologic recovery including that of the photoreceptor reposition of the reattached retina . it is thought that the visual results of pseudophakic detachment depend on the pre - operative macula status and approximately 80% of pseudophakic rd typically presents with the macula detached [ 12 - 14 ] . however , 45% of the rds in this study were macula - on at the time of surgery . we showed that the macula - off rd is not more highly related to poorer outcome , in spite of the worse baseline visual acuity , and that pseudophakic detachment is not present more often with the macula detached . we believe that patients tend to have accurate and frequent eye examinations , which would lead to an earlier diagnosis before there is macular involvement and this is reflected in our final results . in addition , both vitreous loss at the time of the cataract surgery and nd : yag posterior capsulotomy appear to increase the risk of rd when performed after cataract surgery . a defect in the lens capsule may be related to more vitreous involvement and reflect more complex intraocular manipulations . we investigated the defects in the lens capsules and the iol position , but defects in the lens capsules and the iol position were not prognostic factors for post - operative outcomes . although earlier reports suggested that vitrectomy without sb , the use of perflurocarbon liquids during vitrectomy , and the performance of a drainage retinotomy were associated with the existence of more complex rds , and consequently poorer functional results [ 38 - 40 ] , more recent reports have shown that the surgical outcomes appear to be independent of the surgical procedure that is chosen to resolve the rd . in the current study , the type of surgical procedure ( vitrectomy or sb ) and intra - operative factors were not associated with the post - operative results . thus , these results suggest that surgical outcomes are independent of the methods or complexity of the surgery . this retrospective series had a relatively small sample size within one retinal center and the surgical procedures that were used were heterogeneous . we did not describe the size of the retinal breaks , vitreous status , or the intra - operative events that occurred during the cataract surgery . a large , multicenter study is needed to evaluate the precise prognostic factors for surgical outcomes in patients with pseudophakic rds . however , the surgical treatment of all the patients in this study was performed by one experienced retinal specialist . we believe this report is of importance to clinicians who perform surgery for rds in pseudophakic patients . consequently , retinal breaks that were not identified during surgery were related to poor anatomic and functional outcomes . clinicians should perform careful examinations in the superotemporal quadrant to find retinal breaks when visible breaks are not identified . a poorer pre - operative state , including a worse pre - operative visual acuity , the macula - off rd , a larger area involved in the rd , and multiple breaks was not related to a poorer surgical outcome . adequate surgery that is performed according to the preference of the surgeon may lead to successful outcomes in patients with pseudophakic rds . the patient was an 11-year - old boy with no family history of optic neuropathy or dystonia . he presented with focal dystonia in the right upper limb at 3 years of age , and his disease progressed over 4 years to dystonia in both the upper and the lower limbs on his dominant right side . at 5 years of age however , at 7 years of age , he had severe dysarthria and was unable to walk without support owing to dystonia and spasticity . he had neither mental retardation ( verbal iq , 129 ; performance iq , 97 ; full - scale iq , 115 by the weschler intelligence scale for children ) nor short stature . magnetic resonance imaging findings at 3 years of age showed high signal intensity lesions in bilateral putamina on fluid - attenuated inversion recovery images ( figure 1a - c ) and diffusion - weighted images ( figure 1d ) . with time , the high signal intensity lesions on diffusion - weighted images migrated from the putamina to the caudate nuclei ( figure 1e - l ) . the mri findings obtained at 6 years of age showed that the high signal intensity lesions had spread to the bilateral caudate nuclei and the midbrain on fluid - attenuated inversion recovery images ( figure 1i - k ) and diffusion - weighted images ( figure 1l ) . on laboratory examination at 7 years of age , blood cell counts were normal , and the concentrations of lactate and pyruvate were not elevated in serum and cerebrospinal fluid . the serum concentrations of copper and ceruloplasmin were normal . in the serum amino acid analysis , organic acid level in urine , and levels of lysosomal enzyme in white blood cells were found to be normal . sequence analysis of the white blood cell mitochondrial dna from this patient showed the presence of a homoplasmic g > a transition mutation at nucleotide position 14 459 in the reduced nicotinamide adenine dinucleotide dehydrogenase subunit 6 gene ( figure 2 ) . a - d , brain magnetic resonance imaging ( mri ) obtained at 3 years 2 months of age shows high signal intensity lesions with partially cystic lesions in the bilateral putamina on fluid - attenuated inversion recovery images ( a - c ) and diffusion diffusion - weighted imaging ( d ) . e - h , brain mri obtained at 5 years 2 months of age show a high signal intensity lesion that is newly detected in the left caudate nucleus on fluid - attenuated inversion recovery images ( e - g ) . diffusion - weighted imaging shows that the bilateral putamina are iso- or hypointense and the left caudate nucleus is hyperintense ( h ) . i - l , brain mri obtained at 6 years 11 months of age shows high signal intensity lesions spread to bilateral caudate nuclei and the midbrain ( right substantia nigra ) on fluid - attenuated inversion recovery images ( i - k ) and diffusion - weighted imaging ( l ) , in addition to the putaminal hyperintensities . m - o , brain mri obtained at 8 years 6 months of age shows high signal intensity lesions in the bilateral putamina and caudate nuclei on t2-weighted ( m - o ) and diffusion - weighted imaging ( p ) . q - t , brain mri obtained at 10 years 5 months of age shows high signal intensity lesions in the bilateral putamina and caudate nuclei on t2-weighted ( q - s ) and diffusion - weighted imaging ( t ) . a - c , e - g , i - k : fluid - attenuated inversion recovery images ; m - o , q - s : t2-weighted images ; and d , h , l , p , t : diffusion - weighted images . sequence analysis of the white blood cell mitochondrial dna from this patient shows the presence of a homoplasmic g > a transition mutation at nucleotide position 14 459 in the reduced nicotinamide adenine dinucleotide dehydrogenase subunit 6 gene . the patient had been treated with coenzyme q10 ( 2 mg / kg / d ) , l - carnitine ( 90 mg / kg / day ) , and vitamins since the age of 4 years . however , his symptoms and mri abnormalities progressed gradually despite treatment . therefore , oral administration of sodium succinate ( 250 mg / kg / d ) was started from the age of 7 years , on the basis of the presence of g14459a mitochondrial dna mutation ( which can cause complex i deficiency ) and a previous case report of a patient with mitochondrial encephalomyopathy , lactic acidosis , and stroke - like episodes ( melas ) . on the follow - up mri examinations over the subsequent 4 years , progression of mri abnormalities appeared suppressed , but there was no improvement in dystonia ( figure 1m - t ) . in the present case , mitochondrial dna analysis of the entire region revealed a g > a transition mutation at nucleotide position 14 459 , leading to alanine - to - valine amino acid substitution at position 72 of the reduced nicotinamide adenine dinucleotide dehydrogenase subunit 6 of complex i. the g14459a mitochondrial dna mutation has been reported in the following patients : 9 with childhood - onset dystonia , 6 with leber hereditary optic neuropathy , 4 with leber hereditary optic neuropathy plus dystonia , 4 with leigh or leigh - like syndrome , and 4 who were clinically asymptomatic . the general features of cases reported to be of the dystonia phenotype were progressive generalized dystonia and/or optic neuropathy . the clinical findings of leigh disease are optic involvement , early developmental delay , and hypotonia . the patients with leigh - like encephalopathy showed visual dysfunction , ataxia , and hearing loss . t2-weighted magnetic resonance images showed high signal intensity lesions in the medial thalamic nuclei , anterior pons , and cerebral peduncles in one patient and in the dorsal midbrain and red nucleus in other patients , in addition to a basal ganglia lesion . the patient reported in our study presented with the dystonic phenotype and leigh - like findings on neuroimaging , showing degeneration in the bilateral striata and right substantia nigra . the variation in clinical expression indicates that modifying nuclear factors are likely involved , playing a role in the clinical expression of mitochondrial diseases . the diffusion - weighted mri findings in our patient are characteristic . with time , high signal intensity lesions on diffusion - weighted images migrated from the putamina to the caudate nuclei , which closely correlated with his disease progression . diffusion - weighted images enhance the restricted mobility of water molecules in the brain , and it is a useful tool for detecting acute ischemic lesions in patients with stroke . the pathophysiology of the diffusion abnormalities associated with mitochondrial diseases remains unclear , although it has been reported in some patients with leigh disease . considering the finding that diffusion abnormalities migrated from the putamina to the caudate nuclei in parallel with the disease progression in this patient , diffusion abnormalities might be caused by the chronic mitochondrial respiratory dysfunction due to the mutation of the reduced nicotinamide adenine dinucleotide dehydrogenase subunit 6 gene . they include coenzyme q10 , cytochrome c nicotinamide , dichloroacetate , l - arginine , and succinate . these drugs ( except for l - arginine ) are mitochondrial respiratory chain enzymes or its substrates and are considered to compensate for the defects in the corresponding pathways . because the g14459a mitochondrial dna mutation could cause complex i deficiency , oral administration of sodium succinate was started on the basis of a previous case report of a patient with melas . the proposed pharmacological mechanism is that the respiratory capacity with the defect in complex i can be improved or restored by succinate administration through the activation of the complex ii system . it has been reported that succinate enhances electron flow from complex ii to complex iii ( coenzyme q cytochrome c reductase ) and to complex iv ( cytochrome c oxidase ) , enabling these 2 energy coupling sites to operate normally . in the present case , progression of mri abnormalities appears to have been suppressed for 4 years after treatment with sodium succinate . there is a possibility that treatment with sodium succinate is helpful in patients with g14459a mitochondrial dna mutation , although further investigation is necessary .

purposeto evaluate the clinical features and surgical outcomes for primary rhegmatogenous retinal detachments ( rds ) in patients with pseudophakia after phacoemulsification.methodsthe medical records of patients with pseudophakia after phacoemulsification and intraocular lens implantation who had undergone surgery for primary rhegmatogenous rds with a minimum duration of follow - up of 12 months were reviewed retrospectively.resultsa total of 104 patients were enrolled in this study and 106 eyes were analyzed . post - operative retinal attachment was achieved in 87 of the eyes ( 82.1% ) and the final visual acuities ( logarithm of the minimum angle of resolution ) were improved to 0.65 0.49 from the baseline measurement of 1.51 1.14 ( p < 0.001 ) . re - operations were performed in 24 of the eyes ( 22.6% ) and there were no visible retinal breaks in 30 of the eyes ( 28.3% ) . the failure to identify a retinal break during surgery was associated with a lower rate of retinal reattachment , worse final visual acuity , and a higher rate of re - operation ( p = 0.002 , p = 0.02 , and p = 0.002 , respectively ) . the location of the identified retinal break was more common in the superotemporal quadrant than in the other quadrants.conclusionsthe inability to identify a retinal break during surgery was associated with a poor final outcome . other factors were less important for the functional and anatomic success in patients with pseudophakic rds . this article reports the case of an 11-year - old boy with progressive dystonia caused by the homoplasmic g14459a mitochondrial dna mutation . the patient presented with focal dystonia in the right upper limb at 3 years of age , which progressed over 4 years to exhibit dystonia in both the upper and lower limbs . at 7 years of age , high signal intensity lesions in the bilateral striata and the midbrain were observed on fluid - attenuated inversion recovery images . it was observed on diffusion - weighted images that with time , these high signal intensity lesions migrated from the putamen to the caudate nuclei , which closely correlated with disease progression . because his symptoms and abnormal magnetic resonance imaging findings progressed despite treatment with coenzyme q10 and l - carnitine , at 7 years of age he was then started on sodium succinate , hoping to improve his complex i deficiency . after treatment , progression of mri abnormalities appeared to have been suppressed for 4 years , although no improvement was observed in dystonia .

fibrous dysplasia of bone is characterized by the replacement of medullary bone with fibro - osseous tissue . this results in the distortion and overgrowth of bone , leading to characteristic deformities . based on the bony involvement , fibrous dysplasia is divided into three types : monostotic , polyostotic , and mccune albright syndrome ( mas ) . mas is characterized by a triad of monostotic / polyostotic fibrous dysplasia , caf-au - lait macules ( calms ) , and endocrine hyperfunction . the disease is congenital but not hereditary and is due to an activating mutation in g protein - coupled receptor . it manifests usually in childhood , with recurrent fractures , distorted bones , precocious puberty , acromegaly , or hyperthyroidism . untreated disease leads to permanent disfigurement , and the disease is classified as paucibacillary or multibacillary depending on the proliferation of the bacilli . the risk factors for the disease are living in endemic areas with poor hygiene conditions and defects in cell - mediated immunity . we recently encountered a patient with mas who presented with cutaneous features of hansen 's disease . we report the same for the unusual association and to highlight the relevant review of the literature . a 30-year - old man presented with hypoesthetic , depigmented patches over the skin of the chest and back of 1 year duration . he gave history of recurrent fractures since the age of 4 years with shortening of right leg . the parents gave history of hyperpigmented macules over the trunk since birth and denied similar features in family members . the patient attained puberty at the age of 14 years and denies history suggestive of acral enlargement , thyrotoxicosis , cushing 's syndrome , and swellings in any part of the body . examination revealed ( height 152 cm ) asymmetry of the face with expansion of right maxilla and lower jaw and short right leg ( 82 vs 86 cm ) [ figures 1 and 2 ] . cutaneous examination revealed a large calm over the back , extending across the midline and multiple anesthetic patches over scapula and lower back [ figure 3 ] . there was no evidence of goiter , acromegaloid features , and thickened nerves , and the rest of the examination was normal . facial photograph showing craniofacial dysplasia legs showing short right leg ( arrow ) with expansile lesion of tibia ( arrow head ) skin over the back showing a large calm crossing midline ( long arrows ) with depigmented hypoesthetic patches ( short arrow ) hormonal profile revealed normal thyroid adrenal and gonadal axes evaluation . skeletal survey revealed expansile osteolytic lesion over right maxilla , right hip , and shoulder . skin biopsy specimens were obtained from ear lobes , legs , and depigmented lesions over the back , avoiding areas of calm . specimens revealed the presence of acid - fast bacilli along with noncaseating granulomas consistent with the diagnosis of tuberculoid leprosy . the diagnosis of fibrous dysplasia is made by the presence of bone pains , recurrent fractures , deformity of bone , and radiological evidence of fibro - osseous tissue showing expansile lytic lesion . biopsy and tc - mdp scan of the bone were not considered necessary to establish the diagnosis in this classical case . other laboratory workup showed elevated alkaline phosphatase ( 388 u / l ) with normal calcium , phosphorus , parathyroid hormone , and 25-hydroxy vitamin d levels . he was treated with triple drug regimen for leprosy and also given oral calcium carbonate 1.5 g daily along with the first dose of zoledronic acid 4 mg as an intravenous infusion . mas is characterized by the triad of polyostotic fibrous dysplasia , calm , and endocrinopathy . the diagnosis of fibrous dysplasia is based on classical radiological findings supported by histopathology and bone scan . hyperfunctioning of endocrine glands in mas presents with precocious puberty , hyperthyroidism , acromegaly , or cushing 's syndrome . our patient had only two features of the triad and did not have any evidence of endocrine hyperfunction syndromes . the calm is unusual in our patient in that it crossed the midline and was seen on both sides of the body . the calms in mas do not cross the midline and follow the developmental lines of blashko , thus differentiating from those in neurofibromatosis . our patient had an exceptionally large calm that crossed the midline interspersed with anesthetic depigmented macular lesions of hansen 's disease . hansen 's disease is caused by mycobacterium leprae , and the disease spreads via respiratory droplets between close personal contacts over a prolonged duration . immunocompromised situations are the predominant risk factors for the disease , and defects in cell - mediated immunity are implicated in persons with genetic predisposition . the mode of acquisition of hansen 's disease in our patient is possibly due to frequent stay in the hospitals due to multiple fractures and contact with a patient . screening of the persons involved in personal care of the patient did not reveal features of hansen 's disease . the association of hansen 's disease with mas appears coincidental as there is no evidence that mas predisposes to the infectious diseases or leads to immunosuppressed state . extensive literature search did not reveal any previous reports of association between hansen 's disease and mas . they are given as intravenous or oral preparation and act by suppressing the osteoclast activation . treatment of mas involves bisphosphonate therapy to reduce fracture rate apart from treating any associated endocrinopathy . to conclude , we present an interesting case of mas without associated endocrinopathy who had coexisting hansen 's disease . this unusual combination was not reported earlier in world literature . pseudouridine ( ) is the most abundant post - transcriptional rna base modification and the enzymes responsible for this isomerization of uridine and the positions modified in the rnas , are conserved ( 14 ) . this modification is important in the structure and function of small rnas ( 511 ) , including small nuclear rnas ( snrnas ) , cofactors in pre - mrna splicing ( 1216 ) . one pathway is involved in the formation of in rrna and snrna and requires dyskerin or its homologs ( cbf5p in yeast for example ) and rnp cofactors [ most often h / aca small nucleolar ribonucleoprotein particles ( snornps ) ] that enable one enzyme to recognize many different sites for modification on different substrates ( 1725 ) . the other pathway for formation employs site - specific synthases that require no cofactors to recognize and modify the rna substrate . a number of enzymes have been identified in this pathway and are grouped in six families that all share a common basic structure ( 4 ) . guided pathway has received a great deal of attention because of its similarity to aspects of rna editing , but the site - specific pseudouridine synthases accomplish the same task , on more substrates , with fewer tools , arguably making them more interesting from an enzymatic perspective . one of the most studied site - specific enzymes is pseudouridine synthase 1 ( pus1p ) ( 2635 ) . pus1p modifies uridines in several regions of many different trnas ( 27,33,34 ) and one position in yeast u2 snrna ( 36 ) . it is a transcriptional coactivator in nuclear receptor signaling , modifying steroid receptor activator rna ( sra ) ( 37,38 ) , and when mutated or missing , leads to the disorder mitochondrial myopathy and sideroblastic anemia with lactic acidemia ( mlasa ) ( 3942 ) . in vivo and in vitro pus1p modifies positions 1 , 26 , 27 and 28 in many trnas and positions 34 and 36 in the anticodons of certain intron - containing trnas ( 2628,33 ) . in addition , yeast pus1p has been shown to modify positions 65 and 67 in vivo ( 34 ) and mouse ( mpus1p ) and human pus1p ( hpus1p ) modify position 30 in trnas , whereas yeast pus1p does not ( 29,33 ) . the presence of the intron in yeast pre - trna(uau ) is required for the formation of at positions 34 and 36 in the anticodon by pus1p in vitro ( 26,34 ) . the intron allows for the formation of a stem that contains the uridines to be modified ( 26,34,43 ) . residues that are modified in trna by pus1p are part of a base - paired stem , suggesting rna secondary structure is important for recognition by pus1p ( 26,27,3335 ) . even with all of the studies on pus1p , the only sequence consensus ( not requirement ) that has been proposed for pus1p is the presence of a pyrimidine nucleotide 3 of the site of modification ( 44 ) . interestingly , yeast pus1p has been shown to modify position 44 on yeast u2 snrna , a position that is considered to be in a non - base paired region of u2 snrna ( 36 ) . when mouse pus1p is expressed in a pus1 deleted yeast strain , the mouse enzyme can also modify yeast u2 snrna at the same position ( 33 ) . however , the modification of u2 snrna in caenorhabditis elegans does not require pus1p , since the loss of the pus-1 gene product in worms results in the absence of at known pus1p modification sites on trna , but has no effect on the modification of u2 snrna or other snrnas ( 28,45 ) . however , the structure of a member of the same synthase family , bacterial trua , in the presence of trna substrates , has been revealed ( 46,47 ) . trua modifies uridines at positions 3840 in many trnas and the equivalent activity in eukaryotes is associated with pseudouridine synthase 3 , another member of the trua family [ pus3p ; ( 48,49 ) ] . the substrate trna binds across a dimer of trua ( 46,47 ) . the anticodon stem - loop ( asl ) of the trna , as well as the dihydrouridine ( d ) stem and the elbow where the d and tc loops interact , contact the synthase ( 47 ) . trua did not interact with the variable ( v ) stem - loop or the acceptor stem of the trna substrate ( 47 ) . in order to begin to understand how pus1p can modify several positions in trnas and also modify other rnas such as u2 snrna and sra , we undertook an investigation of the sequence and structural requirements for hpus1p activity on human trna . the activity of hpus1p with wild - type and point and deletion mutants of the trna substrate indicates there are structural requirements in the formation of at position 28 in trna . a minimum level of base pairing in the asl ( the location of the modification ) and the presence of an additional 3 stem - loop , constitute the minimal substrate for hpus1p . even when all nucleotides in the asl stem other than u28 were changed in a single minimal substrate , but base pairing was retained , a near wild - type level of modification was observed . site - directed mutagenesis of human trna ( uga ) was performed with a quikchange ii kit ( stratagene ) , utilizing primers containing the mutations and the phts plasmid [ human trna(uga ) ; gift of h. gross ( 50 ) ] to create the mutants . the resulting constructs have a t7 rna polymerase promoter at the 5-end of the trna gene and a bstn1 site at the 3-end of the gene . plasmid dna digested with bstn1 will yield trnas with cca at the 3-terminus after transcription ( 29,50 ) . the primers used for mutagenesis are given below with just the forward primers listed ; the reverse primers are the inverse complement . the forward primer for : 27c was 5cgagtggttaaggcgctggacttgaaatcca,27 g was 5cgagtggttaaggcggtggacttgaaatcca,28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42g,29c was 5gagtggttaaggcgatcgacttgaaatccatt,30c was 5gtggttaaggcgatgcacttgaaatccattg,31c was 5gtggttaaggcgatggccttgaaatccattgg,39 g was 5cgatggacttgaaagccattggggtc,40 g was 5gatggacttgaaatgcattggggtctc,41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template,42c was 5ggacttgaaatcccttggggtctcc,42 g was 5ggacttgaaatccgttggggtctcc,43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template,30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template,31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template, d stem - loop was 5gtagtcgtggcgatggacttgaaatcc, d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . 27c was 5cgagtggttaaggcgctggacttgaaatcca , 27 g was 5cgagtggttaaggcggtggacttgaaatcca , 28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42 g , 29c was 5gagtggttaaggcgatcgacttgaaatccatt , 30c was 5gtggttaaggcgatgcacttgaaatccattg , 31c was 5gtggttaaggcgatggccttgaaatccattgg , 39 g was 5cgatggacttgaaagccattggggtc , 40 g was 5gatggacttgaaatgcattggggtctc , 41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template , 42c was 5ggacttgaaatcccttggggtctcc , 42 g was 5ggacttgaaatccgttggggtctcc , 43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template , 30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template , 31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template , d stem - loop was 5gtagtcgtggcgatggacttgaaatcc , d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . the digested plasmid dnas were transcribed with t7 rna polymerase in the presence of 25 ci [ 5-h]-utp ( 11.1 or 23.9 ci / mmol , moravek ) or 25 ci [ 5,6-h]-utp ( 38 ci / mmol , moravek ) with 100 m cold utp and 1.0 mm , non - labeled , atp , gtp and ctp ( 51 ) . additional rna substrates were synthesized using a sense t7 dna oligonucleotide , an antisense template oligonucleotide and high concentration t7 rna polymerase , as described ( 52,53 ) . the oligodeoxynucleotides used for this type of synthesis are listed below , note that the oligodeoxynucleotides that serve as the template for synthesis are antisense and have antisense t7 primer sequence at the 3-end . the template for the : asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta, tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta, v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta.asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta , v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta . asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a g , allows for efficient synthesis of rnas in vitro . the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . before incubation with hpus1p , the rna substrates were heated to 78c for 3 min and allowed to cool slowly to at least 37c ( 3040 min ) . substrate reactions with the synthases were carried out in 50 mm ammonium chloride , 5 mm dtt , 25 mm tris ( ph 7.5 ) and 1 mm mgcl2 . the amount of synthase used was ~200 ng at 37c for the times indicated . norit a charcoal binds all of the h counts in the sample except those that have been released to water in the process of forming . the released counts are separated from the charcoal - bound counts using a spin - x column ( costar ) , the filtrate is mixed with scintillation fluid , and the released h counts are measured on a scintillation counter . the equilibrium binding assays were carried out as described ( 32 ) using hpus1p concentrations of 0 , 50 , 100 , 200 , 400 and 800 nm in 50 l reactions , with the h - labeled rna substrates at 500 pm . the rnas were synthesized as described above except that the cold utp concentration in the synthesis reaction was 250 m . a slot - blot apparatus ( biorad ) was used for filtration of the samples , the resulting filters were air dried , cut into appropriate pieces and counted in scintillation fluid . site - directed mutagenesis of human trna ( uga ) was performed with a quikchange ii kit ( stratagene ) , utilizing primers containing the mutations and the phts plasmid [ human trna(uga ) ; gift of h. gross ( 50 ) ] to create the mutants . the resulting constructs have a t7 rna polymerase promoter at the 5-end of the trna gene and a bstn1 site at the 3-end of the gene . plasmid dna digested with bstn1 will yield trnas with cca at the 3-terminus after transcription ( 29,50 ) . the primers used for mutagenesis are given below with just the forward primers listed ; the reverse primers are the inverse complement . the forward primer for : 27c was 5cgagtggttaaggcgctggacttgaaatcca,27 g was 5cgagtggttaaggcggtggacttgaaatcca,28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42g,29c was 5gagtggttaaggcgatcgacttgaaatccatt,30c was 5gtggttaaggcgatgcacttgaaatccattg,31c was 5gtggttaaggcgatggccttgaaatccattgg,39 g was 5cgatggacttgaaagccattggggtc,40 g was 5gatggacttgaaatgcattggggtctc,41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template,42c was 5ggacttgaaatcccttggggtctcc,42 g was 5ggacttgaaatccgttggggtctcc,43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template,30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template,31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template, d stem - loop was 5gtagtcgtggcgatggacttgaaatcc, d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . 27c was 5cgagtggttaaggcgctggacttgaaatcca , 27 g was 5cgagtggttaaggcggtggacttgaaatcca , 28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42 g , 29c was 5gagtggttaaggcgatcgacttgaaatccatt , 30c was 5gtggttaaggcgatgcacttgaaatccattg , 31c was 5gtggttaaggcgatggccttgaaatccattgg , 39 g was 5cgatggacttgaaagccattggggtc , 40 g was 5gatggacttgaaatgcattggggtctc , 41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template , 42c was 5ggacttgaaatcccttggggtctcc , 42 g was 5ggacttgaaatccgttggggtctcc , 43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template , 30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template , 31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template , d stem - loop was 5gtagtcgtggcgatggacttgaaatcc , d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc . the digested plasmid dnas were transcribed with t7 rna polymerase in the presence of 25 ci [ 5-h]-utp ( 11.1 or 23.9 ci / mmol , moravek ) or 25 ci [ 5,6-h]-utp ( 38 ci / mmol , moravek ) with 100 m cold utp and 1.0 mm , non - labeled , atp , gtp and ctp ( 51 ) . additional rna substrates were synthesized using a sense t7 dna oligonucleotide , an antisense template oligonucleotide and high concentration t7 rna polymerase , as described ( 52,53 ) . the oligodeoxynucleotides used for this type of synthesis are listed below , note that the oligodeoxynucleotides that serve as the template for synthesis are antisense and have antisense t7 primer sequence at the 3-end . the template for the : asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta, tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta, v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta.asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta , v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta . asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a g , allows for efficient synthesis of rnas in vitro . the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . before incubation with hpus1p , the rna substrates were heated to 78c for 3 min and allowed to cool slowly to at least 37c ( 3040 min ) . substrate reactions with the synthases were carried out in 50 mm ammonium chloride , 5 mm dtt , 25 mm tris ( ph 7.5 ) and 1 mm mgcl2 . the amount of synthase used was ~200 ng at 37c for the times indicated . norit a charcoal binds all of the h counts in the sample except those that have been released to water in the process of forming . the released counts are separated from the charcoal - bound counts using a spin - x column ( costar ) , the filtrate is mixed with scintillation fluid , and the released h counts are measured on a scintillation counter . the equilibrium binding assays were carried out as described ( 32 ) using hpus1p concentrations of 0 , 50 , 100 , 200 , 400 and 800 nm in 50 l reactions , with the h - labeled rna substrates at 500 pm . the rnas were synthesized as described above except that the cold utp concentration in the synthesis reaction was 250 m . a slot - blot apparatus ( biorad ) was used for filtration of the samples , the resulting filters were air dried , cut into appropriate pieces and counted in scintillation fluid . as an initial investigation into the substrate requirements for hpus1p , it was important to choose a known pus1p substrate trna that has only one site modified by the enzyme ( 29,35 ) , to simplify the analysis of the activity . a trna that is typical in structure and has a v stem - loop that was > 6 nt , was preferred in order to be able to test the contribution of all structural components to substrate recognition by hpus1p . in addition , a trna with traditional watson - crick base - pairing in the asl stem was desired . the trna sequence database , trnadb ( 56,57 ) , was consulted to compile consensus sequences for the asl stem where a or u is found at position 28 in sequenced trnas from the fungi and metazoan group in trnadb . at least 36 trna sequences were compiled to generate each consensus shown in figure 1b . first , with a at position 28 , g30c40 is the most conserved base pair ( > 90% conserved ) but when a u is found at position 28 , this sequence is not as well conserved . therefore , one might expect that disruption of this base pair or replacement with the inverse ( c30g40 ) will affect modification . second , for sequenced trnas that have a at 28 , the preferred 2743 bp is u27a43 , whereas with those trnas with a u at 28 , the preferred base pair is c27g43 . third , a a31u39 base pair is more common in those asl stems that have a at position 28 than those with a u at position 28 . finally , the base pairs 2941 and 3139 , below the 2842 bp , are watson - crick only but the rest of the base pairs in the stem can have g - u pairing in either consensus . in the selection of a trna , a good match to this consensus was preferred , and human trna(uga ) meets most of the criteria . the only difference is that this trna has an a27u43 base pair instead of a u a base pair at that position ( figure 1a ) . ( a ) the sequence and proposed secondary structure of human trna(uga ) ( 50 ) . the major aspects of the secondary structure are labeled on the diagram and it is numbered without including most of the variable stem - loop . the single site of pus1p modification , position 28 , is boxed . ( b ) consensus sequences for sequenced trnas from the fungi and metazoa group in the trnadb ( 57 ) that have a or a u at position 28 . a solid line between nucleotides indicates watson - crick pairs only and a dash line indicates g u pairs ( < 50% ) are also found at this position . ( a ) the sequence and proposed secondary structure of human trna(uga ) ( 50 ) . the major aspects of the secondary structure are labeled on the diagram and it is numbered without including most of the variable stem - loop . the single site of pus1p modification , position 28 , is boxed . ( b ) consensus sequences for sequenced trnas from the fungi and metazoa group in the trnadb ( 57 ) that have a or a u at position 28 . the figure is presented in the output style from trnadb . a solid line between nucleotides indicates watson - crick pairs only and a dash line indicates g u pairs ( < 50% ) are also found at this position . the point mutations in the asl of full - length trna are diagrammed in figure 2h and were obtained by site - directed mutagenesis of the plasmid containing wild - type human trna [ phts , gift from h. gross ; ( 50 ) ] followed by in vitro transcription of linear templates ( see materials and methods section ) . time course analyses of the in vitro activity of the substrate rnas and hpus1p are presented in figure 2 ( panels a g ) , with the same wild - type trna curve in all panels ( figure 2a g ) . the formation of in this trna by hpus1p is reduced to background levels ( 0.02 mol /mol rna ) if the u at position 28 is changed to c and a42 is changed to g to preserve base pairing ( table 2 , second experiment ) . this shows that the only u converted to by hpus1p is the one at position 28 . the panels in figure 2 each contain the wild - type trna curve and either two or three curves generated with trna mutant substrates . this style allows for a simplification of the presentation and for the focus in most panels to be on 1 bp at a time . figure 2.time course experiments with single and double mutants in the asl of human trna . ( a g ) , h - labeled rnas were incubated with hpus1p for the times indicated and the amount of formed assayed . each panel has the same time course of wild - type ( wt ) trna values . each mutant is plotted separately and standard deviation ( sd ) bars are displayed for all time points . ( h ) diagram of asl point mutations made in the context of full - length trna that are presented in panels a ( a g ) , h - labeled rnas were incubated with hpus1p for the times indicated and the amount of formed assayed . each panel has the same time course of wild - type ( wt ) trna values . each mutant is plotted separately and standard deviation ( sd ) bars are displayed for all time points . ( h ) diagram of asl point mutations made in the context of full - length trna that are presented in panels a when the a at 27 is changed to c or g ( figure 2a ) , the levels of formed are not significantly different from wild - type trna . the curve that results when the u at 43 is changed to g is distinct from the wild - type trna curve but the difference is not statistically significant because of the relatively large error bars ( sd ) . mutation of the a at the position across from u28 to either c or g has little effect on time course of formed ( figure 2b ) . even though the a at position 42 is preferred ( figure 1b ) , the formation of 28 does not require canonical or even u g base pairing , something unanticipated given the consensus sequence ( figure 1b ) . when g29 is changed to c or c41 is changed to g ( figure 2c ) , there is a significant decrease in formation for both mutations in the formation of at 28 . however , when the base pair is reformed , but is now c - g in the case of the double mutant 29c/41 g , the curve obtained is indistinguishable from that of wild - type trna ( figure 2f ) . this introduction of a base - paired pyrimidine 3 to u28 did not enhance the formation of in the substrate , an expectation if a pyrimidine is preferred at position 29 , as suggested in the introduction . the consensus sequence shown in figure 1b revealed the g30c40 base pair to be highly conserved in the putative pus1p substrates with a at position 28 . it was expected that disruption of this base pair would affect the formation of by hpus1p . in fact , the 30c and 40 g mutants have significantly lower amounts of formed than wild - type trna ( figure 2d ) . if the base pair is reformed but inverted , as with the 30c/40 g double mutant , the time course of formed is the same as that of wild - type trna ( figure 2f ) . this result argues against the requirement for a g at position 30 , even though the 28 consensus suggests it ( figure 1b ) . when the base pair at a31u39 is disrupted by either the 31c or 39 g point mutations , the results are interesting . the curve of formation seen with the 31c substrate is significantly different from wild - type trna ( figure 2e ) , but with the 39 g mutant the curve mimics that seen with wild - type trna ( figure 2e ) . reforming the base pair with the 31c/39 g double mutant results in a curve that matches that of wild - type trna ( figure 2f ) . the only difference between the 28 consensus stem ( figure 1b ) and the stem of the trna ( figure 1a ) is the 2743 bp , which is a u in trna . the formation curves for the 27c , 27 g and 43 g mutants were not significantly different from wild - type trna ( figure 2a ) . this would suggest that the enzyme does not require a base pair at 2743 , and may in fact be indifferent to the presence of a base pair . that statement was supported by the formation curve seen with the 27g/43 g double mutant , which was not significantly different from the curve seen with wild - type trna ( figure 2 g ) , even though there is no canonical base pair formed . the results with the double mutant 27c/43 g , which would form a stronger base pair than the wild - type a u , are also not significantly different from wild - type trna ( figure 2 g ) even though this mutant now mimics the u28 consensus in figure 1b . equilibrium dissociation constants ( kd ) were determined for wild - type trna and the single- and double - mutant substrates found in the panels of figure 2 . the means and standard deviations of two separate kd determinations are presented in table 1 and that of wild - type trna ( 240 nm ) agrees with a previous determination of the kd ( 250 nm ) of hpus1p and yeast pre - trna ( 29 ) . all of the mutants have a higher mean kd than wild - type , with all but 41 g and 43 g > 300 nm . there is not a correlation between the formation levels and the kds for the mutants . it is interesting to note that a trna that is not a substrate for hpus1p , such as the 28c/42 g double mutant , binds to the enzyme with a mean kd of 332 nm . this was also as seen previously with yeast pus1p and a non - substrate trna ( 31 ) . table 1.equilibrium dissociation constants ( kd ) of hpus1p with various rnasrna substratekd mean ( sd)wild - type trna240 ( 14 ) nmsingle point mutants in trna 27c328 ( 46 ) nm 27g370 ( 14 ) nm 29c408 ( 11 ) nm 30c335 ( 120 ) nm 31c358 ( 53 ) nm 39g380 ( 28 ) nm 40g305 ( 49 ) nm 41g282 ( 18 ) nm 42c360 ( 57 ) nm 42g338 ( 46 ) nm 43g248 ( 32 ) nmdouble mutants in trna 27c/43g302 ( 39 ) nm 27g/43g375 ( 7 ) nm 28c/42g332 ( 18 ) nm 29c/41g328 ( 74 ) nm 30g/40c345 ( 21 ) nm 31c/39g315 ( 92 ) nmlisted are the means and standard deviations ( sd ) of two separate binding experiments of hpus1p and the substrates listed ( see materials and methods section for details ) . equilibrium dissociation constants ( kd ) of hpus1p with various rnas listed are the means and standard deviations ( sd ) of two separate binding experiments of hpus1p and the substrates listed ( see materials and methods section for details ) . in order to identify additional recognition elements of the hpus1p substrate , stem or stem - loop deletions of , and mini - substrates derived from trna , were tested in the formation assay . these mutant substrates were generated either by site - directed mutagenesis of phts and subsequent in vitro transcription or by in vitro transcription using oligodeoxynucleotide templates [ see materials and methods section ; ( 53 ) ] . it is understood that , with the deletion of whole stem - loops or the creation of mini - substrates , the tertiary structure of the entire trna is affected , but surprisingly , many of these mutants can function as substrates for hpus1p . the results for 120 min incubations are listed in table 2 relative to the activity seen with wild - type trna . if the d stem - loop is missing in the substrate , there is no decrease in hpus1p activity at position 28 . if the u at position 28 in the d stem - loop mutant is changed to c ( and the a at 42 is changed to g ) so that can not be formed at position 28 , there is essentially no formation ( 2% of wild - type trna formation , see table 2 ) . this result indicates the level of formation seen with the d stem - loop mutant is not due to formation at an additional site created by an altered secondary structure in the deletion mutant . if the tc stem - loop is missing from the trna , there is a significant reduction in the formation of at u28 ( 15% of wild - type trna levels , table 2 ) but the reduction is not as substantial when the v stem - loop is missing ( 67% of wild - type , table 2 ) . table 2.activity of hpus1p on trna(uga ) point and deletion mutants and mini - substratesrna substratemole /mole rna ( sd)percent of wild - type ( sd)wild - type human trna0.60 ( 0.08)100 ( 13) tc stem - loop ( deleted nucleotides 5973)0.09 ( 0.05)15 ( 8) variable stem - loop ( deleted nucleotides 4556)0.40 ( 0.08)67 ( 13)wild - type human trna0.43 ( 0.07)100 ( 16)28c/42 g ( no formation expected)0.02 ( 0)5 ( 0) d stem - loop ( deleted nucleotides 1124)0.44 ( 0.16)102 ( 37) d stem - loop with 28c/42g0.01 ( 0.01)2 ( 2)wild - type human trna0.68 ( 0.02)100 ( 3)asl stem loop alone ( includes nucleotides 2643)0.02 ( 0.01)3 ( 1)asl & d stem - loops ( includes nucleotides 1043)0.07 ( 0.01)10 ( 1)asl & v stem - loops ( includes nucleotides 2647)0.05 ( < 0.01)7 ( 1)asl , d , & v stem - loops ( includes nucleotides 1047)0.14 ( < 0.01)21 ( 1)asl , v , & tc stem - loops ( includes nucleotides 2665)0.48 ( 0.04)71 ( 6)asl , d , v , & tc stem - loops ( includes nucleotides 765)0.64 ( 0.16)94 ( 24)activities are from 2-h incubations , an average of two assays , with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type trna(uga ) with the standard deviation ( sd ) also reported as percent of wild - type . these results are grouped as separate experiments with a separate wild - type control for each experiment . activity of hpus1p on trna(uga ) point and deletion mutants and mini - substrates activities are from 2-h incubations , an average of two assays , with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type trna(uga ) with the standard deviation ( sd ) also reported as percent of wild - type . these results are grouped as separate experiments with a separate wild - type control for each experiment . when a mini - substrate of just the asl stem - loop was incubated with hpus1p , formation was at background levels for 2 h of incubation ( 3% of wild - type levels , table 2 ) . in addition , if either the d stem - loop or the v stem - loop was combined with the asl stem - loop , there was little increase in the amount of formed ( 10% and 7% , respectively , of the formation seen in 120 min with wild - type trna , table 2 ) . this implies that adding a single stem - loop to the 5 or 3 side of the asl stem - loop does not constitute an efficient substrate for hpus1p . if both the d and v stem - loops are added to the asl stem - loop there is a noticeable increase in formed but it is still low ( 21% ) compared to wild - type trna . when the tc stem - loop was combined with the v and asl stem - loops , there was a substantial level of hpus1p activity ( 71% of wild - type trna levels of ) . with just the acceptor stem missing from trna ( asl , d , v and tc stem - loops present ) , the activity is essentially the same as with wild - type trna ( 94% of wild - type formation at 120 min , table 2 ) . these results suggest that the secondary structure of the trna is very important for the modification at position 28 , and a minimal substrate could be created by simply connecting the asl and tc stem - loop as diagrammed ( figure 3a ) . the numbering on the diagram is consistent with full - length trna to allow comparisons with the results reported in figure 2 and table 2 . the level of formation at position 28 in this minimal substrate ( wild - type asl & tc mini - substrate ) was ~0.60 mol /mol rna after 2 h of incubation , a substantial level of modification ( table 3 ) . a mini - substrate incorporating a 28c/42 g double mutation was used in a separate experiment with little or no formation observed ( table 3 ) , providing evidence that in this mini - substrate , the u at position 28 is the only one being converted to , as expected . ( a ) predicted structure of the asl & tc stem loop mini - substrate , retaining the numbering found on the full - length trna and indicating the position of the formed . the structure for the y for y r for r stem mini - substrate mutant and the c28 mutant are also shown . ( b ) diagram of the possible base pairing in the asl stem mutants in the mini - substrate listed in table 3 . the activities in percent of the level observed with wild - type asl & tc stem - loop mini - substrate without the sd are listed above the diagrams . table 3.activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutantsrna substratemole /mole rna ( sd)mole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.60 ( 0.11)0.54 ( 0.16)100 ( 18 ) ( 30)28c/42 g ( no u at expected site of formation)0.01 ( 0.01)2 ( 2)27c/42c0.48 ( 0.04)80 ( 7)27c/39g0.24 ( 0.05)40 ( 8)27c/29c/39g0.10 ( 0.03)17 ( 5)27c/29c/42c0.04 ( 0.01)7 ( 2)27c/39g/40g<0.01 ( 0.01)2 ( 2)27c/29c/39g/40g0.02 ( 0.03)3 ( 5)27c/29c/39g/40g/42c0.02 ( < 0.01)3 ( 2)55 & 560.37 ( 0.10)62 ( 17)55580.17 ( 0.06)28 ( 10)gcaa inserted between 55 and 560.46 ( 0.04)77 ( 7)wild - type asl & tc stem - loop mini - substrate0.30 ( 0.04)100 ( 13)ggacg starting at 610.22 ( 0.03)73 ( 10)aagcuua in tc loop0.34 ( 0.04)113 ( 13)activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . ( a ) predicted structure of the asl & tc stem loop mini - substrate , retaining the numbering found on the full - length trna and indicating the position of the formed . the structure for the y for y r for r stem mini - substrate mutant and the c28 mutant are also shown . ( b ) diagram of the possible base pairing in the asl stem mutants in the mini - substrate listed in table 3 . the activities in percent of the level observed with wild - type asl & tc stem - loop mini - substrate without the sd are listed above the diagrams . activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutants activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . concentrating on the asl stem , a number of mutant substrates were synthesized that pick up where the previous mutations in the full length trna ( figure 2 and table 2 ) left off . the asl stem of trna has five watson - crick base pairs and the mini - substrate mutants listed in table 3 were designed to eliminate two or more of those base pairs in each mutant . the base pairs that may still form in each mutant are diagrammed in figure 3b and the levels of activity , as a percentage of the activity observed with the wild - type asl & tc mini - substrate , are given in table 3 and figure 3b . when there is no base - pairing in the stem there is little formation of , and the possibility of 1 or even 2 bp in the stem , does not substantially elevate the amount of formed . however , with the 27 c/42c mutant substrate , which has 3 bp in the stem below the site of modification , 80% of the level seen with the wild - type asl & tc mini - substrate was observed . the level of formation by hpus1p on the 27c/39 g mutant substrate was only 40% , even though it too has 3 bp in the stem . with respect to the tc stem - loop , if the nucleotides at 55 and 56 are deleted in this asl & tc mini - substrate , there is a decrease in formation relative to the wild - type asl & tc mini - substrate ( 62% of wild - type trna , table 3 ) . if the size of the tc loop is reduced further ( 5558 ) the resulting substrate is a poor one , just 28% of wild - type levels of were observed ( table 3 ) . if 4 nt ( gcaa ) are inserted into the tc loop to make it larger , the substrate still allows for a significant amount of to be formed ( 77% of wild - type asl & tc mini - substrate levels ) . in a separate experiment , if watson - crick base - pairing is disrupted in the tc stem , with the sequence ggacg starting at nucleotide 61 , the level of hpus1p activity was also substantial ( 73% or wild - type asl & tc mini - substrate levels ) , suggesting a stem loop is not required . if the sequence of the tc loop was replaced with its complement ( aagcuua ) , the amount of formed at position 28 was 113% of wild - type asl & tc mini - substrate levels , suggesting that any loop of at least 5 nt will suffice . to summarize the results from the mutation and deletion studies , the levels of formation with the single and double mutants in the context of the full - length trna suggest that the sequence of the asl stem is not an absolute determinant of hpus1p activity , except of course that there must be a u at position 28 to modify ( tables 2 and 3 ) . the preference for a pyrimidine 3 to the site of modification ( at position 29 ; see introduction section ) was not confirmed since even though the 29 c single mutant showed a decreased amount of formed and the double mutant 29 c/41 g exhibited an almost identical curve as wild - type trna , suggesting that a pyrimidine is not preferred , only the base pair . the point mutations in full - length trna show that the base pairing below the modified position ( u28 ) affects formation , whereas the 27 a the results with the asl & tc stem loop mini - substrate mutants also showed that the base pairs below u28 were the most critical . the sequence of the stem nucleotides is of less importance than the presence of base pairs , which agrees with the fact that known substrates of pus1p exhibit no absolute sequence requirement ( 26,27,3335 ) . in terms of the requirements for a particular secondary structure of trna , the d - stem loop and acceptor stem are dispensable for formation at u28 . in fact , a single stem loop , 3 to the asl , is all that is required for substantial formation at position 28 . the sequence , and possibly the structure of this additional rna , is not constrained . given the above summary of the mutation experiments , a mini - substrate where all of the nucleotides in the asl stem have been changed , except of course for the u at position 28 which is modified , should be a substrate for hpus1p . a substrate fitting this description ( figure 3a ) , along with another where the u at position 28 was changed to c , were incubated with hpus1p and assayed for activity . the y for y r for r stem mini - substrate mutant has a replaced by g , g replaced by a , c replaced by u , and u replaced by c when compared with the wild - type asl & tc mini - substrate , except at position 28 . the 28 c mutant has complete replacement of the stem sequence , and should not be modified by hpus1p . the results with these substrates , as well as with the wild - type asl & tc mini - substrate , are presented in table 4 and show that this mutant is modified by hpus1p ( 0.33 mol /mol rna ) at 80% of wild - type levels for this experiment . in addition , the 28c mutant is not modified ( 0.01 mol /mol rna ; 2% of wild - type levels ) , proving that only the u at position 28 is modified . so , even though all the asl stem nucleotides in the mutant are different from the wild - type trna asl stem , and there is a u28g42 base pair in the mutant , the u at position 28 is converted to at near wild - type levels . table 4.activity of hpus1p with wild - type asl & tc stem - loop mini - substrate and stem sequence mini - substrate mutantsrna substratemole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.41 ( 0.05)100 ( 12)y for y r for r stem mini - substrate mutant 27g/29a/30a/31g/39c/40u/41u/42g/43c0.33 ( 0.02)80 ( 5)y for y r for r stem mini - substrate 28c mutant 27g/28c/29a/30a/31g/39c/40u/41u/42g/43c0.01 ( 0.01)2 ( 2)activities are from 2-h incubations , an average of three separate assays , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . activity of hpus1p with wild - type asl & tc stem - loop mini - substrate and stem sequence mini - substrate mutants activities are from 2-h incubations , an average of three separate assays , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type . all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate . a number of studies have determined the sites in trnas that are modified by pus1p ( 26,27,29,33,34 ) . yeast and mammalian pus1p are the most extensively studied and in vivo and in vitro , the enzymes are responsible for the formation of at positions 27 and 28 in many trnas , at 34 and 36 in certain intron containing trnas , and at position 1 in a few substrates ( 29,33,34 ) . the yeast enzyme can also modify uridine at positions 26 , 65 and 67 in vivo but not in vitro ( 34 ) . when the mouse enzyme was expressed in a pus1 yeast strain , position 26 was not modified , and the modification states of positions 65 and 67 were not determined ( 33 ) . sequenced human trnas do not have at positions 26 , 65 , or 67 ( 58 ) , so it is unlikely mpus1p or hpus1p exhibit activity at these sites in vivo . the mammalian enzymes do modify position 30 in a few trnas but the yeast enzyme lacks this activity ( 29,33 ) . for the purposes of the discussion of the current results , the modification at positions 27 and 28 will be considered , since these are formed in vitro in a number of substrates by the mammalian enzymes ( 27,33,35 ) . nearly all ( 17 out of 18 ) of the sequenced yeast and human trnas that have a u at position 27 have converted it to ( 58 ) . however , in the six sequenced yeast trnas that have a u at position 28 , the three that have a 27c43 g base pair lack at position 28 , whereas , the other three have a 2743a base pair and a at position 28 ( 58 ) . this completely agrees with the two consensus sequences shown in figure 1b . in the sequenced human trnas with a u at 28 ( eight total ) three of the trnas have a 2743a base pair , two have a at position 28 , but one does not . a human trna with an 27a43u base pair does have a at position 28 ( 58 ) , suggesting that an u a or a u base pair at 2743 allows for formation at 28 , whereas a 27c43 g base pair is not conducive to formation at position 28 . however , with the four remaining trnas , which have a 27c43 g base pair , two have a at position 28 and two do not ( 58 ) . the current finding that the loss of base pairing at , or the presence of , a c g base pair at 2743 does not significantly affect the formation of at position 28 in trna , correlates with the data from human trnas . human mitochondrial trna is known to be a substrate of hpus1p in vivo ( 41 ) but the sequence of the stem only matches the 28 consensus at the 2743 and 2842 bp . finally , the y for y r for r stem mini - substrate mutant is an efficient substrate for hpus1p in vitro , even though the sequence in the stem does not match the consensus at any position except 28 ( figure 3a and table 4 ) . there must be other determinants allowing for or suppressing formation in these other trnas with a u at 28 . the uridine to be modified does not have to participate in watson - crick base - pairing but nucleotides participating in base - pairing 3 to the modified position appear to be required to constitute an efficient substrate . these results correlate well with the observation that , in vivo , yeast pus1p formed at position 26 in yeast trna(cca ) , where the uridine is in the hinge between the d stem - loop and the asl , and is followed by 5 bp in the asl stem ( 33 ) . in addition , both yeast and mouse pus1p modify the uridine in position 1 on yeast trna(acg ) , with base pairing present in the acceptor stem 3 to the position of modification ( 33 ) . and finally , yeast and mammalian pus1p modifies several uridines in the extensively base - paired asl of yeast pre - trna(uau ) in vitro and in vivo ( 27,29,33 ) . the lack of a strict substrate sequence requirement for hpus1p can be contrasted with the rigid recognition motif of yeast pseudouridine synthase 7 ( pus7p ) , another synthase that can recognize more than one site in trna and other types of rna ( 44 ) . with pus7p there is an absolute requirement for a u at 2 and an a at + 1 relative to the uridine modified ( 44 ) . of the sequenced yeast and human trnas , all that have the proscribed pus7p recognition sequence ( 17 total ) the other site - specific synthases modify only one site in trnas ( pseudouridine synthases 4/10 , 6 , 8/9 ) or one small region in many trnas ( pus3p ) ( 48,49,5962 ) . with these latter synthases the local trna structure appears to be a determining factor for substrate recognition when one considers the results of trua ( pus3p homolog ) and trub ( pseudouridine synthase 4 homolog ) structural studies ( 46,47,63,64 ) . given the requirement that the uridine to be modified be in a base - paired stem , why is the uridine at position 44 in yeast u2 snrna modified by pus1p ( 33 ) when that position is not considered to be in a region that is base paired ? yeast pus1p can modify u2 snrna in vitro and in vivo , but mpus1p only modifies u44 on u2 snrna in vivo , in a pus1 strain ( 33,36 ) . an alternative structure , perhaps in the ribonucleoprotein particle where u44 is base - paired , may form in vivo . it is also possible that another rna , such as a snorna , base pairs in the region of u44 and forms a stem that can be recognized by yeast or mouse pus1p . it is interesting to note that formation in u2 snrna in c. elegans is not dependent on pus1p activity ( 28 ) . the results with mutations in the tc stem - loop suggest that there is a minimum size requirement for this loop but the actual sequence , or base pairing in the stem , are not critical . a minimum size requirement may be a reason that the v stem - loop of trna(uga ) does not substitute for the tc stem - loop when combined with the asl . the v stem - loop in trna(uga ) is predicted to have only 3 nt in the loop . the fact that the tc stem - loop is separated from the asl by 4 nt in the mini - substrate and the v stem - loop has no separation , may also be a factor in why the tc stem - loop can participate in creating a mini - substrate that is recognized by hpus1p . the only synthase in the trua family that has been crystallized is bacterial trua ( see above ) and the structures were obtained in the presence of substrate trnas ( 46,47 ) . trua is a dimer and makes contact with the asl ( positions 3840 are modified if a u is present at those sites ) , the d stem , and the elbow where the d and tc loops interact . the v loop or stem - loop and the acceptor stem did not interact with trua ( 47 ) . in this report the d stem - loop of trna(uga ) was found to be dispensable for hpus1p activity , as was the acceptor stem , but the absence of the v stem - loop from the trna does reduce the amount of formed . in this trna substrate , the tc stem - loop appears to be the more critical component for recognition by hpus1p . therefore , even though there is considerable homology between trua and hpus1p , the substrate contacts with the protein are probably different . yeast pus1p has been shown to be a monomer when bound to trna ( 30 ) and it is reasonable to assume the substrate / enzyme interactions will be fundamentally different between trua and pus1p . crystal structures for any of the pus1p proteins in the presence of trna substrates will go a long way towards reconciling these differences . with what appear to be relatively lax requirements for substrate recognition in trna , what prevents other uridines , such as those at 39 , 43 , 44 , 63 , or 70 in trna ( figure 1a ) from being modified by hpus1p in vitro ? modifications in the asl ( 26 , 27 , 28 , 30 ) of all natural substrates of pus1p are only found on the 5 side of the stem . this specificity is most likely due to the presentation of this side of the asl to the enzyme 's active site cleft , in much the same way the opposite side of the stem is presented to trua for modification of positions 3840 ( 47 ) . this flipping of the orientation of the substrate in the active site cleft could explain why the tc stem - loop is a critical part of the recognition motif for hpus1p rather than the d stem - loop . these other sites that have uridines in a stem are not recognized because they are not presented in a conformation compatible with placing the uridine in the active site . additional natural and mini - substrates will need to be studied to refine the parameters of hpus1p substrate recognition to construct a recognition site that can be used to narrow down the possible modification sites in sra , a relatively large , non - trna substrate of pus1p , where most of the actual residues have not been identified ( 37,38 ) , or to identify additional substrates . the known substrates of pus1p may just be the tip of the iceberg of actual rna substrates for this enigmatic enzyme . national institutes of health ( grant number dk074368 - 01 ) ; south carolina honors college at the university of south carolina ( undergraduate research fellowship and thesis preparation grants to b.s.s . ) .

mccune albright syndrome ( mas ) comprises a triad of fibrous dysplasia of bone , caf - au - lait macule , and endocrinopathy . the disease is due to activating mutation of g protein - coupled receptor leading to hyperfunction of glands . hansen 's disease is caused by infection with mycobacterium leprae and is seen with underlying immunosuppressed conditions in genetically predisposed individuals . we recently encountered a patient with hansen 's disease along with underlying mas and report the same in this report . pseudouridine synthase 1 ( pus1p ) is an unusual site - specific modification enzyme in that it can modify a number of positions in trnas and can recognize several other types of rna . no consensus recognition sequence or structure has been identified for pus1p . human pus1p was used to determine which structural or sequence elements of human trnaser are necessary for pseudouridine ( ) formation at position 28 in the anticodon stem - loop ( asl ) . some point mutations in the asl stem of trnaser had significant effects on the levels of modification and compensatory mutation , to reform the base pair , restored a wild - type level of formation . deletion analysis showed that the trnaser tc stem - loop was a determinant for modification in the asl . a mini - substrate composed of the asl and tc stem - loop exhibited significant formation at position 28 and a number of mutants were tested . substantial base pairing in the asl stem ( 3 out of 5 bp ) is required , but the sequence of the tc loop is not required for modification . when all nucleotides in the asl stem other than u28 were changed in a single mutant , but base pairing was retained , a near wild - type level of modification was observed .

midfacial defects are classified as midline midfacial defects , that include the nose and/or upper lip ; and lateral defects , which include the orbital contents and cheek . facial prosthesis is generally considered over surgical reconstruction to restore function and appearance in patients with facial defects that resulted from cancer resection.1,2 the health of the patient might not permit to undergo another surgery . an intraoral prosthesis such as an obturator is often needed to restore mastication , speech and improves patient appearance.3 fabrication of an extra oral facial prosthesis is a challenging task as it tests the creative ability of the prosthodontist . the choice of material for extraoral prosthesis depends on the need to engage desirable undercuts , mobility of tissue bed , defect size and the weight of the prosthesis . it may be retained by means of medical grade adhesives , resilient attachments , clips and osseointegrated implants . it can also be connected to obturator by magnets.4 this clinical report highlights the rehabilitation of a lateral midfacial defect with a two piece prosthesis that included an intraoral obturator and extra oral facial prosthesis with the use of magnets . a 55-year - old man reported to the department of maxillofacial prosthodontics for a definitive prosthetic rehabilitation with a surgical defect subsequent to radical surgery involving subtotal maxillectomy of left side performed six months before ( figs . radiological examinations revealed loss of floor of left orbit and left half of the maxilla along with the teeth . speech intelligibility and deglutition were severely affected since the patient 's tongue could not make effective functional contacts due to lack of anatomic boundaries during speech and deglutition . fabrication of such prosthesis was planned in two sections to close the intraoral defect and create a partition between the oral cavity and nasal cavity for facilitating speech and deglutition . firstly the extra oral prosthesis was fabricated , followed by fabrication of intraoral obturator prosthesis . a facial moulage impression was made with irreversible hydrocolloid ( algitex , dpi , mumbai , india ) to record the facial defect along with surrounding normal structures ( fig . 2 ) . opened gauze squares were placed on the alginate for mechanical retention for the rigid plaster backing ( 0.25 inch ) , which is necessary for removal of the impression without distortion . when the plaster sets the impression is removed and a definitive cast was poured with type ii gypsum ( kalstone ; kalabhai karson , mumbai , india ) . the facial prosthesis was sculpted with base plate wax and a magnet ( cobalt - samarium , ambica corporation , new delhi , india ) was attached to it on the defect side . the wax pattern with the magnet was evaluated by positioning it on the patient 's face . investing of the wax pattern was done in type ii gypsum to form a mold for packing the silicone . after complete dewaxing , a silicone adhesive ( 2-butanone , voco , germany ) was applied on the magnet . the prosthesis was packed with silicone ( maxillofacial silicone system , factor ii , lakeside , az , usa ) and colored using intrinsic stains and flocks ( silicone coloring kit ; factor ii , lakeside , az , usa ) selected to match the patient 's skin color . after deflasking and finishing , extrinsic stains were applied and the prosthesis was fitted with medical grade adhesive to flush the borders of the prosthesis with the skin ( fig . a preliminary impression of remaining maxillary arch along with the palatal defect was made in irreversible hydrocolloid impression ( algitex , dpi , mumbai , india ) material and a diagnostic cast was retrieved out of it . after adapting a layer of wax spacer an auto polymerizing resin tray was fabricated . definitive impression was made using a medium viscosity poly ( vinyl siloxane ) impression material ( reprosil ; dentsply , konstanz , germany ) and poured in type iii gypsum to get the master cast . a sheet of baseplate wax was adapted and contoured over the framework and jaw relations were recorded . after try - in the waxed - up obturator was made hollow and processed in heat - polymerizing acrylic resin . hollowing of the bulb was done by lost salt technique while the part under the teeth was hollowed with putty viscosity poly ( vinyl siloxane ) impression material ( express xt vps impression materials , 3 m , new delhi , india ) . after processing an aperture was made on the mesial aspect of the bulb and posterior to molar tooth to remove the salt and putty , thus making the obturator hollow ( figs . 4a and 4b ) . cobalt samarium magnet was attached on the supero - lateral aspect of the bulb with autopolymerizing resin so that it can contact the magnet on the extraoral prosthesis for mutual retention ( fig . movement of the prosthesis was minimized by maximal distribution of the occlusal force in centric and eccentric jaw positions . premature occlusal contacts were eliminated and wide distribution of stabilizing components resulted in diminished stress created by horizontal forces . the patient experienced marked improvement in speech intelligibility and deglutition after the placement of the intraoral obturator ( figs . 6 and 7 ) . during the prosthesis insertion appointment the maxillary obturator was placed intraorally and the facial prosthesis was positioned extraorally against the obturator magnet . a facial moulage impression was made with irreversible hydrocolloid ( algitex , dpi , mumbai , india ) to record the facial defect along with surrounding normal structures ( fig . 2 ) . opened gauze squares were placed on the alginate for mechanical retention for the rigid plaster backing ( 0.25 inch ) , which is necessary for removal of the impression without distortion . when the plaster sets the impression is removed and a definitive cast was poured with type ii gypsum ( kalstone ; kalabhai karson , mumbai , india ) . the facial prosthesis was sculpted with base plate wax and a magnet ( cobalt - samarium , ambica corporation , new delhi , india ) was attached to it on the defect side . the wax pattern with the magnet was evaluated by positioning it on the patient 's face . investing of the wax pattern was done in type ii gypsum to form a mold for packing the silicone . after complete dewaxing , a silicone adhesive ( 2-butanone , voco , germany ) was applied on the magnet . the prosthesis was packed with silicone ( maxillofacial silicone system , factor ii , lakeside , az , usa ) and colored using intrinsic stains and flocks ( silicone coloring kit ; factor ii , lakeside , az , usa ) selected to match the patient 's skin color . after deflasking and finishing , extrinsic stains were applied and the prosthesis was fitted with medical grade adhesive to flush the borders of the prosthesis with the skin ( fig . a preliminary impression of remaining maxillary arch along with the palatal defect was made in irreversible hydrocolloid impression ( algitex , dpi , mumbai , india ) material and a diagnostic cast was retrieved out of it . after adapting a layer of wax spacer an auto polymerizing resin tray was fabricated . definitive impression was made using a medium viscosity poly ( vinyl siloxane ) impression material ( reprosil ; dentsply , konstanz , germany ) and poured in type iii gypsum to get the master cast . a sheet of baseplate wax was adapted and contoured over the framework and jaw relations were recorded . after try - in the waxed - up obturator was made hollow and processed in heat - polymerizing acrylic resin . hollowing of the bulb was done by lost salt technique while the part under the teeth was hollowed with putty viscosity poly ( vinyl siloxane ) impression material ( express xt vps impression materials , 3 m , new delhi , india ) . after processing an aperture was made on the mesial aspect of the bulb and posterior to molar tooth to remove the salt and putty , thus making the obturator hollow ( figs . 4a and 4b ) . cobalt samarium magnet was attached on the supero - lateral aspect of the bulb with autopolymerizing resin so that it can contact the magnet on the extraoral prosthesis for mutual retention ( fig . 5 ) . movement of the prosthesis was minimized by maximal distribution of the occlusal force in centric and eccentric jaw positions . premature occlusal contacts were eliminated and wide distribution of stabilizing components resulted in diminished stress created by horizontal forces . the patient experienced marked improvement in speech intelligibility and deglutition after the placement of the intraoral obturator ( figs . 6 and 7 ) . during the prosthesis insertion appointment the maxillary obturator was placed intraorally and the facial prosthesis was positioned extraorally against the obturator magnet . it causes difficulty in deglutition , nasal regurgitation , loss of speech intelligibility and unesthetic appearance , leading to significant psychological problems . with acceptable cosmetic results , a reduced amount of retention can handicap the prosthesis . unlike a conventional prosthesis , additional factors should be taken into consideration such as method of impression , materials to be used in laboratory procedures , prosthesis design , method of connection , direction of insertion and removal , esthetic factors , and maintenance protocol . with the understanding of the remaining anatomic structures , intraoral and extraoral prostheses that mutually retain one various methods of retention for facial prostheses have been described in the literature ; they include eyeglasses , eye patches , extensions from the denture that engage desirable tissue undercuts , medical grade adhesives , magnets , and osseointegrated implants.5 - 7 the introduction of rare - earth permanent magnets made of alloys such as neodymium - ferro - boron and samarium cobalt has resulted in magnets of very small dimensions . tarnish and corrosion can be overcome by coating these magnets with nickel , gold and titanium.8 although osseointegrated implants may provide the most reliable prosthesis retention but additional surgeries , expenses , inadequate available bone , and prior radiation to the area may contraindicate this type of treatment.9,10 mutual retention of the intra- and extra- oral prosthesis can be successfully achieved through magnets . the force needed to separate magnets was designated as breakaway load expressed in terms of grams . the value of breakaway load is inversely proportional to the distance of separation and inclination between the magnets . these magnets are wholly biocompatible in the human body and exert no deleterious effects in human tissue . inert magnetic field is harmless to human body and can be safely used for patients with cardiac pacemaker . there may be a gradual decrease in retentive force upon usage while some open field uncoated magnet exhibits significant cytotoxic effects which may be attributed to the release of corrosion by products from them . the cast - metal framework improves retention , stability , support and bracing of the prosthesis and thus increases prosthesis longevity . problem of two piece prosthesis is that the mobility of intraoral obturator can make facial prosthesis mobile especially during functions but the light weight of the prosthesis can alleviate this problem.11 many methods and materials have been used to fabricate a hollow obturator such as salt , sugar and ice . a lightweight prosthesis which was fabricated in this case by lost salt technique and putty not only counteracted forces due to gravity but also enhanced speech intelligibility.12,13 the rehabilitation of a patient with a combined intraoral - extra oral defect has been presented . a two - piece prosthesis that included an intraoral obturator and extra oral facial prosthesis was fabricated . the incidence of well - differentiated thyroid carcinoma , particularly papillary cancer , has been increasing since the last 2030 years . the surveillance , epidemiology and end results ( seer ) database shows more than a 2-fold increase in thyroid cancer since 1995 . an important contributing factor for the increased incidence of such well - differentiated cancers is the increasing diagnostic rates of papillary thyroid microcarcinoma ( ptm ) . other factors such as iodination programmes in low iodine intake areas , detailed histopathological examination of the excised thyroid tissue , and the increase in bilateral total excision of the thyroid gland during thyroid surgery have also been attributed to the increasing rates of large ( > 10 mm ) and micropapillary carcinoma [ 25 ] . a vast majority of ptm cases are incidentally determined on postoperative histopathological examination of the excised thyroid tissue for the surgical treatment of benign thyroid disorders . incidental ptm ( i - ptm ) is a tumour focus that is clinically unsuspected before thyroid surgery and is identified in the final pathological examination of a thyroidectomy specimen . therefore , several controversies regarding the need for completion surgery for excision of the remaining thyroid tissue and lymph nodes exist . the objectives of this study were to describe the incidence and clinical / pathological characteristics of ptm and discuss our experience with i - ptm cases in an endemic goitre area . the study involved 395 surgical patients with benign disease of the thyroid , without any diagnosis of preoperative malignancy . a total of 37 patients with ptm incidentally diagnosed on postoperative histopathological examination of the excised thyroid tissue were analysed for assessing the rate of incidental diagnosis of ptm and their demographic features and the surgical procedures used for their treatment . histopathological parameters were established by microscopic examination , including the size of ptm , location in the thyroid gland , multifocality and bilateralism in the thyroid lobes , thyroid capsule invasion , presence of lymphovascular invasion ( lvi ) , lymph node metastasis , and tumour recurrence . as an adjuvant treatment , we analysed completion thyroidectomy for i - ptm cases with unilateral hemithyroidectomy , l - thyroxin ( lt4 ) treatment for the suppression of thyroid stimulating hormone ( tsh ) , and radioiodine ( rai ) treatment . patients with i - ptm were followed up for 12 years , mean 102 ( 61144 ) months . biochemical analyses for serum tsh and free thyroxin ( ft4 ) were performed in order to determine the suppressive dose of lt4 ( suppression of tsh at a level of < 0.25 uiu / ml ) . biochemical analyses for serum tsh , ft4 , thyroglobulin ( tg ) , and anti - thyroglobulin antibody ( anti - tg ab ) were performed in total thyroidectomy cases . an ultrasound of the cervical lymph nodes in all patients and the remaining lobes in patients with hemithyroidectomy was also performed . an ultrasound of the cervical lymph nodes in all patients and the remaining lobes in patients with hemithyroidectomy was repeated . biochemical analyses for serum tsh , ft4 , tg , and anti - tg ab were performed . locoregional or distal recurrence of thyroid malignancy in the follow - up period and disease - free or overall survival of patients with i - ptm were the primary outcome parameters . during the study period , total thyroidectomy and right and left hemithyroidectomy were performed in 249 , 75 , and 71 patients , respectively , for the treatment of benign surgical diseases of the thyroid gland among the total 395 patients . i - ptm was diagnosed on histopathological examination in 37 ( 9.4% ) of the 395 patients . moreover , 47% of our i - ptm was diagnosed in 10.8% and 5% of female and male patients , respectively ( table 1 ) . the mean age of the 37 patients with i - ptm was 44.1 ( 1671 ) years . more than half of the patients ( 56.8% ) were aged between 41 and 60 years ( table 2 ) . the prevalence rates of i - ptm were 11.6% ( 29/249 ) in the total thyroidectomy specimens and 5.5% ( 8/146 ) in the unilateral hemithyroidectomy specimens ( table 3 ) . the disease was unifocal in 30 and multifocal in 7 ( 18% ) patients ( 6 cases of two and 1 case of three foci ) . of the 45 foci , 25 ( 55.6% ) were located in the left lobe ( table 4 ) . bilateral location of the ptm foci was observed in 6 ( 20.1% ) of the 29 total thyroidectomy cases ( table 3 ) . the mean size of the ptm foci was 4.88 ( 110 ) mm , and 42.2% , 64.4% , and 71% of the foci were 3 mm , 5 mm , and 6 mm , respectively ( tables 5(a ) and 5(b ) ) . microscopic examination revealed thyroid capsule invasion in 2 ( 5.4% ) of the 37 patients and in 2 ( 4.4% ) of the 45 foci . these two foci measured 3 and 6 mm in two female patients aged 41 and 57 years , respectively . based on the clinical and ultrasound findings of the cervical lymph nodes and thyroid gland , we did not perform completion thyroidectomy in eight ptm cases with unilateral hemithyroidectomy . all patients received a suppressive dose of lt4 to achieve a serum tsh level of < 0.25 uiu / ml . during the follow - up period of 102 ( 61144 ) months , we did not find any negative changes in blood chemistry and ultrasound imaging . we also did not find any unfavourable locoregional or distant events as recurrence in the remaining thyroid lobes , metastasis in the cervical nodes , and distant metastasis . papillary microcarcinoma is accordingly found more frequently and often incidentally upon histopathological examination of surgical specimens from presumed benign thyroid disease . our study region was formerly a low iodine intake ( endemic goitre ) area where preventive iodine supplementation programme has been ongoing since the last 30 years . the significant increase in the diagnostic rates of ptm , particularly the incidental findings after thyroid surgery , raises an important issue . we observed the diagnostic rate of i - ptm to be 78.7% among all the ptm cases , which corroborates our previous results . in our endemic region , the increased frequency of ptm and higher diagnostic rates of i - ptm indicate the occurrence of papillary cancer after iodine supplementation . the presence of incidental thyroid cancer is an important issue in patients with benign thyroid diseases who underwent thyroid surgery . postoperatively , we discovered that the thyroid gland harboured ptm in approximately 1 ( 9.4% ) out 10 of our patients with benign thyroid disease . previous studies have also reported the prevalence of i - ptm to be between 7.1% and 16.3% [ 610 ] . based on the present study and previous results of prevalence , we suggest that the presence of incidental cancer is not an uncommon situation . during our study period in the endemic area , the proportion of ptm was 47% among all papillary thyroid cancer cases , and a vast majority ( 78.7% ) of them showed incidental diagnosis , which indicates the importance of this pathology in the increasing rates of differentiated thyroid cancers . lombardi et al . reported the proportion of i - ptm to be 42% , of which 75.5% were incidental in an area with a high prevalence of goitre . have also reported an increase in the proportion of ptm from 35% to 56% , of which 60% were incidental . in another study , the proportion of ptm in papillary thyroid cancer cases was determined to be 49% , of which 58% were incidental . the proportion of incidental cases among all ptm cases was found to be between 49% and 75.5% [ 8 , 1115 ] . recently reported that age - standardised rates increased from 0.35 per 100,000 per year in 1996 to 0.74 per 100,000 per year in 2008 . about 59% of ptm cases were identified incidentally , and a significant rise in incidence was found only for the incidental cases . the increasing number of total thyroidectomies appears to be an important factor for the higher rate of incidental ptm . in our study , the incidence ( 11.6% ) of i - ptm among total thyroidectomy cases was more than twice the incidence ( 5.5% ) after hemithyroidectomy . rosa pelizzo et al . reported an increase in the proportion of total thyroidectomies ( from 67% to 78% ) and ptm ( from 35% to 56% ) . our incidence rate of 18% for the presence of two or more foci in the gland and the rate of 20% for the location of tumour foci in both lobes confirmed the pathological features of incidental ptm . previous studies have also reported the multifocality rate of i - ptm to be between 13% and 44.1% [ 79 , 1519 ] . some studies have reported multifocality as an independent risk factor for tumour recurrence [ 6 , 8 , 11 , 14 , 18 , 20 , 21 ] . on the other hand , the rate of recurrence is very low in patients with incidental ptm , which has been reported to range from 0% to 5% [ 6 , 8 , 11 , 14 , 15 , 18 , 19 , 22 ] . therefore , i - ptm cases with multifocality should be followed up more closely , despite the low risk of recurrence . another pathological feature arising from our study was the rate of bilateralism ( 20% ) . multiple foci were located in both lateral lobes in 20%27.3% of patients with i - ptm [ 7 , 9 ] . based on the results of both the present and previous studies , the higher rate of bilateral tumour foci in both lobes draws our attention to the remaining lobes in patients with hemithyroidectomy . in such cases , sensitive imaging methods , such as ultrasound , should be used for the detection of nodules in the remaining lobes at both early postoperative and follow - up periods . in general , the average size of the tumour foci was < 5 mm in patients with i - ptm [ 9 , 15 , 16 , 18 ] . our results confirmed the relatively small size ( average 4.88 mm ) of the tumour foci in the thyroid glands of patients with incidental ptm , of which 64% and 71% of foci were 5 mm and 6 mm , respectively . john et al . reported that the tumour foci were 6 mm in 83% of such patients . previous studies have reported tumour size as a risk factor for lymph node metastasis and recurrence . in general , tumour foci diameter > 5 or 6 mm has appeared as an independent risk factor [ 6 , 20 , 21 , 23 , 24 ] . therefore , patients with foci > 6 mm in diameter should be followed up more closely . the recurrence rate of ptm is very low even among tumour foci > 6 mm , which has been reported to range from 0% to 5% [ 5 , 6 , 8 , 14 , 2527 ] . the presence of lvi is a pathological feature showing the metastatic ability of malignancy . in our study , the absence of lvi in all our cases indicates the decreased ability of local and systemic spread of ptm . previous studies have defined thyroid capsule invasion as extrathyroid or extracapsular spread , growth , extension , or invasion . capsular invasion by ptm is an independent risk factor for tumour recurrence in the follow - up period [ 2 , 6 , 11 , 12 , 14 , 2023 , 28 ] . based on the rate of thyroid capsule invasion ( 5.4% ) in the present study , we suggest that ptm with subcapsular localisation uncommonly invades the capsule of the thyroid gland . this rate has been reported to be 3.9% by john et al . and 7.4% by sakorafas et al . . locoregional and distant spread and recurrence of malignancy are the most important factors during the follow - up period . in our study , the absence of recurrence , lymph node , or distant metastasis indicates very low risk of tumour recurrence in patients with incidental ptm . the recurrence rate is 0% in some of the previous studies [ 12 , 14 , 19 , 26 ] . in general , the overall recurrence rate was < 2% in many case series [ 2 , 5 , 15 , 22 , 27 ] . other studies have also reported a low recurrence rate between 2% and 5% [ 6 , 8 , 11 , 13 , 18 , 25 ] . i - ptm is found in the thyroid tissue after surgical treatment of benign thyroid disease . therefore , total thyroidectomy and unilateral hemithyroidectomy are usually performed in these cases . in this situation , completion thyroidectomy for hemithyroidectomy cases , rai treatment , and suppression of tsh levels with lt4 treatment are the primary adjuvant procedures for patients with differentiated thyroid cancer . however , a very low rate of recurrence in a large series of i - ptm cases does not indicate the use of adjuvant treatment , except the suppression of tsh levels with lt4 treatment . after total excision of the gland , thyroidectomized patients receive l - thyroxin ( lt4 ) replacement in order to maintain normal body function and tsh level in normal range . on the other hand , well - differentiated malignant cells of papillary cancer have thyrotropin receptors in which the growth of differentiated malignant cells is controlled by tsh . therefore , inhibition of tsh secretion has beneficial effects on management of tsh - dependent tumour growth of well - differentiated malignancies that this secretion is inhibited by lt4 administration . inhibition of tsh secretion should be provided by suppressive dose of lt4 in all patients with papillary cancer . the suppressive dose of lt4 is adjusted in individual basis by serum tsh level which is maintained between 0.1 and 0.25 uiu / ml according to patients ' tolerability . in older patients , the dose of lt4 and suppression of tsh should be balanced with the cardiac risk . after yearly follow - up the dose of lt4 can be decreased to maintain serum tsh levels between 0.5 and 1 uiu / ml in patients without unfavourable findings . in our study , surgical and nuclear adjuvant treatment was not attempted in any patients after a careful evaluation with imaging modalities and blood analysis . in our series of i - ptm cases , the absence of locoregional and distant recurrence during the follow - up period of 102 months confirmed the adequacy of the surgical treatment . similarly , several previous studies have also reported no tumour recurrences after the surgical treatment of i - ptm [ 12 , 14 , 19 , 26 ] . based on our study and previous studies , we suggest that routine completion thyroidectomy after unilateral excision , lymph node dissection , and rai application are unnecessary in most i - ptm cases . in our endemic goitre area , diagnosis of i - ptm in thyroid tissue is not an uncommon situation after thyroid surgery for benign diseases . the prevalence of i - ptm increases parallel to the increase of total thyroidectomy rate for benign thyroid diseases . size of i - ptm foci in thyroid tissue is relatively small and the majority are smaller than 6 mm . small foci of ptm create very low risk of lymph node metastasis and locoregional or distant recurrences in the follow - up period . routine adjuvant surgical and nuclear treatment as completion thyroidectomy , lymph node dissection , and rai application is unnecessary in vast majority of patients due to low risk of recurrence . such adjuvant procedures should be reserved for small number of recurrent cases discovered in the follow - up period .

facial prosthesis is generally considered over surgical reconstruction to restore function and appearance in patients with facial defects that resulted from cancer resection . retention of the prosthesis is challenging due to its size and weight . retention can be achieved by using medical grade adhesives , resilient attachments , clips and osseointegrated implants . it can also be connected to obturator by magnets . this clinical report highlights the rehabilitation of a lateral midfacial defect with a two piece prosthesis that included an extra oral facial prosthesis and an intraoral obturator with the use of magnets . clinical and pathological characteristics of incidental papillary thyroid microcancer cases , surgical , medical , and nuclear treatment methods , and patients ' outcome were studied during follow - up period of 102 months . we studied 37 patients with incidental papillary thyroid microcancer ( i - ptm ) . the surgical procedure was total thyroidectomy in 29 and hemithyroidectomy in 8 patients . size , multifocality , and bilateralism of ptm foci , thyroid capsule invasion , and presence of lymphovascular invasion were histopathological parameters . we analysed adjuvant medical and nuclear treatment and patients ' outcome during follow - up period of 102 ( 61144 ) months . the prevalence rates of i - ptm were 9.4% in 395 thyroidectomy cases . histopathological examination reported unifocal disease in 30 and multifocal disease in 7 ( 18% ) patients . multifocal disease was bilateral in 6 ( 20.1% ) patients . the mean size of the ptm foci was 4.88 mm . the rate of thyroid capsule invasion was 5.4% . all patients received a suppressive dose of lt4 to achieve a low serum tsh level . adjuvant surgical and nuclear treatment was not performed in our cases . we did not find any negative changes in blood chemistry and ultrasound imaging , and any unfavourable events as locoregional and systemic recurrence . in conclusion , diagnosis of i - ptm is common that multifocality and bilateralism appear as pathologic features . the prognosis is excellent after surgical treatment and tsh suppression . routine adjuvant nuclear treatment is unnecessary in majority of patients .

misdiagnosis of bipolar disorder is a potentially serious problem for patients , which sometimes leads to inappropriate treatment.1 however , it is not unusual for bipolar disorder to be misdiagnosed as a major depressive disorder.2 previous studies have shown that the proportion of cases of bipolar disorder misdiagnosed as a major depressive disorder is 20.8%40%.24 nevertheless , there are few biomarkers for accurate diagnosis . if patients become depressive for the first time and have never experienced a manic episode , it is difficult to determine whether that depressive episode is unipolar or bipolar depression , and the risk of a manic switch is unknown . near - infrared spectroscopy ( nirs ) is a noninvasive and useful method that uses near - infrared light to measure hemoglobin concentration changes in the cortical surface area . although changes in the nirs signals measured on the forehead during the word fluency task may be partially associated with changes in skin blood flow,5 the changes in oxygenated hemoglobin ( [ oxyhb ] ) are associated with changes in the regional cerebral blood volume . nirs enables the assessment of brain function , and recent studies have shown that the frontal hemodynamic patterns detected by nirs can be a biomarker for the diagnosis of major psychiatric disorders.6,7 in the present case , nirs during the word fluency task was measured during the clinical course of bipolar disorder . in brief , [ oxyhb ] and deoxygenated hemoglobin ( [ deoxyhb ] ) were measured with a 52-channel nirs machine ( hitachi etg-4000 ; hitachi medical corporation , tokyo , japan ) at two wavelengths of near - infrared light ( 695 and 830 nm ) , the absorption of which was measured , and [ oxyhb ] and [ deoxyhb ] were calculated as previously described.8 the distance between pairs of source - detector probes was set at 3.0 cm and each measured area between pairs of source detector probes was defined as a channel . the probes of the nirs machine were fixed with 3 11 thermoplastic shells , with the lowest probes located along the fp1-fp2 line according to the international 1020 system used in electroencephalography . we used the word fluency task because it is easy to understand and execute , and it is also an executive task that produces distinct differences in performance and neuroimaging data among each diagnostic group of psychiatric disorders.7 additionally , plasma levels of homovanillic acid ( hva ) , a dopamine metabolite , and 3-methoxy-4hydroxyphenylglycol ( mhpg ) , a noradrenaline metabolite , were measured longitudinally using high performance liquid chromatography . in our hospital , we measured both plasma levels of monoamine metabolites ( every 3 weeks in principle ) and nirs ( before and after the treatment ) in psychiatric inpatients with mood disorders to investigate the association between plasma monoamine metabolites and nirs signals . this protocol was approved by the ethics committee of fukushima medical university , and the patient consented to participate after having been informed of the purpose of the study . the patient was a 22-year - old man who had no history of psychiatric illness . he was admitted to our hospital due to major depression that was diagnosed according to the diagnostic and statistical manual for mental disorders , fourth edition , with moderate sadness , agitation , retardation , and difficulty in concentrating . we treated the patient with paroxetine and lorazepam for about 4 weeks , but treatment was not successful . on day 4 , his montgomery sberg depression rating scale ( madrs ) score was 44 , and his plasma levels of hva and mhpg were 4.6 ng / ml and 1.9 ng / ml , respectively . figure 2 shows the waveform of [ oxyhb ] during the cognitive activation of the word fluency task . nirs signal changes were measured during a 10 second pretask baseline period , a 60 second task period , and a 55 second posttask baseline period . the initial syllables assigned were changed every 20 seconds ( /a/ , /ka/ , and /sa/ , respectively ) during the 60 second task period . during the task period , the patient was instructed to generate as many japanese words beginning with the designated syllable as possible . in the pretask and the posttask baseline periods , the patient was instructed to repeat a train of syllables ( /a/ , /i/ , /u/ , /e/ , and /o/ ) . he sat on a chair in a room with daylight with his eyes open throughout the measurements . we focused on increases in [ oxyhb ] , because this appears to reflect task - related cortical activation more directly than decreases in [ deoxyhb].7 we defined the patterns of nirs waveforms using the grand average waveforms in the frontal region ( ch 2528 , ch 3638 , and ch 4649 ; figure 3 ) and two simple visual indices,7 integral value , and centroid value . the integral value describes the size of the hemodynamic response during the 60 second activation task period , whereas the centroid value serves as an index of time course changes throughout the task , with periods representing the timing of the hemodynamic response.7 a recent study reported that the optimal threshold of the centroid value of the frontal region between major depressive disorder and two other disorders ( schizophrenia and bipolar disorder ) is 54 seconds ( major depressive disorder < other 2 disorders).7 on day 4 , the centroid value of the grand average in the central region was 68.6 seconds ( figure 2 ) , and the nirs waveforms showed an [ oxyhb ] reincrease in the posttask period ( ch 34 , 35 , 36 , 39 , 45 , 46 , 47 , and 49 ; figure 2 ) , which is characteristic of schizophrenia.6,7,911 on day 25 , his madrs score was 40 , his plasma hva level was markedly higher at 49.3 ng / ml , and his plasma mhpg level was 1.6 ng / ml . on day 27 , he was voluntarily discharged from the hospital , but was still in a depressive state . on day 30 , 3 days after leaving the hospital , he became manic and was readmitted to our hospital . he was in a state of psychotic mania with elevated mood , grandiosity , aggression , auditory hallucination , and thought broadcast , with a young mania rating scale ( ymrs ) score of 48 . we subsequently treated him with lithium , risperidone , and zotepine for approximately 3 weeks , and his manic state improved . on day 46 , his ymrs score was 29 , and plasma levels of hva and mhpg were 17 ng / ml and 1.3 ng / ml , respectively . however , he refused drugs because of extrapyramidal symptoms and became manic with psychotic symptoms again . therefore , we tried a combination of lithium , olanzapine , and zotepine , and the treatment was successful . on day 77 , the nirs waveforms turned to a negative pattern ( figure 2 ) , which was defined by an integral value of the grand average in the central region < 0 , whereas there were [ oxyhb ] reincreases in the posttask period ( ch 33 , 41 , 42 , 43 , 44 , 51 , and 52 ) . on day 99 , his ymrs score decreased to 3 , and his plasma levels of hva and mhpg were 8.7 ng / ml and 1.9 ng / ml , respectively . nine months later , he was off medication , became manic again , and was admitted to another psychiatric hospital . to the best of our knowledge , this is the first case report to measure both nirs and plasma levels of monoamine metabolites in a patient with bipolar disorder , although the clinical course in this patient was not usual as the mania occurred within the first weeks of the initiation of serotonin selective reuptake inhibitor ( ssri ) treatment . in this case , it was difficult to diagnose bipolar depression from only clinical symptoms at his first admission . we could not predict the subsequent manic switch with psychotic features , which was likely caused by paroxetine . however , the results of nirs on day 4 showed a pattern of schizophrenia , characterized by a smaller [ oxyhb ] increase during the first half of the task and an [ oxyhb ] reincrease in the posttask period,4,911 but not a unipolar depression pattern , which is characterized by only a smaller [ oxyhb ] increase during the first half of the period.4,9,12,13 care must be taken to interpret these findings as patterns of diseases because of the small number of studies . an [ oxyhb ] reincrease in the posttask period is considered as inefficient activation of the prefrontal cortex,4,9,11 which is one of the quantitative abnormalities in schizophrenia.14 these results may have predicted a possible risk of a manic switch in this patient . a recent study7 reported that the frontal hemodynamic patterns detected by nirs accurately distinguished between patients with major depressive disorder and those with bipolar disorder or schizophrenia with depressive symptoms . although the clinical status of bipolar depression may be different from that of unipolar depression,1 it is not easy to clearly distinguish without a previous manic episode . in fact , most bipolar disorder patients with depressive symptoms are initially diagnosed as having major depression.2 because there are few biomarkers for diagnosis , nirs findings may help for distinguishing unipolar and bipolar disorders . however , nirs signals changed after treatment with antipsychotics and lithium in this patient . although it is believed that nirs waveforms are a possible trait marker , nirs signals may change with the state or the medication . interestingly , nirs findings in this case showed a pattern of schizophrenia , not a pattern of bipolar disorder , which is characterized by a smaller [ oxyhb ] increase during the early period , and larger [ oxyhb ] increase during the late period of this task.15 this result is consistent with a manic episode in this case that included psychotic symptoms such as hallucination and thought broadcast . furthermore , 5 days before the manic switch , the patient s plasma hva level was almost ten times higher than just after admission . because the hva level change occurred in tandem with the manic symptoms during the clinical course , it is estimated that the manic episode in this case was associated with dopaminergic function . plasma hva levels have been considered as a possible indicator in the treatment of schizophrenia16,17 and bipolar disorder.18,19 furthermore , because ssris act on dopaminergic and serotonergic neural systems,20 the increase in the plasma hva level in this patient may have been induced by paroxetine . a previous study reported that psychosis or mania induced by ssris showed a pattern of increased plasma hva levels.21 this is consistent with our findings of the changes in plasma hva levels in this patient . taken together , the nirs findings in this patient may be based on dopaminergic dysfunction . however , the changes in the nirs may not be directly associated with the changes in plasma hva levels , because nirs findings are based on frontal brain functions , whereas plasma hva levels are related to psychotic symptoms,22 which seem to be a reflection of the mesolimbic dopamine system . in this case , nirs findings in a depressive state may predict the possible risk for a manic switch . longitudinal monitoring of plasma hva levels may be useful in the treatment of bipolar disorder , in particular in patients with psychotic symptoms like this case . chronic subdural hematoma ( csdh ) is a rare complication of unruptured aneurysm clipping surgery5,12 ) . because csdh signs and symptoms may appear slowly , it can cause problems such as headache , dizziness , seizure , and hemiparesis , and requires surgical drainage . mori et al.11 ) reported severe complications of surgical drainage of postoperative csdh , such as tension pneumocephalus and acute subdural hematoma , which can cause permanent neurologic deficits or death . the general incidence of csdh after brain surgery is reported to be approximately 0.3 - 1.5%8,12 ) however , aneurysm clipping surgery is regarded as an important risk factor of postoperative csdh7,10,11 ) . it is known that advanced age ( > 70 years ) and male gender may contribute to csdh after clipping surgery5,12 ) . additionally , postclipping csdh considering the relatively benign course of unruptured aneurysms , it is important to prevent csdh and identify its risk factors after clipping surgery in order to minimize treatment - related morbidity . the purpose of this study was to determine the incidence and risk factors of postoperative csdh after surgical clipping for unruptured anterior circulation aneurysms . between january 2008 to december 2013 , 980 consecutive patients with unruptured aneurysm underwent surgical clipping in a single tertiary institute . to reduce technical variables that depend on the surgeon , inclusion criteria were unruptured small to large aneurysms ( size<2.5 cm ) on anterior circulation . exclusion criteria were giant or complex aneurysms requiring complicated surgical techniques such as bypass surgery . we also excluded distal anterior cerebral artery ( aca ) aneurysms and posterior circulation aneurysms that were approached by other than pterional approach , such as anterior frontal craniotomy , subtemporal approach , and etc . we reviewed their medical records as well as radiological data including age , sex , size and location of aneurysm , size of craniotomy depending on the surgical approach , premature rupture , presence of arachnoid plasty , degree of preoperative brain atrophy , usage of antithrombotics and amount of immediate postoperative subdural fluid collection ( sdfc ) . we categorized the location of aneurysm as aca , middle cerebral artery ( mca ) , and internal carotid artery ( ica ) according to the parent artery . aneurysm sizes were divided into three groups based on diameter : tiny ( < 5 mm ) , small ( 5 - 10 mm ) , and large ( 10 - 25 mm ) . the size of all aneurysms was its maximal diameter as measured by the three - dimensional reconstructed images of digital subtraction angiography . the degree of preoperative brain atrophy was estimated using the bicaudate ratio ( bcr ) , which is defined as the ratio of the width of ventricles between the caudate nuclei and internal diameter of the vault at the same level2 ) . bcrs were measured on brain computed tomography ( ct ) scans that were taken within 6 months prior to surgery . in patients without ct scans , we used magnetic resonance t2-weighted imaging scans that were obtained within same period . postoperative sdfc was defined as the maximal distance between the inner table of the skull and the cerebral cortex . we measured its maximal vertical depth on the ipsilateral side of the clipped aneurysm on brain ct angiography that was performed 3 or 4 days after surgical clipping . we compared two groups who had postoperative sdfc less than or greater than 10 mm . the indication of surgery was determined by patient 's symptom . in the case of symptomatic patients with headache or focal neurological deficit , a surgical intervention was performed , whereas in case of asymptomatic patients were treated with conservative management with a clinical follow - up . we excluded the cases of csdh did not require surgical drainage because we did not perform routine radiological follow - up if they had no progressive symptoms . after clipping surgery , brain non - contrast ct and intracranial ct angiography was performed on postoperative days 1 and 3 or 4 , respectively . after discharge , clinical follow - ups were routinely performed 1 , 3 , and 6 months after clipping surgery at our outpatient clinic . scheduled radiologic follow - ups using intracranial ct angiography were performed after 1 or 2 years . however , imaging studies were employed when any patient showed clinical symptoms or signs that required further evaluation . surgical treatments were performed using a standard pterional approach or lateral supraorbital ( lso ) approach . the surgical techniques and average craniotomy size of these two approaches have been described previously3,16 ) . if necessary , temporary clips were placed in order to soften the aneurysm and to reduce intraoperative rupture . arachnoid plasty has been used since april 2011 . the arachnoid plasty technique applied fibrillar [ surgical fibrillar ( ethicon llc , san lorenzo , puerto rico ) ] and fibrin glue at the dissected arachnoid surface at the end of surgery ( fig . 1 ) . mean and frequency comparisons were performed using student 's t - tests , chi - square ( ) tests , mann - whitney u tests , or fisher 's exact tests as appropriate . univariate statistical analyses ( tests or fisher 's exact tests ) were performed to assess associations between variables and csdh , the categorical variable . the variables with p - values < 0.10 in univariate analyses were selected for multivariate models using multiple logistic regression analyses . statistical analyses were performed using spss 19.0 software ( spss inc . , chicago , il , usa ) . surgical treatments were performed using a standard pterional approach or lateral supraorbital ( lso ) approach . the surgical techniques and average craniotomy size of these two approaches have been described previously3,16 ) . if necessary , temporary clips were placed in order to soften the aneurysm and to reduce intraoperative rupture . arachnoid plasty has been used since april 2011 . the arachnoid plasty technique applied fibrillar [ surgical fibrillar ( ethicon llc , san lorenzo , puerto rico ) ] and fibrin glue at the dissected arachnoid surface at the end of surgery ( fig . mean and frequency comparisons were performed using student 's t - tests , chi - square ( ) tests , mann - whitney u tests , or fisher 's exact tests as appropriate . univariate statistical analyses ( tests or fisher 's exact tests ) were performed to assess associations between variables and csdh , the categorical variable . the variables with p - values < 0.10 in univariate analyses were selected for multivariate models using multiple logistic regression analyses . the patients enrolled in this study ( n=518 ) consisted of 173 males and 345 females aged 56.579.03 years ( meanstandard deviation ) . table 1 summarizes the baseline characteristics of patients . of these , 16 ( 3.1% ) patients suffered from postoperative csdh , which required burr hole drainage surgery . in univariate analyses , multivariate analyses also showed similar results [ p=0.017 , odds ratio ( or ) 4.037 , range 1.287 - 12.688 ] . in this study , we observed no statistically significant correlation with advanced age ( 65 years vs. < 65 years , 4.4% vs. 2.7% , p=0.257 based on fisher 's exact test ) . the group with higher bcr indices tended to show larger postoperative sdfc in this study ( 4.0 vs. 4.2 , p=0.083 based on mann - whitney u test ) . in addition , the bcr index was associated with postoperative csdh whereas sdfc was not ( table 2 ) . the location and diameter of aneurysms were positively associated with risk of symptomatic csdh in univariate analyses . compared to other locations , aca aneurysms had higher incidences of csdh in fisher 's exact test ( 6 cases , 5.4% , p=0.051 ) . additionally , larger aneurysms resulted in a higher incidence of csdh in fisher 's exact test ( 3 cases , 12% , p=0.030 ) . however , these differences were not significantly different in multivariate analyses using logistic regression test ( p=0.231 and p=0.243 , respectively ) . these findings were statistically significant in uni- and multivariate analyses ( p=0.004 , or 4.854 , range 1.658 - 14.085 ) . all patients who received surgical treatment recovered completely and experienced no complications or permanent neurologic deficits . no patient had recurrence of csdh , and complete csdh resorption was confirmed by follow - up brain ct scans . in this study , we found that male gender was associated with csdh , which agrees with the findings of previous studies5,12 ) . but , the reason of male preponderance ca n't be explained by the results of this study . kanat et al.6 ) suggested that the male preponderance of csdh in adults may be due to more brain atrophy and increased in cerebrospinal fluid ( csf ) in male gender and estrogens may have a protective effect on the capillaries . brain atrophy was also associated with postoperative csdh in this study , but other factors such as increment of csf or hormonal effect should be considered again in future study . in addition , previous studies have demonstrated that postoperative sdfc and advanced age were associated with postoperative csdh5,12,15 ) . we hypothesized that the degree of preoperative brain atrophy estimated by bcr and the degree of postoperative sdfc affect the development of postoperative csdh17 ) . postoperative sdfc can result from a newly developed artificial csf tract from the torn arachnoid membrane to the subdural space created by surgical dissection9 ) . if a patient has a higher degree of brain atrophy , the maximal depth of postoperative sdfc could also be higher . csdh tends to occur in elderly people because brain atrophy causes enlargement of the subarachnoid space and stretching of the bridging veins . these pre - existing conditions facilitate tearing of the arachnoid membrane and leakage of bloody csf into the subdural space after a neurosurgical procedure , thus producing overdrainage of csf from the subarachnoid space or the ventricular system13 ) . a previous study demonstrated that sdfc after aneurysm clipping might contribute to csdh12 ) . in theory , these explanations appear to be acceptable . our data indicate that higher bcr indices tended to show larger sdfc . however , sdfc itself was not associated with csdh . these differences can be explained by the following : first , the maximal vertical depth of the sdfc did not represent its total volume . if the exact volume of sdfc could be measured , the results might be different . second , the timing of brain ct scans differed and we did not checked serial follow - up ct scans , which can determine whether sdfc increased . changes in sdfc might be more predictive of the incidence of csdh rather a single measurement of sdfc . in this study , the degree of brain atrophy using bcr is a better predictor than age or sdfc to predict postoperative csdh . in order to prevent csf leakage from the subarachnoid space to the subdural space , arachnoid plasty the arachnoid plasty is not a generally accepted procedure in unruptured aneurysm surgery , but sealing of dissected arachnoid plane as an effort to prevent csf leakage seems to be convincing . if sdfc after craniotomy was related to csdh occurrence , effective arachnoid plasty would reduce postoperative csdh . mino et al.9 ) reported that arachnoid plasty using fibrin glue prevented sdfc after ruptured aneurysm surgery . differences between the mino et al.9 ) study and our study included aneurysm status ( ruptured vs. unruptured ) and number of patients . additionally , fibrin glue could spontaneously deteriorate over time , thus resulting in continuous csf leakage . however , the number of patients included in the previous study was relatively small ; therefore , it is difficult to conclude that arachnoid plasty has significant general efficacy . further studies are required to determine the detailed technical procedure as well as the role of arachnoid plasty in aneurysm surgery . the reported incidence of postoperative csdh after brain tumor removal is 0.4% , and the incidence after aneurysm clipping surgery is 2.4%12 ) . the main difference between these types of surgery is the extent of arachnoid dissection . in this study , aca location tended to be associated with csdh in univariate analyses and may have correlated with the range of cisternal dissection to soften the brain and expose the aneurysm . aca aneurysm surgery usually requires dissection of the sylvian fissure , chiasmatic cistern , and interhemispheric cistern ; however , other procedures require less . these procedures create a new csf tract from the subarachnoid space to the subdural space4 ) . although immediate sdfc was not associated with the incidence of postoperative csdh , considering the higher incidence of csdh in clipping surgery , perhaps the continuous leakage of csf through the surgically - dissected cisternal space may contribute to delayed csdh that requires surgical intervention . the maximal depth and amount of immediate postoperative sdfc can be larger in mass debulking surgeries , such as brain tumor removal , compared to aneurysm clipping . indeed , most subdural fluid collections are asymptomatic and disappear spontaneously and have thus not received much attention15,17 ) . authors thought that the association of aca aneurysms with csdh could suggest that more extensive cisternal dissection would have a clinical role in the development of postoperative csdh . in our data set , we observed a strong association between csdh and the use of antithrombotic medication . in our perioperative antithrombotic management strategy , we stopped medications such as aspirin or clopidogrel 7 days before surgery . in patients using antithrombotics , we corrected the bleeding tendency just before surgery . antithrombotic therapy was started again after we confirmed there was no evidence of hemorrhage on brain non - contrast ct scans 1 day post - surgery . the use of antithrombotic medication is known as an independent risk factor for csdh in general1,14 ) . therefore , patients using antithrombotic medication require special attention due to the potential risk of csdh . also optimal timing of resuming antithrombotics to reduce postoperative csdh should be identified in further study . we did not set routine radiological follow - up protocol for observing natural changes of asymptomatic postoperative csdh . second , the history of coexisting medical diseases was not fully considered , and could increase the risk of bleeding . prospective studies are necessary to further elucidate the incidence and risk factors of postoperative csdh after surgical clipping . this study shows that male gender , degree of brain atrophy , and the use of antithrombotic medication were associated with postoperative csdh .

misdiagnosis of bipolar disorder is a serious , but not unusual problem for patients . nevertheless , there are few biomarkers for distinguishing unipolar and bipolar disorder . near - infrared spectroscopy ( nirs ) is a noninvasive and useful method for the measurement of hemoglobin concentration changes in the cortical surface area , which enables the assessment of brain function . we measured nirs and plasma monoamine metabolite levels in a patient with bipolar disorder . a 22-year - old man was admitted due to major depression . at admission , nirs findings showed oxygenated hemoglobin reincrease in the posttask period , which is characteristic of schizophrenia . after treatment with paroxetine , he became manic with psychotic symptoms . his plasma level of homovanillic acid just before the manic switch was ten times higher than that just after paroxetine initiation . treatment with lithium and antipsychotics was successful , and plasma homovanillic acid decreased after treatment . in this case , the nirs findings may predict a possible risk of a manic switch , which is likely induced by paroxetine . nirs may be able to help distinguish unipolar and bipolar disorder in clinical settings . objectivechronic subdural hematoma ( csdh ) is a rare complication of unruptured aneurysm clipping surgery . the purpose of this study was to identify the incidence and risk factors of postoperative csdh after surgical clipping for unruptured anterior circulation aneurysms.methodsthis retrospective study included 518 patients from a single tertiary institute from january 2008 to december 2013 . csdh was defined as subdural hemorrhage which needed surgical treatment . the degree of brain atrophy was estimated using the bicaudate ratio ( bcr ) index . we used uni- and multivariate analyses to identify risk factors correlated with csdh.resultssixteen ( 3.1% ) patients experienced postoperative csdh that required burr hole drainage surgery . in univariate analyses , male gender ( p<0.001 ) , size of aneurysm ( p=0.030 ) , higher bcr index ( p=0.004 ) , and the use of antithrombotic medication ( p=0.006 ) were associated with postoperative csdh . in multivariate analyses using logistic regression test , male gender [ odds ratio ( or ) 4.037 , range 1.287 - 12.688 ] , high bcr index ( or 5.376 , range 1.170 - 25.000 ) , and the use of antithrombotic medication ( or 4.854 , range 1.658 - 14.085 ) were associated with postoperative csdh ( p<0.05 ) . postoperative subdural fluid collection and arachnoid plasty were not showed statistically significant difference in this study.conclusionthe incidence of csdh was 3.1% in unruptured anterior circulation aneurysm surgery . this study shows that male gender , degree of brain atrophy , and the use of antithrombotic medication were associated with postoperative csdh .

helicobacter pylori have infected nearly half of the world s population . in the developing countries , more than 90% of adults are typically infected during their early childhood , and once acquired the infection would last for several decades ( 1 ) . helicobacter pylori infection is the main cause of gastritis and persistent chronic inflammation may lead to peptic ulcers or gastric cancer ( 2 - 4 ) . helicobacter pylori gastritis is associated with the expression of various cytokines and immune cells infiltration ( 1 ) . it is recently demonstrated that il-17 , il-8 and il-18 are associated with h. pylori - related gastritis diseases ( 5 - 7 ) . recently , it is found that il-17 , a pro - inflammatory cytokine that can recruit neutrophils by inducing the production of cxc chemokines , plays a vital role in the inflammatory response to the h. pylori - related diseases ( 8) . furthermore , it is also reported that il-17 , il-8 , and il-18 are important to maintain the chronic gastric inflammation in the mongolian gerbils model induced by the h. pylori ( 8 - 10 ) . considering that up - regulation of il-17 , il-8 and il-18 plays a crucial role in h. pylori infection , it was explored that the expression of il-17 , il-8 and il-18 in peripheral blood may be involved in the inflammatory response to h. pylori . the current study aimed to obtain evidence regarding the association between serum levels of il-17 , il-8 and il-18 and the stomach pathological changes induced by h. pylori infection in mongolian gerbils . three - week - old male specific pathogen - free mongolian gerbils were purchased from wenzhou medical university animal experiment center / china ( approximately 25 g ) . they were kept in polypropylene cages on saw dust bedding in groups of two per cage , and caged under a ld 12:12 cycle . the animals used in the current study were cared for according to the institutional guidelines . the experimental protocol was approved by the animal care and use committee of wenzhou medical university / china . the bacteria were cultivated in columbia agar ( oxoid england ) with 10% sheep blood ( wenzhou kont biology and technology , ltd , china ) for two to three days at 37c under microaerobic conditions ( 15% co2 ) and high humidity , on a shaker at 100 rpm . after fasting , animals were inoculated every other day for three counts with 1 ml medium of h. pylori ( 109 cfu / ml ) using a metal stomach catheter as one cycle . mongolian gerbils were randomly divided into four groups ( n = 6 in each ) . groups a , b and c were firstly inoculated by gavage with h. pylori nctc 11637 for one cycle . group b was enhanced inoculation for two counts , and group c was enhanced inoculation for three counts after the first inoculation . all the animals were sacrified under anesthesia at 35 weeks . immediately after scarifying , the stomach was quickly removed and used for macroscopic , histological and electron microscopy examinations . the serum levels of il-17il-8 and il-18 were measured using an elisa kit ( invitrogen , carlsbad , ca , usa ) according to the manufacturer s instructions . differences between the groups were tested by an anova with subsequent scheffe post - hoc test and the relationship between il and gastritis serious were tested using spearman test . three - week - old male specific pathogen - free mongolian gerbils were purchased from wenzhou medical university animal experiment center / china ( approximately 25 g ) . they were kept in polypropylene cages on saw dust bedding in groups of two per cage , and caged under a ld 12:12 cycle . the animals used in the current study were cared for according to the institutional guidelines . the experimental protocol was approved by the animal care and use committee of wenzhou medical university / china . the bacteria were cultivated in columbia agar ( oxoid england ) with 10% sheep blood ( wenzhou kont biology and technology , ltd , china ) for two to three days at 37c under microaerobic conditions ( 15% co2 ) and high humidity , on a shaker at 100 rpm . after fasting , animals were inoculated every other day for three counts with 1 ml medium of h. pylori ( 109 cfu / ml ) using a metal stomach catheter as one cycle . mongolian gerbils were randomly divided into four groups ( n = 6 in each ) . groups a , b and c were firstly inoculated by gavage with h. pylori nctc 11637 for one cycle . group b was enhanced inoculation for two counts , and group c was enhanced inoculation for three counts after the first inoculation . all the animals were sacrified under anesthesia at 35 weeks . immediately after scarifying , the stomach was quickly removed and used for macroscopic , histological and electron microscopy examinations . the serum levels of il-17il-8 and il-18 were measured using an elisa kit ( invitrogen , carlsbad , ca , usa ) according to the manufacturer s instructions . differences between the groups were tested by an anova with subsequent scheffe post - hoc test and the relationship between il and gastritis serious were tested using spearman test . the assessment was performed for inflammation , neutrophil infiltrate , mucosal atrophy and intestinal metaplasia using the semi quantitative scales ( , none ; + , mild ; + + , moderate ; + + + , severe ) of the updated sydney system ( 11 ) ( table 1 ) . note . n= number of animals ; = none ; + = mild ; + + = moderate ; + + + = severe . for control and the other groups ( n = 6 ) . the results of the analysis showed the correlation between enhance inoculation counts and gastritis serious . the serum levels of il-17 , but not il-8 and il-18 showed a correlation with the severity of gastritis ( figure 1 ) there was a correlation between serum levels of il-17 and the degree of gastritis . the current study showed that the serum levels of il-17 , il-8 and il-18 obviously increased compared to those of control group . moreover , it indicated that the serum levels of il-17 continuously increased after repetitive intragastric administration of h. pylori and were obviously correlated with the severity of gastritis . however , the serum levels of il-8 and il-18 were up - regulated after intragastric administration of h. pylori for one cycle and without increase after intragastric administration ( figure 2 ) . a : il-17 ; b : il-8 ; c : il-18 . the gastric mucosa of the control group and group a showed no visible changes . in group b , the stomach had several small ulcers and mucosa expanded and thickened slightly . in group c , erosive lesions with more ulcers were detected , covered by abundant mucus ( figure 3 ) a : control group ; b : group a ; c : group b ; antral mucosa slightly expanded and several ulcers were detected ( arrows ) ; d : group c ; mucosa was covered by abundant mucus ( arrows ) . histology examination was taken in the gastric mucosa of mongolian gerbils . in the control group , inflammatory cell infiltration in the lamina propria was inconspicuous . in group the normal gland cells disappeared , leaving elongated pseudopyloric glands with a little of mucins in the transitional zone mucosa . in the lamina propria of the pyloric mucosa , moderate amount of inflammatory infiltrating cells was observed . in group the submucosa also located similar mucous glands surrounded by interstitium with a conspicuous infiltration of lymphocytes . in group c , several ulcers were found near the transitional zone and some of them reached the muscularis propria . in the vicinity of transitional mucosa between pylori and fundic mucosa large erosion was observed , which consisted of numerous inflammatory cells ( figure 4 ) . a : control group ; b : group a ; few neutrophils infiltrated the epithelium in the transitional zone ; c : group b ; erosion was observed ; d : group c ; the glandular structures were damaged ( h & e x400 ) . electron microscopy observation was conducted on the mongolian gerbils gastric mucosa . in the control group , a , the nucleus showed mild pyknosis , mild expansion could be observed in rough endoplasmic reticulum , and the mitochondria edema could be observed in cellular . moreover , gastric mucosa microvilli showed edema and falling off . in group b , the nucleus showed pyknosis and deformation , the mitochondria vacuoles appeared , and the mucus particles of glands epithelial cells became fusion and aggregation . in group c , gap of nuclear membrane and rough endoplasmic reticulum expanded , the edema , degeneration and necrosis could be observed in mitochondria ( figure 5 ) . b : group a ; the gastric mucosa microvilli edema and falling off ( arrows ) ; c : group b. the mucus particles of glands epithelial cells became fusion and aggregation ( arrows ) ; d : group c ; autophagosome and autophagic vacuoles formatiom ( arrows ) . in the present study , it was known that the regulation of cytokines in the gastric mucosa plays a significant role in the h. pylori infections such as il-17 , il-6 , il-8 , il-18 , and tnf- ( 1 , 5 , 9 , 10 , 12 ) . mongolian gerbils are perfect animals to study gastric disease pathogenesis details ( 13 , 14 ) . the current research enriches the cytokine profiles of the chronic phases of h. pylori infections . to the authors ` best knowledge , there are no reports on the serum levels of il-17 , il-8 , il-18 and the gastric mucosa pathological changes to date . helicobacter pylori infections in the mongolian gerbils induced an antral - predominant gastritis and gradually progressed to the whole corpus ( 14 , 15 ) . some mucosal cytokines expression was paralleled with the acute inflammation such as il-1 ( 16 ) . in addition , gastric corporal mucosal chronic inflammation was paralleled with ifn- expression ( 17 ) . pro - inflammatory gene activity was highly sublimated and the risk of gastric disease was promoted for the increased inflammatory cytokines . il-17 as an emerging family member expressed in the t memory cells plays a key role in the pathogenesis of some chronic inflammatory diseases ( 18 - 20 ) . il-17 can also stimulate the expression level of il-8 and facilitate the chemotaxis of neutrophils ( 20 , 21 ) . some clinical studies found that the levels of il-17 increased in gastric mucosa of the patients infected with h. pylori ( 20 ) . it was also known that the gastric mucosal levels of il-17 increased in mongolian gerbils with h. pylori infection , especially in the chronic inflammatory phase ( 8 , 18 - 20 ) . up to now , almost all investigators only focused on studying the gastric mucosal levels of il and the mrna expression . the current study focused on studying the serum levels of il-17 and it was confirmed to be bound up with the amount of neutrophils as well as the development of gastric disease . numerous small ulcers appeared in the animals stomach and mucosa expanded and thickened slightly in the macroscopic observation . an obvious neutrophils infiltration on the background of chronic inflammation could be perceived by the ordinary microscope and the edema , degeneration and necrosis in subcellular organelles could be observed through electron microscopy . the contemporaneous appearance of pathology changes and il-17 level suggest that the inflammation of cells infiltration may be linked to the serum cytokines levels . these results suggest that il-17 plays a significant role in the inflammatory response to h. pylori infection and may serve as a potential biomarker to diagnose and predict the chronic active gastritis . il-8 acts as a chemo attractant which could enhance the migration of neutrophils and leukocytes , and regulate the processes of chronic inflammation ( 22 ) . it is also engaged in human gastric cancer cells adhesion , migration , and invasion in vitro ( 23 ) and highly expressed in gastric cancer cells ( 24 ) . yamaoka y , et al . reported that il-8 and il-18 are associated with h. pylori - related gastritis diseases ( 5 , 7 ) . the current study also demonstrated that il-8 was a marked up regulation compared with the control group , and the increased serum levels of il-8 may not promote the inflammatory response . il-18 as a member of il-1 superfamily induced by ifn- was correlated with the severity of gastric inflammation ( 25 , 26 ) . some researchers reported that il-18 is important to maintain the chronic gastric inflammation in the mongolian gerbils model induced by the h. pylori ( 8 - 10 ) . however , other studies indicated that the mrna levels of il-18 are independent of h. pylori infections ( 25 , 27 ) . in the current study , however , the serum levels of il-18 were not correlated with counts of enhanced inoculation or the severity of pathology changes . therefore , in order to understand the role of il-18 in human h. pylori infections further studies are needed . in conclusion , the current study results demonstrated that the serum levels of il-17 , il-8 and il-18 play significant roles in the progression and regulation of mongolian gerbils with h. pylori infections . moreover , the serum levels of il-17 differed among each group of animals , and depended on the severity of gastritis disease . therefore , il-17 might be related to the gastritis clinical prognosis and be used as a marker to predict the prognosis . however , in order to understand more correlation of the cytokines in h. pylori infections additional studies are required . tuberous sclerosis complex ( tsc ) is a genetic disorder that can affect almost all organ systems . renal manifestations occur with high frequency , the most prevalent being angiomyolipoma ( aml ) . tsc - associated amls tend to be larger , more numerous , and more likely to cause spontaneous hemorrhage compared with sporadic amls . removal of renal calculi from kidneys with amls is particularly difficult because these tumors increase the potential for hemorrhage . we report the case of a 65-year - old man with tsc - associated aml and a renal calculus greater than 4 cm in his left kidney . the calculus was successfully removed by retrograde flexible ureterorenoscopy ( f - urs ) with holmium laser lithotripsy . he was previously diagnosed with tsc after being identified with the typical features , such as dermatological lesions ( facial angiofibroma , ungual fibromas , and hypomelanotic macules on the toes ) , subependymal nodules , and multiple amls in both kidneys . while there was no family history of tsc , the patient had childhood epilepsy . he also experienced two episodes of hypovolemic shock concomitant with extensive retroperitoneal hemorrhage caused by spontaneous aml rupture , once in each kidney . following the second episode , which occurred in the left kidney , he developed sepsis caused by staphylococcus aureus , which was isolated from both venous blood and urine . blood counts were within normal limits except for a low red blood cell count ( 331 10/mm ; normal , 430 10570 10/mm ) , low hemoglobin ( 9.5 g / dl ; normal , 13.517.0 g / dl ) , and a low hematocrit value ( 30.4% ; normal , 40%50% ) . serum creatinine level was 1.0 mg / dl ( normal , 0.61.1 mg / dl ) . ureter bladder ( kub ) radiography revealed a 43 16 mm renal calculus in the left kidney [ figure 1a ] . intravenous pyelography revealed that the calculus filled the pelvis and lower calyx , causing dilatation of the upper calyx [ figure 1b ] . abdominal enhanced computed tomography ( ct ) demonstrated a significant bilateral renal aml burden in addition to the renal calculus in the left kidney [ figure 2 ] . we decided to remove the renal calculus for several reasons , including the present clinical symptoms and a previous history of urinary tract infection . minimally invasive retrograde f - urs with holmium laser lithotripsy was chosen because multiple renal amls have hemorrhagic potential . ( a ) kub radiography reveals a lower pole renal calculus in the left kidney . ( b ) intravenous pyelography shows the calculus causing dilatation of the upper calyx abdominal enhanced computed tomography demonstrates significant bilateral renal aml burden and a large renal calculus in the left kidney a 6.9-fr f - urs , a 10-fr ureteral access sheath , and a 200- holmium : yag laser fibers were used for treatment . we attempted to vaporize the stone by painting the laser fiber over the stone surface . a 1.9-fr nitinol basket , which minimizes the loss of deflection during f - urs , was used to extract large fragments and reposition them from the lower pole calyx into a position more readily accessible by the laser fiber , such as the renal pelvis or upper pole calyx . no evidence of greater than 2-mm residual stones was observed before removal of the ureteral stent in postoperative kub radiography . three months after surgery , kub radiography confirmed the absence of any residual stones [ figure 3 ] , and urinalysis was also revealed normal findings . three months after flexible ureterorenoscopy , follow - up kub radiography confirms the absence of any residual stones rakowski et al . reported renal lesions in 57.5% tsc patients ; of these , aml was present in 85.4% , cysts in 44.8% , and renal cell carcinoma in 4.2% . tsc - associated aml is more likely to cause spontaneous hemorrhage compared with its sporadic counterpart , with a hemorrhagic risk of 25% to 50% . tsc patients are also predisposed to nephrolithiasis because of both the disease and its treatments . for example , drugs like topiramate have proven to be effective for some forms of tsc - associated epilepsy ; however , they also increase the risk of nephrolithiasis . patients are also at higher risk for renal cystic disease because disruption of distal tubular function by a significant cyst burden leads to hypocitraturia . our patient was not undergoing any treatment for epilepsy , and ct demonstrated no evidence of renal cystic lesions . traditional surgical treatment of renal calculi , including minimally invasive procedures like percutaneous nephrolithotomy ( pnl ) or extracorporeal shockwave lithotripsy ( eswl ) , may lead to bleeding in patients with renal amls . pnl remains a milestone technique with a high success rate and with a low percentage of major complications ; however , the blood transfusion rate reported varies from 11.2% to 17.5% . pnl involves puncture and dilation of the renal collecting system via the renal parenchyma and poses an unacceptable risk of hemorrhage in kidneys with aml , although there are two reported cases of pnl performed directly through a renal aml . on the other hand , eswl requires large numbers of shock waves , which can lead to capillary damage with parenchymal or subcapsular hemorrhage ; eswl - induced retroperitoneal bleeding has been reported in a patient with renal aml . recent studies have evaluated f - urs as an alternative treatment for patients with contraindications to pnl or eswl , such as pregnant woman , obese patients , or those with coagulopathy . indeed , f - urs using pulsed laser energy can fragment stones with less risk of hemorrhage . furthermore , f - urs is a minimally invasive procedure that can treat < 2-cm renal calculi with a low morbidity and high success rate . our case and other reported cases indicate that f - urs can also safely treat larger stones . however , the management of lower pole renal calculi with f - urs remains debatable because of limited access and poor clearance of stone fragments . by the current literature , pnl should be considered the first line of treatment for greater than 2-cm lower pole renal calculi . further advances in endoscopic technology and techniques are required to resolve the limitations of f - urs , including the technical difficulty in removing large stones , intrarenal influx with infected stones , and prolonged surgical duration . in conclusion , retrograde f - urs with holmium laser lithotripsy is an effective , minimally invasive therapy for patients at high risk of renal hemorrhage .

background : persistent helicobacter pylori infection confers an increased risk for serious illnesses such as peptic ulcers and gastric cancer . various cytokines are involved in the regulation of inflammatory immune response in h. pylori - infected gastric mucosa.objectives:the current study aimed to obtain evidence regarding the association between il-17 , il-8 and il-18 expression in peripheral blood and h. pylori infection in mongolian gerbils.materials and methods : mongolian gerbils were inoculated with h. pylori by a metal stomach catheter . after sacrifice , their gastric mucosae were examined in macroscopic , histological and electron microscopy levels . in addition , enzyme linked immunosorbent assay ( elisa ) assay was performed on the il-17 , il-8 and il-18 cytokines in the blood samples.results:serum levels of il-17 , il-8 and il-18 were remarkably up - regulated compared to those of the control group . there was an obvious correlation between the increase of il-17 and the serious extent of gastritis in the current study . however , the serum levels of il-8 and il-18 without getting increasingly more for repetitive intragastric administration . there were plenty of neutrophils infiltrating in the infected group mucosal . intestinal metaplasia and gastric ulcers were also founded in h. pylori infected animals after enhanced inoculation . the edema , degeneration and necrosis changes could be found in organelles by transmission electron microscopy . more serious pathological changes were detected in the enhanced inoculation groups compared to the cycle group.conclusions:the serum levels of il-17 , but not il-8 and il-18 may serve as a potential biomarker for diagnosis and predicting the prognosis of gastritis caused by h. pylori . renal calculi in patients with renal angiomyolipomas are difficult to treat because of the hemorrhagic potential of these tumors . we describe the case of a 65-year - old man having tuberous sclerosis complex - associated multifocal renal angiomyolipomas with a large renal calculus . the patient presented with left flank dullness and a previous history of spontaneous angiomyolipoma rupture . intravenous pyelography revealed a 43 16 mm calculus in the pelvis and lower calyx of the left kidney . the calculus was successfully removed by retrograde flexible ureterorenoscopy and holmium - yag lithotripsy . flexible ureterorenoscopy is an effective , minimally invasive therapy for patients at high risk of renal hemorrhage .

the diagnosis of unexplained syncopal episodes and sustained palpitations remains a difficult task in clinical cardiology . standard 24 h holter monitoring has a low diagnostic yield , while implantable loop recorder ( ilr ) has a higher diagnostic yield , but it is expensive and mildly invasive , making its role in diagnostic work - up of unexplained syncope and palpitations questionable . the utility of external prolonged electrocardiogram ( ecg ) monitoring in work - up of syncope is still undefined . the few available studies , generally single - centre and retrospective , provided conflicting results , mainly due to heterogeneity of patient populations and devices capabilities . in sustained palpitations , the external loop recorders ( elrs ) utility was more established . earlier elrs with relatively low - storage capacity and brief patient - activated ecg tracings had limited usefulness in syncope or asymptomatic arrhythmias . recently , newer elrs with auto - trigger capabilities showed 30% diagnostic yield for syncope and 75% for palpitations . external ecg recorders were also utilized to evaluate the burden of asymptomatic atrial fibrillation ( af ) , as in cryptogenic transient ischaemic attacks or after transcatheter ablation . syncope and palpitations tend to occur in clusters , with higher reoccurrence rate early after an event , thus an early use of long - term ecg monitoring may increase the likelihood of clinical diagnosis . the synarr - flash study ( monitoring of syncopes and/or sustained palpitations of suspected arrhythmic origin ; clinicaltrial.gov nct02253134 ) is the first international , multicentre , prospective trial designed to evaluate the feasibility and usefulness of external prolonged ecg monitoring in the early clinical work - up of unexplained syncope and/or sustained palpitations of suspected arrhythmic origin . at each enrolling centre , consecutive patients screened for enrolment had to meet both the following inclusion criteria : ( i ) recent ( within 1 month ) episode of syncope or sustained palpitations ( index event ) , after being discharged from emergency room or hospitalization without a conclusive diagnosis , and ( ii ) suspected arrhythmic origin according to the clinical features defined in the 2009 syncope guidelines , including the presence of cardiovascular disease or channelopathy , family history of unexplained sudden death , or abnormal ecg findings . syncope was defined as complete loss of consciousness with spontaneous recovery , while sustained palpitations as long - lasting sensation of irregular or fast heart rhythm . abnormal ecg findings included atrio - ventricular conduction disorders [ first - degree atrio - ventricular block ( avb ) i , second - degree avb ii type mobitz 1 , left or right ventricular bundle branch block ( lbbb / rbbb ) ] , supraventricular rhythm disorders [ sinus arrest or pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia > 100 b.p.m . , paroxysmal af , frequent premature atrial contractions ( pacs ) ( > 10/min ) , chronic af , paroxysmal atrial flutter / tachycardia ] , and ventricular rhythm disorders [ frequent premature ventricular contractions ( pvcs ) ( > 10/min ) , couplets , non - sustained vt ] ( table 1 ) . table 1demographic characteristicsparameters ( number , % ) itt population with syncopen = 110itt population with palpitationn = 282p - valuedemographics age , mean ( sd)65.1 ( 17.2)53.9 ( 18.4)<0.001 male gender64 ( 59.3%)101 ( 35.8%)<0.001nyha , n68191 i / ii66 ( 97.1%)190 ( 99.5%)0.170 iii / iv2 ( 2.9%)1 ( 0.5%)previous diagnosis test , n106262 24 h holter recording64 ( 60.4%)159 ( 60.7%)0.956 hut21 ( 19.8%)13 ( 5.0%)<0.001 csm27 ( 25.5%)10 ( 3.8%)<0.001 exercise stress test12 ( 11.3%)41 ( 15.6%)0.284history of previous rhythm disorders supraventricular rhythm disorders40 ( 57.1%)103 ( 66.0%)0.200 av conduction disorders32 ( 45.7%)63 ( 40.4%)0.453 ventricular rhythm disorders29 ( 41.4%)65 ( 41.7%)0.973history of cardiovascular disease , n70155 systemic hypertension41 ( 58.6%)68 ( 43.9%)0.041 coronary artery disease8 ( 11.4%)12 ( 7.7%)0.368 cerebrovascular accident8 ( 11.4%)7 ( 4.5%)0.054 ischaemic cardiomyopathy5 ( 7.1%)9 ( 5.8%)0.701 previous myocardial infarction3 ( 4.3%)6 ( 3.9%)1.000family history of sudden cardiac death9 ( 8.2%)7 ( 2.5%)0.006cardiovascular medication at the time of the test82 ( 74.5%)190 ( 67.4%)0.166 demographic characteristics sd , standard deviation ; nyha , new york heart association functional class ; av , atrio - ventricular . includes sinus arrest / pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , and paroxysmal atrial flutter / tachycardia . include frequent pvcs ( > 10/min ) , couplets , and non - sustained vt . by study inclusion criteria , all patients were enrolled within 30 days after the index event , after being discharged from emergency room or hospitalization without a conclusive diagnosis . at enrolment , patient medical history was collected , including a detailed report of the index event ( syncope or palpitation ) and the history of events in the preceding year and of the previously performed diagnostic tests . patients were instructed by the study nurse on how to use the recorder and to report symptoms in the diary . patients had to wear the recorder for 4 weeks , and a clinic visit was scheduled at the end of the recording period ( m1 ) . patients had to contact the centre if any symptom occurred before m1 ( in case an additional clinic visit was scheduled ) or if any technical problem occurred or the recording stopped prematurely ( in order to re - initiate the recording ) . at m1 , patients were interviewed , diaries were collected , and information was cross - checked . clinical and device information , including events , device acceptance , device malfunction , adverse events , and medications , were reported in case report forms ( crfs ) . the study was conducted in accordance with guidelines for good clinical practice and declaration of helsinki and approved by relevant local ethics committees . all enrolled patients provided written informed consent to study participation . at enrolment , a prolonged high - capacity elr ( spiderflash - t , sorin group srl , saluggia , italy sized 75 50 19 mm , powered by lithium battery ) was provided to each patient . the three - wire recorder was connected to the chest using disposable adhesive electrodes , which the patient had to change daily . recorders store two leads ecg tracings on a high - capacity removable digital card capable of up to 40 day monitoring . three recording modalities are available : ( i ) manual patient activation in case of symptoms ; ( ii ) automatic activation at predefined intervals ; and ( iii ) auto - trigger activation at preselected rhythm disturbances , such as pauses or bradycardia or supraventricular tachycardia ( svt ) and ventricular tachycardia ( vt ) . all ecg monitorings were reviewed by two separate investigators ( e.t.l . and a.m. ) and categorized based on both patient diary and interview and ecg data , as follows . diagnostic monitorings include the following : conclusive events , when syncope or palpitations were reported and a readable ecg recording was available at the time of symptom ( independently of the presence or absence of arrhythmias);significant events , when the following predefined arrhythmias were detected by auto - trigger function in the absence of reported symptoms : ( i ) advanced avb ( third - degree avb or second - degree avb mobitz type 2 ) , sinus bradycardia ( < 30 b.p.m . ) , pauses 6 s ; ( ii ) fast sustained supraventricular tachycardia ( svt ) , af , or flutter ( afl ) ( rate > 180 b.p.m . , duration > 3 min ) ; and ( iii ) non - sustained ventricular tachycardia ( nsvt , duration > 10 s ) or sustained ventricular tachycardia ( > 30 s ) . those arrhythmias were considered diagnostic even in the absence of recurrent syncope according to the 2009 syncope guidelines.non-diagnostic monitorings include the following : suggestive events , when the following predefined arrhythmias were detected by auto - trigger function , in the absence of reported symptoms : ( i ) sinus bradycardia ( 3040 b.p.m . ) , 36 s pauses ; ( ii ) brief burst of svt , af , or afl ( 15180 s ) ; and ( iii ) nsvt < 10 s;negative monitorings : ( i ) monitorings without event recurrence reported in the patient diary and without recording of asymptomatic arrhythmia by auto - trigger function ; ( ii ) monitorings with recurrence of symptoms different from the index event ; and ( iii ) monitorings with recurrence of syncope or palpitations , but without available ecg recording . conclusive events , when syncope or palpitations were reported and a readable ecg recording was available at the time of symptom ( independently of the presence or absence of arrhythmias ) ; significant events , when the following predefined arrhythmias were detected by auto - trigger function in the absence of reported symptoms : ( i ) advanced avb ( third - degree avb or second - degree avb mobitz type 2 ) , sinus bradycardia ( < 30 b.p.m . ) , pauses 6 s ; ( ii ) fast sustained supraventricular tachycardia ( svt ) , af , or flutter ( afl ) ( rate > 180 b.p.m . , duration > 3 min ) ; and ( iii ) non - sustained ventricular tachycardia ( nsvt , duration > 10 s ) or sustained ventricular tachycardia ( > 30 s ) . those arrhythmias were considered diagnostic even in the absence of recurrent syncope according to the 2009 syncope guidelines . suggestive events , when the following predefined arrhythmias were detected by auto - trigger function , in the absence of reported symptoms : ( i ) sinus bradycardia ( 3040 b.p.m . ) , 36 s pauses ; ( ii ) brief burst of svt , af , or afl ( 15180 s ) ; and ( iii ) nsvt < 10 s ; negative monitorings : ( i ) monitorings without event recurrence reported in the patient diary and without recording of asymptomatic arrhythmia by auto - trigger function ; ( ii ) monitorings with recurrence of symptoms different from the index event ; and ( iii ) monitorings with recurrence of syncope or palpitations , but without available ecg recording . at each enrolling centre , consecutive patients screened for enrolment had to meet both the following inclusion criteria : ( i ) recent ( within 1 month ) episode of syncope or sustained palpitations ( index event ) , after being discharged from emergency room or hospitalization without a conclusive diagnosis , and ( ii ) suspected arrhythmic origin according to the clinical features defined in the 2009 syncope guidelines , including the presence of cardiovascular disease or channelopathy , family history of unexplained sudden death , or abnormal ecg findings . syncope was defined as complete loss of consciousness with spontaneous recovery , while sustained palpitations as long - lasting sensation of irregular or fast heart rhythm . abnormal ecg findings included atrio - ventricular conduction disorders [ first - degree atrio - ventricular block ( avb ) i , second - degree avb ii type mobitz 1 , left or right ventricular bundle branch block ( lbbb / rbbb ) ] , supraventricular rhythm disorders [ sinus arrest or pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia > 100 b.p.m . , paroxysmal af , frequent premature atrial contractions ( pacs ) ( > 10/min ) , chronic af , paroxysmal atrial flutter / tachycardia ] , and ventricular rhythm disorders [ frequent premature ventricular contractions ( pvcs ) ( > 10/min ) , couplets , non - sustained vt ] ( table 1 ) . table 1demographic characteristicsparameters ( number , % ) itt population with syncopen = 110itt population with palpitationn = 282p - valuedemographics age , mean ( sd)65.1 ( 17.2)53.9 ( 18.4)<0.001 male gender64 ( 59.3%)101 ( 35.8%)<0.001nyha , n68191 i / ii66 ( 97.1%)190 ( 99.5%)0.170 iii / iv2 ( 2.9%)1 ( 0.5%)previous diagnosis test , n106262 24 h holter recording64 ( 60.4%)159 ( 60.7%)0.956 hut21 ( 19.8%)13 ( 5.0%)<0.001 csm27 ( 25.5%)10 ( 3.8%)<0.001 exercise stress test12 ( 11.3%)41 ( 15.6%)0.284history of previous rhythm disorders supraventricular rhythm disorders40 ( 57.1%)103 ( 66.0%)0.200 av conduction disorders32 ( 45.7%)63 ( 40.4%)0.453 ventricular rhythm disorders29 ( 41.4%)65 ( 41.7%)0.973history of cardiovascular disease , n70155 systemic hypertension41 ( 58.6%)68 ( 43.9%)0.041 coronary artery disease8 ( 11.4%)12 ( 7.7%)0.368 cerebrovascular accident8 ( 11.4%)7 ( 4.5%)0.054 ischaemic cardiomyopathy5 ( 7.1%)9 ( 5.8%)0.701 previous myocardial infarction3 ( 4.3%)6 ( 3.9%)1.000family history of sudden cardiac death9 ( 8.2%)7 ( 2.5%)0.006cardiovascular medication at the time of the test82 ( 74.5%)190 ( 67.4%)0.166 demographic characteristics sd , standard deviation ; nyha , new york heart association functional class ; av , atrio - ventricular . includes sinus arrest / pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , and paroxysmal atrial flutter / tachycardia . include frequent pvcs ( > 10/min ) , couplets , and non - sustained vt . by study inclusion criteria , all patients were enrolled within 30 days after the index event , after being discharged from emergency room or hospitalization without a conclusive diagnosis . at enrolment , patient medical history was collected , including a detailed report of the index event ( syncope or palpitation ) and the history of events in the preceding year and of the previously performed diagnostic tests . patients were instructed by the study nurse on how to use the recorder and to report symptoms in the diary . patients had to wear the recorder for 4 weeks , and a clinic visit was scheduled at the end of the recording period ( m1 ) . patients had to contact the centre if any symptom occurred before m1 ( in case an additional clinic visit was scheduled ) or if any technical problem occurred or the recording stopped prematurely ( in order to re - initiate the recording ) . at m1 , patients were interviewed , diaries were collected , and information was cross - checked . clinical and device information , including events , device acceptance , device malfunction , adverse events , and medications , were reported in case report forms ( crfs ) . the study was conducted in accordance with guidelines for good clinical practice and declaration of helsinki and approved by relevant local ethics committees . at enrolment , a prolonged high - capacity elr ( spiderflash - t , sorin group srl , saluggia , italy sized 75 50 19 mm , powered by lithium battery ) was provided to each patient . the three - wire recorder was connected to the chest using disposable adhesive electrodes , which the patient had to change daily . recorders store two leads ecg tracings on a high - capacity removable digital card capable of up to 40 day monitoring . three recording modalities are available : ( i ) manual patient activation in case of symptoms ; ( ii ) automatic activation at predefined intervals ; and ( iii ) auto - trigger activation at preselected rhythm disturbances , such as pauses or bradycardia or supraventricular tachycardia ( svt ) and ventricular tachycardia ( vt ) . all ecg monitorings were reviewed by two separate investigators ( e.t.l . and a.m. ) and categorized based on both patient diary and interview and ecg data , as follows . diagnostic monitorings include the following : conclusive events , when syncope or palpitations were reported and a readable ecg recording was available at the time of symptom ( independently of the presence or absence of arrhythmias);significant events , when the following predefined arrhythmias were detected by auto - trigger function in the absence of reported symptoms : ( i ) advanced avb ( third - degree avb or second - degree avb mobitz type 2 ) , sinus bradycardia ( < 30 b.p.m . ) , pauses 6 s ; ( ii ) fast sustained supraventricular tachycardia ( svt ) , af , or flutter ( afl ) ( rate > 180 b.p.m . , duration > 3 min ) ; and ( iii ) non - sustained ventricular tachycardia ( nsvt , duration > 10 s ) or sustained ventricular tachycardia ( > 30 s ) . those arrhythmias were considered diagnostic even in the absence of recurrent syncope according to the 2009 syncope guidelines.non-diagnostic monitorings include the following : suggestive events , when the following predefined arrhythmias were detected by auto - trigger function , in the absence of reported symptoms : ( i ) sinus bradycardia ( 3040 b.p.m . ) , 36 s pauses ; ( ii ) brief burst of svt , af , or afl ( 15180 s ) ; and ( iii ) nsvt < 10 s;negative monitorings : ( i ) monitorings without event recurrence reported in the patient diary and without recording of asymptomatic arrhythmia by auto - trigger function ; ( ii ) monitorings with recurrence of symptoms different from the index event ; and ( iii ) monitorings with recurrence of syncope or palpitations , but without available ecg recording . conclusive events , when syncope or palpitations were reported and a readable ecg recording was available at the time of symptom ( independently of the presence or absence of arrhythmias ) ; significant events , when the following predefined arrhythmias were detected by auto - trigger function in the absence of reported symptoms : ( i ) advanced avb ( third - degree avb or second - degree avb mobitz type 2 ) , sinus bradycardia ( < 30 b.p.m . ) , pauses 6 s ; ( ii ) fast sustained supraventricular tachycardia ( svt ) , af , or flutter ( afl ) ( rate > 180 b.p.m . , duration > 3 min ) ; and ( iii ) non - sustained ventricular tachycardia ( nsvt , duration > 10 s ) or sustained ventricular tachycardia ( > 30 s ) . those arrhythmias were considered diagnostic even in the absence of recurrent syncope according to the 2009 syncope guidelines . suggestive events , when the following predefined arrhythmias were detected by auto - trigger function , in the absence of reported symptoms : ( i ) sinus bradycardia ( 3040 b.p.m . ) , 36 s pauses ; ( ii ) brief burst of svt , af , or afl ( 15180 s ) ; and ( iii ) nsvt < 10 s ; negative monitorings : ( i ) monitorings without event recurrence reported in the patient diary and without recording of asymptomatic arrhythmia by auto - trigger function ; ( ii ) monitorings with recurrence of symptoms different from the index event ; and ( iii ) monitorings with recurrence of syncope or palpitations , but without available ecg recording . all analyses were performed on the intention - to - treat ( itt ) population , both overall and separately per index event ( syncope or palpitations ) . since the distribution of the data was normal , continuous data were expressed as mean standard deviation ( sd ) , while categorical variables as frequencies and percentages and differences in categorical variables were tested by test or by fisher exact test as appropriate . the time - to - first occurrence of a diagnostic event during 4-week monitoring was analysed by kaplan meier survival analysis , and survival curves of patients with syncope and palpitations were compared by log - rank test . actuarial diagnostic yields for diagnostic events were retrieved from kaplan meier statistics and compared by analysis when applicable . predictors of occurrence of diagnostic events during the 4-week monitoring were identified by multivariable cox proportional - hazard model , after proportional - hazard assumptions were verified . a stepwise procedure was applied : first , univariable analyses determined statistically significant variables at 20% , and then significant variables were introduced in the multivariable model . two - sided p values of < 0.05 were considered significant in all evaluations . analyses were performed using sas software release 9.2 ( sas institute , cary , nc , usa ) . the synarr - flash study enrolled 395 patients ( 42.3% males , age 56.9 18.7 years ) from 10 centres in five countries between august 2010 and june 2013 . as index event information was unavailable for three patients , the itt population included 392 patients ; 282 patients ( 71.9% ) enrolled for palpitations and 110 ( 28.1% ) for syncope . patients with syncope were older , more frequently males , with previous history of cardiovascular disease , family history of sudden death , hypertension , and use of cardiovascular medication ( table 1 ) . the mean duration of ecg monitoring was 23.0 8.1 days , similar for syncope and palpitations . in 22 patients , the recording was stopped for the occurrence of a major event . in four patients ( 1% ) , the recording was interrupted due to intolerance to electrodes , while two patients ( 0.5% ) withdrew from the study . skin reactions to electrodes were reported in 12 patients ( 3% ) , but all were defined as non - serious and solved spontaneously . twenty - seven patients with prior syncope ( 24.5% ) had a diagnostic event within the 4-week monitoring . conclusive events with ecg - documented syncope occurred in 11 patients ( 10% , table 2 ) : in 6 patients , supraventricular bradyarrhythmias were recorded during syncope , while in 5 cases , regular sinus rhythm or sinus tachycardia was recorded ( table 2 ) . auto - trigger function identified asymptomatic significant arrhythmias in 16 patients ( 14.5% , table 2 ) . table 2electrocardiogram findings during monitoring in patients with syncope and palpitationpatientssyncope ( n = 110)palpitation ( n = 282)n%n%diagnostic tests2724.520271.6conclusive events ( recurrence reported on patient diary)1110.019368.4non - arrhythmic ( sinus rhythm , sinus tachycardia)54.56623.4arrhythmic65.512745.0 pauses ( 6 s ) , avb 3 or avb 2 mobitz 243.610.4 pauses ( 36 s ) , sinus bradycardia21.872.5 paroxysmal af , afl ( > 3 min)00.0227.8 paroxysmal svt ( > 3 min)00.0155.3 non - sustained svt ( 15 s3 min)00.04014.2 nsvt < 10 s00.051.8 nsvt > 10 s00.010.4 frequent vpbs or apbs00.03612.7asymptomatic significant arrhythmias1614.593.2 fast sustained af / afl / svt > 3 min > 180 b.p.m.87.382.8 sinus bradycardia < 30 b.p.m . , pauses 6 s , avb 3 or avb 2 mobitz 254.510.4 nsvt > 10 s or sustained ventricular tachycardia > 30 s32.700.0non - diagnostic tests8375.58028.4negative monitoring7366.45920.9asymptomatic suggestive arrhythmias109.1217.5 sinus bradycardia 3040 b.p.m . , sinus pauses 36 s54.5113.9 nsvt < 10 s54.593.2 af / afl / svt 15 s3 min00.010.4avb 2 , avb 3 , atrio - ventricular block ( grade 2 or 3 ) ; af , atrial fibrillation ; afl , atrial flutter ; svt , supraventricular tachycardia ; nsvt , non - sustained ventricular tachycardia ; vpbs , ventricular premature beats ; apbs , atrial premature beats . electrocardiogram findings during monitoring in patients with syncope and palpitation avb 2 , avb 3 , atrio - ventricular block ( grade 2 or 3 ) ; af , atrial fibrillation ; afl , atrial flutter ; svt , supraventricular tachycardia ; nsvt , non - sustained ventricular tachycardia ; vpbs , ventricular premature beats ; apbs , atrial premature beats . the actuarial diagnostic yield , calculated by the kaplan meier cumulative occurrence of diagnostic events , was 13.2% at 1 week , 19.1% at 2 weeks , and 29.4% at 4 weeks ( figure 1 ) . figure 1kaplan meier analysis of recurrence - free rate in patients with syncope ( continuous line ) and palpitations ( dashed line ) of first diagnostic event during prolonged ecg monitoring ( log rank : p < 0.001 ) . kaplan meier analysis of recurrence - free rate in patients with syncope ( continuous line ) and palpitations ( dashed line ) of first diagnostic event during prolonged ecg monitoring ( log rank : p < 0.001 ) . auto - trigger function detected asymptomatic suggestive arrhythmias in 10 patients ( 9.1% , table 2 ) , although per definition were not included as diagnostic events . multivariable analyses to identify potential predictors of diagnostic events included age , gender , previous recording of supraventricular or ventricular arrhythmia , time from index event to enrolment ( 015 days vs. > 15 days after index syncope ) , the number of events in preceding year , history of cardiovascular disease , and family history of sudden cardiac death . the only two significant predictors for diagnostic findings during the 4-week monitoring were ( i ) early start of monitoring after index event and ( ii ) previous history of supraventricular arrhythmias [ sinus arrest or pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia > 100 b.p.m . , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , paroxysmal atrial flutter / tachycardia table 3 ] . monitoring was started within 15 days from index event in 74 patients : 4-week actuarial diagnostic yield was 33.0% in patients with early initiation compared with 15.6% in later initiation ( p = 0.021 , figure 2a ) . history of supraventricular arrhythmias was present in 39 patients : 4-week actuarial diagnostic yield was 43.6% in patients with supraventricular arrhythmias compared with 21.3% in those without ( p = 0.018 , figure 2b ) . table 3predictors for diagnosis during the 4-week ecg monitoringvariablesp - valueodds ratio95% wald confidence limitsa patients studied for syncope time between index event and enrolment015 days vs. > 15 days0.0216.21.329.6 history of supraventricular arrhythmiasyes vs. no0.0183.61.49.7b patients studied for palpitations history of previous palpitations ( in the previous 12 months)25 vs. 10.0362.61.16.3610 vs. 10.0292.81.17.311 vs. 1<0.0014.32.09.4 time between index event and enrolment07 days vs. 815 days0.0043.01.46.407 days vs. > 15 days0.0312.31.14.9supraventricular arrhythmias include sinus arrest / pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , and paroxysmal atrial flutter / tachycardia . figure 2(a ) kaplan meier analysis of recurrence - free rate in patients with syncope enrolled at 015 days after the index syncope ( continuous line ) vs. > 15 days after the index syncope ( dashed line ) , or = 6.222 , 95% ci ( 1.30929.573 ) , p = 0.02 ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with syncope without previous history of supraventricular rhythm disorder ( continuous line ) vs. patients with previous history of supraventricular rhythm disorder ( dashed line ) , or = 3.631 , 95% ci ( 1.3569.724 ) , p = 0.01 ( see table 3 for details ) . predictors for diagnosis during the 4-week ecg monitoring supraventricular arrhythmias include sinus arrest / pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , and paroxysmal atrial flutter / tachycardia . ( a ) kaplan meier analysis of recurrence - free rate in patients with syncope enrolled at 015 days after the index syncope ( continuous line ) vs. > 15 days after the index syncope ( dashed line ) , or = 6.222 , 95% ci ( 1.30929.573 ) , p = 0.02 ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with syncope without previous history of supraventricular rhythm disorder ( continuous line ) vs. patients with previous history of supraventricular rhythm disorder ( dashed line ) , or = 3.631 , 95% ci ( 1.3569.724 ) , p = 0.01 ( see table 3 for details ) . a total of 202 patients ( 71.6% ) had a diagnostic event within the 4-week monitoring ( table 2 ) . was present at patient - activated recording during palpitation in about two - thirds of the cases , while svt , af or afl , bradycardia , pauses or nsvt were present in the remaining cases . significant asymptomatic arrhythmias were detected by auto - trigger function in nine patients ( 3.2% ) . asymptomatic suggestive arrhythmias were detected by auto - trigger function in 21 patients ( 7.5% , table 2 ) , which per definition were not included as diagnostic events . meier cumulative occurrence of diagnostic events was 42.4% at 1 week , 57.2% at 2 weeks , and 77.0% at 4 weeks ( figure 1 ) . previous frequent palpitations and early start of recording after the index event were significant predictors of diagnostic events ( table 3 ) . the diagnostic yield progressively increased with the number of palpitations during the year preceding the enrolment ( figure 3a ) . the diagnostic yield was 46.2% in patients with 1 or no palpitation , 68.1% in patients with 25 palpitations , 76.7% in patients with 610 palpitations , and 83.1% in patients with 11 palpitations ( figure 3a ) . the 4-week diagnostic yield was higher in patients starting the recording 07 days after the index event ( table 3 , figure 3b ) . figure 3(a ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the previous number of events in the last 12 months ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the time between index event and enrolment ( see table 3 for details ) . ( a ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the previous number of events in the last 12 months ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the time between index event and enrolment ( see table 3 for details ) . the synarr - flash study enrolled 395 patients ( 42.3% males , age 56.9 18.7 years ) from 10 centres in five countries between august 2010 and june 2013 . as index event information was unavailable for three patients , the itt population included 392 patients ; 282 patients ( 71.9% ) enrolled for palpitations and 110 ( 28.1% ) for syncope . patients with syncope were older , more frequently males , with previous history of cardiovascular disease , family history of sudden death , hypertension , and use of cardiovascular medication ( table 1 ) . the mean duration of ecg monitoring was 23.0 8.1 days , similar for syncope and palpitations . in 22 patients , the recording was stopped for the occurrence of a major event . in four patients ( 1% ) , the recording was interrupted due to intolerance to electrodes , while two patients ( 0.5% ) withdrew from the study . skin reactions to electrodes were reported in 12 patients ( 3% ) , but all were defined as non - serious and solved spontaneously . twenty - seven patients with prior syncope ( 24.5% ) had a diagnostic event within the 4-week monitoring . conclusive events with ecg - documented syncope occurred in 11 patients ( 10% , table 2 ) : in 6 patients , supraventricular bradyarrhythmias were recorded during syncope , while in 5 cases , regular sinus rhythm or sinus tachycardia was recorded ( table 2 ) . auto - trigger function identified asymptomatic significant arrhythmias in 16 patients ( 14.5% , table 2 ) . table 2electrocardiogram findings during monitoring in patients with syncope and palpitationpatientssyncope ( n = 110)palpitation ( n = 282)n%n%diagnostic tests2724.520271.6conclusive events ( recurrence reported on patient diary)1110.019368.4non - arrhythmic ( sinus rhythm , sinus tachycardia)54.56623.4arrhythmic65.512745.0 pauses ( 6 s ) , avb 3 or avb 2 mobitz 243.610.4 pauses ( 36 s ) , sinus bradycardia21.872.5 paroxysmal af , afl ( > 3 min)00.0227.8 paroxysmal svt ( > 3 min)00.0155.3 non - sustained svt ( 15 s3 min)00.04014.2 nsvt < 10 s00.051.8 nsvt > 10 s00.010.4 frequent vpbs or apbs00.03612.7asymptomatic significant arrhythmias1614.593.2 fast sustained af / afl / svt > 3 min > 180 b.p.m.87.382.8 sinus bradycardia < 30 b.p.m . , pauses 6 s , avb 3 or avb 2 mobitz 254.510.4 nsvt > 10 s or sustained ventricular tachycardia > 30 s32.700.0non - diagnostic tests8375.58028.4negative monitoring7366.45920.9asymptomatic suggestive arrhythmias109.1217.5 sinus bradycardia 3040 b.p.m . , sinus pauses 36 s54.5113.9 nsvt < 10 s54.593.2 af / afl / svt 15 s3 min00.010.4avb 2 , avb 3 , atrio - ventricular block ( grade 2 or 3 ) ; af , atrial fibrillation ; afl , atrial flutter ; svt , supraventricular tachycardia ; nsvt , non - sustained ventricular tachycardia ; vpbs , ventricular premature beats ; apbs , atrial premature beats . electrocardiogram findings during monitoring in patients with syncope and palpitation avb 2 , avb 3 , atrio - ventricular block ( grade 2 or 3 ) ; af , atrial fibrillation ; afl , atrial flutter ; svt , supraventricular tachycardia ; nsvt , non - sustained ventricular tachycardia ; vpbs , ventricular premature beats ; apbs , atrial premature beats . the actuarial diagnostic yield , calculated by the kaplan meier cumulative occurrence of diagnostic events , was 13.2% at 1 week , 19.1% at 2 weeks , and 29.4% at 4 weeks ( figure 1 ) . figure 1kaplan meier analysis of recurrence - free rate in patients with syncope ( continuous line ) and palpitations ( dashed line ) of first diagnostic event during prolonged ecg monitoring ( log rank : p < 0.001 ) . kaplan meier analysis of recurrence - free rate in patients with syncope ( continuous line ) and palpitations ( dashed line ) of first diagnostic event during prolonged ecg monitoring ( log rank : p < 0.001 ) . auto - trigger function detected asymptomatic suggestive arrhythmias in 10 patients ( 9.1% , table 2 ) , although per definition were not included as diagnostic events . multivariable analyses to identify potential predictors of diagnostic events included age , gender , previous recording of supraventricular or ventricular arrhythmia , time from index event to enrolment ( 015 days vs. > 15 days after index syncope ) , the number of events in preceding year , history of cardiovascular disease , and family history of sudden cardiac death . the only two significant predictors for diagnostic findings during the 4-week monitoring were ( i ) early start of monitoring after index event and ( ii ) previous history of supraventricular arrhythmias [ sinus arrest or pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia > 100 b.p.m . , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , paroxysmal atrial flutter / tachycardia table 3 ] . monitoring was started within 15 days from index event in 74 patients : 4-week actuarial diagnostic yield was 33.0% in patients with early initiation compared with 15.6% in later initiation ( p = 0.021 , figure 2a ) . history of supraventricular arrhythmias was present in 39 patients : 4-week actuarial diagnostic yield was 43.6% in patients with supraventricular arrhythmias compared with 21.3% in those without ( p = 0.018 , figure 2b ) . table 3predictors for diagnosis during the 4-week ecg monitoringvariablesp - valueodds ratio95% wald confidence limitsa patients studied for syncope time between index event and enrolment015 days vs. > 15 days0.0216.21.329.6 history of supraventricular arrhythmiasyes vs. no0.0183.61.49.7b patients studied for palpitations history of previous palpitations ( in the previous 12 months)25 vs. 10.0362.61.16.3610 vs. 10.0292.81.17.311 vs. 1<0.0014.32.09.4 time between index event and enrolment07 days vs. 815 days0.0043.01.46.407 days vs. > 15 days0.0312.31.14.9supraventricular arrhythmias include sinus arrest / pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , and paroxysmal atrial flutter / tachycardia . figure 2(a ) kaplan meier analysis of recurrence - free rate in patients with syncope enrolled at 015 days after the index syncope ( continuous line ) vs. > 15 days after the index syncope ( dashed line ) , or = 6.222 , 95% ci ( 1.30929.573 ) , p = 0.02 ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with syncope without previous history of supraventricular rhythm disorder ( continuous line ) vs. patients with previous history of supraventricular rhythm disorder ( dashed line ) , or = 3.631 , 95% ci ( 1.3569.724 ) , p = 0.01 ( see table 3 for details ) . predictors for diagnosis during the 4-week ecg monitoring supraventricular arrhythmias include sinus arrest / pauses <3 s , sinus bradycardia > 40 b.p.m . , sinus tachycardia , paroxysmal af , frequent pacs ( > 10/min ) , chronic af , and paroxysmal atrial flutter / tachycardia . ( a ) kaplan meier analysis of recurrence - free rate in patients with syncope enrolled at 015 days after the index syncope ( continuous line ) vs. > 15 days after the index syncope ( dashed line ) , or = 6.222 , 95% ci ( 1.30929.573 ) , p = 0.02 ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with syncope without previous history of supraventricular rhythm disorder ( continuous line ) vs. patients with previous history of supraventricular rhythm disorder ( dashed line ) , or = 3.631 , 95% ci ( 1.3569.724 ) , p = 0.01 ( see table 3 for details ) . a total of 202 patients ( 71.6% ) had a diagnostic event within the 4-week monitoring ( table 2 ) . was present at patient - activated recording during palpitation in about two - thirds of the cases , while svt , af or afl , bradycardia , pauses or nsvt were present in the remaining cases . significant asymptomatic arrhythmias were detected by auto - trigger function in nine patients ( 3.2% ) . asymptomatic suggestive arrhythmias were detected by auto - trigger function in 21 patients ( 7.5% , table 2 ) , which per definition were not included as diagnostic events . meier cumulative occurrence of diagnostic events was 42.4% at 1 week , 57.2% at 2 weeks , and 77.0% at 4 weeks ( figure 1 ) . previous frequent palpitations and early start of recording after the index event were significant predictors of diagnostic events ( table 3 ) . the diagnostic yield progressively increased with the number of palpitations during the year preceding the enrolment ( figure 3a ) . the diagnostic yield was 46.2% in patients with 1 or no palpitation , 68.1% in patients with 25 palpitations , 76.7% in patients with 610 palpitations , and 83.1% in patients with 11 palpitations ( figure 3a ) . the 4-week diagnostic yield was higher in patients starting the recording 07 days after the index event ( table 3 , figure 3b ) . figure 3(a ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the previous number of events in the last 12 months ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the time between index event and enrolment ( see table 3 for details ) . ( a ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the previous number of events in the last 12 months ( see table 3 for details ) . ( b ) kaplan meier analysis of recurrence - free rate in patients with palpitations according to the time between index event and enrolment ( see table 3 for details ) . the synarr - flash study is the first international , multicentre , observational , prospective trial designed to prove the feasibility and usefulness of external prolonged ecg monitoring in early clinical work - up of unexplained syncope and/or sustained palpitations of suspected arrhythmic origin . in unexplained syncope , prolonged external ecg monitoring achieved a diagnosis in about one - third of the patients , and most diagnoses occurred in those who started the monitoring within 15 days from index syncope ( figure 2a ) and in those with history of svt ( figure 2b ) . these findings suggest two new clinical criteria in order to increase the diagnostic likelihood in unexplained syncope , namely early initiation of monitoring after an event and previous history of supraventricular arrhythmias . in unexplained palpitations , the 4-week actuarial diagnostic yield was 77.0% , increasing up to 81.3% in patients with previous frequent palpitations ( figure 3a ) , supporting its utility as first - line diagnostic tool in palpitation work - up . all enrolled patients had unexplained syncope of suspected arrhythmic origin according to the criteria outlined in 2009 guidelines for diagnosis and management of syncope . in this study , the actuarial diagnostic yield of the 4-week ecg monitoring was 29.4% , higher than the 110% diagnostic yield generally reported for 24 h holter recording , and consistent with previous studies utilizing external long - term ecg monitoring , reporting diagnostic yields of 1520% at 1 month . in our study , the 4-week diagnostic yield for syncope was similar or higher than in studies using ilr , when considering a similar monitoring period . in the picture registry , the 3-month diagnostic yield was 19% , and in most studies , the diagnostic yield of ilr ranged from 30 up to 50% , indicating that syncope often remained unexplained even after 3-year ilr monitoring . these data support the concept that diagnostic findings in most patients with unexplained syncope occur relatively early after an index event , making prolonged external ecg monitoring sufficient to obtain a clinical diagnosis in most cases , with only a few selected patients requiring a much longer monitoring period . in our study , arrhythmias were documented during syncope in about half of the patients , typically bradycardia or pauses leading to pacemaker implant , while in the remaining cases , no arrhythmias were detected , excluding an arrhythmic origin of syncope . these patients should then be referred for further clinical evaluation , possibly including carotid sinus massage ( csm ) or head - up tilt test ( hut ) or neurological work - up , while further prolonged ecg monitoring is not necessary . asymptomatic arrhythmias predefined as significant were detected by auto - trigger function in 16 patients ( 14.5% , table 2 ) . significant arrhythmias were considered diagnostic even in the absence of recurrent syncope following the 2009 syncope guidelines , with evidence class 1 level c. this classification was specifically proposed by the international study on syncope of unknown etiology ( issue ) investigators , with the aim to group the observations from prolonged ecg monitoring into homogeneous patterns in order to define an acceptable standard useful for future studies and clinical practice . of note , this classification has been utilized in most studies with ilr , where unsuspected asymptomatic arrhythmias in the absence of syncope were generally considered diagnostic findings . to avoid potential bias , for this study , we utilized more stringent criteria for definition of significant asymptomatic arrhythmias than those proposed by the 2009 syncope guidelines . asymptomatic suggestive arrhythmias were detected in 9.1% of patients ( table 2 ) , which per definition were not included in the diagnostic yield , although some of these arrhythmias , such as 36 s pauses , would have been considered diagnostic according to 2009 syncope guidelines . patients with asymptomatic suggestive arrhythmias seem to be the ideal candidates to continue prolonged ecg monitoring by ilr , to confirm or exclude the presence of significant arrhythmias . in syncope , early initiation of recording after an index event was a significant predictor of diagnosis during the 4-week monitoring ( figure 2a ) . this is consistent with some preliminary observations suggesting that syncopal events occur in clusters , making it crucial to initiate the monitoring as soon as possible after an event . a second significant predictor of diagnostic events was history of supraventricular arrhythmias , suggesting that patients with previous paroxysmal af are reasonable candidates to prolonged ecg monitoring after an unexplained syncope . of note , af was not included among ecg criteria suggesting an arrhythmic origin for unexplained syncope in 2009 guidelines . in patients with unexplained sustained palpitations , the diagnostic yield of external monitoring was 42.4% at 1 week , 57.2% at 2 weeks , and 71.6% at 4 weeks . these data are consistent with previous studies , making the 4-week external ecg monitoring the first - choice tool in the diagnostic workflow of unexplained palpitations . only patients with palpitations remaining unexplained after the 4-week monitoring may require further observation , either by ilr or by event recorders . the diagnostic yield was 50% at 1 week and 70% at 2 weeks in patients with previous frequent palpitations ( figure 3a ) or in patients studied early after an index event ( figure 3b ) . these data support the utility of 1-week and 2-week external recordings , especially when utilized in high - risk patients and started early after an event . sinus rhythm or sinus tachycardia , or short episodes of svt were observed during palpitations in most patients , while sustained and fast svt , af , or afl were documented in 13% of the cases ( table 2 ) . in fewer cases , sinus bradycardia and pauses the clinical significance or the clinical management of such findings was beyond the scope of this study . although the main objective of prolonged external ecg monitoring was to correlate symptoms and arrhythmias , asymptomatic paroxysmal af ( either brief or sustained episodes ) was detected in 10% of the patients with unexplained palpitations . these findings support the discrepancy between perceived symptoms and documented arrhythmias , and confirm that silent af is relatively frequent even in patients with history of palpitations . one possible limitation was that not all centres contributed with the same amounts of patients , as three centres ( valld'hebron , milan , and lavagna ) enrolled 75% of the patients . the information provided in patients ' diary was used to categorize for the absence or presence and kind of symptom ( syncope or palpitation ) at the time of arrhythmias . it is impossible to verify the accuracy of events reported in the diary by each patient , although the diary information was cross - checked at m1 clinic visits . predefined criteria were utilized to distinguish between significant and suggestive asymptomatic arrhythmias detected by auto - trigger function , based on recommendation from current guidelines and on our best clinical judgement . due to lack of consensus on the minimum duration required to define an episode of paroxysmal af , we utilized a restrictive criteria ( duration > 180 s ) for significant events , while shorter events were only considered suggestive . although the presence of fast and sustained supraventricular arrhythmias may suggest a syncope of arrhythmic origin , the precise mechanism provoking the syncope in the individual patient remains unknown , and the attribution of a specific aetiology for syncope in the individual patients is beyond the scope of this study . more stringent criteria were used also for pauses and nsvt , although some suggestive asymptomatic arrhythmias may represent meaningful findings in the diagnostic workflow and would have been considered diagnostic according to the 2009 syncope guidelines . information about previous diagnostic tests , specifically csm and hut , was collected wherever available , but it was beyond the power of this study to verify why a specific diagnostic test was performed or not performed in the single patient . this study population was too small to draw any definitive conclusion on the possible correlation between elr - negative and hut - negative findings . the cumulative diagnostic yield observed in this study both for syncope and palpitation may be an overestimation of the true clinical benefit . the capability of prolonged early monitoring to influence therapeutic decisions or to improve clinical outcomes was beyond of the scope of this study and remains to be demonstrated by an appropriately designed study . all enrolled patients had unexplained syncope of suspected arrhythmic origin according to the criteria outlined in 2009 guidelines for diagnosis and management of syncope . in this study , the actuarial diagnostic yield of the 4-week ecg monitoring was 29.4% , higher than the 110% diagnostic yield generally reported for 24 h holter recording , and consistent with previous studies utilizing external long - term ecg monitoring , reporting diagnostic yields of 1520% at 1 month . in our study , the 4-week diagnostic yield for syncope was similar or higher than in studies using ilr , when considering a similar monitoring period . in the picture registry , the 3-month diagnostic yield was 19% , and in most studies , the diagnostic yield of ilr ranged from 30 up to 50% , indicating that syncope often remained unexplained even after 3-year ilr monitoring . these data support the concept that diagnostic findings in most patients with unexplained syncope occur relatively early after an index event , making prolonged external ecg monitoring sufficient to obtain a clinical diagnosis in most cases , with only a few selected patients requiring a much longer monitoring period . in our study , arrhythmias were documented during syncope in about half of the patients , typically bradycardia or pauses leading to pacemaker implant , while in the remaining cases , no arrhythmias were detected , excluding an arrhythmic origin of syncope . these patients should then be referred for further clinical evaluation , possibly including carotid sinus massage ( csm ) or head - up tilt test ( hut ) or neurological work - up , while further prolonged ecg monitoring is not necessary . asymptomatic arrhythmias predefined as significant were detected by auto - trigger function in 16 patients ( 14.5% , table 2 ) . significant arrhythmias were considered diagnostic even in the absence of recurrent syncope following the 2009 syncope guidelines , with evidence class 1 level c. this classification was specifically proposed by the international study on syncope of unknown etiology ( issue ) investigators , with the aim to group the observations from prolonged ecg monitoring into homogeneous patterns in order to define an acceptable standard useful for future studies and clinical practice . of note , this classification has been utilized in most studies with ilr , where unsuspected asymptomatic arrhythmias in the absence of syncope were generally considered diagnostic findings . to avoid potential bias , for this study , we utilized more stringent criteria for definition of significant asymptomatic arrhythmias than those proposed by the 2009 syncope guidelines . asymptomatic suggestive arrhythmias were detected in 9.1% of patients ( table 2 ) , which per definition were not included in the diagnostic yield , although some of these arrhythmias , such as 36 s pauses , would have been considered diagnostic according to 2009 syncope guidelines . patients with asymptomatic suggestive arrhythmias seem to be the ideal candidates to continue prolonged ecg monitoring by ilr , to confirm or exclude the presence of significant arrhythmias . in syncope , early initiation of recording after an index event was a significant predictor of diagnosis during the 4-week monitoring ( figure 2a ) . this is consistent with some preliminary observations suggesting that syncopal events occur in clusters , making it crucial to initiate the monitoring as soon as possible after an event . a second significant predictor of diagnostic events was history of supraventricular arrhythmias , suggesting that patients with previous paroxysmal af are reasonable candidates to prolonged ecg monitoring after an unexplained syncope . of note , af was not included among ecg criteria suggesting an arrhythmic origin for unexplained syncope in 2009 guidelines . in patients with unexplained sustained palpitations , the diagnostic yield of external monitoring was 42.4% at 1 week , 57.2% at 2 weeks , and 71.6% at 4 weeks . these data are consistent with previous studies , making the 4-week external ecg monitoring the first - choice tool in the diagnostic workflow of unexplained palpitations . only patients with palpitations remaining unexplained after the 4-week monitoring may require further observation , either by ilr or by event recorders . the diagnostic yield was 50% at 1 week and 70% at 2 weeks in patients with previous frequent palpitations ( figure 3a ) or in patients studied early after an index event ( figure 3b ) . these data support the utility of 1-week and 2-week external recordings , especially when utilized in high - risk patients and started early after an event . sinus rhythm or sinus tachycardia , or short episodes of svt were observed during palpitations in most patients , while sustained and fast svt , af , or afl were documented in 13% of the cases ( table 2 ) . in fewer cases , sinus bradycardia and pauses the clinical significance or the clinical management of such findings was beyond the scope of this study . although the main objective of prolonged external ecg monitoring was to correlate symptoms and arrhythmias , asymptomatic paroxysmal af ( either brief or sustained episodes ) these findings support the discrepancy between perceived symptoms and documented arrhythmias , and confirm that silent af is relatively frequent even in patients with history of palpitations . one possible limitation was that not all centres contributed with the same amounts of patients , as three centres ( valld'hebron , milan , and lavagna ) enrolled 75% of the patients . the information provided in patients ' diary was used to categorize for the absence or presence and kind of symptom ( syncope or palpitation ) at the time of arrhythmias . it is impossible to verify the accuracy of events reported in the diary by each patient , although the diary information was cross - checked at m1 clinic visits . predefined criteria were utilized to distinguish between significant and suggestive asymptomatic arrhythmias detected by auto - trigger function , based on recommendation from current guidelines and on our best clinical judgement . due to lack of consensus on the minimum duration required to define an episode of paroxysmal af , we utilized a restrictive criteria ( duration > 180 s ) for significant events , while shorter events were only considered suggestive . although the presence of fast and sustained supraventricular arrhythmias may suggest a syncope of arrhythmic origin , the precise mechanism provoking the syncope in the individual patient remains unknown , and the attribution of a specific aetiology for syncope in the individual patients is beyond the scope of this study . more stringent criteria were used also for pauses and nsvt , although some suggestive asymptomatic arrhythmias may represent meaningful findings in the diagnostic workflow and would have been considered diagnostic according to the 2009 syncope guidelines . information about previous diagnostic tests , specifically csm and hut , was collected wherever available , but it was beyond the power of this study to verify why a specific diagnostic test was performed or not performed in the single patient . this study population was too small to draw any definitive conclusion on the possible correlation between elr - negative and hut - negative findings . the cumulative diagnostic yield observed in this study both for syncope and palpitation may be an overestimation of the true clinical benefit . the capability of prolonged early monitoring to influence therapeutic decisions or to improve clinical outcomes was beyond of the scope of this study and remains to be demonstrated by an appropriately designed study . the early use of external 4-week ecg recorders in unexplained syncope of suspected arrhythmic origin proved to be a feasible stepwise strategy , starting the recording as soon as possible after an event in order to maximize the diagnostic yield of external ecg monitoring . in patients with unexplained syncope of suspected arrhythmic origin , in whom prolonged ecg monitoring is considered appropriate according to current guidelines , the 4-week external ecg monitoring should be utilized as first step , while more expensive and minimally invasive ilr should be reserved to those cases who remained undiagnosed after the 4-week external monitoring . in patients with unexplained palpitation , the 4-week external ecg monitoring can be considered as first - line diagnostic tool , providing a conclusive diagnosis in most cases , avoiding useless repetition of standard 24 h holter monitoring , with longer monitoring by ilr would be required only in a few selected cases . the results of this study , which utilized external loop recording with auto - trigger function , may be extended to new systems of long - lasting external ecg recordings providing continuous ecg monitoring , that may have an even higher capability of detecting asymptomatic arrhythmias . in patients with unexplained palpitation , the 4-week external ecg monitoring can be considered as first - line diagnostic tool , providing a conclusive diagnosis in most cases , avoiding useless repetition of standard 24 h holter monitoring , with longer monitoring by ilr required only in few selected cases . this study was supported by a research grant of sorin group , which supplied the spiderflash - t recorders free of charge to all enrolling centres and provided logistic support and assistance for database management and statistical analysis . r.w . was supported as a clinical researcher by the fund for scientific research flanders ( fwo ) . funding to pay conflict of interest : jjf is a consultant for nkt therapeutics and receives research funding from nkt therapeutics and astellas . red cell transfusions are frequently used to treat severe anemia and to reduce the effects of sickle cells . unlike many patient populations , patients with scd have a much higher risk of adverse events from transfusion therapy , due in part to known antigen incompatibility between blood donors , who are mostly of european descent , and the blood recipients , who are of african descent . alloantibody formation in this population can be life - threatening , because delayed hemolytic transfusion reactions ( dhtr ) may sometimes initiate a hyperhemolytic process whereby there is not only hemolysis of transfused allogeneic cells but also the patient s own autologous cells . there are also descriptions of hyperhemolysis associated with autoantibodies , as well as cases with no antibodies . regardless of whether an antibody is present or not , the end result of these hyperhemolytic events is a profoundly low hemoglobin , even lower than pre - transfusion levels . despite profoundly low hemoglobin values in addition to avoidance of transfusions , therapies are often directed at suppressing the immune system ( i.e. corticosteroids and ivig ) . we identified an associated autoantibody that caused robust hemolysis and severe anemia , which was temporally associated with end - organ dysfunction and death . what makes this case unique is that the predominant organ affected was not the brain or the lung , but the bone marrow . bone marrow ischemia , the cause of vaso - occlusive pain , is common in scd ; however , extensive bone marrow necrosis is rare . this case underscores the risks associated with transfusion in scd , especially in a heavily alloimmunized patient . a 61 year - old female with hemoglobin sc disease on hydroxurea , a history of multiple red cell alloantibodies ( anti - e , -jk , -k , -n , -s , -cw , and js ) and previous delayed hemolytic transfusion reactions ( last recorded red cell transfusion was 3 years before the current admission ) , a chronic pain syndrome , and g6pd deficiency , presented to the hospital due to a vaso - occlusive pain crisis . on admission , her pain was reported to be located in her chest and back , which was similar to her previously reported chronic pain crises . her presenting laboratory tests were notable for a hemoglobin of 5.3 g / dl ( baseline of 8 - 9 g / dl ) , a platelet count of 120,000 cells / ul ( baseline of 140,000 cells / ul ) , and normal renal function ( baseline : 0.8 mg / dl ) . the blood bank work - up , however , was significant for a newly positive direct antiglobulin test ( dat ) ( polyspecific : 1 + , anti - igg : weak , anti - c3 : negative , eluate : negative ) , and a new anti - mcc , which was considered not clinically significant and has not been reported to cause hemolysis ( table 1 ) . she was started on a hydromorphone patient - controlled analgesia pump for pain management , and she was transfused two units of least incompatible ( incompatibility was from the new anti - mcc ) , abo compatible ( patient : b+ ) , leukocyte - reduced , sickle negative , and antigen - matched ( e- , jk(b- ) , k- , s,- by serology , and c- and js(a- ) by genotype ) red blood cells ( rbc ) , with no complications and an appropriate increase in hemoglobin to 7.4 g / dl . on her second day of admission ( hd 2 ) , her platelet count unexpectedly decreased to 61,000 cells / ul , and her count continued to decline through her hospitalization . as her reticulocyte count was also inappropriately low at the time ( 0.012 e6/l ) , her worsening thrombocytopenia was felt most likely to be due to myelosuppression from hydroxyurea . consequently , in addition to halting the hydroxyurea , she also received epoetin alfa ( 40,000 units sq ) for marrow stimulation . despite holding hydroxyurea and the dose of epoetin , she required an additional 4 units of phenotype - matched rbcs ( 1 unit hd 5 , 1 unit hd 8 , 2 units hd 12 ) during the hospitalization to maintain her hemoglobin level . although immune hemolysis was suspected at the time , no additional antibodies were identified ( table 1 ) . after her pain improved , she was discharged on hospital day 12 with a hemoglobin of 9.1 g / dl and a platelet count of 20,000 cells / ul ( a discharge lactate dehydrogenase ( ldh ) and total bilirubin were not performed ) . at her first outpatient follow - up , 6 days after discharge ( about 7 days from her last red cell transfusion ) , she reported new symptoms of fatigue , shortness of breath , and worsening pain . her hemoglobin at this visit was 2.7 g / dl , and platelet count was 20,000 cells / ul . of note , her reticulocyte percent was below the level of detection ( < 0.4% ) despite an erythropoietin level that was elevated at 132 miu / ml ( reference : 3.7 - 31.5 miu / ml ) . her laboratory results were consistent with intravascular red cell hemolysis : ldh was > 2500 u / l ( ref : 100 - 190 u / l , her baseline was 342 u / l ) , haptoglobin was 18 mg / dl ( ref : 30 - 200 mg / dl ) , and her total bilirubin was 5.0 mg / dl ( ref : 0.2 - 1.0 mg / dl ) with hemoglobinuria detected on urine screen . there was no evidence of disseminated intravascular coagulation , and no schistocytes were observed on her peripheral smear . her dat was now strongly positive , with 3 + polyspecific ahg , 1 + igg , 2 + c3 reactivity , and an eluate that was positive with all cells . of interest , the patient also had a new cold autoantibody with broad specificity and shown to react at body temperature ( 30 and 37 c ) as defined by thermal amplitude studies , suggesting a clinically - significant mixed ( igg / igm ) autoantibody . she was hospitalized again , and was given 2 units of compatible rbcs ( with an appropriate immediate post - transfusion response , post - transfusion hemoglobin 5.2 g / dl ) , epoetin alpha ( 40,000 units sq ) , and prednisone ( 1 mg / kg daily ) for a presumed mixed - type ( igg / igm ) hyperhemolysis syndrome ( figure 1 ) . . on hd 3 of readmission , she was started on intravenous immunoglobulin ( ivig ) at 500 mg / kg , received another dose of epoetin alpha ( 40,000 units ) , and 2 units of rbcs . she further developed evidence of marrow failure ( white count , 2,200 cells / ul ) on hd 4 prompting a bone marrow biopsy , which revealed diffuse and widespread bone marrow necrosis ( figure 2 ) . due to worsening of her thrombocytopenia , romiplostim ( 2 mcg / kg ) and 2 platelet transfusions were attempted without any meaningful effect on hd 10 ( figure 1 ) . she developed multisystem organ failure and continued to decline cognitively during the hospitalization , and was transitioned to comfort care . we present a unique case of transfusion - induced hyperhemolysis in an adult with scd . in this heavily alloimmunized , recently - transfused patient , an autoantibody that triggered hyperhemolysis was likely the key insult that ultimately led to her death . upon presentation with vaso - occlusive pain , the dat was only weakly positive , but after the patient received red cell transfusions , there was evidence that the autoantibody strengthened . later in the clinical course , laboratory testing clearly demonstrated complement - coated red cells , as well as a new clinically - significant mixed warm and cold autoantibody . the strengthening of the autoantibody was associated with worsening hemolysis and a clinical picture consistent with acute hyperhemolysis : a recent transfusion ( about 7 days before the hemoglobin nadir ) , a new red cell antibody , reticulocytopenia , and a severe anemia ( well below pre - transfusion levels ) . unfortunately , in this case , the severe anemia and resultant ischemia from red cell hemolysis likely fatally exacerbated extensive , clinically - significant bone marrow necrosis ; a process that likely began during her first hospitalization as reflected by her progressively strengthening dat and worsening thrombocytopenia during that time period . hyperhemolysis syndrome is a well - reported and life - threatening complication of red cell transfusions for patients with scd . clinically , the typical presentation of acute hyperhemolysis is severe anemia , developing about 7 days after a previous transfusion , with a hemoglobin that is lower than pre - transfusional levels . other common findings include reticulocytopenia , pain , fever , and signs of hemolysis , including jaundice , increased ldh , hyperbilirubinemia , and hemoglobinuria . while hyperhemolysis has no definitive diagnostic test , there is sometimes evidence of a new allo- or autoantibody . one potential reason for the autologous rbc destruction in these patients is the formation of a nonspecific antibody that binds complement and destroys both donor and recipient red cells , known as bystander hemolysis . as in our patient , previous studies have shown a strong association between the formation of nonspecific autoantibodies and the history of previous transfusions . another explanation for excess patient rbc destruction is the hyperactive phagocytic activity of macrophages . of note , sickled rbcs expose some antigens ( e.g. phosphatidylserine ) on their outer surface , which some the - orize can allow recognition by activated macrophages without antibody interactions . others consider the interaction between vcam-1 and 41 on sickled red cells , and cd11c and icam-4 on transfused donor red cells , to be critical for antibody - independent phagocytosis by activated macrophages in patients with scd . lastly , suppression of erythropoiesis has also been proposed as a cause or modulator of the disease . the severity of this patient s anemia ( hemoglobin of 2.7 g / dl ) , was notable . first , as mentioned above , this patient had an ongoing reticulocytopenia that prevented an appropriate response in the face of hemolysis . the reticulocytopenia may have developed from bone marrow necrosis and , potentially , the immune - mediated hemolysis of red cell precursors as well . second , the cold autoantibody active at 30c likely contributed to a more severe hemolysis . a recent study suggests that mixed - type immune hemolytic anemias may be more severe than other forms of immune hemolysis , with 63% of mixed hemolysis patients presenting with a hemoglobin < 6 g / dl in comparison to 29% of patients with typical warm autoimmune hemolytic anemia , and 9% of patients with cold agglutinin disease , respectively . moreover , hyperhemolysis in a patient with scd and a clinically significant cold autoantibody , anti - ih , has contributed in at least one reported fatal outcome from hyperhemolysis . our case , however , is the first where the hemolysis from a mixed - type auto agglutinin led to profound bone marrow failure . extensive bone marrow necrosis that affects hematopoiesis is a rare , but established , complication in patients with scd . greater than 90% of cases of bone marrow necrosis occur in the context of other diseases such as cancer , most often hematological malignancy , infection or autoimmune conditions . bone marrow necrosis in scd encompasses a broad spectrum of severity , from a localized process to one that is widespread . given that one of the most common sites for vaso - occlusion is in the bone marrow , aspirates often reveal patches of necrotic marrow , although rarely extensive enough to impact hematopoiesis , as was seen in our case . common symptoms of bone marrow necrosis include back pain , fever , cytopenias and a leukoerythroblastic picture ( immature white blood cells and nucleated red cells in peripheral blood ) . large autopsy studies suggest that the prevalence of diffuse bone marrow necrosis can range from 0.2 - 15 percent of susceptible patients . as of 2016 , about 60 cases of severe bone marrow necrosis in patients with scd has been reported in the literature . to our knowledge , none of these were reported to be precipitated by a previous red cell transfusion . of those reported cases , 64% had a fatal outcome and only 12 patients ( 21% ) documented complete recovery , all after receiving red blood cell exchanges . death from extensive marrow necrosis is secondary to the dissemination of fat emboli , acute multi - organ system failure , and acute chest syndrome episodes . there is no standard approach to the treatment of hyperhemolysis or extensive bone marrow necrosis . in the case of hyperhemolysis , transfusion of red cells is usually avoided , because it may precipitate increased bystander hemolysis and macrophage activation . our patient did receive red cell transfusions , especially when the clinical picture was unclear during her first admission . otherwise , our patient was treated with steroids and ivig to decrease antibody production and reduce the clearance of antibody - laden red cells , as has been reported to be beneficial in the literature . growth factors are also a mainstay , especially epoetin alpha , since reticulocytopenia is often a presenting feature . erythropoietin can correct anemia by stimulating red cell precursors and also mitigate the destruction of young red cells . additionally , rituximab ( anti - cd20 ) has been used in some cases with success . while we had some evidence that the patient s immune hemolysis was improving ( her direct antiglobulin test was showing weakening of her igg and c3 values during her second hospitalization ) , these therapies likely had no impact on her final outcome as none of these therapies will aid in bone marrow recovery . simple or exchange red cell transfusions may be beneficial for bone marrow necrosis to improve oxygenation and blood flow to the ischemic marrow ; unfortunately , aggressive transfusion therapy was not an option for our patient . this case demonstrates the unique risks of red cell transfusions in a patient with scd . severe anemia associated with hyperhemolysis was likely what caused the bone marrow necrosis that ultimately led to this patient s death . the bone marrow biopsy , which ultimately clarified her pancytopenia , should be seriously considered in patients with scd when severe anemia , reticulocytopenia , and thrombocytopenia are observed together . in conclusion , an awareness of the potential for serious complications , such as hyperhemolysis , is necessary when determining the need for transfusion in a patient with scd .

aimssynarr - flash study ( monitoring of syncopes and/or sustained palpitations of suspected arrhythmic origin ) is an international , multicentre , observational , prospective trial designed to evaluate the role of external 4-week electrocardiogram ( ecg ) monitoring in clinical work - up of unexplained syncope and/or sustained palpitations of suspected arrhythmic origin.methods and resultsconsecutive patients were enrolled within 1 month after unexplained syncope or palpitations ( index event ) after being discharged from emergency room or hospitalization without a conclusive diagnosis . a 4-week ecg monitoring was obtained by external high - capacity loop recorder ( spiderflash - t , sorin ) storing patient - activated and auto - triggered tracings . diagnostic monitorings included ( i ) conclusive events with reoccurrence of syncope or palpitation with concomitant ecg recording ( with / without arrhythmias ) and ( ii ) events with asymptomatic predefined significant arrhythmias ( sustained supraventricular or ventricular tachycardia , advanced atrio - ventricular block , sinus bradycardia < 30 b.p.m . , pauses > 6 s ) . synarr - flash study enrolled 395 patients ( 57.7% females , 56.9 18.7 years , 28.1% with syncope , and 71.9% with palpitations ) from 10 european centres . for syncope , the 4-week diagnostic yield was 24.5% , and predictors of diagnostic events were early start of recording ( 015 vs. > 15 days after index event ) ( or 6.2 , 95% ci 1.329.6 , p = 0.021 ) and previous history of supraventricular arrhythmias ( or 3.6 , 95% ci 1.49.7 , p = 0.018 ) . for palpitations , the 4-week diagnostic yield was 71.6% and predictors of diagnostic events were history of recurrent palpitations ( p < 0.001 ) and early start of recording ( p = 0.001).conclusionthe 4-week external ecg monitoring can be considered as first - line tool in the diagnostic work - up of syncope and palpitation . early recorder use , history of supraventricular arrhythmia , and frequent previous events increased the likelihood of diagnostic events during the 4-week external ecg monitoring . in patients with sickle cell disease , hyperhemolysis is a rare but life - threatening complication of transfusion . in this case report , we describe a 61 year - old woman with hemoglobin sickle cell ( sc ) disease and history of alloimmunization who developed hyperhemolysis associated with a transfusion . she was found to have a warm and a clinically - significant cold autoantibody . severe anemia ( hb 2.7 g / dl ) with reticulocytopenia and thrombocytopenia prompted a bone marrow biopsy , which demonstrated extensive bone marrow necrosis . despite treatment , the bone marrow failure did not improve and the patient died on hospital day 38 . this case illustrates the potential risks of transfusion in a patient with sickle cell disease , especially one with previous hemolytic reactions . while uncommon , hyperhemolysis can cause death , in this case by extensive bone marrow necrosis . in patients with sickle cell disease , judicious use of red cell transfusions with phenotypically - matched units can diminish , but never completely abrogate , the risks associated with transfusion .

the vertebral fractures is the most common complications of osteoporosis.13 ) these fractures result in significant mortality and morbidity including prolonged and intractable pain.4,13 ) percutaneous vertebroplasty , a therapeutic procedure for filling the collapsed vertebral body with polymethylmethacrylate , provides pain relief.2,4 ) in general , percutaneous vertebroplasty is simple and safe if performed under continuing fluoroscopic control and technical precautions . vertebroplasty has the potential risk of serious complications such as leakages of bone cement , cardiopulmonary complications , infection and the new fractures of the adjacent vertebrae.6,7 ) we present a case of extraspinal leakage after vertebroplasty at our hospital . a 73-year - old male was admitted to our hospital for low back pain after slip down at one day earlier . the patient had experienced l1 vertebroplasty and t12-l2 screw fixation for l1 compression fracture at another hospital before 2 years ago . after incision of the skin , an 11-guage vertebroplasty needle was placed percutaneously on the posterior part of the vertebral body via bilateral transpedicular approach . the needle was pushed through the cortex , situated the center of the pedicle as possible , and then directed into the vertebral body . the contrast medium ( iohexol ) was injected to estimate bone cement distribution and minimize bone cement leakage and intraoperative complication such as pulmonary thromboembolism . when a thin toothpaste consistency was achieved , the bone cement was injected into the vertebral body under continuous fluoroscopic control . the filling process was stopped immediately when leakage of bone cement was observed into lateral space of body . lumbar simple x - rays after procedure showed leakage of bone cement to right lateral side of the l4 vertebral body ( figure 1 ) . lumbar computed tomography ( ct ) scanning revealed extraforaminal leakage of bone cement to outside of right l4 pedicle and it caused significant compression of l4 nerve root ( figure 2 ) . the leaked bone cement along the l4 nerve root was removed carefully via paraspinal muscle - splitting approach . during surgery , two pieces of bone cements were removed carefully . two whitish bone cements were located in the right lateral space of l4 - 5 body and the intervertebral foramen ( figure 3 ) . postoperative lumbar spine plain images show that the leaked bone cement along right l4 nerve root was disappeared ( figure 4 ) . complications related with vertebroplasty are not uncommon such as cement extravasation , pulmonary embolism , infection , epidural hematoma , systemic toxicity , and vertebral body fractures.1,7,8 ) among the commonest complications , cement extravasation has been estimated in 70% cases for vertebroplasty procedures.3,5 ) most of the cement extravasation is asymptomatic but , disastrous complications causing paraparesis have been also reported in the literature.5,7,12 ) the biplanar fluoroscopy is the best way to monitor the leakage of cement in procedure of vertebroplasty , but sometimes it is difficult to judge the leakage of cement after injection of the contrast medium . yeom et al.14 ) described three different types of leakages of cement after vertebroplasty such as type b ( via basivertebral vein ) , type s ( via segmental vein ) , and type c ( via cortical defect ) . nieuwenhuijse et al.9 ) recommended that the high viscosity of bone cement should decrease the rate of cement leakage during vertebroplasty . due to high risk of pedicle fracture during vertebroplasty , cement leakage via pedicle violation can cause significant morbidity.11 ) park and kim10 ) mentioned technical tips while taking entry into the pedicle and the vertebral body . the bone access needle can be advanced through the center of pedicle with use of a tunnel vision technique ( turn the c - arm obliquely until pedicle can be visualized at its widest and roundest ) and verification of needle trajectory at midpedicular level and posterior surface of vertebral body on lateral view . on anterior - posterior view , if the tip is located too medially , the medial cortical wall of pedicle may be damage and leading to spinal canal violation . if the tip is located too far laterally , lateral cortical bone damage and lateral violation could occurred . in our case , the reason of cement leakage might be presumed . the tip of needle located more laterally when needle tip is located in the pedicle and vertebral body . we experienced a case of lumbar nerve root compression by leaked bone cement after vertebroplasty . proper entry point of trocar , keeping trocar within pedicle and proper position of trocar between the pedicle and spinous process under biplanar fluoroscopy view would be help to prevent leakage of bone cement after vertebroplasty . stridor in children is usually due to airway pathology but neurological conditions causing stridor are not infrequent . in cases with an underlying neurological pathology causing stridor , the treatment of the underlying cause usually relieves the stridor . we report the association of stridor in a child presenting with hydrocephalus and raised intracranial pressure following the repair of a lumbosacral myelomeningocele ; where the stridor resolved after the placement of a ventriculo - peritoneal ( v - p ) shunt . to the best of our knowledge , an 8-month - old female baby born at term by normal vaginal delivery , from a second - degree consanguinous marriage ; presented with noisy breathing and progressive increase in the size of the head for 4 weeks . the child underwent lumbosacral myelomeningocele repair 2 months ago . on examination , she was awake , irritable and had noisy breathing that was aggravated on crying . her heart rate was 123/min , blood pressure was 80/50 mm of hg , respiratory rate was 38/min and oxygen saturation was 99% on room air . glasgow coma scale was 15/15 , head circumference was 48 cm and the anterior fontanelle was bulging and tense . both upper limbs were spastic and the lower limbs were flaccid with grade 0 power . contrast enhanced computed tomography ( cect ) brain showed dilatation of all the ventricles s / o obstructive hydrocephalus [ figure 1 ] . she was posted for an emergency v - p shunt insertion . contrast enhanced computed tomography brain showing dilatation of all the ventricles s / o obstructive hydrocephalus informed high - risk consent was obtained from the parents for the procedure . standard american society of anesthesiologists monitors , including electrocardiogram , noninvasive blood pressure , spo2 , etco2 and temperature monitoring were used . the left side v - p shunt ( medium pressure chabbra system ) was placed , and tracheal extubation was uneventful . postoperatively the child 's stridor decreased , head circumference was 47 cm , anterior fontanelle became lax , and she became active , playful and started feeding normally . the association of myelomeningocele with hydrocephalous and stridor has been reported earlier . in the report by kirsch et al . , the child underwent myelomeningocele repair and a ventriculoatrial shunt was placed at the age of 23 days but presented with cyanosis and stridor at the age of 16 months requiring tracheostomy . described a child presenting with hydrocephalous and stridor , 5 months after she underwent lumbar myelomeningocele repair . solan and glaisyer described a neonate with stridor after the development of hydrocephalus secondary to surgical repair of the lumbosacral myelomeningocele . in this report , the stridor resolved completely after an emergency ventricular tap was performed , and the vp shunt procedure was performed only later . similar to our case , adeloye et al . described a child who presented initially with lumbar myelomeningocele and later developed hydrocephalus and severe laryngeal stridor , that resolved dramatically after the placement of a v - p shunt stridor is defined as the sound caused by abnormal air passage into the lungs and may be caused by extra thoracic ( nose , pharynx , larynx , trachea ) or intra - thoracic airway ( tracheobronchial tree ) obstruction . other causes include secondary airway lesions , vocal cord paralysis ( vcp ) , subglottic stenosis , tracheal anomaly , laryngeal cleft , vascular and lymphatic malformation , laryngeal papillomas , craniofacial abnormalities , and even head and neck tumors . the stridor in our case was probably due to unilateral vocal cord palsy due to stretching of the vagus nerve as it resolved promptly after the v - p shunt was performed . the differential diagnoses in our case could be bilateral vocal cord palsy or other congenital or infective laryngeal pathology causing stridor . vocal cord paralysis may be idiopathic , iatrogenic , and secondary to neurological abnormality including arnold chiari malformation [ acm ] , cerebral palsy , hydrocephalus , myelomeningocele , spina bifida , or hypoxia or birth trauma . described 21 infants and children with bilateral abductor vcp and associated meningomyelocele , acm , and hydrocephalus . they reported a temporal relationship between increased intracranial pressure ( icp ) and vcp , apnea , aspiration , and dysphagia . according to chen and inglis in infants with bilateral vcp , acm with hydrocephalous and myelomeningocele is the commonest neurologic finding . various causes have been proposed for the development of vcp in patients with raised icp with neurological disorders including ; the downward displacement of the brainstem secondary to increased icp ; stretching , compression or ischemia of the vagus nerves during their course from the nuclei ambiguii to the jugular foramen ; circulatory impairment of the brainstem and primary brainstem dysgenesis with hypoplasia of the cranial nerve nuclei . the treatment options include decompression of increased icp by temporary measures such as cerebrospinal fluid tap followed by a definitive v - p shunt or decompression of the foramen magnum to reduce the pressure difference between intracranial and intra spinal compartments . usually , the stridor will be relieved after the decompression procedure but some authors have reported worsening of symptoms after the surgical procedure . ideally the ventricular shunt should be performed within 48 h of onset of hindbrain dysfunction . tracheostomy may be required if the stridor does not resolve by 48 h after the definitive surgical procedure . in the perioperative period the avoidance of further increase in icp at the time of induction and intubation can not be over emphasized . early visualization of the larynx is required in patients who become stridulous postoperatively , especially those who have undergone prior thoracic procedures or with neurologic disorders associated with intracranial hypertension . stridor in children is not always due to airway pathology and clinicians should be aware of the neurological associations of this symptom . when associated with neurological conditions , relieving the neurological condition often decreases the stridor and hence may avoid unnecessary airway intervention and evaluation .

we experienced a 73-year - old male with lumbar nerve root compression due to leakage of bone cement after vertebroplasty . he was underwent vertebroplasty for acute osteoporotic l4 compression fracture at our hospital . after vertebroplasty , his back pain was improved but right leg pain was newly developed . lumbar computed tomography scanning showed that bone cements were leaked along the l4 nerve root . the leaked cements around l4 nerve root were removed carefully via paraspinal muscle - splitting approach . after operation , severe right leg radiating pain was improved . we recommend proper entry point , high viscosity of polymethylmethacrylate and constant monitoring can reduce complication . stridor in children is usually , but not always caused by airway pathology . the anesthesiologists should have a sound knowledge of the neurological associations of stridor and its management . in such cases , prompt treatment of the neurological pathology usually resolves the stridor and may prevent unnecessary airway evaluation and intervention in the child .

the success of dental implant is primarily based on good osseointegration , which depends on the biocompatibility of the implant material and implant surface properties , as well as on the quantity and quality of remaining tissue.1 commercially pure titanium and titanium alloys are traditional materials used in most commercially available endosseous implants because of favorable mechanical properties and excellent biocompatibility.2 and titanium has been proven to be safe and efficient as an implant material in many studies.3,4 but recently , the biostability of titanium is increasingly questioned . corrosion products in inner organs5 and galvanic side effects6 have been reported while using titanium implants . and allergic reactions and sensitivities to titanium have been also reported.7 esthetic problems may arise due to dark metallic color of titanium when the thickness of soft tissue is insufficient to mask it and soft tissue resorption is expected . long - term stability of esthetic implant restorations in anterior dentition is a challenge due to thin buccal bone and gingiva . while the survival rate and function of implant were main issues in implant dentistry in the past , nowadays the esthetic aspect of it became more focused . the success rate of the anterior maxillary implants was lower than other zones of the dental arch due to esthetic failure , while the survival rate of it showed no significant difference.8 thus , tooth - colored materials have been considered as an implant fixture materials and zro2 might be the choice of material as the tooth - colored implant . zirconia is a bioinert nonresorbable material that has good chemical and dimensional stability , and a high bending strength and fracture toughness.9 covacci et al . tested the potential toxicity of zirconia and the cell capability to grow and adhere upon the ceramic surfaces , and reported that zirconia showed good cytocompatibility and had no mutagenic and carcinogenic effects on cells.10 zirconia shows less accumulation of plaque than titanium , and results in less irritation through the so - called bio - film . reported the low potential for bacterial colonization of zirconia.11,12 much study on the biocompatibility and mechanical properties of zirconia has been done and zirconia has come to be used commercially as implant fixture materials . however , the gene expression levels between titanium and zirconia ceramic related to osteoblast adhesion and signal transduction are unknown . this study was performed to define attachment and growth behavior of osteoblast - like cells mc3t3-e1 cultured on zirconia discs by mtt assay , scanning electron microscope , and alp activity assay , and evaluate the gene expression of mc3t3-e1 cells on zirconium oxide surfaces compared to titanium surface using the cdna microarray . co. , ltd , suwon , korea ) used for the cell culture were machined from grade 4 titanium alloy . the discs were prepared to be 10 mm diameter and 2 mm thick and used as the culture substrate in the control group ( titanium disc group ) . paul , mn , usa ) of y - tzp ( yttria - stabilized tetragonal zirconia polycrystal ) were prepared in 10 mm diameter and 2 mm thick ( zirconia disc group ) . the surface roughness values for each group were measured using 3d - interactive display ( wyko nt8000 , veeco instruments inc . , the surface of titanium discs ( a ) had an average roughness ( ra ) value of 322 nm , while that of zirconia discs ( b ) had an average roughness ( ra ) value of 5.09 nm ( fig . disc samples were rinsed twice in absolute alcohol and once in demineralized water in ultrasonic , before sterilization by autoclave . mc3t3-e1 cells , osteoblast - like cells from rat calvaria were obtained ( korean cell line bank , seoul , korea ) and cultured in sterile cell culture plate containing alpha minimal essential medium . cells were collected and seeded at a density of 1 10 cells / ml by using 0.1% trypsin , 0.02% edta in ca and mg -free eagle 's buffer for cell release . one set of wells contained sterile 24 discs of titanium , whereas another contained 24 discs of zirconia at 37 in 5% co2 for 24 hours and the samples were moved to new dishes and media was added and the plated discs were cultured at 37 in 5% co2 for 24 hours . all cell culture media were supplemented with 100 units / ml penicillin - g , 100 g / ml streptomycin , and 0.25 g / ml fungizone ( gemini bio - products , inc . , woodland , ca , usa ) . total rna extraction was performed with qiagen mini kit ( qiagen , chatsworth , ca , usa ) for microarray assay . cell suspension ( 1 10 cell / ml ) was seeded to titanium discs and zirconia discs . the cultures were incubated for 4 and 24 hours to evaluate the influence of the substrate geometry on titanium and zirconia using sem . at the end of the various incubation times , the non - attached cells on the different substrates were removed by rinsing twice with 0.1 m sodium cacodylate buffered solution , dehydrated in a series of ethanol , dried by tetramethylsilane ( merck , darmstadt , germany ) and sputter - coated with au - pd ( bio rad , sc-500 , hertfordshire , uk ) . finally , they were observed in a sem ( jsm-6430f , jeol , tokyo , japan ) at an accelerating voltage of 15kv . three runs of experiments were carried out , which included all different samples in threefold . the mtt test assay ( sigma , st . louis , mo , usa ) was used for examining attachment of cells after culturing the cells on titanium discs and zirconia discs for 4 hours and 24 hours . the substance used for mtt test was a 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyl - tetrazolium salt , which turns into a blue formazan product due to the viable mitochondria in active cells . the cells were cultured on the 12 discs placed in 24-well culture plates and incubated at 37 in 5% co2 for 4 hours and 24 hours . the discs were moved to new 24- well culture plates after 4 hours and 24 hours of incubation and new media was added . mtt solution ( 5 mg / ml ) was added and incubated at 37 for 4 hours in 5% co2 . the media was discarded and 400 l of isopropanol with 0.04 n hcl was added in each well . the product solution was moved into 96-well plates . the absorbance at 570 nm was measured using microplate reader ( bio - rad , hercules , ca , usa ) . cell suspension ( 1 10 cell / ml ) was seeded to titanium discs and zirconia discs . the cells were added with 0.1% triton x-100 ( polyscience , warrington , pa , usa ) and cultured for 30 minutes . finally , the cells were added with 100 mm p - nitrophenyl - phosphate in 0.1 m glycine - naoh ( ph 10.4 ) and cultured for 10 minutes . the absorbance at 409 nm was measured using microplate reader ( bio - rad , hercules , ca , usa ) . the cdna microarray agilent rat 22k chip ( digital genomics , seoul , korea ) was used to monitor the expression of 21575 genes . the samples ( 10 g total rna per condition ) were processed according to the manufacturer 's recommendation . fluorescent - labeled cdna for oligo microarray analysis was prepared by amplification of total rna in the presence of aminoallyl - utp followed by the coupling of cy3 or cy5 dyes ( amersham pharmacia , uppsala , sweden ) . rna extracted from cells grown on titanium discs was labeled with cy3 and used as control against the cy5-labeled rna extracted from cells grown on zirconia discs in the first experiment and then switched . oligo microarray kit was hybridized with the fluorescently labeled rna at 60 for 16 hours and then washed . this microarray contains 21575 distinct sequences ( 1075 control spots and 20500 gene spots ) . dna chips were scanned using genepix 4000b ( axon instruments , union city , ca , usa ) . scanned images were analyzed with genepix pro 3.0 software ( axon instruments , union city , ca , usa ) to obtain gene expression ratios . test mean values and standard deviation ( sd ) were computed for mtt test and alp activity assay . nonparametric test for two group comparison by mann - whitney test was used to assess the significance level of the differences between the experimental groups . all statistical analyses were performed using spss software ( version 12.0 , spss inc . , the general shape and growth pattern of the osteoblast - like cells mc3t3-e1 was observed using sem for each group . 3 shows representative sem images of mc3t3-e1 cells cultured for 4 hours and 24 hours on titanium discs and zirconia discs . after 4 hours of culture , cells on both discs were irregularly triangular or elongated in shape with formation of filopodia that means good attachment to the substrate . after 24 hours of culture , cells on both discs were observed to be more flattened and well spread . the density of mc3t3-e1 cells was measured over 2 different time - periods ( 4 hours and 24 hours ) by using the mtt assay . 4 shows the optimal density of formazan produced by the mc3t3-e1 cells on zirconia and titanium surfaces . after 4 hours of adhesion , the osteoblast - like cell density on titanium surfaces and zirconia surfaces showed no significant difference ( p>.05 ) . after 24 hours of adhesion , the optical density of both groups significantly increased but there was no significant difference between both groups ( p>.05 ) . the optical density of cells on titanium discs was slightly higher than that of cells on zirconia discs but there was no significant difference ( p>.05 ) . 5 shows the optimal density of the differentiation rate of the cells on zirconia and titanium surfaces . up regulated genes were classified as cdna clones that exhibited a 2-fold or greater change in expression level . the up- and down - regulated genes in zirconia surface are reported in table 1 and table 2 . when an implant is inserted into bone , it is expected that an apposition of bone to the implant surface will occur , a process called osseointegration.13 for this osteoinductive event , gaps between bone and implant must be sealed , and damaged bone by preparation of implant site must be repaired . the initial responses on implant in the bone are comprised of two aspects : the response of the host to the implant and the behavior of the material in the host . the almost immediate event that occurs upon implantation of biomaterials , is adsorption of proteins.14 this biological protein layer formed on the surface of implant biomaterials is believed to be responsible for host cell response . the ability of protein adsorption depends on the surface macro- and micro - topography , chemistry , and energy of implant biomaterials.15 the host response to implants placed in bone involves a series of cell and matrix events involving many physiologic , chemical and genetic pathways.16 this study emphasizes the cell response to implant biomaterials , especially zirconia ceramics . reported that direct bone apposition was observed at bone - zirconia interfaces in histological and ultrastructural studies , and suggested that zirconia may also be a suitable implant material.17,18 ko et al . investigated the initial osteoblast - like hos cell response to yttrium - stabilized tetragonal zirconia polycrystal ( y - tzp ) and reported that y - tzp showed at least equivalent or slightly better biological response of osteoblast - like hos cells than pure titanium during a short - time cell culture period.19 in our experiment , mtt assay and sem examination showed that cellular attachment and proliferation were comparable between titanium and zirconia on a short - time cell culture period , which suggests that zirconia implants may be as favorable as titanium implants in attachment of osteoblasts on initial healing period after implant placement . alp activity is an important parameter typically used as markers of osteoblastic differentiation . in our experiment , the differentiation rate of e1 cells on cultured titanium and zirconia discs was similar each other . we used the cdna microarray technique for identifying the expression of important genes in cells adhesion , signal transduction , and transcription , based on previous literatures . cell adhesion to materials play important roles in biological processes including cell motility , cell proliferation , cell differentiation , regulation of gene expression and cell survival . there are three major parts , involved in cell adhesion to materials ; extracellular matrix ( ecm ) proteins , cytoskeletal molecules , cell adhesion molecules ( cams ) . some ecm proteins like fibronectin , osteopontin , bone sialoprotein , thrombospondin , type i collagen , vitronectin have chemotactic or adhesive properties due to their arg - gly - asp ( rgd ) sequence which is specific to the fixation of cell transmembrane receptors like integrin.20 in our experiment , fibronectin type iii , type i collagen , and vitronectin showed similar expression levels between titanium and zirconia . at the site of contact between tissue - cultured cell and extracellular matrix , absorbed onto substrate surface , specialized structures , so - called " focal contacts " or " adhesion plaque " , this adhesions are closed junctions where the distance between the substrate surface and the cell membrane is about 10 - 15 nm.21 on the intracellular part , cytoskeletal proteins are involved in signal transduction with integrin , proteases , protein kinases , kinase phosphatases , and other signaling molecules.22 among the genes related to regulation of actin cytoskeleton , integrin 8 ( itga8 ) were upregulated , while protein tyrosine kinase 2 ( ptk2 ) , integrin 3 ( itga3 ) were downregulated in zirconia . integrins are integral cell - surface proteins , which are known to participate in cell - surface adhesion , as well as cell - surface mediated signaling.23 integrin - mediated adhesion is a highly regulated process involving receptor ligand interactions and subsequent adhesion strengthening and cell spreading . in our experiment , integrin 8 ( itga8 ) was upregulated , while integrin 3 ( itga3 ) , 5 ( itga5 ) were downregulated in zirconia . it was reported that adhesion to fibronectin was inhibited by antibodies to the itga5.24 among other genes involved in focal adhesion , laminin 1 , 5 , 1 subunit 1 were down - regulated in zirconia . laminin is a major non - collagenous protein of the basal lamina and provides excellent conditions for the promotion of neurite outgrowth.25 among the cell adhesion molecules ( cams ) observed in this experiment , thiopurine methyltransferase ( tpmt ) , an enzyme that methylates thiopurine compounds , and cd6 antigen ( cd6 ) , surface molecule that may regulate cd5 tyrosine phosphorylation , were downregulated in zirconia . syndecan 1 ( sdc1 ) , a member of the syndecan proteoglycan family , was also downregulated in zirconia . the syndecans mediate cell binding , cell signaling , and cytoskeletal organization.26 tight junctions ( or zonula occludens ) are composed of transmembrane protein and a cytoplasmic " plaque " consisting of many different proteins that form large complexes . the transmembrane proteins mediate cell adhesion and constitute the intramembrane and paracellular diffusion barriers.27 tight junction protein complex , consist of cytoplasmic " plaque " of tight junctions , appears to organize the transmembrane proteins and couple them to other cytoplasmic proteins and to actin microfilaments.28 in our experiments , upregulation of tight junction protein 1 ( tjp1 ) was observed . protein tyrosine phosphatases ( ptps ) may promote the disassembly or turnover of focal contact and regulate the phosphorylation state of many signalling molecules , such as the mitogen - activated protein ( map ) kinase family.29 the expression of most of ptps showed similar expression level between titanium and zirconia . , while protein tyrosine phosphatase , receptor type , f polypeptide ( ptprf ) , interacting protein , 1 , protein tyrosine phosphatase receptor type , m ( ptprm ) , and protein tyrosine phosphatase receptor type , u ( ptpru ) were downregulated in zirconia . growth hormone receptor ( ghr ) was up regulated , while transforming growth factor , leukemia inhibitory factor ( lif ) , and fibroblast growth factor were down regulated in zirconia . using cdna microarray , total of 20215 gene expressions were observed and only 147 genes were up regulated and 838 genes were down regulated in zirconia . however , most genes related to cell adhesion showed similar expression level between titanium and zirconia , which correlated with the results of sem and mtt assay . also , most genes related to signal transduction including the activation of several pathways showed similar expression level between titanium and zirconia . thus , these results suggested that zirconia ceramics could regulate genetic effect of osteoblast - like cells , as titanium did . recently , surface treatments , like the change of surface topography , chemistry , and material composition , are on trial to improve cell adhesion and osteoinductive potential on titanium implants . further study is necessary to investigate biological response of cells on zirconia surface with various surface treatments . zirconia ceramic showed comparable biological responses of osteoblast - like cells to titanium during a short - time cell culture period . most of the genes related to cell adhesion and signal transduction including the activation of several pathways showed similar expression level between titanium and zirconia . this suggests that zirconia implants could be as favorable as titanium implants in attachment of osteoblasts on initial healing period after implant placement . techniques for instrumented fixation and fusion of the cervical spine have rapidly advanced in the past 30 years . these techniques have helped surgeons adequately manage complex trauma , tumor , and degenerative pathologies . hadra in 1890 described the use of wiring and graft techniques to perform the first spinal fusion procedures . these techniques were standard until the start of modern cervical spinal fusion with roy - camille in the 1970s . since this time there have been improvements in technique and implants , which have made the procedures safer , thus leading to increased adoption of instrumented cervical fusion . within the united states , approximately 150 000 cervical fusion procedures are performed each year . around 90% of cervical fusions the use of plate and screw instrumentation for anterior cervical fusion has become a mainstay , so much so that in 2010 the american medical association combined 2 current procedural terminology codes into a comprehensive code . the indications for cervical fusion are well established , based on an abundance of studies showing clinical and biomechanical efficacy . despite these studies there is a paucity of literature detailing the complications associated with cervical spinal fusion . most studies are retrospective in design , contain a small numbers of patients , report single - institution data , relate to a single indication , or contain information with outdated procedures . the overall complication rate for cervical fusion procedures is low , and these earlier studies may not reliably provide data on less common complications . while rare , screw backout or misplaced screws can lead to morbidity , the need for revision surgery , and increased costs . we have conducted a retrospective multicenter case series study involving 21 high - volume surgical centers from the aospine north america clinical research network , selected for their excellence in spine care and clinical research infrastructure and experience . medical records for 17 625 patients who underwent cervical spine surgery ( levels from c2 to c7 ) between january 1 , 2005 , and december 31 , 2011 , inclusive , were reviewed to identify occurrence of 21 predefined treatment complications . the complications included reintubation requiring evacuation , esophageal perforation , epidural hematoma , c5 palsy , recurrent laryngeal nerve palsy , superior laryngeal nerve palsy , hypoglossal or glossopharyngeal nerve palsy , dural tear , brachial plexopathy , blindness , graft extrusion , misplaced screws requiring reoperation , anterior cervical infection , carotid artery injury or cerebrovascular accident , vertebral artery injuries , horner s syndrome , thoracic duct injury , tetraplegia , intraoperative death , revision of arthroplasty and , pseudomeningocele . trained research staff at each site abstracted the data from medical records , surgical charts , radiology imaging , narratives , and other source documents for the patients who experienced one or more of the complications from the list . copies of case report forms were transferred to the aospine north america clinical research network methodological core for processing , cleaning , and data entry . test was used to analyze changes in clinical outcomes at follow - up compared to preoperative status . a total of 12 903 cervical procedures qualified for screening for the complication of misplaced screw requiring reoperation . there were 11 instances of misplaced screws requiring reoperation , for an incidence of 0.085% , translating to a rate of 8.5 per 10 000 cervical spine surgeries ( table 1 ) . a total of 8887 cases screened were specifically only anterior procedures making the incidence 3/8887 ( 0.034% ) . we do not have enough information on the remaining cases to make a determination of the incidence of misplacement of posterior screws requiring reoperation , but suspect it would a higher rate . the average age of the patients was 53 years , and there were 4 female patients . myelopathy and radiculopathy were the most common reasons for initial operation , 6/11 ( 55% ) and 5/11 ( 45% ) , respectively , with 1 patient having both symptoms . importantly , there were no changes in the health - related quality - of - life metrics due to this complication . there were no new neurologic deficits ; patients most often presented with pain , and screw backout was diagnosed on postoperative plain x - ray or computed tomography scan . five of 11 ( 45% ) and 3/11 ( 27% ) patients were diagnosed within the original hospital admission or postoperative clinic follow - up , respectively , while we were unable to obtain these data on 3 cases . the most common location for screw backout was c6 , 4/11 ( 36% ) ; 2 patients had screw backout at c2 . patient 4 had aggressive removal of an osteophyte complex and an anterior cervical screw penetrated into the central canal ( figure 1a and b ) . the patient had symptoms of right - sided proximal upper extremity weakness , which resolved with removal and revision of the c5 penetrating screw ( figure 1c ) . ( c ) postoperative lateral cervical spine x - ray demonstrating removal of misplaced c5 screw . due to the risk and complexity associated with cervical spinal surgery , current techniques and approaches did not become popular until the mid-20th century . advances in imaging , operative microscopes , and instrumentation have allowed cervical spinal surgery to progress to the current state . since 1891 when the first cervical fusion procedure was performed there have been sequential advances described by many authors to develop the early fixation techniques . several biomechanical animal and cadaver studies have been performed during the rapid advancement in cervical spinal instrumentation over the past 30 years . posterior instrumented fusion studies have consistently proven the stiffness of the constructs to be equal to or greater than that of an intact spine . the most common failure in these studies was screw pullout at a predictable amount of force . anterior plating studies have strongly guided the development of each type of system and have shown continued improvement in the efficacy of constructs . the advancements and improvements in cervical spinal instrumentation would be expected to decrease the rate of hardware - related complications . the overall complication rate of cervical spinal surgery is estimated to be 0.2% to 17.8% . after 30 days , the most common complication is related to instrumentation dislodgement , breakage , or both , and occurs in 2% to 25% of cases of spinal fusion procedures . reoperation rate for instrumented spinal fusions is reported between 2% and 25% , with the majority related to pseudoarthrosis . the majority of information is based on case reports , retrospective reviews , and small series of patients . there are few recent articles addressing the rate of screw backout or misplacement requiring reoperation , and many older studies include the use of unrestricted devices that permitted screw backout . fehlings et al prospectively collected 302 cervical spondylotic myelopathy patients and found a perioperative complication rate of 15.6% , with 11.6% minor and 7.0% major perioperative risk . three patients had instrumentation malposition / migration for a rate of 1% in the perioperative period . three patients had instrumentation / graft migration or instrumentation failure , making the risk of delayed hardware complications 1.1% . of 195 anterior cervical plating procedures , 5 ( 2.6% ) required reoperation for hardware failure . al used the marketscan database and identified 13 662 patients who underwent anterior or posterior cervical fusion for degenerative disc disease . the rate of any complication excluding dysphagia was 12.3% and 17.8% for anterior and posterior approaches , respectively . there is , however , no specific mention of the reason for reoperation in the study . in 2011 , katonis et al reported on 1662 consecutively placed lateral mass screws . screw pullout occurred in 3 ( 1.3% ) patients , but they did not mention if these patients required reoperation or if the system was a screw - plate or polyaxial screw - rod implant system . the report also mentioned 11 ( 0.6% ) screws had a suboptimal trajectory but no patient required reoperation for removal or replacement of hardware . al barbarawi et al had 2 screw pullout cases out of 430 lateral mass screw - rod fixation cases . two ( 4.0% ) cases were found to have screw pullout in the study by kim et al using yoon s method . kasimatis et al included 74 patients from 1990 to 2005 who underwent anterior cervical treatment of cervical spine trauma in their study looking at complications . screw backout was reported in 4 ( 5.4% ) cases , all of which were unrestricted backout plates . seven cases ( 9.5% ) had less - than - ideal positioning of the construct with only one case requiring reoperation because of a malpositioned screw being completely in the intervertebral space . veeravagu et al looked at the marketscan database from 2006 and 2010 and found in 28 777 patients a reoperation rate of 9.13% and 10.7% for single - level and multilevel anterior cervical fusion , respectively . unfortunately , their data did not include data about the rate of reoperation related specifically to hardware failure . shapiro et al found no screw pullout when using locking screw fixation in 246 consecutive anterior cervical fusion cases . several reports exist in the literature of screw pullout resulting in dysphagia and sometimes erosion into the gastrointestinal tract . they continue to document the incidence of screw pullout related to misplacement to be uncommon , but can result in complications with serious morbidity and increased costs . coe et al performed a systematic review of lateral mass screw fixation , and implant removal was defined as a surgical procedure to correct malpositioned screws , screw breakout , or loosening . reoperation as defined was necessary for 27 ( 1.2% ) of 2185 screws placed in 294 patients across 5 studies . limited information was available to determine the number of patients with screw misplacement that did not require reoperation . there was also insufficient information within the screening population to determine the number of screws placed for comparison . the screening population was divided into anterior only and posterior or anterior / posterior combined groups . this does not allow for calculation of the incidence of posterior misplacement requiring revision , and it can only be speculated that this number would be much higher than the rate for anterior alone procedures . the advancements and improvements in cervical spinal instrumentation would be expected to decrease the rate of hardware - related complications . the overall complication rate of cervical spinal surgery is estimated to be 0.2% to 17.8% . after 30 days , the most common complication is related to instrumentation dislodgement , breakage , or both , and occurs in 2% to 25% of cases of spinal fusion procedures . reoperation rate for instrumented spinal fusions is reported between 2% and 25% , with the majority related to pseudoarthrosis . the majority of information is based on case reports , retrospective reviews , and small series of patients . there are few recent articles addressing the rate of screw backout or misplacement requiring reoperation , and many older studies include the use of unrestricted devices that permitted screw backout . fehlings et al prospectively collected 302 cervical spondylotic myelopathy patients and found a perioperative complication rate of 15.6% , with 11.6% minor and 7.0% major perioperative risk . three patients had instrumentation malposition / migration for a rate of 1% in the perioperative period . three patients had instrumentation / graft migration or instrumentation failure , making the risk of delayed hardware complications 1.1% . of 195 anterior cervical plating procedures , 5 ( 2.6% ) required reoperation for hardware failure . cole et al used the marketscan database and identified 13 662 patients who underwent anterior or posterior cervical fusion for degenerative disc disease . the rate of any complication excluding dysphagia was 12.3% and 17.8% for anterior and posterior approaches , respectively . there is , however , no specific mention of the reason for reoperation in the study . in 2011 , katonis et al reported on 1662 consecutively placed lateral mass screws . screw pullout occurred in 3 ( 1.3% ) patients , but they did not mention if these patients required reoperation or if the system was a screw - plate or polyaxial screw - rod implant system . the report also mentioned 11 ( 0.6% ) screws had a suboptimal trajectory but no patient required reoperation for removal or replacement of hardware . al barbarawi et al had 2 screw pullout cases out of 430 lateral mass screw - rod fixation cases . two ( 4.0% ) cases were found to have screw pullout in the study by kim et al using yoon s method . kasimatis et al included 74 patients from 1990 to 2005 who underwent anterior cervical treatment of cervical spine trauma in their study looking at complications . screw backout was reported in 4 ( 5.4% ) cases , all of which were unrestricted backout plates . seven cases ( 9.5% ) had less - than - ideal positioning of the construct with only one case requiring reoperation because of a malpositioned screw being completely in the intervertebral space . veeravagu et al looked at the marketscan database from 2006 and 2010 and found in 28 777 patients a reoperation rate of 9.13% and 10.7% for single - level and multilevel anterior cervical fusion , respectively . unfortunately , their data did not include data about the rate of reoperation related specifically to hardware failure . shapiro et al found no screw pullout when using locking screw fixation in 246 consecutive anterior cervical fusion cases . several reports exist in the literature of screw pullout resulting in dysphagia and sometimes erosion into the gastrointestinal tract . they continue to document the incidence of screw pullout related to misplacement to be uncommon , but can result in complications with serious morbidity and increased costs . coe et al performed a systematic review of lateral mass screw fixation , and implant removal was defined as a surgical procedure to correct malpositioned screws , screw breakout , or loosening . reoperation as defined was necessary for 27 ( 1.2% ) of 2185 screws placed in 294 patients across 5 studies . limited information was available to determine the number of patients with screw misplacement that did not require reoperation . there was also insufficient information within the screening population to determine the number of screws placed for comparison . the screening population was divided into anterior only and posterior or anterior / posterior combined groups . this does not allow for calculation of the incidence of posterior misplacement requiring revision , and it can only be speculated that this number would be much higher than the rate for anterior alone procedures . this study represents the largest series to analyze and tabulate the incidence of screw backout or misplacement requiring reoperation following cervical spine surgery . the data suggest this is a very rare adverse event , despite the widespread use of cervical fixation . due to the methodology of this study , there is a potential for underestimation of screw misplacement requiring reoperation within this series of patients . this potential for underreporting is more likely to occur in the case of posterior cervical fusion as this could not be calculated based on this study . the data therefore represents a lower limit for the incidence of screw misplacement requiring reoperation . patients suffering this complication require hardware revision , but do not usually suffer neurologic sequelae . meticulous technique and thorough knowledge of the relevant anatomy are the best means of preventing this complication .

purposethis study was performed to define attachment and growth behavior of osteoblast - like cells and evaluate the gene expression on zirconia compared to titanium.materials and methodsmc3t3-e1 cells were cultured on ( 1 ) titanium and ( 2 ) zirconia discs . the tetrazolium - based colorimetric assay ( mtt test ) was used for examining the attachment of cells . cellular morphology was examined by scanning electron microscopy ( sem ) and alkaline phosphatase ( alp ) activity was measured to evaluate the cell differentiation rate . mann - whitney test was used to assess the significance level of the differences between the experimental groups . cdna microarray was used for comparing the 20215 gene expressions on titanium and zirconia.resultsfrom the mtt assay , there was no significant difference between titanium and zirconia ( p>.05 ) . from the sem image , after 4 hours of culture , cells on both discs were triangular or elongated in shape with formation of filopodia . after 24 hours of culture , cells on both discs were more flattened and well spread compared to 4 hours of culture . from the alp activity assay , the optical density of e1 cells on titanium was slightly higher than that of e1 cells on zirconia but there was no significant difference ( p>.05 ) . most of the genes related to cell adhesion showed similar expression level between titanium and zirconia.conclusionzirconia showed comparable biological responses of osteoblast - like cells to titanium for a short time during cell culture period . most of the genes related to cell adhesion and signal showed similar expression level between titanium and zirconia . study design : a multicenter , retrospective case series.objective:in the past several years , screw fixation of the cervical spine has become commonplace . for the most part , this is a safe , low - risk procedure . while rare , screw backout or misplaced screws can lead to morbidity and increased costs . we report our experiences with this uncommon complication.methods:a multicenter , retrospective case series was undertaken at 23 institutions in the united states . patients were included who underwent cervical spine surgery from january 1 , 2005 , to december 31 , 2011 , and had misplacement of screws requiring reoperation . institutional review board approval was obtained at all participating institutions , and detailed records were sent to a central data center.results:a total of 12 903 patients met the inclusion criteria and were analyzed . there were 11 instances of screw backout requiring reoperation , for an incidence of 0.085% . there were 7 posterior procedures . importantly , there were no changes in the health - related quality - of - life metrics due to this complication . there were no new neurologic deficits ; a patient most often presented with pain , and misplacement was diagnosed on plain x - ray or computed tomography scan . the most common location for screw backout was c6 ( 36%).conclusions : this study represents the largest series to tabulate the incidence of misplacement of screws following cervical spine surgery , which led to revision procedures . the data suggest this is a rare event , despite the widespread use of cervical fixation . patients suffering this complication can require revision , but do not usually suffer neurologic sequelae . these patients have increased cost of care . meticulous technique and thorough knowledge of the relevant anatomy are the best means of preventing this complication .

biological variation allows for a unique craniofacial character and many asymmetries found in each individual . a neurodevelopmental disorder has been defined as an impairment of the growth and development of the brain or central nervous system . a narrower use of the term refers to a disorder of the brain function that affects emotion , learning ability , and memory , which unfolds as the individual grows . disorders considered neurodevelopmental in origin or that have neurodevelopmental consequences when they occur in infancy and childhood include : autism and autism spectrum disorders such as asperger syndrome , fetal alcohol spectrum disorder , traumatic brain injury , ( including congenital injuries such as those that cause cerebral palsy ) , communication , speech , and language disorders , genetic disorders , such as , fragile - x syndrome , and down syndrome . the definition of children with a neurodevelopmental disorder or a neurodevelopmental impairment has varied in several studies . for the purpose of this study , we have tried as far as possible to apply a definition of children with abnormal brain development , due to neurodevelopmental disorder , to include all children who have been fully diagnosed with any form of abnormality that appears likely to affect day - to - day function , such as , a developmental quotient more than two standard deviations below the normal mean , a diagnosis of neuromotor impairment , including cerebral palsy ( cp ) , speech and language disorders , hearing and vision impairment , attention - deficit hyperactivity disorder ( adhd ) , autism spectrum disorder ( asd ) , epilepsy , learning disorders ( ld ) , and mental retardation ( mr ) . neurodevelopmental disabilities ( ndds ) among children have always been an important concern , but yet to be considered as a significant public health problem , especially in the low- and middle - income countries , including nigeria . this is probably due to the fact that attention and resources are focused on more widely prevalent and visible vaccine - preventable childhood diseases , infections , nutritional deficiencies , and neonatal issues . it is implicated that 85% of the children with ndds live in low- and middle - income countries ( lmics ) however ; there is very little evidence to support this . paucity of documented data regarding the prevalence of ndds and their risk factors in lmics , further aggravates the problem . there is also no comprehensive , valid , reliable , and culturally sensitive screening tool for multiple ndds that can be used in resource - constraint environments . the importance of conducting craniometric or cephalometric studies can not be overemphasized especially in the areas of pediatrics , plastic surgery , oral surgery , and forensic medicine . cranial index ( ci ) is one of the clinically recognized anthropometric parameters used in the investigation of a craniofacial skeleton , because of its validity and practicability . the cranial index is the most investigated craniofacial parameter , as it utilizes the length and breadth of the head which are very helpful in the study of secular trends . the cranial index is used to determine variations in the shape of the head and face in newborns , and to determine the head dimensions in fetuses . however , due to paucity of data on the physical or morphometric cranial indices of children with abnormal brain development due to neurodevelopmental disorders ( ndds ) in the low- and middle - income countries , including nigeria , this study aims to discern whether abnormal brain development in the form of a neurodevelopmental disorder can cause any deviation in the cranial index of affected children , and thus , probably , serve as a baseline reference measurement for growth monitoring and evaluation . this study was conducted in the pediatric neurology clinic ( pnc ) of the institute of the child health unit , usmanu danfodiyo university teaching hospital , sokoto , for over a period of six months , by applying the inclusion and exclusion criteria for the study . cranial measurements such as head lengths and head widths were taken from 330 children of the pediatric age group 0 - 18 years , who visited the clinic during the period of study . the subjects comprised of 112 children who had been diagnosed with at least one form of abnormal brain development as a result of neurodevelopmental disorder by the pediatric neurology department of uduth , and who attended the pediatric neurology clinic . two hundred and eighteen normal growing children without any abnormal brain development on routine check up at the center were also recruited in this study . children with craniofacial malformations or a history or signs of craniofacial syndromes were excluded from this study . the bio - data of the subjects were obtained and this includes their names , hospital card numbers , sex , and age . the head length ( greatest anteroposterior diameter ) and head width ( maximum transverse diameter between two fixed points ) were measured with the help of a spreading caliper . the tips were made to touch the cranial points and the reading was observed on the scale . the cranial index was calculated as the ratio of the head width to head length multiplied by 100 . the cranial index was used to classify the head shapes into four international categories namely dolichocephalic ( ci = < 74.9 ) , mesocephalic ( ci = 75 - 79.9 ) brachycephalic ( ci = 80 - 84.9 ) and hyperbrachycephalic ( ci = > 85 ) . the research was approved by the ethics committee of the college of health sciences , usmanu danfodiyo university , sokoto . ethical consideration included taking informed consent from the parents or guardians of study participants , using hospital card numbers , to maintain confidentiality and making recommendations to the appropriate authorities . the data obtained from the measurements of the head length and head width were used in determining the cranial index . the measurement values were entered and analyzed using the statistical package for social sciences ( spss ) version 17 for windows . the student 's t test was used for comparison between the means and the statistical significance was set at p < 0.05 . summary of the neurodevelopmental disorders seen during the course of this study comparison of the mean values of the cranial index of male children with ndds and normal growing males without ndds according to age group comparison of the mean values of the cranial index of female children with ndds and normal growing females without ndds according to age group this study was conducted in the pediatric neurology clinic ( pnc ) of the institute of the child health unit , usmanu danfodiyo university teaching hospital , sokoto , for over a period of six months , by applying the inclusion and exclusion criteria for the study . cranial measurements such as head lengths and head widths were taken from 330 children of the pediatric age group 0 - 18 years , who visited the clinic during the period of study . the subjects comprised of 112 children who had been diagnosed with at least one form of abnormal brain development as a result of neurodevelopmental disorder by the pediatric neurology department of uduth , and who attended the pediatric neurology clinic . two hundred and eighteen normal growing children without any abnormal brain development on routine check up at the center were also recruited in this study . children with craniofacial malformations or a history or signs of craniofacial syndromes were excluded from this study . the bio - data of the subjects were obtained and this includes their names , hospital card numbers , sex , and age . the head length ( greatest anteroposterior diameter ) and head width ( maximum transverse diameter between two fixed points ) were measured with the help of a spreading caliper . the tips were made to touch the cranial points and the reading was observed on the scale . the cranial index was calculated as the ratio of the head width to head length multiplied by 100 . the cranial index was used to classify the head shapes into four international categories namely dolichocephalic ( ci = < 74.9 ) , mesocephalic ( ci = 75 - 79.9 ) brachycephalic ( ci = 80 - 84.9 ) and hyperbrachycephalic ( ci = > 85 ) . the research was approved by the ethics committee of the college of health sciences , usmanu danfodiyo university , sokoto . ethical consideration included taking informed consent from the parents or guardians of study participants , using hospital card numbers , to maintain confidentiality and making recommendations to the appropriate authorities . the data obtained from the measurements of the head length and head width were used in determining the cranial index . the measurement values were entered and analyzed using the statistical package for social sciences ( spss ) version 17 for windows . the student 's t test was used for comparison between the means and the statistical significance was set at p < 0.05 . summary of the neurodevelopmental disorders seen during the course of this study comparison of the mean values of the cranial index of male children with ndds and normal growing males without ndds according to age group comparison of the mean values of the cranial index of female children with ndds and normal growing females without ndds according to age group table 1 presents a summary of the neurodevelopmental disorders seen during the course of this study . tables 2 and 3 present the mean sd values of the cranial index of children with neurodevelopmental disorders ( ndds ) and that of normal growing children without ndds . all measurements were age - related . on an average , children with ndds had a lower cranial index ( mean ci = 77.78 2.95 ) than those of normal growing children without ndds ( mean ci = 79.82 3.35 ) . however , the mean cranial index for the males with ndds was 77.54 3.19 as against 79.16 3.19 of the normal growing males without ndds , while that of the females varied from 78.21 2.42 for those with ndds to 80.63 3.36 in normal growing females without ndds . statistically , all the age groups in males with ndds when compared to the normal growing children , showed no significant difference ( p > 0.05 ) except for the age group of 16 - 18 years , where the difference was statistically significant ( p < 0.05 ) as presented in table 1 . the female children with ndds did not show any statistical significant difference in comparison to the normal growing female children without ndds , as shown in table 3 . furthermore , the differences in cranial index between the sexes in children with ndds was not significant ( p = 0.2131 ) , but it was statistically significant in the normal growing children without ndds ( p = 0.002 ) . the present study shows that the cranial index values in children with ndds are similar to those of the normal growing children without ndds , in every age group . generally , the children with ndds had a lower cranial index ( mean ci = 77.78 2.95 ) than those of normal growing children ( mean ci = 79.82 3.35 ) , but this difference was not statistically significant . these values were higher when compared to the 72.96 6.12 cephalic index mean value found in the ogbia children from nigeria and lower when compared to 80.42 from chile . the overall minimum value for cranial index reported in this study was 68.39% and the maximum value was 89.29% . in the group of children with ndds , the mean cranial index was 77.54 3.19 in males and 78.21 2.42 in females , while in the group of children without ndds , the mean cranial index was 79.16 3.19 in males and 80.62 3.36 in females [ table 4 ] . it was observed from both groups that the mean values of females in this present study was higher than that of males and these differences were not statistically significant , with a p value > 0.05 . these agreed with a previous study , which reported higher cephalic index values in females than males , while our findings did not agree with any previous study that reported a statistically significant difference between the cephalic index of both sexes . the mean values for head length , head width , and cranial index observed in both sexes of children with ndds and normal growing children without ndds the mean cranial index values in both sexes are lower than the mean cephalic index values of 82.42 5.38 ( higher value ) and 81.00 5.35 ( lower value ) in males and females , respectively , found in children from bangalore , and also lower than the mean values of 83.10 in males and 84.60 in females from the gurung community . this implies that the cephalic index can be higher in any sex , depending on the peculiarity of the population under study . the mean cranial index values observed in both males and females without ndds are higher than those reported in the ogbia children , who showed values of 73.68 6.53 for males and 72.24 5.60 for females . although , the mean cranial value for females in the group of children without ndds is higher and very similar to the 80.08 mean cephalic index value found in females from port harcourt , nigeria , those of their male counterparts is lower than the 79.73 mean cephalic index value for males from port harcourt . the maximum cranial index value of females in this study is slightly higher than that reported in a previous study , whose minimum and maximum cranial index values in females was 72 and 87% , respectively , while the minimum value of females in the present study is also slightly lower than that reported by maria et al . for the normal growing males without ndds , the minimum and maximum cranial index values in this study ( 68.39 and 86.96% , respectively ) are not the same as those in the study by maria et al . , even though the maximum cranial index value in males , 93% , is higher than the maximum value of cranial index of males in this study ( 86.96% ) and the minimum cephalic index reported in male children , ( 72% ) , is higher than the minimum cranial index value of 68.39% for males reported in this study . however , for both sexes in the group of children without ndds , the minimum cranial index values in the present study are slightly lower when compared to the minimum values reported in previous studies . an explanation for this is that it may be due to the difference in the range of the children 's age group studied . however , the unsteady increase and decrease in the mean cranial index of females with ndds , with an increase in age , observed in this study , agrees with the earlier study , while the steady decrease in cranial index of females without ndds does not agree with the study of ligha and fawehinmi . the increase in mean cranial index across all age groups observed in males with ndds agrees with the reported increase in mean cephalic index with increase in age of normal growing male children from port harcourt , nigeria , while the unsteady increase and decrease seen in males without ndds does not agree with the study by ligha and fawehinmi . there was no statistically significant difference observed in the mean cranial index between the two groups and across the age ranges . on an average , the mean cranial index values of females in the present study are higher when compared to those of males across all age groups . these higher mean head width values observed in females could be due to the nondominant lateral direction of expanding of the neural mass , by neural fibers , in females . conclusively , we can deduce from this present study that the cephalic index in children does not change due to neurodevelopmental problems . the present data can be of clinical importance in determining the timing and etiology of brain insults , especially during the critical period of brain development , as well as , help in the early identification and management of neurological abnormalities . it is recommended that further studies be conducted , possibly with a larger sample size , to corroborate these findings , so as to make further inference with regard to the clinical and anthropometric importance of such studies . the verbal informed patient consent was acquired for the case report publication during the acute rehabilitation . institutional review board approval was not sought for this case report . whether a patient visits a clinic or is admitted to the hospital , the importance of a detailed physical examination must be emphasized as a resource leading to an appropriate diagnosis and treatment . every medical student starts his or her medical career with learning how to take a good history from the patient and perform a thorough physical examination . this report details such a case where a thorough initial physical examination resulted in the diagnosis of subacute hemorrhagic stroke in a patient with esophageal adenocarcinoma . a 65-year - old male , an experienced civil engineer , with a history of gastroesophageal reflux disease and hiatal hernia with a new complaint of progressive difficulty swallowing associated with a recent 20-pound weight loss was referred to an oncologist . the patient was evaluated by the oncology specialist 2 weeks later , and esophagogastroduodenoscopy was performed that revealed a distal esophageal mass , which was identified through a biopsy as moderately differentiated adenocarcinoma . subsequent positron emission tomography and computed tomography ( ct ) scan studies performed 2 weeks later revealed distal esophageal wall thickening with an increased uptake extending into the proximal stomach but no brain lesions . the patient received neoadjuvant chemotherapy and en bloc esophagectomy with cervical esophagostomy , pyloroplasty , and jejunostomy feeding tube placement during the subsequent admission to a tertiary medical center . the patient was medically stabilized and evaluated for admission to a rehabilitation unit . during the rehabilitation admission examination , the patient was found to have left homonymous hemianopsia as a new physical examination finding . the patient also had some confusion with decreased initiation , not being able to perform at his educational level . considering these new findings along with his recent history of invasive and possibly metastatic esophageal adenocarcinoma , the admission team decided to proceed with a magnetic resonance imaging ( mri ) study of the brain . the differential diagnosis of brain metastasis versus a new stroke was proposed by the primary treating team . according to the radiology department report , it was likely a primary brain neoplasm , such as a neural or glial tumor , or lymphoma , but less likely a focus of metastatic disease , given its appearance . however , according to the oncology department evaluation , the lesion was more consistent with a metastatic tumor than a primary brain lesion . he was treated with oral antiepileptics . at the same time , the patient complained of having shortness of breath and was diagnosed as having pleural effusions . a pathological examination of thorasentesis concluded that no malignant cells were present in the pleural effusion fluid . a series of two head ct scans within 2448 hours were performed to rule out any acute hemorrhage ( figure 2a ) . the tumor was found to be accessible for resection per neurosurgery evaluation with a plan for tumor resection when the patient was medically stable . a subsequent craniotomy and lesion biopsy followed by pathology investigation surprisingly identified the lesion as a subacute cortical ischemic stroke negative for neoplasm . he was provided with appropriate treatment and secondary stroke prophylaxis and was able to complete the rehabilitation on the acute rehabilitation floor . in the case presented , the diagnosis of subacute ischemic stroke was made on the basis of focal findings during a detailed admission physical examination in the university - based acute rehabilitation unit . the patient was found to have mild chronic delirium with an inability to concentrate , decreased initiation , affected short - term memory , and homonymous hemianopsia . all new focal deficit findings , such as homonymous hemianopsia , require additional attention and further investigation . there are several case reports in the literature strongly suggesting that physicians have to conduct a complete medical history and arrange a specialist consultation for all patients focal findings such as visual field cuts.1 the top differential diagnostic considerations for a presentation of homonymous hemianopsia for this patient were metastatic lesion from a known esophageal carcinoma , stroke , and a primary brain tumor . according to the mri report , the lesion was likely primary brain neoplasm , such as a neural or glial tumor , or lymphoma and less likely a focus of metastatic disease given the appearance on the scan images ( figure 1a and b ) . it is very important to use the knowledge of the morphological presentation , and probability for each type of malignancy , to perform the second step in the evaluation of the patient when the clinical presentation led to the discovery of pathology . this case presents a complicated identification of the pathology , due to a very unusual presentation of the lesion leading radiology , oncology and neurosurgery departments to believing that the lesion was a tumor with unspecified etiology . it was only surgical resection that allowed identifying the correct pathology of the brain lesion as a subacute stroke . the incidences of metastatic lesions from esophageal adenocarcinoma were previously reported in the literature , estimating 2% of patients with esophageal carcinoma having brain metastasis.2 however , a more recent cohort study found 16% of patients with esophageal carcinoma having metastasis to the brain.3 visual field defects after stroke are also well reported in the literature . the incidence of homonymous field defects was estimated at 8% in all patients with stroke with the majority ( 54% ) located in occipital lobes.4 the estimated lesions located in the lateral geniculate body ( 1% ) are published in the literature.4,5 patients impairments after stroke are significantly exacerbated by visual field defect.6 the most common clinical sign of infarctions in the posterior cerebral arteries distribution are visual field defect followed by neuropsychological deficits and higher order dysfunction including memory impairment as has been presented in this case.7 acute confusional state and delirium are also common especially in the elderly population.7 recent data suggested that confusion and delirium from a stroke in the right posterior cerebral artery distribution is significantly lower in comparison to the left or bilateral posterior artery distribution.8 delirium was reported to remain for an appreciable poststroke period and was a marker of a poor prognosis.9 in this case , the patient is a well - educated and experienced engineer and the owner of an engineering company . the slow initiation and inferior performance , in light of his level of education , on the admission physical examination , and reported delirium state during the treatment before the admission to the rehabilitation floor , could be explained by the delirium and memory impairment after the posterior artery stroke . the poststroke visual field defect can result in significant disability and reduction in quality of life . patients with visual field cut can have a severely reduced quality of life and require additional neuropsychological and visual rehabilitation.4,6 only a thorough physical examination during admission helped to challenge prior negative positron emission tomography scan data and led to the diagnosis of subacute stroke , and following appropriate treatment , secondary stroke prophylaxis and rehabilitation , instead of brain radiation and chemotherapy .

background : abnormal brain development due to neurodevelopmental disorders in children has always been an important concern , but yet has to be considered as a significant public health problem , especially in the low- and middle - income countries including nigeria.aims:the aim of this study is to determine whether abnormal brain development in the form of neurodevelopmental disorders causes any deviation in the cranial index of affected children.materials and methods : this is a comparative study on the head length , head width , and cranial index of 112 children ( 72 males and 40 females ) diagnosed with at least one abnormal problem in brain development , in the form of a neurodevelopmental disorder ( ndd ) , in comparison with that of 218 normal growing children without any form of ndd ( 121 males and 97 females ) , aged 0 - 18 years old seen at the usmanu danfodiyo university teaching hospital , sokoto , over a period of six months , june to december , 2012 . the head length and head width of the children was measured using standard anatomical landmarks and cranial index calculated . the data obtained was entered into the microsoft excel worksheet and analyzed using spss version 17.results:the mean cephalic index for normal growing children with normal brain development was 79.82 3.35 and that of the children with abnormal brain development was 77.78 2.95 and the difference between the two groups was not statistically significant ( p > 0.05).conclusion : it can be deduced from this present study that the cranial index does not change in children with neurodevelopmental disorders . this case report describes the detection of homonymous hemianopsia and delirium during the admission physical examination of a patient with esophageal adenocarcinoma , resulting in the new diagnosis of subacute hemorrhagic stroke . the poststroke visual field defect can result in significant disability and reduction in quality of life . patients with visual field cut show a severely reduced quality of life and require additional neuropsychological and visual rehabilitation . only thorough physical examination is able to challenge prior negative positron emission tomography scan , leading to the diagnosis of subacute stroke and , following appropriate treatment , secondary stroke prophylaxis and rehabilitation , instead of brain radiation and chemotherapy .

higher energy intake than expenditure causes excessive accumulation of fat and leads to the development of obesity and associated metabolic disorders , which has reached epidemic proportions not only in developed countries but worldwide . furthermore , obesity can lead to the development of various co - morbidities , like type 2 diabetes , cardiovascular diseases , neurodegenerative disorders and some types of cancers , that severely impact life quality and expectancy . while treatments for the obesity related co - morbidities are available , albeit with varying success , no therapeutic avenues , with the exception of highly invasive bariatric surgery , exist to treat obesity directly . several approaches that target the central nervous system to reduce food intake have failed in this context due to massive side effects . as it can both store and burn calories , adipose tissue has gained renewed attention recently as a possible target for the treatment of obesity due to its janus - faced character . in this context , adipose tissue can be functionally divided into two main depots , namely white and brown adipose tissue . white adipose tissue is characterized by the presence of large unilocular adipocytes whose main function is the storage of energy and the release of fatty acids during fasting and starvation . brown adipose tissue is characterized by the presence of smaller cells with multilocular lipid droplets . in contrast to that of white adipose tissue , the primary function of brown adipose tissue is to produce heat , a feature which is achieved through the function of uncoupling protein 1 ( ucp1 ) . ucp1 effects a proton leak from the mitochondrial intermembrane space to the mitochondrial matrix , effectively short circuiting the electron transport chain . thus , as its name suggests , ucp1 uncouples oxidation from phosphorylation , driving a futile cycle that produces heat . in addition , to classical brown adipocytes , which are mainly localized to the interscapular depot , other brown like adipocytes , termed either beige or brite adipocytes , can be found in predominantly white adipose tissue depots . these cells have received substantial attention in recent years due to the fact that their appearance is dependent on induction . the origin of these cells , however , remains a matter of debate [ 810 ] . during cold exposure the sympathetic system activates brown fat via secretion of catecholamines that stimulate 3-adrenergic receptors on the adipocyte surface . in rats , brown adipose tissue has been shown to be responsible for over 60 percent of the excess heat production during cold stimulation ; also in newborn humans , the role of brown fat in protecting against hypothermia has been appreciated for a long time . in addition , several independent groups recently demonstrated that brown fat is also activated by cold and contributes to heat production through non - shivering thermogenesis in adult humans [ 1418 ] . . showed in 2013 that repeated cold exposure leads to recruitment of brown fat in humans and increases cold - induced gains of energy expenditure . further corroborating a functional role for brown fat in the adult human was gained from a study by cypess et al . , which demonstrated that 3-adrenergic receptor agonists can not only increase glucose uptake of brown fat but also increase resting metabolic rate by approximately 13% . brown fat activation is achieved by a variety of factors , including but not limited to natriuretic peptide a , 3-adrenergic receptor agonists and other circulating factors such as fibroblast growth factor 21 ( fgf21 ) . until recently , the energy expenditure enhancing and weight loss effects of fgf21 were thought to be mediated mainly by brown fat thermogenesis , because fgf21 has been demonstrated to directly stimulate thermogenic gene expression and to induce browning of white fat . thus , fgf21 seemed to be a suitable candidate for both the recruitment of brown cells in the white fat , as well as for the activation of endogenous brown fat . fgf21 is a member of the prominent fibroblast growth factor ( fgf ) superfamily , which currently encompasses 22 members . based on various genetic mutations , fgfs were first implicated in embryonic development , regulating proliferation and differentiation as well as organ morphogenesis . the spectrum has been expanded recently , and it is now accepted that fgf signaling plays an important role in the pathogenesis of several diseases . this is especially true for fgf21 , which has emerged as a new therapeutic target for the regulation of whole body metabolism . only five years later it was shown by the lilly research laboratories that fgf21 is an important factor regulating glucose uptake as fgf21 knockout mice exhibit a mild weight gain , increased adipocyte size and slightly impaired glucose homeostasis . furthermore , when challenged with a ketogenic diet , fgf21ko mice develop hepatosteatosis and gain weight concomitant with an impairment in ketogenesis and glucose homeostasis , as well as a downregulation of pgc1 and pgc1 both in liver and white fat . conversely , systemic injection or transgenic overexpression of fgf21 leads to protection from diet - induced obesity , concomitant with a significant reduction in blood glucose and triglyceride levels . interestingly , fgf21 overexpression also causes a marked increase in food intake under chow and high fat conditions , suggesting that either energy expenditure and/or nutrient uptake in these animals is induced . taken together , these facts identify fgf21 as a major regulator of metabolic utilization of energy containing metabolites ; therefore fgf21 is considered a novel therapeutic target for the treatment of obesity and its associated metabolic disorders [ 6,2629 ] . fgf21 is expressed in liver , pancreas , fat tissue and muscle . in adult humans , fructose ingestion acutely stimulates circulating fgf21 levels and in liver , fgf21 expression is induced after birth in response to intake of milk during suckling through the action of ppar. due to its high expression in brown adipose tissue several studies have addressed the regulation of fgf21 expression and secretion in response to activation of this organ . for example , hondares et al . showed that higher expression leads to increased secretion by analyzing arteriovenous differences in fgf21 concentration across interscapular brown fat . in contrast , work from chartoumpekis et al . could not demonstrate an increase in fgf21 serum levels after an acute cold induction at the end of a 12 h starvation period . compared fgf21 mrna levels in muscle , heart , liver , subcutaneous white and brown fat from animals at different housing temperatures . interestingly , at thermoneutrality , only liver seems to express fgf21 at a level comparable to that in activated brown adipose tissue . in ucp1 genetically ablated mice ( ucp1ko ) , fgf21 expression is induced in brown adipose tissue as well as in circulation . this increase only occurs during cold adaptation , suggesting that brown fat communicates with other organs , maybe to compensate for the loss in thermogenic capacity . in contrast , transgenic overexpression of ucp1 switches on fgf21 production in muscle suggesting that this phenotype is specific for brown fat and might be diametrically opposite in other organs . fgf21 signaling utilizes the classical intracellular fgfr signaling pathway , but , in contrast to other fgfs , it does not bind directly to the fgfr . instead , fgf21 mediates its function via the transmembrane receptor -klotho , which is a fgfr co - receptor with a high expression in liver , fat and the central nervous system . whole body deletion of -klotho leads to a complex phenotype including developmental defects such as growth retardation and a counterintuitive increase in glucose tolerance and insulin sensitivity . in brown adipose tissue of these mice , however , ucp1 levels and body temperature are reduced , suggesting that -klotho deficient mice have less energy expenditure than wild - type mice . since it was shown that other fgfs such as fgf19 can also signal via -klotho , this finding could be explained by a broader ligand spectrum for this surface receptor . the cell autonomous effect of fgf21 on adipocytes was first reported in 2005 by kharitonenkov , et al . , who demonstrated an fgf21 mediated induction of glucose uptake in mature adipocytes . in 2012 , ding et al . showed that ap2-cre mediated ablation of -klotho leads to blockade of the acute insulin - sensitizing effect . however , given the fact that ap2-cre mediated deletion targets multiple cells within the adipose tissue including adipocyte precursors , macrophages and endothelial cells [ 3941 ] , it is unclear whether this effect is due to fgf21 signaling in adipocytes . since brown fat activity is tightly regulated by the sympathetic nervous system it remains unclear whether the systemic effects of fgf21 are due to a cell autonomous regulation of brown fat activity or if this effect is mediated via the cns . a central effect of fgf21 has been demonstrated by intracerebro - ventricular ( i.c.v . ) injection of fgf21 , which , similar to systemic fgf21 injection , led to increased energy expenditure , food uptake and insulin sensitivity . however , central fgf21 administration failed to reduce body weight and size of adipose tissue , suggesting that fgf21 effects are mediated both peripherally and centrally . further evidence for the importance of fgf21 in regulating central pathways comes from animal models with hypothalamic- and hindbrain - specific genetic deletion of -klotho in conjunction with an fgf21 overexpression model , which demonstrated that centrally or peripherally administered fgf21 increases sympathetic activation in brown adipose tissue and that this effect can be blunted by i.c.v . in addition to its endocrine function , fgf21 may have paracrine or autocrine functions , as fat - specific knockout of fgf21 inhibits browning of white adipose tissue in adaptive thermogenesis . investigate the causal link between fgf21 increased browning and the loss of body weight . in accordance with previous results , the study by veniant et al . demonstrates that fgf21 injection leads to an increase in brown adipose tissue mass concomitant with an increase in ucp1 expression in brown adipose tissue , while the effect in inguinal white adipose tissue is lost at thermoneutrality . interestingly , the effect on weight loss is retained even when fgf21 is injected into ucp1ko mice , suggesting that the effects of fgf21 on weight loss are independent of brown fat activation . a closer look at the data , however , reveals that this is a simplified assumption . scrutiny of the energy expenditure data suggests that in both studies the effect of fgf21 is reduced in ucp1ko mice , a finding that is especially evident in the study by samms et al . in which fgf21 induced energy expenditure seems to be almost completely abolished in ucp1ko mice . while the effect of fgf21 on ffa and cholesterol are retained in ucp1ko mice , the effect on circulating glucose seem to be reduced or completely blunted . one important point to note is the effect of fgf21 on food intake in the context of ucp1 ablation . observes a reduced induction of food intake in ucp1ko while the study by samms et al . does n't observe an induction of food intake by fgf21 in wild type mice and actually reports a reduction of food intake in ucp1ko mice when treated with fgf21 ; the discrepancy could be due to differences in the dosing paradigm and housing temperature . taking into account that ucp1ko mice have a reduced food intake upon fgf21 treatment , one would expect them show a reduction in body weight , a finding that is not observed . therefore , it is possible that this difference is due to the effect of fgf21 on weight loss is mediated through the activation of brown adipose tissue . in ucp1ko mice both brown fat fgf21 mrna levels and circulating levels of fgf21 are increased significantly . since some effects of fgf21 are abolished in ucp1ko animals the question remains how fgf21 reduces body weight independent of ucp1 . first of all , most tissues contribute to the metabolic rate and oxygen consumption of an organism and in the adult state the majority of calories ingested , are finally lost as heat . the contribution of different tissues to mammalian metabolic rate ( independent of exercise ) mainly depends on the genetic background and the environmental temperature . based on the reported functions for fgf21 and given the literature of fgf21 and brown fat activation , the main question that arises from the recent studies is whether fgf21 acts as a physiological feedback molecule from thermogenically active tissues to integrate information about the available capacity and , if so , whether this alteration sensitizes other tissues to the action of fgf21 . to answer these questions it is necessary to review the literature on ucp1 mediated and brown fat mediated uncoupling in detail . it is well established that the genetic ablation of ucp1 induces obesity in mice living at thermoneutrality . furthermore , overexpression of ucp1 under the ap2 and the human skeletal actin promoter protects against development of genetic and diet - induced obesity , respectively . these data , which have been reviewed comprehensively , suggest that ucp1 function is the main driver of brown adipose tissue thermogenesis and that loss of ucp1 leads to a decrease in energy expenditure and concomitant obesity . that demonstrates that loss of ucp1 can protect from diet induced obesity under certain conditions . based on this controversy , several studies have addressed the ucp1-independent adrenergic heat production with somewhat controversial results and opinions range from 20 to 50% of brown fat capacity and it has been suggested that these effects could be due to ucp1-independent increases in thermogenesis in white adipose tissue . how these thermogenic processes are regulated is unclear at the moment ; however , futile cycling as well as fatty acid mediated uncoupling of the mitochondrial membrane have been suggested as possible mechanisms . for example , in mammalian muscle , sarcolipin was identified as a regulator of non - shivering thermogenesis . increased energy expenditure associated with the activation of a futile protein turnover cycle in peripheral branched - chain amino acid metabolism . furthermore , simultaneous activation of lipogenesis and lipolysis as well as prolonged beta3-adrenergic receptor activation can induce futile cycling both in white and brown adipose tissue . fgf21 induces weight loss and leads to reduction of some key metabolic parameters in ucp1ko mice . while some of these effects seem to be conserved and thus independent of ucp1 , others seem to be blunted . in particular , the fgf21 mediated changes in food intake seem to be dependent on ucp1 , suggesting a compensatory central regulation maybe through the induction of circulating fgf21 . in line with this notion recent work by schultz et al . might be important , as it suggests a fixed thermogenic capacity , which is tightly regulated by compensatory effects . thus , it is possible that under physiological conditions the effects of fgf21 are mainly mediated by brown adipose tissue through ucp1 , while under abnormal conditions such as ucp1 ablation , these effects might be mediated via other tissues . in light of these findings , and although ucp1-dependent thermogenesis is the most potent and possibly the most relevant physiological regulator of non - shivering thermogenesis , other mechanisms that have been proven to play a role in thermogenesis should be considered in the context of fgf21 signaling . endometriosis affecting the rectum accounts for a small but significant group of women with severe pelvic pain . perhaps because of the inadequacy of conservative or medical measures , many authors advocate the complete surgical removal of rectovaginal disease , even if surgery involving the rectum is required . we report herein a modification of existing techniques to enable a small anterior resection to be done without having to open either the abdomen or the rectal lumen . the patient was a 28-year - old woman with a 10-year history of pelvic pain . the most recent had suggested tethering of the rectum to the back of the cervix . when filling out a visual analogue pain survey , she gave pain scores for dysmenorrhea ( 40/100 ) , dyspareunia ( 20/100 ) , pain on defecation ( 85/100 ) , and daily pain ( 65/100 ) . she had considerable impairment in her quality of life , and using the eq-5d quality of life assessment , she gave herself a self - rated evaluation of 31 out of 100 ( population mean=88.82 ) for the quality of her health and had a calculated health state of 0.09 out of a maximum of 1.0 ( population mean=0.93 ) . on examination , a tender nodular area was noted at the top of the posterior fornix of the vagina . she was extensively counseled prior to the procedure and received liquid oral bowel preparation the day before the surgery . the patient received a general anesthetic , during which an epidural was sited for postoperative pain relief . ureteric catheters ( 6 french gauge ) were inserted cystoscopically to facilitate identification of the ureters . after insufflating carbon dioxide to a pressure of 18 mm hg via an intraumbilical disposable verress needle , the following ports were sited : a 10-mm umbilical port , through which a 10-mm laparoscope was passed , 5-mm ports in the right and left iliac fossae , lateral to the inferior epigastric vessels , and a 12-mm port with 5-mm reducer was inserted suprapubically , to the right of the midline but medial to the inferior epigastric vessels . we found the rectum tethered to the back of the cervix and the left pelvic side wall with incomplete obliteration of the pouch of douglas ( figure 1 ) . for most of the procedure , dissection was performed using maryland grasping forceps and metzenbaum scissors with a single - patient use replaceable tip . hemostasis , suction , and irrigation were achieved using a surgiflex wave ( acmi , usa ) and 20 watts of bipolar power . the technique of opening of the peritoneum lateral to any disease and reflecting the tethered rectum off the back of the cervix has already been described and illustrated by redwine and is therefore not illustrated herein . using this technique , the peritoneum was opened laterally on the pelvic sidewall below the level of the ureters . the tethered rectum was then reflected down , allowing dissection into the rectovaginal space and leaving any disease on the surface of the rectum . the peritoneal incisions are extended medially and again are joined at the midline by incising over the serosal surface of the rectum , proximal to the diseased area . this technique allows the en - bloc excision of any diseased peritoneum either lateral to or overlying the rectum . at this point , we had isolated a 3-cm patch of nodular endometriosis that infiltrated the muscular layer of the rectum . it was apparent that this area was too large to be excised without creating a large defect in the rectum , and a decision was made to proceed to a segmental anterior resection . the mesorectum was dissected using the ultracision harmonic scalpel ( ethicon , usa ) , exposing a 6-cm length of rectum that incorporated the diseased area ( figure 2 ) . a circular end - to - end anastomosis instrument ( 28-mm premier plus ceea , tyco healthcare , usa ) was inserted through the anus and advanced up the rectum to a point proximal to the disease . by unscrewing the handle , the anvil of the ceea device was made visible through the rectal wall , and the shaft was grasped by using a soft bowel grasper ( figure 3 ) . the handle of the device was then removed , leaving the anvil held in place within the lumen of the rectum , proximal to the disease . an endogia universal stapling device ( tyco healthcare , usa ) was used to transect the rectum above and below the diseased area ( figure 4 ) . the excised tissue was removed through the 12-mm port site with the assistance of a retrieval bag . the point of the shaft of the anvil was then pushed through the transverse staple line on the proximal end of the colon ( figure 5 ) . the ceea device was reinserted through the anus , and the anvil reattached ( figure 6 ) . to reduce adhesion formation , 1000 ml of icodextrin solution ( adept , shire pharmaceuticals , uk ) was left in the abdomen . the ceea device is inserted up the rectum ; the anvil is removed and held proximal to the diseased segment before the rectal segment is excised . the rectal segment is excised using the endo - gia stapler . the point of the shaft of the anvil is pushed through the proximal end of the colon . the rectal tube was removed after liquid stool was passed on the fourth postoperative day , and the patient was discharged on the fifth day . histological examination of the rectum demonstrated endometriosis within the muscularis propria of the rectal wall . the patient was a 28-year - old woman with a 10-year history of pelvic pain . the most recent had suggested tethering of the rectum to the back of the cervix . when filling out a visual analogue pain survey , she gave pain scores for dysmenorrhea ( 40/100 ) , dyspareunia ( 20/100 ) , pain on defecation ( 85/100 ) , and daily pain ( 65/100 ) . she had considerable impairment in her quality of life , and using the eq-5d quality of life assessment , she gave herself a self - rated evaluation of 31 out of 100 ( population mean=88.82 ) for the quality of her health and had a calculated health state of 0.09 out of a maximum of 1.0 ( population mean=0.93 ) . on examination , a tender nodular area was noted at the top of the posterior fornix of the vagina . she was extensively counseled prior to the procedure and received liquid oral bowel preparation the day before the surgery . the patient received a general anesthetic , during which an epidural was sited for postoperative pain relief . ureteric catheters ( 6 french gauge ) were inserted cystoscopically to facilitate identification of the ureters . after insufflating carbon dioxide to a pressure of 18 mm hg via an intraumbilical disposable verress needle , the following ports were sited : a 10-mm umbilical port , through which a 10-mm laparoscope was passed , 5-mm ports in the right and left iliac fossae , lateral to the inferior epigastric vessels , and a 12-mm port with 5-mm reducer was inserted suprapubically , to the right of the midline but medial to the inferior epigastric vessels . we found the rectum tethered to the back of the cervix and the left pelvic side wall with incomplete obliteration of the pouch of douglas ( figure 1 ) . for most of the procedure , dissection was performed using maryland grasping forceps and metzenbaum scissors with a single - patient use replaceable tip . hemostasis , suction , and irrigation were achieved using a surgiflex wave ( acmi , usa ) and 20 watts of bipolar power . the technique of opening of the peritoneum lateral to any disease and reflecting the tethered rectum off the back of the cervix has already been described and illustrated by redwine and is therefore not illustrated herein . using this technique , the peritoneum was opened laterally on the pelvic sidewall below the level of the ureters . the tethered rectum was then reflected down , allowing dissection into the rectovaginal space and leaving any disease on the surface of the rectum . the peritoneal incisions are extended medially and again are joined at the midline by incising over the serosal surface of the rectum , proximal to the diseased area . this technique allows the en - bloc excision of any diseased peritoneum either lateral to or overlying the rectum . at this point , we had isolated a 3-cm patch of nodular endometriosis that infiltrated the muscular layer of the rectum . it was apparent that this area was too large to be excised without creating a large defect in the rectum , and a decision was made to proceed to a segmental anterior resection . the mesorectum was dissected using the ultracision harmonic scalpel ( ethicon , usa ) , exposing a 6-cm length of rectum that incorporated the diseased area ( figure 2 ) . a circular end - to - end anastomosis instrument ( 28-mm premier plus ceea , tyco healthcare , usa ) was inserted through the anus and advanced up the rectum to a point proximal to the disease . by unscrewing the handle , the anvil of the ceea device was made visible through the rectal wall , and the shaft was grasped by using a soft bowel grasper ( figure 3 ) . the handle of the device was then removed , leaving the anvil held in place within the lumen of the rectum , proximal to the disease . an endogia universal stapling device ( tyco healthcare , usa ) was used to transect the rectum above and below the diseased area ( figure 4 ) . the excised tissue was removed through the 12-mm port site with the assistance of a retrieval bag . the point of the shaft of the anvil was then pushed through the transverse staple line on the proximal end of the colon ( figure 5 ) . the ceea device was reinserted through the anus , and the anvil reattached ( figure 6 ) . to reduce adhesion formation , 1000 ml of icodextrin solution ( adept , shire pharmaceuticals , uk ) was left in the abdomen . the ceea device is inserted up the rectum ; the anvil is removed and held proximal to the diseased segment before the rectal segment is excised . the rectal segment is excised using the endo - gia stapler . the point of the shaft of the anvil is pushed through the proximal end of the colon . the rectal tube was removed after liquid stool was passed on the fourth postoperative day , and the patient was discharged on the fifth day . histological examination of the rectum demonstrated endometriosis within the muscularis propria of the rectal wall . almost all the methods involve separating the adherent rectum from the back of the vagina and cervix and dissecting down into the rectovaginal septum . as this is done , the disease can be left on the side of the rectum to be stripped off after the dissection or can be left on the vaginal side of the septum and excised vaginally . shaving the disease off the front of the rectum invariably thins the serosal surface , and procedures to plicate the uterosacral ligaments and lateral rectal peritoneum across the midline have been described . where deeper involvement of the rectal muscularis or mucosa occurs , it is necessary to excise a full - thickness part of the rectum . this can be done by excising a disc of rectal wall or performing an anterior segmental rectal resection . concerns about the safety of performing such aggressive surgery for benign disease have been raised . temporary colostomy formation has been performed when the anastomosis is extremely close to the anal margin and to manage a postoperative obstruction , but is otherwise rarely required . clinically detectable anastomotic leaks occur after 7.3% of resections for carcinoma but to our knowledge have not been reported after resections for endometriosis . other risks include fistula formation , intestinal obstruction , and pelvic abscess formation , but also appear to be very uncommon . advances in laparoscopic techniques and equipment have allowed segmental rectal resections to be performed whilst remaining loyal to the principles of laparoscopic surgery and minimizing the need for the traditional midline laparotomy . total laparoscopic anterior resection has been described in animal models and has been used extensively for benign and malignant colorectal conditions . at some points during the several described techniques , the port sites need to be enlarged or a mini - laparotomy needs to be performed to remove the specimen or insert the anvil of the circular end - to - end anastomosis device . other techniques describe the use of a 33-mm port or a hand port to facilitate the reanastomosis . most descriptions involve opening a small part of the proximal stump of sigmoid or rectum to insert the anvil into the proximal stump . at this point , the risk of fecal spillage is present , even with adequate bowel preparation , and a purse string suture is required to secure the hole in the proximal stump around the shaft of the anvil . our procedure requires a maximum port size of 12 mm , does not require a mini - laparotomy or a large port for the insertion of the anvil , and prevents any risk of fecal spillage during the operation . we hope this technique will speed postoperative recovery and improve the safety of a laparoscopic anterior resection for patients with rectal endometriosis . we believe that this sort of surgery should be performed in units where sufficient colorectal surgical backup is available and where facilities are in place to ensure that meticulous follow up and audit of these procedures occurs .

backgroundfibroblast growth factor 21 ( fgf21 ) belongs to the large family of fibroblast growth factors ( fgfs ) . even though fgf signaling has been mainly implicated in developmental processes , recent studies have demonstrated that fgf21 is an important regulator of whole body energy expenditure and metabolism , in obesity.scope of reviewgiven the fact that obesity has developed epidemic proportions , not just in industrialized countries , fgf21 has emerged as a novel therapeutic avenue to treat obesity as well as associated metabolic disorders . while the metabolic effects of fgf21 are undisputed , the mechanisms by which fgf21 regulate weight loss have not yet been fully resolved . until recently it was believed that fgf21 induces brown fat activity , thereby enhancing energy expenditure , which concomitantly leads to weight loss . novel studies have challenged this concept as they could demonstrate that a part of the fgf21 mediated effects are retained in a mouse model of impaired brown adipose tissue function.major conclusionsthe review illustrates the recent advances in fgf21 research and discusses the role of fgf21 in the regulation of energy expenditure linked to brown fat activity . background : anterior rectal resection is sometimes necessary to treat deeply infiltrating rectovaginal endometriosis . we describe a completely laparoscopic approach as a new way of excising rectal endometriosis that can be used without opening any part of the rectum . this avoids opening the abdomen or any risk of fecal spillage.methods:the patient received preoperative oral bowel preparation . ureteric stents ( 6 f ) were inserted cystoscopically . the peritoneum in the ovarian fossae was opened lateral to any disease and the rectum reflected off the back of the cervix , leaving any endometriosis on the front of the rectum . the pelvic peritoneum was reflected medially , below the level of the ureters . the mesorectum was then dissected off a 6-cm length of rectum by using a harmonic scalpel . a circular end - to - end anastomosis instrument was passed anally until the outline of the anvil was visible , inside the colon , above the diseased rectum . the anvil was detached and held by a soft grasper before the rectum was then divided above and below the disease using a laparoscopic stapling device . the tip of the anvil was pushed through the proximal end of the colon allowing reanastomosis of the rectal stump.conclusion:the patient was discharged after 5 days without complications .

thymic carcinoma is an aggressive anterior mediastinal malignancy that can cause severe chest pain , although its incidence is rare . echocardiography is useful for assessing chest pain in the emergency room , but the diagnosis of an extracardiac mass can be missed . the purpose of this report is to describe the validation of echocardiography for accessing an extracardiac mass that presented with chest pain . an 80-year - old male visited the emergency room for sharp chest pain that had lasted for 1 month . he had a history of inferior wall myocardial infarction and percutaneous coronary intervention on the proximal - middle right coronary artery 2 years ago . on the physical examination , his temperature was 36.6 , his blood pressure was 100/60 mmhg , and his pulse was 81 beats / min . laboratory examinations showed that the levels of ck - mb , troponin - i , and brain natriuretic peptide were not significantly elevated ( 2.8 ng / ml , 0.05 ng / ml , and 143 ng / ml , respectively ) . electrocardiography revealed atrial fibrillation with q - waves in lead ii , iii , and avf . emergent coronary angiography was performed because of his history of previous myocardial infarction , but mild in - stent restenosis of the proximal right coronary artery and no new lesions of significant coronary artery stenosis for chest pain were shown . two - dimensional transthoracic echocardiography showed akinesia of the basal inferior wall , but no newly detected regional wall motion abnormality . this extracardiac mass was located in the upper anterior mediastinal area and showed vascularity by color doppler imaging ( fig . 2c ) . the right ventricle was slightly compressed by this mass , but there was no detectable pericardial effusion . the previous echocardiography 1 year ago had shown no echogenic mass in the extracardiac area ( fig . a huge anterior mediastinal mass was enhanced heterogeneously and directly invaded the sternal cartilage , costal cartilage , aortic arch , and pulmonary artery ( fig . these ct findings were suggestive of a thymic carcinoma , and the severe chest pain of this patient was due to the huge anterior mediastinal mass with bone invasion . histological findings by sonography - guided fine - needle aspiration demonstrated that the great majority of cells had tumoral necrosis and a very small number of cells had a high nucleus to cytoplasm ratio with hyperchromatin . these findings represented malignancy of the thymus ; however , it was impossible to classify the subtype of tumor because of severe necrosis ( fig . 5 ) . the patient was referred to an oncologist for further management of the left anterior mediastinal mass . however , further specific tests were refused . according to the clinical presentation , the anterior mediastinal mass with invasiveness , and ptosis and muscle weakness , however , palliative radiotherapy was discontinued because the patient complained of significant anorexia and weakness . only 15% to 20% of patients with acute chest pain actually have acute coronary syndrome.1,2 to discriminate patients with acute coronary syndrome from patients with non - cardiac chest pain is an immediate challenge to the primary clinician , especially in patients with a history of myocardial infarction and percutaneous coronary intervention . the accuracy and efficiency of the differential diagnosis of acute chest pain can be improved by combining various approaches based on vigilant history taking , electrocardiography , biochemical markers , and imaging modalities such as echocardiography , coronary computerized tomography , and coronary angiography . because transthoracic echocardiography is widely available , with bedside utility and high sensitivity and negative predictive value in patients with acute chest pain for ischemia therefore , after ruling out life - threatening conditions , it is necessary to consider other possible causes . especially when chest pain is sharp and lasts for hours , it is frequently suspicious that the chest pain originates from a musculoskeletal cause . when a mediastinal mass such as a thymic carcinoma invades the chest wall , pleura , or heart , patients frequently present with chest pain.3 our case illustrates several considerations in the initial evaluation of chest pain . first , chest ct was needed more than emergent coronary angiography because of the characteristics of the chest pain , which was sharp and had lasted for a month . although a few cases of thymic cyst and malignancy have been documented,4 - 7 there has been no report that echocardiography detected an extracardiac mediastinal mass in the course of evaluating chest pain . thymic carcinoma is a heterogeneous group of aggressive and epithelial malignancies , and its incidence is rare . most patients present with cough , dyspnea , or chest pain . while these symptoms are being evaluated , the malignancy is often incidentally detected by chest radiographs or ct scan.3 when an anterior mediastinal mass shows the presence of an irregular contour , necrotic or cystic component , heterogeneous enhancement , lymphadenopathy , and great vessel invasion on a ct scan , the mass is likely to be a thymic carcinoma.8 mediastinal tumors , including thymoma , thymic carcinoma , and seminoma have been incidentally detected by thallium-201 ( tl 201 ) imaging.9 considering that the mediastinal mass is adjacent to the cardiac chamber , echocardiography can evaluate a mediastinal mass like a ct scan or tl 201 uptake . the mediastinal mass can distort or partially displace one or more cardiac chambers , and an anterior mediastinal mass can compress the right heart chamber , which can be shown by echocardiography.5 it has been reported that thymic cancer was detected by echocardiography presenting as cardiac tamponade or supra vena cava ( svc ) echocardiography is useful for the evaluation of chest pain in emergency circumstances and can detect a mediastinal mass because mediastinal masses are just adjacent to the heart ; however , the diagnosis of an extracardiac mass can be missed . therefore , it is important to look outside the heart in addition to the heart itself in the assessment of chest pain . bromodomains are epigenetic readers that specifically recognize acetyl - lysine ( kac ) marks on proteins . targeting this protein interaction module with small molecules has recently emerged as a potential therapeutic strategy for the treatment of several diseases including cancer and inflammation . to date , most inhibitor development efforts have been focused on the bet family of bromodomain proteins for which several inhibitors have now entered clinical testing . bromodomains have good predicted druggability , and selective chemical tool compounds have been developed even for less attractive binding sites that possess open or highly charged acetyl - lysine binding pockets such as baz2 and atad2 . in addition , potent inhibitors have been developed for highly druggable bromodomains present in brpf , cbp , and brd9 . interestingly , several recent inhibitor development projects have highlighted the success of fragment - based approaches identifying inhibitors , in particular for poorly druggable bromodomains . p300/cbp - associated factor ( pcaf , also known as histone acetyltransferase kat2b ) is a multidomain protein that harbors an acetyltransferase ( hat ) and e3 ubiquitin ligase domains as well as a c - terminal bromodomain that may associate with the hats p300 and cbp . while the roles of the acetyltransferase and the e3 ubiquitin ligase activities have been shown to be required for cell proliferation and apoptosis , little is known about the regulatory function of the pcaf bromodomain in cellular processes . selective inhibitors , so - called chemical probes , would therefore be interesting reagents to unravel the functions of the pcaf bromodomain and to assess its therapeutic potential as a targeting site for drug development . an important role of the pcaf acetyl - lysine recognition module has already been demonstrated for the replication of aids viruses . the pcaf bromodomain targets the hiv tat protein acetylated at k50 , an essential association that activates hiv-1 transcription and promotes the integrated proviral replication . development of pcaf bromo - domain inhibitors has therefore been proposed as a potential strategy for the treatment of aids , and this strategy has been confirmed by early n1-aryl - propane-1,3-diamine - based compounds that have been shown to prevent pcaf / tat association . this compound class has recently been followed up by the development of a series of 2-(3-aminopropylamino ) pyridine 1-oxide derivatives that however did not lead to improvement of the micromolar potency of current pcaf bromodomain inhibitors . this prompted us to identify more diverse chemical starting points using fragment screening by thermal shifts assays and nmr as well as structure - based approaches . to extend structural knowledge from our previous apo crystal structure , we initially determined the crystal structure of the pcaf - acetyl - lysine complex to provide insights into acetyl - lysine recognition . the binding mode of the acetyl - lysine in pcaf strongly resembled that observed in other bromodomains with the kac carbonyl forming two canonical hydrogen bonds to a conserved n803 and a conserved water molecule . this water is part of a conserved intricate network of structural waters located at the bottom of the pocket that bridge interaction with the za loop y760 and the backbone carbonyl of e750 ( figure 1a ) . despite sharing the conserved interactions , the kac binding groove in pcaf was fenced on one side by the bulky residue y809 , resulting in a tight and restricted pocket ( figure 1b ) . large aromatic residues in this position are not common for human bromodomains , present in less than a third of proteins in the family . ( a ) detailed interactions between the bound acetyl - lysine ( kac , shown in yellow stick ) and the pcaf bromodomain . ( b ) electrostatic surface representation reveals a tight central kac pocket that is extended by wide cavities lined by the za and bc loop regions . another interesting structural feature was the extension of the binding pocket by a shelf created by the za loop harboring a mixture of hydrophobic and polar surface residues ( figure 1b ) . however , recently the development of baz2 inhibitors demonstrated that such large binding pockets can be efficiently occupied for instance by aromatic ring systems of the baz2-icr probe that are aligned by intramolecular aromatic -stacking . the low affinities of the previously developed compounds prompted us to search for other kac mimetics that would be compatible with the constraint nature of the central pcaf pocket . we employed different techniques to screen two fragment libraries . in one set of experiments , we used a thermal stability shift assay to screen a small fragment set at 1 mm and identified several fragments that resulted in positive melting temperature ( tm ) shifts in the range of 12 c ( figure 2 ) . the highest tm shifts were recorded for 1-methylquinolin-2(1h)-one ) ( 1 , br004 ) , 1h - indole-5-carboxamide ( 2 , br005 ) , 2-methylisoindolin-1-one ( 3 , br013 ) , 4-methoxybenzo[d]isoxazol-3-amine ( 4 , mb360 ) , 2,3-dihydrobenzo[b]dioxine-5-carboxamide ( 5 , mb364 ) , and ( 4-(1,2,3-thiadiazol-4-yl)phenyl)methanamine ( 7 , mb093 ) , most of which comprised known kac mimetic scaffolds . in parallel , we used target immobilized nmr screening ( tins ) to screen a diverse collection of approximately 950 commercially available fragments . we first confirmed that binding of kac to immobilized pcaf could be observed in 2% dmso ( supporting information , figure s1 ) . subsequently mixtures of fragments were screened with a maximum of four compounds per mix such that the dmso concentration was never more than 2% . consistent with earlier predictions , we observed a tins profile indicating that pcaf is highly ligandable ( figure 2c ) . on the basis of the profile , 63 hits were selected , a 6.6% hit rate . in contrast to tins , binding of kac to immobilized pcaf in the presence of dmso could not be detected by spr . consequently , only those hits that were readily soluble in an aqueous buffer were orthogonally validated by spr . the binding of 17 hits could be fully characterized by spr , and three were selected for structural studies based on novel features ( supporting information , figure s2 and table s1 ) . these fragments ( 9 , 10 , and 11 ) also resulted in substantial tm shifts ( figure 2 ) . fragments containing kac mimetics identified as potential binders for pcaf bromodomain . chemical structures of the identified fragments are shown in ( a ) together with average tm shifts ( sem ) using triplicate experimental data listed in ( b ) . the t / r ratio of each compound screened was calculated as a height weighted average of the ratio of the peak amplitude of each nmr resonance in the presence of pcaf over that in the presence of the reference protein . the t / r ratios were then binned , and the frequency is plotted above . the asymmetry and tailing to the left ( i.e. , a large number of compounds displaying preferential binding to pcaf ) are indicative of high ligandability of pcaf . we next attempted to verify the binding modes of the identified fragments and successfully determined the complex crystal structures for seven kac mimetic fragments . as expected from their chemical structure , all of these fragments occupied the kac binding site through groups that mimicked the hydrogen bond interaction of acetyl - lysine . because the co - crystallized compounds were small , the contacts with the bromodomain were limited only to the canonical hydrogen bond with n803 and the typical water - mediated contact with y760 ( figure 3a ) . however , additional interactions were also observed for fr11 , of which the 1-ethanol decoration was oriented toward the open za cavity and formed both direct and water - mediated hydrogen bonds to the backbones of the za loop v752 and p751 . superimposition of all structures revealed that the co - crystallized fragments fit tightly into the narrow kac pocket , and most fragments formed aromatic interactions with y809 that lines the central acetyl - lysine binding groove of the pcaf bromomdomain ( figure 3b ) . no significant structural alterations were observed when comparing all complexes , suggesting that the pcaf bromodomain contains a rigid acetyl - lysine binding pocket . some kac mimetic groups of the identified fragments were not specific for pcaf and have been previously shown to bind to other bromodomains , for example , 1 also interacts with atad2 and the isoxazole 8 with brd4 and crebbp with highly conserved binding modes . ( a ) detailed interactions between the bound fragments ( yellow stick ) within the pcaf kac binding site . the conserved water molecules at the bottom of the pocket are shown in pink spheres , and an additional water molecule involving in additional water - mediated interactions observed in the complex with 11 is highlighted by magenta sphere . ( b ) superimposition of the bound fragments and kac revealed canonical acetyl - lysine mimetic binding modes and no significant conformational changes upon inhibitor binding within the pocket . coordinates have been deposited under accession codes : 5fe1 , 5fe2 , 5fe3 , 5fe4 , 5fe6 , 5fe7 , and 5fe5 . with the success in identifying the kac mimetic fragments and obtaining the complex structures , we next expanded the ligand series to more decorated compounds by choosing two diverse scaffolds ( figure 4a ) . two compounds , cpd1 ( 12 ) ( n-(1,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)acetamide ) and cpd2 ( 13 ) ( n-(1,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)methanesulfonam ) , were derivatives based on fragment hit 1 containing an additional 4-methyl group and two different decorations at the seventh position , either with n - linked acetamide or sulfonamide . a third inhibitor , cpd3 ( 14 ) ( n - methyl-5-(2-oxo-2-(phenylamino)ethoxy)-2-((tetrahydro-2h - pyran-4-yl)oxy)benzamide ) , contained instead a benzene core harboring an acetamide extension , a kac mimetic group resembling fragment 1 . in addition , this latter compound was also further decorated by tetrahydropyran and n - phenylacetamide linked to the benzene ring via an ethoxy linkage at the ortho and meta positions relative to the acetamide group , respectively . ( a ) chemical structures of three in - house available compounds that contain the fragment - related kac mimetic scaffolds . ( b ) average tm shift values from triplicate experiments for the selected compounds . ( c ) itc binding data for the interactions between 13 and 14 with pcaf with the isotherms of raw titration heat shown on the top and the normalized binding heat with the single - site fitting ( red line ) shown at the bottom . the binding of these three compounds was initially confirmed by dsf with 12 and 13 , showing a similar degree of stabilization with tm shifts of 2.62.9 c at 1 mm concentration ( figure 4b ) . because of signal interference , 14 was used at half the concentration of the others but nonetheless produced a similar tm shift of 3.3 c . as expected , the increase of the tm shift values of these three ligands suggested stronger binding to pcaf bromodomain . we used isothermal titration calorimetry ( itc ) to determine binding affinities in solution . in agreement with the dsf results , the measured kd of 13 was observed to be 21 m , which was 3-fold higher than that of the more decorated 14 ( kd of 7 m ) ( figure 4c ) . the increase in the affinity of the latter was due to significantly larger negative binding enthalpy , suggesting more favorable polar interaction of this inhibitor . however , the ligand efficiency ( le ) decreased to 0.24 for 14 compared to le of 0.34 for 13 . to gain insight into the selectivity of the most potent fragment hits , we performed a temperature shift screen using a panel of 48 recombinant bromodomains ( supporting information , figure s3 ) . this selectivity screen revealed strong interaction of 14 to bromodomains present in cecr2 ( 7.1 c ) , brd7 ( 6.5 c ) , suggesting that this fragment can also be optimized targeting other bromodomains . , both inhibitors showed strong inhibition for brd9 ( supporting information , table s2 ) . this is not surprising because 1-methylquinoline-2-ones have been developed into highly specific brd9 inhibitors . the crystal structures of the ligand pcaf complex allowed us to evaluate the interactions of the introduced decorations . all compounds could be unambiguously positioned based on the well - defined electron density map ( figure 5 ) . as expected in complexes with 12 and 13 , the 1-methylquinoline-2-one group occupied the kac binding groove and formed similar canonical interactions as observed in the pcaf complex with 1 , while the decoration of both compounds oriented toward the za pockets . in contrast , the 13 n - linked sulfonamide was observed to replace a water molecule that bridged interactions between e756 and k753 in the kac complex ( figure 1a ) , enabling the formation of two direct hydrogen bonds to these two residues as well as a water - mediated contact to the carbonyl backbone of w746 ( figure 5 ) . interestingly , a sulfonamide at this analogous position also engaged within the kac site of baz2b in the gsk2801 inhibitor complex . crystal structures of pcaf in complexes with 12 , 13 , and 14 . detailed interactions between the bound compounds with the pcaf bromodomain are shown . the conserved water at the bottom of the pocket is displayed in pink spheres , while an additional water molecule involving in contact between the 13 and protein is shown in magenta sphere . coordinates have been deposited under accession codes : 5fe8 , 5fe9 , and 5fdz , respectively . the binding mode of 14 was slightly different potentially due to its larger decorations and its different acetyl - lysine mimetic moiety ( figure 5 ) . the acetamide interacted with pcaf in a typical kac mimetic manner , with the benzene core stacking against the y809 in a similar fashion observed also for the other fragments . the tetrahydropyran expanded into the open za pocket , while the n - phenylacetamide extension occupied the bc pocket with the phenyl ring located above n803 and oriented perpendicularly to y802 and p804 within a distance for -stacking . considering its affinity , such limited interactions were rather surprising when compared to the binding mode of the weaker 13 , of which the extended group engaged in more hydrogen bond interactions . we hypothesize therefore that 14 may gain its increased binding potency through a larger contact surface . hence , an extension of the kac - mimetic fragments resulted in improved binding affinities despite the lack of direct polar interaction with the protein . in addition , the large surrounding space adjacent to the za and bc loops provides few spatial constraints , enabling the binding of diverse chemical moieties , albeit with little shape complementarity . it is likely that elongated , flexible , and uncharged substituents that could span the interface as well as engage a few interactions may be beneficial for increasing ligand potency through targeting the za and bc pockets in the pcaf bromodomain . the fragment - based approach coupled with structural information presented here offers a platform expanding the knowledge on pcaf kac - mimetic scaffolds and binding modes to aid development of potent and selective pcaf bromodomain inhibitors . the structural models provide valuable data for the rational design of inhibitors to the rigid and poorly enclosed pcaf kac binding pocket . comparison of inhibitor binding modes highlights the importance of aromatic interactions with y809 and provides several diverse acetyl - lysine mimetic chemotypes . these chemotypes may serve as attractive starting points for inhibitor development efforts targeting this interesting bromodomain . all fragments that were cocrystallized formed an acetyl - lysine mimetic hydrogen bond to n803 . thus , for future development of these fragments into more potent hits the fragment need to be grown rather than linked in order to increase potency . the pcaf bromodomain ( aa 715831 ) containing an n - terminal his6-tag was recombinantly expressed in escherichia coli rosetta . the protein was initially purified by ni - affinity chromatography and subsequently cleaved the tag with tev protease . the cleaved protein was further purified using size exclusion chromatography and stored in 25 mm hepes , ph 7.5 , 150 mm nacl , and 0.5 mm tcep . fragments were purchased from chemical vendors and were at least 95% pure according vendor specifications . the purity of the synthesized compounds 12 , 13 , and 14 was assessed by analytical hplc using an agilent 1100 equipped with photodiode array detector ( dad ) , quaternary gradient pump , and micro plate sampler ( agilent 220 ) . separation was performed upon centurysil c18-aq + 5 m , 50 mm 4.6 mm ( johnson ) . the flow rate of the mobile phase was kept at 3.5 ml / min . mobile phases b and c were acetonitrile with 0.35% cf3co2h and water with 0.35% cf3co2h , respectively . the gradient conditions were as follows : 00.5 min 1% b and 99% c , 3.7 min 90% b and 10% c , 5 min 99% b and 1% c. the injection volume was 10 l . 12 and 13 were 90% pure by hplc at 254 nm and 99% by evaporative light scattering detection ( elsd ) . the protein at 2 m in 10 mm hepes , ph 7.5 and 250 mm nacl was mixed with the fragments / compounds at 1 mm concentration ( unless stated ) . the assays , data evaluation , and melting temperature ( tm ) calculation were performed using a real - time pcr mx3005p machine ( stratagene ) with the protocols described previously . all isothermal calorimetry ( itc ) experiments were carried out on nanoitc ( ta instruments ) at 15 c in the buffer containing 25 mm hepes , ph 7.5 , 150 mm nacl , and 0.5 mm tcep . titration was performed by injecting pcaf ( 250 m ) into the reaction cell containing the compounds ( 1520 m ) . the corrected data of the integrated heat of titration were fitted to an independent single binding site model based on the manufactor protocol , from which the thermodynamics parameters ( h and ts ) , equilibrium association and dissociation constants ( ka and kd ) , and stoichiometry ( n ) were calculated . purified , biotinylated pcaf was captured onto a neutraavidin - coated cm5 sensor chip surface in 20 mm sodium phosphate ph7.5 , 150 mm nacl , 2 mm dtt , and 0.05% p20 tween . an acetyl - lys 16 peptide derived from h4 was used to monitor the ligand binding capacity of the immobilized pcaf bromodomain at regular intervals . apo crystals of pcaf ( 1418 mg / ml ) were obtained using the vapor diffusion method at 4 c and either single - peg - based ( 2135% peg 3350 , 0.1 m bis - tris ph 5.57.0 ) or peg - smear - based ( 2140% medium - molecular - weight peg smears buffered either with 0.1 m bis - tris ph 6.07.5 or 0.1 m tris ph 7.58.8 ) reservoir solutions . ligand complex crystals , soaking was performed using the mother liquor supplemented with the fragments / compounds at 1020 mm concentration and 20% ethylene glycol . diffraction data were collected either in - house or at the diamond light source . initial structure solution was achieved using molecular replacement with phaser from ccp4 suite and the published coordinates of pcaf . manual model building alternated with refinement were performed in coot and refmac , respectively .

we report a case of thymic carcinoma that was initially detected by echocardiography in an 80-year - old male who visited the emergency room for chest pain and had a history of myocardial infarction and percutaneous coronary intervention . transthoracic echocardiography showed a huge extracardiac mass that was located in the anterior mediastinum and was diagnosed as a thymic carcinoma by biopsy . the p300/cbp - associated factor plays a central role in retroviral infection and cancer development , and the c - terminal bromodomain provides an opportunity for selective targeting . here , we report several new classes of acetyl - lysine mimetic ligands ranging from mm to low micromolar affinity that were identified using fragment screening approaches . the binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective pcaf inhibitors .

every human voice is unique as it was found to be quantitatively composed of components called phonemes that have a pitch , cadence , and inflection . hence human voice has been used as one of the popular biometrics in biosecurity applications ; it can be used to authenticate a person 's identity ( identification ) and control access ( authentication and verification ) to a protected resource . unlike other biological traits , like fingerprints and iris scans , voiceprints are relatively vulnerable to replay attack . much of the research works have been devoted to finding improved solutions in the hope of strengthening voiceprints for meeting high demands of security applications . some popular techniques include multimodal authentication that fused audio , visual , and other forms of biometrics into one . since then , voice biometrics has been largely geared towards the security directions of biometric identification and biometric verification . voice biometrics is used either alone or in combination with other biometrics . in voice verification ( vv ) , a voiceprint of a speaker who claims to be who he is , is presented to the biometrics system for a one - to - one checking of the reference voiceprint which is stored in a database . once he is successfully verified with a match , subsequent access rights would be granted to him . the other type of checking called voice identification ( vi ) relies on a one - to - many checking for identifying a previously unknown voiceprint . the unlabelled voiceprint under question is searching through the whole database with the aim of finding a match of an already known sample . we can see that both vv and vi require a priori condition that a set of voiceprints must have already been known for the matching of new samples to proceed . this is akin to database query or supervised learning where preknown samples must be initially used to train up a decision model , so testing and matching of new sample can follow . what if in a scenario where a handful of unknown voiceprints are collected , but we wish to obtain some information about them ? such scenarios may include but not limited to security surveillance problems where a list of voice traces are captured from a monitored area , how many unique speakers there are , their ages , and genders , and from their speech accents which ethnic backgrounds these people belong to ; customer - service applications where callers will be automatically classified from their tones to categories of their needs and emotions . it was only until recently , voice classification ( vc ) that attempts to determine if a speaker should be classified to a particular characteristic group rather than to a particular individual has gained popularity . vc can help complement the security of vv and vi systems too . in figure 2 an example of a voice biometric system is being compromised ; through hacking , the content of a voiceprint b is modified to that of another voiceprint ( let us say a ) that has a higher access authority . because the database of the voiceprints just like an encrypted list of passwords in a file system is accessed individually , each voiceprint is protected independently ; allowing the existence of two same voiceprints goes undetected . so an imposter with b can cheat gaining a restricted access right by matching b to a in a vi system . vc could be used to prevent this fraud by checking how many unique items there are in different groups . if extra voiceprints suddenly emerge or have gone missing from a group , the integrities of the voiceprints must have changed . for developing a vc system , several approaches have been studied , such as artificial neural networks ( ann ) , support vector machines ( svms ) , hidden markov models ( hmms ) and gaussian mixture models ( gmms ) . they have been used heavily for training up a model with predefined voice samples for voice recognition . table 1 shows a summary of the techniques by which majority of research works used . these techniques generally function like a black box ; for instance , the weights for mapping the relations of the inputs to the outputs are in plain numeric , the kernel parameters are used for low - level computation , and so forth . they require a full set of known samples to be available before they can be tuned up for actual use . in this paper voiceprints who share similar features will be placed into distinctive groups that represent some labels about the speakers . subsequently a decision tree ( classifier ) can be built after studying and confirming the characteristic groups . the advantage of decision tree is that easily comprehensible rules in terms of if - then - else conditions can be generated when the decision tree is constructed . using the features of a human voice as a voice classifier for classifying speakers has not been researched a great extent although it has a large implication in voice biometrics applications . to the best of the author 's knowledge , nobody has applied such techniques of vc before . this is the research focus of this paper ; a collection of algorithms are introduced for supporting grouping unlabeled voiceprints and then subsequently classifying new incoming voiceprints . they can be used for checking the integrity of the groups of voiceprints for solving the security problem that is illustrated in figure 2 . the contribution of this paper is an alternative computation platform for realizing voice classification ; the algorithms are relatively simpler than the existing ones and fellow researchers that can easily adopt them for implementing vc systems . the model that we proposed aims at providing a generic voice classification framework under which a collection of algorithms such as hierarchical time series clustering , dynamic time wrap transform , discrete wavelet transform and decision tree would have to work together . the prominent advantage is its generic property that can be applied across a variety of applications that capitalize on voice classification . while the inputs are previously unknown voices , the voices would be automatically grouped together according to their own characteristics . each group or cluster being formed as an output represents one pronounced characteristic which is shared in common by all the voice samples inside ( total = n ) . our model assumes that the collected voices in waveforms would be recorded in the format of time series . each time series is a vector of numeric data points that can be represented by a set of m attribute values , such as a time series s = [ x1 , x2 , , xm ] . in a collected dataset whose speakers ' identities are not known , a sufficient amount of voice samples are gathered from each speaker and these samples can then be clustered by using hierarchical time series clustering algorithm . clustering is done based on the characteristics of the voice samples themselves . at this point no classification area was sought specifically , for instance gender or ethnic background , as it was preferred to allow the results to decide the characteristics that lead to a particular clustering group . as shown in figure 3 , an example scenario by the proposed model is a surveillance eavesdropper that collects from a secret meeting a total of n voice traces . the voice traces may be spoken by more than one speaker , one trace per speaker at a time , and each voice trace can be encoded by m coefficient attributes regardless of how long the conversation is . the voices that are in the form of time series can be submitted for hierarchical clustering for self - grouping . hierarchical clustering instead of others is applied because it gives a layered structure of groupings which we do not know in advance in different resolutions . after the clustering , not only we know how the speakers whose voices are distinctively grouped , the number of unique voices ( hence the number of speakers ) can also be identified . in essence , it may be possible to infer from the groupings that how many speakers there are in the meeting , what characteristics they have in each group . however , it requires further verification and probably extra information to infer detailed assertions such as gender , age , and the emotions of the speech . with the groupings available a voice classifier can be developed after the unlabeled voice traces labeled with the classes derived from the characteristics of the groups . so that in our model , unsupervised learning by clustering comes first , and then supervised learning for building the decision tree follows . the voice traces in the form of labeled time series can be used as training data to build a classifier . however , in our experiment , we opt to transform the voice traces from time domain to frequency domain , as our experiment results show that the accuracy performance of the classifier can be significantly improved . when the classifier is ready , future new voice samples can be automatically classified into the characteristic groups . if necessary , the process of hierarchical clustering can be applied on the new samples again in case new characteristics from the voice samples may be discovered . the goal of time series clustering is to identify the speaker category to which a voice belongs given the multivariate time series points of each voice trace . in our experiments the synthetic control wave dataset and empirical datasets from uci data archive the wave has 60 coefficients and the live japanese vowel data each wave is characterized by 12 coefficients . similar waves cling together to form a cluster , and dissimilar waves tend to stay far apart in separate clusters . iteratively the time series clustering algorithm relocate the data points one step at a time to ensure that the data points inside the same cluster have the minimum intradissimilarity and data points across different clusters have the maximum inter - similarity . the similarity is defined as the multidimensional distance between two data points whose multiple attributes are measured as how close they are in values . two variables exist for time series clustering algorithm , one is for choosing the similarity function for measuring the distance between each pair of data points , and the other is the overall operation that converge from an initial assignment of data points to clusters to a converged or optimal assignment of data points to clusters . many similarity measures are available such as manhattan , euclidean and minkowski just to name a few . in our experiments , a range of popular similarity functions are compared in performance in order to observe which one performs the best . table 2 shows a list of performance results in the percentage of correctly clustered groups by using various similarity functions . because the nature of the data points that we are working with is time series , we choose to use dynamic time warping function ( dtw ) as a distance measure that finds optimal alignment between two sequences of time series data points . dtw a pairwise comparison of the feature ( or attribute ) vectors in each time series . it finds an optimal match between two sequences that allows for stretched or compressed sections of the sequences . in other words it allows some flexibility for matching two sequences that may vary slightly in speed or time . the sequences are warped nonlinearly in the time dimension to determine a measure of their similarity independent of certain nonlinear variations in the time dimension . it is popular in the application of signal processing where two signal patterns are to be matched in similarity . particularly suitable dtw is for matching sequences that may have missing information or various lengths , on condition that the sequences are long enough for matching . in theory , dtw is most suitable for voice wave patterns because exact matching for such patterns often may not occur , and voice wave patterns may vary slightly in time domain . they range from simple ones like k - means and k - medoids , to sophisticated algorithms like dbscan , density - based clustering for clustering structures . in our case , hierarchical clustering is desirable because it allows the time series which are voice waves to be grouped in different levels automatically that helps a user to explore the structure of the groupings from coarse to refined . like most of the clustering algorithms which operate by unsupervised learning , hierarchical clustering does not require the number of clusters to be predefined at the beginning ; it allows the data to decide the suitable number of groups by themselves . in our experiment , agglomerative mode which is also known as the bottom up approach is used . initially each observation starts in its own cluster , and pairs of clusters are merged as one moves up the hierarchy . for deciding which clusters should be merged , a similarity function is used between sets of observations . a variety of similarity functions are used here for experiments , they are canberra , dtw , euclidean , manhattan , and minkowski from power 1 to 10 . the clustering algorithm constructs the hierarchy from the individual time series by progressively merging clusters up . the basic process of hierarchical clustering comprises of the following steps , given n time series , and a two dimensional n n similarity matrix s. step 1each time series is assigned to a cluster of its own , with a total of n clusters for n time series . initialize s with similarity measures between the clusters which are the same as the similarity measure between the time series that they contain . each time series is assigned to a cluster of its own , with a total of n clusters for n time series . initialize s with similarity measures between the clusters which are the same as the similarity measure between the time series that they contain . retain the current level of clusters and move up a level in the hierarchy . retain the current level of clusters and move up a level in the hierarchy . step 3calculate the new similarity measures in s between the new clusters and each of the old clusters . calculate the new similarity measures in s between the new clusters and each of the old clusters . step 4finally repeat step 2 and step 3 until all the time series are clustered into a single cluster of size n. when this happens , the highest level of the hierarchy is attained . finally repeat step 2 and step 3 until all the time series are clustered into a single cluster of size n. when this happens , the highest level of the hierarchy is attained . two experiments are conducted for testing the performance of the algorithm over a synthetic control dataset and a live dataset . the synthetic control dataset contains 600 examples of time series wave forms synthetically generated by the process in alcock and manolopoulos ( 1999 ) . there are six different classes of control charts that represent generally different shapes of time series waves . each wave is characterized by 60 coefficients in the form of temporal data points , each different group ( or class ) has 100 samples and total there are 600 samples in the dataset . each class has an essentially unique bunch of waveforms that are different from those of the other classes ; hence , we can assume the six classes represent six different types of speakers who have different voiceprints from one another . for example , it could be speakers who come from six different geographical locations therefore different ascents , speakers who speak in six different emotions / languages , or speakers of six distinctively different age groups . the other empirical dataset contains 640 time series of 12 linear prediction cepstrum coefficients ( lpcs ) taken from nine male speakers . the data was initially collected by a japanese research team for examining a newly developed classifier for multidimensional curves ( multidimensional time series ) . a 12-degree linear prediction analysis was applied to obtain a discrete - time series with 12 lpc cepstrum coefficients . this means that one utterance by a speaker forms a time series whose length is in the range 729 , and each point of a time series is of 12 features ( 12 coefficients ) . analysis parameters are as follows : sampling rate = 10 khz , frame length = 25.6 ms , and shift length = 6.4 ms . blocks 130 represent speaker 1 , blocks 3160 represent speaker 2 , and so on up to speaker 9 . a sample of the time series taken from one of the voice trace is shown in figure 4 . just as shown in our model in figure 3 , the raw voice series are formatted and processed into records that have exactly 12 coefficients ( attributes ) . hierarchical time series clustering is applied over the processed data , so that each data point that the clustering algorithm works with has identical attributes and scales for similarities measures . by plotting the processed data with x - axis as the first column of a consecutive block against the rest of the series with values within the range at the y - axis , we generate some visualization of the time series points with distinguishable shapes . figure 5 shows three groups of voice series that are taken from the dataset blocks from three different speakers . just by visual inspection the four voice utterances on the top row sit at about three quarters on the x - axis , the cap of the data clusters is dominated by small square dots ( that just represent one of the coefficient values of the block of the sample ) , then followed by other shapes of dots and diamond shaped dots at the bottom . though each of the four clusters on the top row is not exactly identical to each other , they roughly have a similar structure . in the middle row , the voice visualization by another speaker has the data near the middle of x - axis , and the outlined structure has the cross - shaped markers on the cap . and the visualization on the bottom row has an obviously different formation than the other two . that shows the voices of the three speakers are essentially different as by their voice characteristics , and the differences can be visually spotted . however , computationally , the differences in voice characteristics would have to be revealed by clustering algorithm . the hierarchical clustering algorithm that we applied in the experiment is implemented in r which is a free software environment for statistical computing and graphics ( http://www.r-project.org/ ) . the synthetic data are first sampled with a ratio of 10% for producing the first iteration of data points and clusters . dtw that serves as the similarity function is embedded in the clustering algorithm for processing the time series data till convergence . a snapshot of the resulting dendrogram by using dtw similarity function is shown in figure 6(a ) . it can be seen that the dendrogram by dtw can effectively partition the time series into six distinct groups that represent six speech types . the groupings , by the dtw dendrogram as shown in figure 6(b ) , can be used to map over to the actual row number of the dataset that has a total of 600 rows . in other words , the time series that are indexed by the row numbers can be allocated to the six groups by the dendrogram as a result of time series clustering . in another counter example , similarity function by euclidean distance is applied in the experiment ; we can easily see that the groupings at the dendrogram as shown in figure 7(a ) are not in perfect order at all . we can safely remark that dtw is superior to euclidean in the clustering . from a bird - eye view , figure 8 shows clearly on the efficacy of the two similarity functions in hierarchical time series clustering . without showing the dendrogram for each of the other varieties of similarity functions , a comparison table below shows the performance of grouping of each technique . the performance is estimated by counting the number of mislocated groups in the dendrogram by that particular similarity function . dtw performs consistently well , while minkowski shows an optima when the power was increasing from 1 to 10 . this observation confirms that dtw is suitable for time series clustering as time series do vary in time domain more or less to certain extent . the subsequent experiment is to build a decision tree after the grouping has been formed by hierarchical time series clustering . ripper function is suggested to be run for generated comprehensible rules that are in the form of if - then - else . the rules specify a sequence of conditions meeting which in order lead to a predefined class label . when a new voiceprint is received , pass it over the rules by checking its coefficient values that can determine which class label the voiceprint fits in . the other decision tree algorithm is the classical c5.0 or j48 with pruning mode on , in weka which is an open source of machine learning algorithms for solving data mining problems implemented in java and open sourced under the gpl ( http://archive.ics.uci.edu/ml ) . the time series data , however , are converted to their corresponding frequency domain by discrete wavelet transformation ( dwt ) . dwt applies the the haar wavelet transform which was invented by kristian sandberg from university of colorado at boulder , usa in year 2000 . dwt in principle works better than time series points in classification because dwt can find where the energies are concentrated in the frequency domain , and remarkable coefficients called haar attributes are well describing the characteristics of the time series . a comparison of the original coefficients in time domain and transformed coefficient in frequency domain can be seen that wavelets after the transformation have sharper and narrower statistical distribution than the time series points , in figure 9 . dwt is implemented in the plug - in filter in weka called weka.filters.unsupervised.attribute.wavelet . the performance of the decision tree which is a voice classifier is defined as a composite of accuracy measures . they generally come in the following indices in data mining as ( 1 ) the percentage of correctly classified instances , ( 2 ) the root relative squared error , ( 3 ) coverage of cases , ( 4 ) overall precision ; in a classification task , the precision for a class is the number of true positives ( i.e. , the number of items correctly labeled as belonging to the positive class ) divided by the total number of elements labeled as belonging to the positive class ( i.e. , the sum of true positives and false positives , which are items incorrectly labeled as belonging to the class ) , ( 5 ) overall recall which is defined as the number of true positives divided by the total number of elements that actually belong to the positive class ( i.e. , the sum of true positives and false negatives , which are items which were not labeled as belonging to the positive class but should have been ) , and ( 6 ) f - score , which is a measure of a test 's accuracy by considering both the precision and the recall of the test to compute the score . the performance comparison table is given in table 3 . it compares mainly the classification accuracy by using a j48 decision tree in weka of the time series version and the wavelet version of the two testing datasets . it can be noticed that in general wavelets have improvement over the time series in terms of classification accuracy . the results of the empirical data are generally lower in accuracy than the synthetic control data probably due to its complex and less uniform in the time series structures , plus the normalization effect for limiting the time series into fixed length from its original variable length . a sample of the decision tree generated from the experiment is shown in figure 10 . by using the decision tree as classifier using voice as a biometrics has its advantage because it is a noninvasive nature process in human interaction , and human voice has been proven to contain biological traits that can uniquely identify an individual . in the past many studies have focused on applying voice biometrics into security - related applications such as user verification and biometric identification . voice classification is recently becoming popular as it serves as the underlying technique for monitoring different types of speakers and providing supreme customer service by estimating the natures of phone / web calls ; these applications potentially have high values in security surveillance and commercial uses . in this paper , a set of relatively simple and transparent techniques are described for enabling voice classification . fellow researchers are encouraged to test out the collection of algorithms as recommended in this paper for experimenting voice datasets pertaining to voice classification applications . in particular , we showed via experiments that hierarchical time series clustering algorithm with various similarity functions can yield different levels of accuracy . it is shown possible that time series can be grouped into different clusters , just as if some unknown voices are grouped together by their common characteristics . wavelets after transforming of time series samples into frequency domain demonstrate an improved accuracy performance in decision tree . the future works include fine - tuning the mentioned algorithms in the paper for even better performance . the algorithms should be programmed into a single software program in order to support as a core classification engine for voice biometric application systems . medical doctors move across borders being motivated by higher salaries , better working conditions , new professional experience , and training and career opportunities . in europe , migration of medical doctors has been observed since the 1940s and has shown various dynamics over the years . european integration has offered new possibilities for medical doctors to improve their skills , to study or to work in other countries . however , outflows from eastern europe started before accession , due to the political transitions of the late 1980s and the last 1990s . the eu enlargement , first in 2004 and then in 2007 , has generated increased mobility , especially from east to west , namely from eu-12 to eu-15 . studies have shown that migration of physicians from new member states has been lower than the leaving intentions . given the increasing trends in health professionals mobility , the european union has established a legal framework to regulate both the recognition of professional qualification and the free mobility of doctors and patients within europe . however , a legal framework for the recognition of professional qualification for physicians willing to work outside europe and for those coming from non - european countries is still lacking . migration of health professionals in generally and of medical doctors in particular has its roots in current problems of healthcare systems . medical doctors decide to move from one country to another not only because of higher incomes , but in search of better working environments , career opportunities and social recognition . thus , mobility of medical doctors is seen as a symptom of more fundamental health systems problems . these need to be addressed by policy makers in an integrated manner because health professional mobility can not be considered in isolation . while some european countries have to deal with major shortages of medical doctors , other are confronted with increasing pressures to manage maldistribution , both geographically and in terms of specialities needed . usually the positive effects occur when mobility is temporary and with the purpose of achieving new experiences , new specialities and training , followed by return in home country . in addition , mobility of doctors impacts positively the patients access to medical treatment and medical service , because patients can benefit of the knowledge and training achieved by medical doctors in other countries . if mobility is for long - term and outflows occur in countries struggling with shortages of medical doctors , then the negative impacts on the healthcare systems are felt both at macro level - financial loss for the country that has paid for the education of the physicians , national health system has to be adapted to the new situation , and at micro level lack of sufficient medical doctors or maldistribution will impact patients safety and access to care , and finally patients health . among the most - cited factors for physicians mobility is the financial motivation . as regards the salaries , major differences between european countries can be observed . especially since the eu became more diverse in socio - economic terms , with larger salary differentials , incentives to seek employment in another member state have increased . for example , an estonian medical doctor can earn six times more in finland while a romanian general practitioner can earn ten times more in france . in 2009 , a 25% cut in the salaries of health professionals in romania has led to an increase in the number of doctors seeking work abroad . a reverse effect has been observed in poland , lithuania and slovenia , where annual salary increases have helped to diminish the outflow of medical doctors . high salary differentials exist not only between eu-12 and eu-15 , but also between eu-15 and non - eu countries , such as us , new zeeland and australia . therefore , many doctors from western european countries ( eu-15 ) may be motivated to seek work overseas . the economic situation of a country has a major impact on the quality and standards of healthcare facilities and on the social benefits offered for health professionals . while some european countries , mostly from eu-15 , have developed high standards for healthcare such as better equipped hospitals , introduction of high technologies for diagnosis and surgery , and new tools for testing , other countries struggle with major shortages as regards the facilities named above . this is usually the case of new member states , which had to pass through a difficult transition period and are affected by economic problems that do not leave room for further development of the existent healthcare facilities . therefore , when medical doctors are forced to deal with a lower number of hospital beds than the number of patients , with old diagnostic tools , with shortage of medicines and instruments available in hospital pharmacies , or with inappropriate conditions for carrying out complex surgeries , they often decide to work in an environment that can offer them better conditions for doing their job . however , better working conditions do not only refer to access to better healthcare facilities but also to social and economic incentives , working schedule and promotion opportunities . training and career opportunities are also among the relevant decision - making factors for physicians who consider leaving their country of origin , either temporary or for a long period of time . again , the available training opportunities vary considerably across europe and there are also differences between europe and countries like us or japan . therefore , doctors migrate because they may wish to specialise in a certain medical filed which is not available in their country or , if available , it is not yet sufficiently developed . but doctors may seek training and career opportunities in other countries not only because such opportunities lack in their home country , but because the experience and the know - how achieved abroad is often very enriching and acts as a boost for their career . in addition , new professional and personal experiences achieved across the borders may widen their horizon in the medical field . finally , some medical doctors also wish to migrate because they simply want to make a change in their lifestyle . for instance , a physician from denmark or sweden could seek working in spain or in italy , because the southern european countries are well known for their warm climate , relaxed daily life , rich culture and tasty foods . similarly , a physician from bulgaria could wish to work in austria not necessarily for a higher salary but for the well organised system and infrastructure , for the austrian high living standards or for the great mountains . according to a who report on healthcare workforce migration in europe , there are also other factors associated with migration flows that can stimulate migration and affect the choice of a destination country : organizational factors , such as heavy workload , occupational risks , poor management , favouritism or lack of due process , lack of recognition;healthcare system factors , such as the absence or inadequacy of human resource policies , insufficient funding of health services , and centralised decision - making;general environmental factors , such as poor economic conditions and lack of security . organizational factors , such as heavy workload , occupational risks , poor management , favouritism or lack of due process , lack of recognition ; healthcare system factors , such as the absence or inadequacy of human resource policies , insufficient funding of health services , and centralised decision - making ; general environmental factors , such as poor economic conditions and lack of security . the mobility of doctors within the european region was to a large extent influenced by the eu enlargement in 2004 and 2007 . the eu enlargement triggered east west asymmetries in terms of inflows and outflows of health professionals , with migrants from new member states moving to countries from eu-15 . however , many of the eu-15 countries have outflows of the same magnitude as the eu-12 , but unlike the eu-15 , the eu-12 countries have only negligible inflows . in europe , among the major destination countries are germany , france , italy , uk and spain . they receive doctors from countries like poland , greece , romania , switzerland and the czech republic . germany is at the same time a big source and destination country , german doctors choosing to migrate mainly to the united kingdom and italy , followed by switzerland and us . outflows have also been observed in the uk , mainly toward neighbouring countries like ireland and france , but also to spain and the us . according to a who study on health personnel migration , the flow of migrant doctors is very dynamic in the european region . this situation is illustrated in the fig . 1 , which shows the inflows and outflow of physicians from selected european countries .fig . 1migration of physicians in the who european region ( red arrows indicate two - way flows ) , taken from migration of physicians in the who european region ( red arrows indicate two - way flows ) , taken from in fig . 1 one can observe not only a flow from east to west , but also a dynamic exchange between western countries . thus , the flow of migrant doctors among belgium , france and the netherlands is also multidirectional . although there have been several recent studies examining the migration of healthcare workforce in europe , data are still limited . policy makers , workforce planners and healthcare managers need to better understand the mobility of trends in order to react with the right measures . due to the european integration and eu enlargement , which brought more flexibility for travelling and working in europe , it became obvious that an eu framework for professional mobility is needed . in response to this situation , the european parliament and the european council adopted the directive 2005/36/ec on recognition of professional qualifications , which affects more than 800 different professions regulated by member states across the eu , including medical doctors , nurses , midwives , and dentists . the directive sets the european legal framework for the recognition of professional qualifications obtained in a member state other than the one where the person wishes to work . with directive 2005/36/ec the eu has reformed the system for recognition of professional qualifications , in order to help make labour markets more flexible , further liberalise the provision of services , encourage more automatic recognition of qualifications , and simplify administrative procedures . the most recent consolidated version of the directive was made available on 24 march 2011 . until 20 october 2007 when the transposition period ended , this directive has replaced 15 existing directives in the field of recognition of professional qualifications , providing the first comprehensive modernisation of the eu system since its introduction over 40 years ago . according to the european commission , only 22 out of 27 member states fully transposed the directive within the given timeframe . considering the complexity of directive and the degree it impacts national legislation , some member states had to adapt a significant number of measures in order to complete the transposition . directive 2005/36/ec facilitates temporary provision of services ( tps ) by replacing the previous system of prior check of qualifications by the simpler , optional , system of prior declaration . however , due to the need to protect consumers , a prior check of qualifications may be maintained for professions having public health or safety implications ( article 7(4 ) of the directive ) . according to the european commission , 26 member states have implemented article 7(4 ) of the directive by april 2010 , apart from greece where the situation was still unclear . , professionals can in principle work on the basis of a declaration made in advance . the directive also applies to professionals wishing to establish themselves in an eu country other than that in which they obtained their professional qualifications , either as an employed or self - employed person , either on a permanent basis . directive 2005/36/ec sets out three systems for the recognition of qualifications : automatic recognition for professions for which the minimum training conditions have been harmonised ( health professionals , architects , veterinary surgeons).basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4)specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1)the general system for other regulated professions ( including non - automatic recognition - art . . automatic recognition for professions for which the minimum training conditions have been harmonised ( health professionals , architects , veterinary surgeons).basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4)specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1 ) basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4 ) specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1 ) the general system for other regulated professions ( including non - automatic recognition - art . 10 to 15 ) . recognition on the basis of professional experience for certain professional activities . title ii of directive 2005/36/ec governs the recognition of professional qualifications in the context of a temporary move to the territory of another eu country . the temporary and occasional nature of the activities of a self - employed or employed person is assessed on a case - by - case basis , in light of the duration of the activity , its frequency , regularity and continuity . but the current system must be evaluated in order to verify whether full use has been made of all the opportunities offered by directive 2005/36/ec . the system must also take account of the considerable changes that have occurred in the member states educational and training systems . for this reason , the commission has begun working on evaluating the 2005 directive which culminated in a green paper modernising the professional qualifications directive in june 2011 . the public consultation on the green paper was launched by the commission in january 2011 and a summary report with responses is already available . specifically the mobility of health professionals , the european commission issued a green paper on european workforce for health in december 2008 and invited all interested organisation to a public consultation , which closed on 31 march 2009 . in addition to directive 2005/36/ec addressing cross - border workforce mobility across europe , on 9 march 2011 the european parliament and council adopted directive 2011/24/eu on the application of patients rights in cross - border healthcare . the directive addresses in particular the mobility of patients seeking treatment in another member state and introduces rules regarding the reimbursement of treatments and medical care or investigations received abroad . as regards the impact of directive 2011/24/eu on the mobility of health professionals , the directive stipulates that member states should facilitate cooperation between healthcare providers , purchasers and regulators of different member states at national , regional or local level in order to ensure safe , high - quality and efficient cross - border healthcare . this could be of particular importance in border regions , where cross - border provision of services may be the most efficient way of organising health services for the local population , but where achieving such cross - border provision on a sustained basis requires cooperation between the health systems of different member states . according to directive 2011/24/eu , cooperation between member states may concern practical mechanisms to ensure continuity of care or practical facilitating of cross - border provision of healthcare by health professionals on a temporary or occasional basis . however , in directive 2011/24/eu it is mentioned that this directive should be without prejudice to directive 2005/36/ec on the recognition of professional qualifications . this means that free provision of services of a temporary or occasional nature , including services provided by health professionals in another member state is not subject to specific provisions of union law and is to be restricted for any reason relating to professional qualifications . although the mobility of doctors within the european union is to an extent regulated by eu law , it is also necessary to take into consideration the national legislation from european member states . as mentioned in previous paragraphs , the existing legal framework addresses only the mobility of health professionals ( and other ) when they cross borders within europe . but thus , if a german physician would like to work in china he / she may encounter difficulties in acquiring the recognition of his / her qualifications allowing working in this country . or , if a physician from georgia is seeking work in france , again , there is to date no european legal act which can help in such cases . therefore , the european union should consider introducing further regulations or developing legal instruments aiming at solving this challenge healthcare workforce migration beyond europe s borders . besides the legislative framework aiming at regulating the mobility of doctors in europe , the world health organisation developed a non - regulatory instrument on the international recruitment of health personnel - who global code of practice on the international recruitment of health personnel . the who code of practice was adopted at the sixty - third world health assembly in geneva on 21 may 2010 and its key components are : in destination countries : ethical recruitment practices;protection of the rights of foreign healthcare workers;increased education and training for health sector students;pairing of needs and supply.in source countries : improved conditions for healthcare professionals;continued medical training and increased opportunities;incentives to retain physicians and nurses in countries and regions with human resource shortages . in destination countries : ethical recruitment practices;protection of the rights of foreign healthcare workers;increased education and training for health sector students;pairing of needs and supply . ethical recruitment practices ; protection of the rights of foreign healthcare workers ; increased education and training for health sector students ; pairing of needs and supply . in source countries : improved conditions for healthcare professionals;continued medical training and increased opportunities;incentives to retain physicians and nurses in countries and regions with human resource shortages . improved conditions for healthcare professionals ; continued medical training and increased opportunities ; incentives to retain physicians and nurses in countries and regions with human resource shortages . this initiative of who comes also in response to the consequences of the financial crisis on labour markets and addresses the need to mitigate the negative effects of migration on health systems in developing countries and to ensure equitable access to health care services while minimizing the need to rely on the immigration of health personnel from other countries . the resolution ( eur / rc59/r4 ) adopted by the regional committee urges member states to increase their efforts to develop and implement sustainable health workforce policies , strategies and plans as a critical component of health systems strengthening and to advocate the adoption of a global code of practice on the international recruitment of health personnel in line with the european values of solidarity , equity and participation , both within the who european region and globally . in order to assist the medical doctors ( and other professionals ) who intend to work across borders , national contact points have been established at the initiative of the european commission s service free movement of professionals , directorate general internal market . there are contact points in every eu country that can give information on the recognition of professional qualifications according to the national law and procedures to be followed . contact points may be ministries of education , research or science or other national institutions . the contact points also serve as a guide for the applicants , helping them to complete the required administrative formalities . but contact points can only assist the applicants with their requests and are not in charge for deciding whether the recognition of a certain profession should be granted . the decision - makers are the national competent authorities , who decide whether or not to recognise professional qualifications obtained in other eu countries , in accordance with european and national legislation . the competent authorities in member states are expected to use a set of common rules laid down by a code of conduct . as regards the procedure to be followed for the recognition of professional qualifications , the applicant must apply to the authority that oversees the doctor profession in that country and provide the authority with the proof of qualifications . the competent authority must acknowledge the application within 1 month of receiving it , and ask for missing but necessary documents to process the application . then the authority assesses the qualifications and decides whether to grant the application within 3 months . for complicated cases in the area of non - automatic recognition this procedure may last 4 months . if the applicant does not accept the decision of the competent authority , he / she can appeal to the relevant court in that country . with the aim of ensuring better coordination both among member states and between member states and ec , the tasks of this group include helping national authorities and the commission work together better , monitoring policies with a bearing on qualifications for regulated professions , and exchanging experiences and good practices in the recognition of qualifications . group s members and alternate members are appointed by national governments and they meet several times a year . experts and observers are invited to take part in the group s meetings , which are chaired by the european commission . further support for workforce mobility within europe was made available by the european commission directorate general on employment , social affairs and equal opportunities , who has established eures the european job mobility portal . the purpose of eures is to provide information , advice and recruitment / placement ( job - matching ) services for the benefit of workers and employers as well as any citizen wishing to benefit from the principle of the free movement of persons . there are currently over 20 eures cross - border partnerships , spread geographically throughout europe and involving more than 13 countries . eures plays an important especially in cross - border regions , areas in which there are significant levels of cross - border mobility . more than 600 000 people live in one eu country and work in another and they have to cope with different national practices and legal systems . they may come across administrative , legal or fiscal obstacles to mobility on a daily basis . eures advisers in these areas provide specific advice and guidance on the rights and obligations of workers living in one country and working in another . the impacts on the performance of health systems are subtle , meaning that they are indirect or hard to discern . moreover , some areas from eastern european countries ( e.g. romanian rural areas ) may be particularly vulnerable , showing some of the highest emigration rates among medical doctors and nurses . for the healthcare systems in source countries emigration can contribute to a major shortage of health professionals . this can be observed in various forms , such as loss of training capacity ( when trainers leave ) , heavier workloads for doctors who decide to stay or disruption of services when a key staff member leaves . in addition , the source country also loses the investment in the education of health professionals as well as the contribution they would have otherwise made for the healthcare system . some source countries may have also some benefits from the emigration of doctors , like reduction in staff surpluses and access to new knowledge and skills , in case the emigration is temporary . in some cases , source countries can benefit from collaborative training programmes , research projects or teaching activities which are initiated by emigrant doctors with their home country . unlike in source countries , in destination countries the benefits tend to be more obvious . for instance , migrant physicians may accept lower salaries than native ones , and they may also accept working in geographic areas avoided by national workers . however , destination countries may also encounter some difficulties related to inflow of medical doctors . cultural differences may hinder communication and lack of familiarity with advanced equipment may lead to higher error rates . for temporary migrants , investment in workplace induction can be relatively high compared to the time of service provided by the migrant physicians . as regards the policy implications of doctor s migration , first , the uncertainties surrounding the impact of the economic crises which forced some countries to drastically reduce their healthcare budgets , while in other countries budgets remained unaffected . a second factor impacting healthcare policies is the uncertainty of the development of healthcare workforce in europe . according to a european commission forecast , a shortage of around 1 million health professionals compensation of health workforce shortages by recruiting from third countries is to an extent restricted by ethical concerns , as stipulated in the who global code of practice for the international recruitment of health personnel adopted in 2010 . for these reasons , the european countries dealing with a high demand for medical doctors will face increasing difficulties in filling their vacancies with doctors from abroad . according to the european observatory on health systems and policies , there are three main sets of policy implications linked with the mobility of medical doctors : the first refers to the amount of data , intelligence and evidence , which are currently not sufficiently developed . therefore , in the absence of inflow and outflow data , policy makers can not take appropriate measures to manage doctors migration.the second set of policy implications refers to the strengthening the general workforce strategies . symptom of underlying domestic workforce aspects , such as working conditions , salaries , and training opportunities.the third policy implication is related to sustaining the re - emerging interest in workforce planning methods and techniques , by taking into account especially the dynamics and the need of healthcare force in future . the first refers to the amount of data , intelligence and evidence , which are currently not sufficiently developed . therefore , in the absence of inflow and outflow data , policy makers can not take appropriate measures to manage doctors migration . symptom of underlying domestic workforce aspects , such as working conditions , salaries , and training opportunities . the third policy implication is related to sustaining the re - emerging interest in workforce planning methods and techniques , by taking into account especially the dynamics and the need of healthcare force in future . in responding to the inflow and outflow of medical doctors , today and in future , countries may take a series of measures aiming at an appropriate management of their healthcare workforce . for instance , they may sign bi - lateral agreements or facilitate the recognition of diplomas from non - european countries or they may consider developing twinning schemes and joint training programmes . although these policy responses may facilitate a better management of doctors mobility , the european countries should seek to solve mainly the domestic problems liked to doctors migration . thus , they should seek to strengthen existing healthcare strategies by improving retention , increasing salaries and providing more advanced training opportunities . in addition , countries could develop new healthcare strategies which can better respond to the current and future mobility trends .

voice biometrics has a long history in biosecurity applications such as verification and identification based on characteristics of the human voice . the other application called voice classification which has its important role in grouping unlabelled voice samples , however , has not been widely studied in research . lately voice classification is found useful in phone monitoring , classifying speakers ' gender , ethnicity and emotion states , and so forth . in this paper , a collection of computational algorithms are proposed to support voice classification ; the algorithms are a combination of hierarchical clustering , dynamic time wrap transform , discrete wavelet transform , and decision tree . the proposed algorithms are relatively more transparent and interpretable than the existing ones , though many techniques such as artificial neural networks , support vector machine , and hidden markov model ( which inherently function like a black box ) have been applied for voice verification and voice identification . two datasets , one that is generated synthetically and the other one empirically collected from past voice recognition experiment , are used to verify and demonstrate the effectiveness of our proposed voice classification algorithm . this chapter aims at providing an insight into some major aspects linked to migration of medical doctors within europe . the article describes main factors which contribute to doctors migration . further , the current and future mobility trends in europe are discussed . a major part of this chapter is dedicated to an overview of the eu legal framework impacting healthcare professionals mobility , followed by some useful information related to the procedures for recognition of professional qualifications and offices in charge of mobility . finally , the impacts on healthcare systems and the policy implications of doctor s mobility are described in context of personalised medicine .

imipramine was found to improve mood while being used in a protocol to search for an antipsychotic : ' iproniazid , a drug used in the treatment of tuberculosis , was likewise found to have beneficial effects on mood . the former , a tricyclic antidepressant ( tca ) , and the latter , a monoamine oxidase inhibitor ( maoi ) , belong to two classes of drugs still in use today . depressive mood appears to be attributable to diminished activity of the dopaminergic , noradrenergic , and serotonergic neurotransmitter systems . antidepressants restore the activity of these transmitters by inhibiting reuptake in the presynaptic neurons . additionally , the classic antidepressants have effects on other neurons ( eg , histamine , acetylcholine ) , resulting in major side effects limiting their broader use . depressive symptoms have been described in as many as 40 different disorders , which would imply that they could be used in all of them . although the efficacy of tcas has been well established , the high incidence of side effects and the high number of nonresponders or treatment - resistant patients represent drawbacks that have made it necessary to search for new drugs . the development of selective serotonin reuptake inhibitors ( ssris ) was the first attempt based on a pathophysiological approach . these drugs , which have similar efficacy , but less side effects than the tcas , have become the preferred pharmacological treatment for depression . however , the high number of nonresponders and the delay in onset of response have limited their value . today , up to seven different classes of antidepressants are available , which mainly differ in their selectivity for the respective monoamines and their receptors . these discoveries have intensively stimulated biochemical - pharmacological research into the mechanism of action of antidepressants . findings from these investigations suggest that enhanced activity of the central noradrenergic and/or serotonergic transmitter system is essential for the clinical antidepressant action . such enhancement could be achieved either presynaptically by blocking 2-adrenergic receptors , or in the synaptic cleft by inhibiting the transmitter reuptake or the main metabolic enzyme monoamine oxidase ( mao ) . the increased transmitter concentration in the synaptic cleft after chronic treatment leads to a downregulation of postsynaptic -receptors , sometimes modulated by interaction with neuropeptides and hormones . in addition , depending on the antidepressant used , the sensitivity of 5-ht2a , somatodendritic 5-ht1a , or noradrenergic 1 receptors may be reduced , leading to an overall increase in serotonin transmission . such receptor alterations appear to provide the best explanation for the delay in clinical antidepressant response . the introduction of new classes of antidepressants has led to renewed thinking about their mechanism of action . recent investigations of second messenger systems such as the adenylate cyclase system and the phosphatidylinositol system are very promising . antidepressant drugs , including the mood stabilizers lithium and carbamazepine , modulate both of these second messenger systems , which in turn modulate the phosphorylation status of neuronal proteins via protein kinase . the outcome is a positive alteration of the gene expression of the relevant biochemical structures ( enzymes , transporters , receptors ) , thus restoring the normal function of the respective neuronal systems . thanks to clearer understanding of the function of this complex serotonergic system we now know that a great number of normal and abnormal behaviors can be attributed to dysfunction of the serotonergic neurons , in addition to their role in depression . the limited number of serotonin neurons in the brain ( approximately 300 000 ) suggests that their role is mainly a modulating one . this implies that they act to either dampen or accelerate a given type of behavior . drugs targeting the serotonergic system are therefore able to influence many kinds of behavior abnormalities ( figure 1 ) . concerning the norepinephrine system as already mentioned , some forms of depression are assumed to be accompanied by reduced noradrenergic activity . however , this is a matter for discussion , and some forms of depression may even be accompanied by increased noradrenergic function . it is hypothesized that noradrenergic neurons in the locus ceruleus are activated or increased in anxiety and panic disorders . conversely , a norepinephrine deficit is invoked to explain disturbances of attention , psychomotor retardation , and impaired vigilance . some antidepressants also increase dopaminergic neuron activity , either directly or indirectly , by acting on serotonergic and noradrenergic pathways . dopamine , a major transmitter of the reward system also plays a role in depressive states . dopaminergic antidepressants could be of interest in some subgroups of depression , but so far no such drugs are available in europe . however , in some patients with refractory depression , dopaminergic drugs like amphetamine have some beneficial effects . neuronal circuits establish functional relationships between serotonergic , noradrenergic , and dopaminergic systems , which explains why deficiency in one system impairs the other systems as well , and why even specific drugs like the ssris arc also able to modulate the other systems . the variety of the clinical uses for the newer antidepressants may necessitate a reexamination of traditional diagnostic categories and of theories on the way antidepressants work . antidepressant drugs are used in a wide range of psychiatric disorders . empirical evidence in the 70s suggested that the nonselective serotonin antidepressant clomipramine improved symptoms of ocd . newer generations of antidepressants with fewer side effects have proved to be even more active in ocd . furthermore , 5-ht1a serotonin agonists are being investigated in general anxiety disorders . other potential indications for ssris and the new generation of antidepressants are panic disorders , premenstrual dysphoric disorder , eating disorders , substance abuse disorder , chronic pain , dementia , and personality disorders with aggression or impulse disturbances , and general anxiety disorders . depressive symptoms arc frequently diagnosed in patients with schizophrenia and have been described in schizoaffective disorders . . this may be the reason why such patients are frequently ( 50% of cases ) treated simultaneously with antipsychotics and antidepressants . antidepressants are also useful in the treatment of a group of disorders that may be phenomenologically and genetically related to major depression , such as fibromyalgia , chronic fatigue syndrome , migraine , irritable bowel syndrome , atypical facial pain , and premenstrual dysphoric disorder . it is likely that the etiology of depression ( as a symptom ) in these disorders is similar to that of major depression as an entity , and therefore would explain the efficacy of ssris . although not impossible , it would be contrary to expectation if the mechanism of antidepressant effect was independent of the mechanism of depression in migraine , premenstrual dysphoric disorder , and other conditions . and it would be even more difficult to believe that different , chemically unrelated antidepressant drugs , share the same pharmacological properties while having different mechanisms of action . disturbances in neurodevelopment and/or abnormal immune function may be responsible for schizophrenic symptoms . additionally , abnormal dopamine , norepinephrine , and serotonin transmitter activities in some areas of the brain may be pathophysiological relevant to some schizophrenic syndromes . other theories put forward disturbances in the glutamatergic and gabaergic circuits . because of this heterogeneity and the impossibility of characterizing clinical subgroups of schizophrenic patients , none of these theories has been conclusively proved so far . the discovery of chlorpromazine for the treatment of schizophrenia opened new perspectives for the care of psychiatric patients . unfortunately , chlorpromazine and the other classic neuroleptics produce side effects that limit their widespread use . for many years , the dopamine hypothesis , based on the assumed mechanism of action of these compounds , was the predominant theory . the introduction of new atypical neuroleptics such as clozapine , which was the first one , paved the way for revisiting the dopamine hypothesis of schizophrenia and related theories on the mechanism of action of neuroleptics . to explain the unique features of clozapine , new theories have been put forward , partly in relation to interference with dopamine receptor subtypes and partly in relation to interference with other neurotransmitters such as norepinephrine and serotonin . the nonspecificity of second - generation atypical neuroleptics for the dopaminergic system , the therapeutic ineffectiveness of some selective dopaminergic drugs , the lack of success of genetic studies targeted to the dopaminergic system , and the disappointing biochemical findings in schizophrenic patients have resulted in alternative theories of pathogenic causes of schizophrenia being proposed , opening up new perspectives for the development of future drugs . based on neuropath ological and neuroanatomical findings and in concordance with the revised dopamine hypothesis , new models have been postulated focusing attention on the excitatory amino acid y - aminobutyric acid ( gaba ) and the most ubiquitous amino acid transmitter in the brain , glutamate . psychotic symptoms of schizophrenia have been divided into negative symptoms ( blunted affect , anhedonia , asociality , inability to initiate and carry out complex tasks to completion ) , which seem to be related to cortical hypofunction , and which , in turn , may be associated with decreased mesocortical dopaminergic activity and positive symptoms ( hallucinations , delusions , and thought disorders ) . they also appear in disorders other than schizophrenia as well as many nonpsychotic disorders , and are related to increased activity of the subcortical striatal dopaminergic neurons . was considered as the standard treatment for bipolar depressive and manic patients , the pharmacological strategies for bipolar disorder included neuroleptics and antidepressants . they are now mainly used to treat the psychotic symptoms present during one of the poles of the disorder , or as an adjunctive treatment when other alternatives have failed . there have been several reports that clozapine may be more effective in patients with mania and schizoaffective disorder than in patients with schizophrenia . preliminary reports suggest that the newer atypical antipsychotics olanzapine and sertindole may also be effective in stabilizing mood or in the management of affective symptoms . the occurrence of psychotic symptoms is frequent during the evolution of idiopathic parkinson 's disease and other parkinsonian syndromes . they seem to be related to interactions between the underlying neuropathologies manifestations of the syndromes and the adverse effects associated with chronic antiparkinsonian drug administration . in patients with advanced parkinson 's disease olanzapine has been reported to be effective in the suppression of psychotic symptoms in these patients , but the currently available dose increments may result in an exacerbation of motor disability . as mentioned above , since no solid alternatives have emerged from biological research to replace the current hypothesis regarding the pathogenesis of psychiatric disorders , the development of new psychotropic drugs remains based on the restoration of the imbalance in the monoamincrgic system . this is exemplified by the development of the new antidepressants . the postulate that depression results from a dysfunction in the noradrenergic , serotonergic , and dopaminergic systems leads logically to the attempt to design antidepressants that act mainly on one of the neurotransmitter systems . the idea is to increase selectivity without compromising efficacy , while at the same time reducing the side effects that result of interactions with these and other neurotransmitter systems . thus , blockade of serotonin reuptake gave rise to the now well - known ssris . a new class of drugs , which selectively inhibit the reuptake of norepinephrine , was recently introduced onto the market however , experience with psychotropic drugs acting on either the noradrenergic or the serotonergic systems suggest how important it is ( at least in certain situations ) to act on both systems at once . research was therefore undertaken to develop new antidepressants with a dual action on these systems . although drug development tries to focus on specific mechanisms involved in depression and its symptoms , clinical research is not nosologically but transnosologically oriented . the tools used to monitor therapeutic response in clinical trials are usually rating scales that evaluate the depressive or psychotic state rather than treatment efficacy on a specific entity . efficacy , nosology , and duration of treatment are based on the antidepressant effect , and , therefore , in many of the specific entities where they are presently used , these variables have not been confirmed . similarly , in most trials focusing on therapeutic outcome , there are no differences between different drugs belonging to the same therapeutic group . the current situation is therefore characterized by disharmony prevailing between psychotropic drug development , nosological classification of diseases , clinical research , and therapeutic uses of psychotropic drugs . africa , the population size of the city of tshwane in 2011 was roughly equal to 3 million . the city is home to over 5,000 businesses out of which 1,603 businesses operate within the central business district of tshwane , known as pretoria . according to the annual report issued by the city of tshwane metropolitan municipality for the budget year 2010 - 2011 , about 1,734,295 tons of solid waste the solid waste produced by businesses in the city includes trash or garbage such as wood , product packaging , empty bottles , used tyres and car parts , and cans , garden refuse , furniture , clothing , leftover food , newspapers , wires , grease , appliances , paint , pieces of metal , broken containers , sheet metal , used medicine , and the like . these businesses produce massive volumes of solid and liquid waste on a daily basis . taxi ranks , bus stations , open flea markets , food outlets , and small businesses located in pretoria are synonymous with litter , uncontrolled solid and liquid waste , as well as lack of capacity in the efficient management of waste . the annual report released by the city of tshwane metropolitan municipality for the year 2011 shows that massive waste is accumulated during strike action by municipal workers responsible for the removal of waste from households and businesses . the collection , disposal , and processing of waste produced by businesses and households are regulated by legislative policies set out and enforced by the municipality of tshwane and the south african national department of environmental affairs and tourism [ 2 , 3 ] . the use of an integrated municipal solid waste management system [ 47 ] has been shown to be essential for improving overall efficiency in municipal waste management in almost all developed nations of the world . in order for an integrated waste management system to perform efficiently , all relevant stakeholders of the waste chain must play a mutually collaborative role in the collection , disposal , processing and management of waste . a review of the relevant literature shows that such a measure is essential for reducing the overall cost of waste management , and for the protection of the environment [ 810 ] . overall efficiency in the management of waste depends on adherence to municipal acts on waste disposal and management [ 1113 ] . research work carried out in several developing nations of the world has shown that health education on environmental sanitation and primary health care , socioeconomic incentives , as well as a strict enforcement of municipal bylaws are all needed for ensuring overall environmental cleanliness and the efficient management of waste in metropolitan cities such as tshwane . the study was conducted against the background of a host of factors that are well known to undermine overall efficiency in the management of solid waste in almost all metropolitan cities of the developing world . these background factors are lack of infrastructural capacity required for the efficient management and processing of solid waste [ 2 , 3 , 17 , 18 ] , poor awareness about the benefits of proper waste management , lack of socioeconomic incentives to stakeholders relevant to the waste management chain , failure to adequately utilize modern waste management and processing technology [ 21 , 22 ] , failure to vigorously enforce municipal bylaws [ 2 , 3 ] , and the absence of an integrated waste management system in tshwane [ 23 , 24 ] . the study aims to identify and quantify the key predictors of inefficiency in the management of solid waste in the city of tshwane . with a view to ensure optimal efficiency in the management of solid waste , this research work will assess the degree to which municipal bylaws and regulations that are relevant to solid waste management are adhered to by businesses conducting business in the city of tshwane . the bylaws and regulations are relevant to all stages of the waste management chain ( sorting of waste at source , collection , treatment , disposal , and processing ) . the model considers 4 categories of solid waste ( municipal , organic , plastics , and electronic ) and is designed for realizing a sustainable and optimally efficient solid waste management system for the city of tshwane . the model is accompanied by a comprehensive monitoring and evaluation plan that could be used for assessing the current status - quo , and for assessing and evaluating efficiency in waste management at each stage of the waste management chain . norms and standards set out by the south african national department of environment and tourism are used for benchmarking . although more than 25% of small and medium - sized businesses as well as informal traders operating in the city of tshwane generate waste in the course of conducting routine business [ 2 , 3 , 17 ] , the pace at which such waste is properly collected and disposed of by municipal workers employed by the city of tshwane is significantly lower than the pace at which waste is generated . this situation is exacerbated at times of civil action carried out by municipal workers responsible for routine waste collection and disposal . taxi ranks , the various sources of solid and liquid waste and pollution , bus stations , liquor stores , petrol stations , schools , shops , open markets , garages , and so forth are well known contributors for the generation of high volume of solid and liquid waste in the cbd of pretoria . there is room for improvement of the current level of efficiency in the management of waste produced by the various sources of solid and liquid waste and pollution . in terms of the strategic plan adopted by the south african department of environmental affairs and tourism ( deat ) to improve efficiency in the management of waste , the various sources of solid and liquid waste and pollution are a major stakeholder , and no progress can be made unless otherwise the various sources of solid and liquid waste and pollution are involved in the implementation of the plan drawn up by the deat [ 4 , 17 ] . there is a significant accumulation of solid waste especially in townships such as mamelodi and marabastad , and at the various taxi ranks in and around the city of pretoria . there is a shortage of essential facilities such as trash cans , seats , shelters , public toilets , and clean tap water at taxi ranks and public gathering arenas . some of the various sources of solid and liquid waste and pollution do not have properly functioning toilets and washrooms although the proper functioning of such facilities is an essential requirement for business . the various sources of solid and liquid waste and pollution , taxi ranks , and public gathering arenas are often characterized by bad smell and a large accumulation of solid and liquid waste . such places are conducive for the spread of communicable diseases such as food poisoning and diarrhoea unless they are controlled and managed efficiently by the ctmm . not enough is known so far about the extent to which efficiency in waste disposal can be improved in view of the fact that there is lack of empirical evidence in this area of research interest . not enough research has been done in the cbd of pretoria to assess and identify factors that affect efficiency in proper waste disposal . there is a shortage of empirical evidence based on a properly designed multivariate modelling in areas related to the quantification of factors that contribute to poor environmental sanitation and the accumulation of solid and liquid waste in the cbd of pretoria . low level of awareness in areas related to recycling , classification of waste at source and composting plays a major role in compounding the current lack of efficiency in the efficient utilization of resources such as broken bottles , empty cans , used goods , plastic bags , and so forth . far from being put to economic use , broken bottles are harming pedestrians and school children in addition to polluting the environment and decreasing the beauty of the city in the eyes of visitors and residents . some irresponsible inhabitants of the city and visitors who travel to the city from outside do not have adequate respect for environmental sanitation and demonstrate total disregard for cleanliness of the streets of the city . . examples of such rubbish are beer bottles , empty cans , and used food packages . empty bottles break into harmful pieces as they are thrown onto the streets from moving taxis and private cars . there is a need to have irresponsible inhabitants and visitors educated and disciplined so that they show respect for environmental sanitation and cleanliness . experience drawn from large municipalities in sweden , the united states , and kenya shows that these deeply ingrained problems could be addressed adequately by enforcing the relevant municipal bylaws with vigour , by promoting health and environmental education , and by providing socioeconomic incentives to stakeholders and role - players in the waste management chain . a review of the literature shows that in cities as big as tshwane , modern waste treatment technologies such as composting , incineration , landfills , recycling , and window composting should be used for solid waste management [ 2023 ] . modern solid waste management techniques such as landfills , incineration , recycling , biological processing , energy recovery , radio frequency identification tags , global positioning system tracking integrated software packages , rear vision cameras , and the like are used in cities such as geneva and stockholm . examples of advanced solid waste treatment technologies that are suitable for the city are anaerobic digestion , ethanol production , biodrying , gasification , in - vessel composting , mechanical biological treatment , mechanical heat treatment , plasma arc waste disposal , pyrolysis , sewage treatment , and tunnel composting are also highly appropriate technologies for the city [ 19 , 20 ] . in the city of tshwane , the management of solid waste involves collection , transportation , processing or disposal , management , and monitoring of waste materials . the south african department of environmental affairs and tourism promotes the use of modern technologies and evaluation techniques that are necessary for the efficient management of waste although advanced technologies that are used for waste management and evaluation are poorly utilized in south african cities including the city of tshwane . globally , diverse methods have been used for improving efficiency in the collection and disposal of solid waste . the most commonly used methods are designed for ensuring efficiency in solid waste management and are outlined in the un - habitat 's third global report of 2010 . in the world 's cleanest cities , the following methods are used : integrated sustainable waste management , the enforcement of municipal bylaws , the promotion of primary health care services and environmental sanitation , the promotion of health education on proper waste management , financial sustainability , institutional framework , good governance , community based campaigns of awareness , the provision of incentives for good performance in the collection of solid waste , process flow diagrams , as well as monitoring and evaluation programmes designed for the assessment of municipal service delivery programmes [ 2225 ] . in this study , assessment of efficiency in waste management was made by using the classic structural time - based model constructed by tchobanoglous et al . as well as adherence to municipal bylaws . the city of tshwane uses the model for assessing overall efficiency in the collection of solid waste by municipal service providers . the model is suitable for assessing efficiency in the collection of solid waste from fixed containers that are used for depositing solid waste by businesses and ordinary residents . the model assesses overall efficiency as a function of time taken for the management of waste . the aim of this research is to identify and quantify factors that affect overall efficiency in the management of solid waste generated by businesses that operate in the city of tshwane . based on data collected from a random sample of n = 1,034 businesses operating in the city of tshwane , the research article aims to : assess the current state of solid waste management with regards to type of wastes and quantities of waste generated , identify key factors that affect efficiency in the management of solid waste , andassess the degree to which businesses adhere to norms and standards that are recommended for businesses in terms of solid waste disposal . assess the current state of solid waste management with regards to type of wastes and quantities of waste generated , identify key factors that affect efficiency in the management of solid waste , and assess the degree to which businesses adhere to norms and standards that are recommended for businesses in terms of solid waste disposal . data was collected in 2010 from a random sample of 1,034 businesses operating in the city of tshwane . managers and owners of the businesses as well as employees were asked a total of 89 questions related to the type of business they were operating , the type and volume of waste generated , how waste was collected , sorted out , disposed of and managed , the extent of cleanliness and sanitation at the workplace , their socioeconomic and demographic characteristics , as well as their personal opinion on the benefits of the proper management of waste . efficiency in solid waste management was assessed by using the model proposed by tchobanoglous et al . for estimating the median time required for the collection of solid waste from the businesses that took part in the study . the model measures efficiency in the collection of solid waste from fixed locations and containers based on the mathematical expression shown below : ( 1)e=1a+b . in ( 1 ) , a denotes an off - route factor , and b is a measure of off - route activity time by individuals collecting waste . in cases where a = b , the collection of solid waste becomes perfectly efficient . the larger the difference between a and b , the less efficient becomes the waste collection procedure . the time required per trip is given by the expression shown below : ( 2)t = t1+t2+y1a . in ( 2 ) , t is the time required for waste collection from a fixed site per trip , t1 is equal to pick up time of container at the site of collection , t2 is equal to on - site time per trip , y denotes the total haul time during waste collection , and x denotes the average round - trip haul distance per trip . since the relationship between y and x follows simple linear regression , we have ( 3)y=0+x . in ( 3 ) , 0 is the constant term in the simple linear regression of y on x , and is the regression coefficient in the simple linear regression of y on x. in cases where waste is collected from fixed sites , the time required to collect waste per trip follows a stationary model , and is expressed by the expression shown below : ( 4)t1=p+q+r . in ( 4 ) , p is the time taken to pick up a container that is full of solid waste ; q is the time taken to unload an empty container ; r is the time taken for driving from one container to the next container . the number of trips per day is given by the expression shown below : ( 5)n = y(1a)(t3+t4)t . in ( 5 ) , variable t is the time required for waste collection from a fixed site per trip ; t3 is the time required to drive from dispatch station to first container location to be served by the day ; t4 is the time required to drive from the last container location to be served by the day to the dispatch station . the time taken per trip is given by the expression shown below : ( 6)t5=t6ty . in ( 6 ) the number of trips made per week is given by the expression shown below : ( 7)t6=krq . in ( 7 ) , variable k is a measure of the volume of waste generated in a week in cubic meters per kg ; r is the average size of the container in cubic meters per kg ; q is the weighted average container utilization factor . since t = ( t1 + t2 + y)/(1 a ) as shown above in ( 2 ) efficiency can thus be measured by the following relationship : ( 9)e = y+t1+t2+btt=(t1+t2+y)+btt=(1a)t+btt=1a+b . values of e in the expression e = 1 a + b were calculated for each of the 1,034 businesses that took part in the study . businesses for which values of e fell below the median were used for identifying businesses in which efficiency in waste management was inadequate . businesses for which values of e were greater than or equal to the median were used for identifying businesses in which efficiency in waste management was deemed adequate . that is , ( 10)overall efficiency={inadequate if score is belowthe median of eadequate if score is greater thanor equal to the median of e. pearson 's chi - square tests of association were used for the screening of variables . this was done by performing two - by - two tests of associations between overall efficiency in the management of waste and the various socioeconomic , demographic , sanitary , environmental , and health - related variables on which data was gathered as part of the study . at the 5% level of significance , an association was deemed significant if the p value was below 5% . the dependent variable of study is a measure of overall efficiency in the management of waste . the variable has only 2 possible values ( inadequate , adequate ) . since the dependent variable of study is dichotomous , binary logistic regression analysis was used in order to identify key predictors of inefficiency in the management of waste . multilevel analysis was used in order to estimate the extent of variation with regards to efficiency in the management of waste by geographical zone and category of business enterprise . multilevel analysis is a statistical procedure that enables planners and policy makers to allocate resources that are needed for intervention based on the extent of variation observed at various hierarchical levels . in this study , the two hierarchical levels depending on which efficiency in waste management varies are geographical location and category of business . the degree of adherence to municipal bylaws and guidelines for the disposal of solid waste was measured by using a 2-point scale . the guidelines used for measuring adherence were the iso 14000 and iso 14031 guidelines for environmental management and performance monitoring in the management of waste and the environment [ 3032 ] . at each of the 1,034 businesses selected for the study , the degree of adherence to municipal bylaws and procedures recommended for solid waste management by businesses by the city of tshwane was graded based on iso 14000 and iso 14031 guidelines . that is , at each business enterprise , binary grades ( adequate , not adequate ) were allocated as a measure of compliance according to criteria stipulated in iso 14000 and iso 14031 guidelines . the binary variable created for assessing degree of adherence to iso guidelines or municipal bylaws was used as one of the predictor variables of study . category 1 of the binary variable represented inadequate adherence to iso guidelines or municipal bylaws . category 2 of the binary variable represented adequate adherence to iso guidelines or municipal bylaws . the purpose of conducting binary logistic regression analysis was to identify influential predictors of inefficiency in the management of waste among businesses operating in the city of tshwane . the use of binary logistic regression analysis was appropriate as the dependent variable of study had only 2 possible values ( 1 , 0 ) . in binary logistic regression analysis , odds ratios were used as an epidemiological measure of effect . at the 5% level of significance , influential predictors of inefficiency in waste management are characterized by estimated odds ratios that differ from 1 significantly , p - values that are smaller than 0.05 , and 95% confidence intervals of odds ratios that do not contain 1 . the adequacy of the fitted logistic regression model was assessed by using standard diagnostic procedures such as the classification table , the hosmer and lemeshow goodness - of - fit test , receiver operating characteristics ( roc ) plots , and sensitivity / specificity plots . table 1 shows a comparison between businesses that were efficient with regards to solid waste management with those that were not . it can be seen from the table that 857 of the 1,034 businesses ( 83% ) were efficient , while the remaining 177 businesses ( 17% ) were inefficient . the table shows that a significant percentage of businesses located in the central and western parts of the city were inefficient , whereas businesses located in the eastern and northern parts of the city were by and large efficient . the table shows that 76% of operators who managed businesses that were efficient with regards to waste management have acquired formal education at college level or better . in general , businesses that are inefficient in the management of waste are relatively younger , poor in personal hygiene and cleanliness of premises , and are by and large commercial . the majority of old businesses ( 6 years or more ) are efficient in waste management . businesses that are operated by owners are more efficient in comparison with businesses that are operated by employed managers . utilization of private contractors for waste removal and management , regular inspection of premises by municipal workers , familiarity with the south african white paper on waste management , source reduction of waste , good perception on the benefits of proper waste management , and adherence to waste management regulations recommended by the municipality are much more common among businesses that are efficient in waste management . two - by - two pearson chi - square tests of associations was used for performing a preliminary screening of influential factors that were significantly associated with inefficient management of waste . table 2 shows a list of 15 factors that are significantly associated with poor or less than satisfactory waste disposal at the 0.001 level of significance . in each of the tests , the outcome variable of study , y , was defined as follows : ( 11)overall efficiency={inadequate if score is belowthe median of eadequate if score is greater thanor equal to the median of e. at the 0.001 level of significance , significant associations are characterized by large observed chi - square values and p - values that are smaller than 0.001 . table 2 provides a list of 15 variables that are significantly associated with inefficient waste management . at the 0.001 level of significance , all 15 variables shown in table 2 are significantly associated with overall efficiency in the management of waste . it can be seen from the table that the top 5 significant variables are : lack of adherence to municipal bylaws and regulations , wrong perception on the potential benefits of proper waste management , failure of businesses to provide customers with enough trash cans , the status of the business operator ( owner or employee ) , and the frequency at which business premises are inspected by municipal sanitation and health workers , in a decreasing order of strength . results from binary logistic regression analysis are theoretically more reliable than results from pearson 's chi - square tests of association . this is because the measure of effect in binary logistic regression is the odds ratio , and not two - by - two significant associations . logistic regression analysis allows multivariate analysis involving several variables that are influential over waste disposal . it is also possible to assess the reliability of the fitted logistic regression model based on highly reliable diagnostic tests such as the classification table , the likelihood ratio test , the hosmer - lemeshow goodness - of - fit test , as well as receiver operating characteristic ( roc ) and sensitivity / specificity plots . at the 0.05 level of significance , influential predictor variables are characterized by estimated odds ratios that differ from 1 significantly , p - values that are smaller than 0.05 , and 95% confidence intervals of odds ratios that do not contain 1 . accordingly , it can be seen from table 3 that all 4 predictor variables are significant at the 0.05 level . the results show that efficiency in the proper management of solid waste is significantly influenced by 4 predictor variables . these 4 influential predictor variables are lack of adherence to municipal bylaws and regulations ( or = 9.17 ; 95% c.i . = ( 6.42 , 12.54 ) ) , wrong perception ( or = 8.81 ; 95% c.i . = ( 6.01 , 11.35 ) ) , failure to provide customers with enough trash cans ( or = 3.15 ; 95% c.i . = ( 1.46 , 5.87 ) ) , and the operation of businesses by employed managers ( or = 2.69 ; 95% c.i . = this shows that a business that is managed by an operator who fails to adhere to guidelines set out for waste management by the city of tshwane is 9.17 times as likely to be inefficient in the proper management of solid waste in comparison with a business that is managed by an operator who adheres to recommended guidelines . this shows that a business operator who has the wrong perception on the benefits of proper management of solid waste is 8.81 times as likely to be inefficient in comparison with another business operator with the correct perception on the benefits of proper waste management . the adjusted odds ratio of the variable failure to provide customers with enough trash cans is 3.15 . this shows that a business in which there are not enough trash cans for customers is 3.15 times as likely to be inefficient in the management of solid waste in comparison with a business in which enough trash cans are provided to customers . this shows that an outlet that is operated by someone who does not own the business being operated is 2.69 times as likely to beinefficient in the proper management of solid waste in comparison with an outlet that is operated by someone who actually owns the business . adjusted odds ratios are more reliable than unadjusted odds ratios in epidemiological studies of this kind . in this study , the estimated odds ratios were adjusted for two potential confounding variables ( level of education of business operator and the physical location of business ) . the adjusted odds ratios did not differ much from the unadjusted odds ratios , thereby showing that none of the variables used for adjustment was a confounding variable . the reliability of the fitted logistic regression model was assessed using standard goodness - of - fit tests suitable for binary logistic regression analysis . the classification table showed that the fitted model had an overall percentage of correct classification of 88.78% , a percentage sensitivity of 57.06% , and a percentage specificity of 95.33% . the hosmer - lemeshow goodness - of - fit test gave a p - value of 0.0701 , a figure which is greater than 0.05 , thereby showing that there was no reason to doubt the reliability of the fitted logistic regression model . figure 1 below shows a plot of sensitivity / specificity versus probability cut - off point . the magnitude of the area that lies under the roc plot is a measure of variation explained by the fitted logistic regression model . in this case , the area under the roc plot is 88.82% , a figure that is significantly above 75% . the large proportion of explained variation and the small proportion of unexplained variation show that the fitted model is highly reliable in explaining variability in waste disposal as a function of the explanatory variables used for logistic regression analysis . the likelihood ratio test is used for assessing the collective efficiency of the 8 predictor variables used for performing binary logistic regression analysis . at the 5% level of significance , a p - value that is smaller than 0.05 shows that the 8 predictor variables used for performing binary logistic regression analysis are jointly efficient . in this case , the p - value from the likelihood ratio test is equal to 0.000 , a figure that is smaller than 0.05 . this small p - value shows that the 8 predictor variables used for binary logistic regression analysis are collectively efficient in accounting for failure in the proper disposal of waste . multilevel analysis was used in order to estimate the extent of variation with regards to efficiency in the management of waste by geographical zone and category of business enterprise . table 1 provides frequency distributions for each of the 7 categories of business ( agricultural , commercial , construction , industrial , institutional , municipal , and processing and manufacturing ) as well as 5 geographical locations ( central , east , west , north , south ) of the city of tshwane that were considered in performing multilevel analysis . results obtained from multilevel analysis showed that there were significant differences among the 7 categories of waste . the results showed that 23.05% of the total variation in efficiency is due to differences among the 7 categories of waste produced by business enterprises operating in the city of tshwane . the results also showed that businesses within the same category of waste and geographical location were equally efficient in the management of solid waste . the key objective of research was to identify and quantify factors that affect efficiency in the management of solid waste by businesses that are operated in the city of tshwane . the study has shown that efficiency in the management of solid waste is significantly influenced by 4 key predictor variables of study . these 4 influential predictor variables are lack of adherence to municipal bylaws and regulations , wrong perception on the potential benefits of proper waste management , failure to provide customers with enough trash cans for waste disposal , and the practice of operating businesses by employed managers , in a decreasing order of importance . in the city of tshwane , municipal solid waste consists of everyday items such as product packaging , empty bottles and cans , grass clippings , furniture , clothing , left - over food , newspapers , appliances , paint , batteries , pieces of metals , and so forth . such solid waste is generated by businesses , households , schools , hospitals , and visitors travelling into the city on foot or by other modes of transport such as cars , train , taxi , or bus . the health department of the city of tshwane has a municipal bylaw that stipulates how solid waste should be packaged , sorted , collected , and disposed of by inhabitants of the city . the bylaw encourages inhabitants of the city to practice source reduction of solid waste , recycling , and composting ( collecting organic waste such as left - over food and garden refuse , storing these wastes under conditions that are designed to help them break down naturally , and then use the resulting compost as a natural fertilizer ) . according to the south african national department of environmental affairs and tourism , the disposal and combustion of municipal solid waste is conducted by the use of landfills , the conversion of non - recyclable waste materials into useable heat , electricity , or fuel , combustion , and transfer stations . although the use of such mechanisms is consistent with the municipal bylaw in the city of tshwane , the mechanisms have been poorly utilized mostly due to lack of infrastructural development and technical skills . the results are expected from a typical sub - saharan african country in which poverty , unemployment , and massive immigration into urban centres prevail . environmental hazards arising from the decomposition of solid waste under oversaturated conditions and dumping of solid waste in illegal landfills often cause sludge . such problems are particularly evident in suburbs of tshwane such as mamelodi and marabastad . in this regard , the city of tshwane can be viewed as a combination of clean white suburbs and dirty black townships . the other key environmental hazard is caused by arbitrary landfill sites that dot the peripheries of the city of tshwane . the study by snyman and vorster has found that composting and the pretreatment of municipal waste before landfilling are viable options for the city of tshwane . have found that composting and pretreatment of municipal waste before landfilling significantly reduce the volume of solid waste and contributes for overall environmental sanitation . at the moment , the city of tshwane does not have adequate capacity for large scale composting , and there is an acute need for addressing this shortcoming . the study has shown that a combination of technical and administrative solutions is required in order to improve efficiency in the management of solid waste in the city of tshwane . to separate waste generated at the various businesses , it is necessary to provide businesses with custom - made containers that are suitable . the frequency of collecting waste should be balanced with the volume of waste generated by the various businesses . studies conducted in various parts of the developed world have shown that there are economic benefits in outsourcing the collection and disposal of recyclable waste to the informal sector , and that sanitary landfills should be used for the final disposal of solid waste . in addition to enforcing the law with vigour , it is equally important to provide community based health education on environmental sanitation by collaborating with the department of health . awareness campaigns and socioeconomic incentives could be provided by civic society and nongovernmental organizations that have a vested interest in improving environmental sanitation and cleanliness . since most of the waste generated in the city of tshwane , the provision of incentives and education at the grass - roots level carries a clear socioeconomic merit . composting is a form of aerobic treatment , and is suitable for treating organic waste in the city of tshwane . according to barlaz , facilities used for storage and collection of waste must be compatible with each other . waste disposal and processing sites must be located strategically so that the cost of waste collection , disposal and processing becomes optimal . the city of tshwane needs to make the necessary initial investment available in order to benefit from composting in the long - run . extensive waste management research conducted in various parts of china has shown that educating rural as well as urban people on how to produce compost by using low technology has long - term economic benefits to big metropolitan municipalities such as tshwane . research conducted in nigeria has shown that strategic partnerships and collaboration among academic and research institutions and municipalities have the potential for enhancing overall efficiency in waste management , skills development , and the creation of employment opportunities in municipalities such as tshwane . one particular area of waste that stands to benefit out of such partnership is the management and processing of plastic and e - waste . the collection and recycling of plastic waste is characterized by serious challenges and difficulties in the city of tshwane . the city of tshwane has no coherent policy on the collection of e - waste . neither does it provide clear incentives to entrepreneurs who wish to collect , classify , and process e- and plastic waste . since e - waste could be hazardous , there is a dire need to build capacity in the classification of e - waste into one of two categories ( harmful or hazardous ) , and processing each category of waste by utilizing an appropriate form of technology . there is an acute need for providing incentives , enforcing municipal bylaws , the provision of health and sanitary education , and a comprehensive monitoring and evaluation programme for assessing the progress made in this regard regularly . a well - functioning integrated solid waste management system can only be realized in the city of tshwane by providing clear incentives for good practice and behaviour , and by severely penalizing irresponsible behaviour in the city . liu et al . have found that the provision of direct socioeconomic incentives , clear guidelines on the collection , disposal , and processing of e- and plastic waste , as well as a strict enforcement of municipal bylaws is required for improving overall efficiency in the management of e and plastic waste in the city of tshwane . the efficient management of solid waste produced by enterprises that conduct business in the various parts of the city of tshwane has numerous economic , sanitary , and health - related benefits to the inhabitants of the city . up until recently , emphasis has been placed on waste disposal , and not on management , recycling , and composting . poor management of waste has an adverse impact on the environment and public health , particularly in townships such as mamelodi , marabastad , soshanguve , and attridgeville . in these townships , waste is managed poorly , and landfills are inappropriately sited , designed , managed , and operated . until recently , the management of waste generated by businesses operating in the city has not been given due consideration . the waste management that took place focused mainly on waste disposal and was reactive in that it addressed needs as they occurred . the low priority that was historically accorded to waste management has resulted in waste impacting detrimentally on the south african environment and on human health . efficient waste disposal is a process that requires the full collaboration of all stakeholders on a community based collaborative approach . in addition to providing sanitary education and inspection services to the businesses , clear incentives must be provided to ensure maximum success . the enforcement of regulations , the provision of incentives , adequate logistical resources , additional manpower , financial rewards , public - private partnerships , and awareness campaigns are all essential . the treatment of waste produced by the businesses in the city of tshwane is similar to waste produced in a typical developing nation in the sense that treatment of this waste involves simply a reduction of its volume by use of methods such as baling or shredding although incineration and composting is practiced at a small scale . the emphasis remains on disposal of general waste by landfill without treatment as the lowest cost disposal option , as landfill airspace is still available in south africa . the lack of pretreatment of general waste before disposal is therefore currently not regarded as a problem in south africa . incineration of general waste and hazardous waste is not acceptable to many stakeholders due to the poor operation of many existing facilities and noncompliance with existing by - laws . incineration is not economically feasible in south africa since its warm climate limits the market for the energy derived from the incineration process . the majority of operating incinerators in south africa are used for the treatment of infectious medical waste . increasing general awareness about the benefits of proper waste disposal is a key requirement for success . the number of waste disposal sites is limited , and the disposal of waste is expensive . since there are not enough of these facilities , hazardous waste is often transported over long distances , resulting in increased risks of accidents and higher transport costs . some other helpful steps are to undertake an integrated plan in which waste is gathered and disposed of efficiently based on mutual collaboration among stakeholders , strengthening the technical , financial , administrative , and operating capacity of the institutions in the basic environmental sanitation sector , and encouraging health education and community promotion activities , which are basic to the success of waste collection and disposal , especially at taxi ranks and the streets at the central business district of pretoria , to provide clear incentives to businesses that improve the quality of waste collection and disposal based on generally accepted standards . technical cooperation among stakeholders must be directed toward the strengthening of institutions in the basic environmental sanitation sector and emphasis should be given to the following activities : operation and maintenance , community promotion , training , administration and management , the preparation of plans and studies helpful for efficient waste disposal , and the application of technologies that are helpful for efficient waste disposal . in - depth interviews conducted with some of the managers of the 1,034 businesses that took part in the study have revealed that the businesses suffer enormously during prolonged strike actions . waste material gets vandalized by scavengers as a result of delay in collection by service providers . it is too risky to leave waste uncollected during rainy seasons as waste could easily be mixed up with excreta that could be washed away by rain - water , ending up in wells and streams . the germs in the excreta could then easily contaminate drinking or washing water . in such situations failure to dispose of waste can have a significant effect on the health of communities . where refuse is not disposed of properly , it can lead to pollution of surface water , as rain washes refuse into rivers and streams . refuse disposed of in storm drains may cause blockages and encourage fly and mosquito breeding . all business premises operating in the city of tshwane must be inspected for environmental sanitation and cleanliness regularly with a view to encourage and reward good practice , and to penalize irresponsible behaviour . municipal bylaws on the collection , disposal and management of waste must be enforced with enough vigour and commitment . failure to do so can easily result in frustration among businesses that obey municipal guidelines and regulations on waste management . the study showed that 17% of the 1,034 businesses were not efficient enough with regards to the proper collection , disposal , and management of solid waste . the study has shown that efficiency in the management of solid waste is adversely affected by lack of adherence to municipal bylaws and regulations on proper waste management , wrong perception on the potential benefits of proper waste disposal , failure to provide customers with enough trash cans at business premises , and the operation of businesses by employed managers , in a decreasing order of importance . lack of adherence to municipal bylaws and regulations that are essential for proper waste management constitutes a key challenge in the city of tshwane . the presence of wrong perception on the potential benefits of waste disposal is also a well known hurdle . to rectify this issue , the second intervention is to provide incentives to businesses that manage solid waste properly in accordance with guidelines provided by the city of tshwane . regulatory and legislative actions must be taken against those who fail to respect municipal bylaws that are related to cleanliness and proper waste management . the efficient disposal of waste generated by businesses operating in the city has direct economic benefits to all inhabitants of the city . accordingly , waste should be gathered efficiently and disposed of in accordance with the waste collection and management plan produced by the city of tshwane . the third intervention is to actively promote an education campaign in all parts of the city of tshwane with a view to ensure the full collaboration of businesses conducting business in the city . based on findings of this particular study , the following recommendations are made to the city of tshwane in order to improve overall efficiency in the management of solid waste that is generated by businesses operating in the city.the city of tshwane must produce and implement an integrated plan for the management of solid waste in collaboration and partnership with the relevant stakeholders in the city so that each of the role players in the waste management chain can investment adequately in basic environmental sanitation.an initial infrastructural investment needs to be made by the city of tshwane in order to build adequate capacity for commercial composting . this should be done in collaboration with business enterprises so that they can share the financial burden at the initial stage , and benefit from compost - related business opportunities in the long-run.the city of tshwane must strive to increase awareness about the potential benefits of proper waste collection and disposal by promoting health education on environmental sanitation and techniques that are useful for collecting and sorting waste . incentives must be provided to businesses that do a good job in terms of the proper collection and disposal of solid waste . the city must also support research initiatives conducted by academic and research institutions in areas that are related to waste management , environmental sanitation , and personal hygiene by funding them partially or fully.the city of tshwane must improve the conditions of employment of municipal workers responsible for waste collection and disposal.the city of tshwane and gauteng department of health must provide technical assistance to businesses that do not have their own waste management plans so that such businesses can contribute for overall efficiency in environmental sanitation . the city of tshwane must produce and implement an integrated plan for the management of solid waste in collaboration and partnership with the relevant stakeholders in the city so that each of the role players in the waste management chain can investment adequately in basic environmental sanitation . an initial infrastructural investment needs to be made by the city of tshwane in order to build adequate capacity for commercial composting . this should be done in collaboration with business enterprises so that they can share the financial burden at the initial stage , and benefit from compost - related business opportunities in the long - run . the city of tshwane must strive to increase awareness about the potential benefits of proper waste collection and disposal by promoting health education on environmental sanitation and techniques that are useful for collecting and sorting waste . incentives must be provided to businesses that do a good job in terms of the proper collection and disposal of solid waste . the city must also support research initiatives conducted by academic and research institutions in areas that are related to waste management , environmental sanitation , and personal hygiene by funding them partially or fully . the city of tshwane must improve the conditions of employment of municipal workers responsible for waste collection and disposal . the city of tshwane and gauteng department of health must provide technical assistance to businesses that do not have their own waste management plans so that such businesses can contribute for overall efficiency in environmental sanitation .

the current clinical use of psychotropic drugs is transnosologically oriented . this is facilitated by the current classification of mental disorders ( international classification of diseases , 10th revision [ icd-10 ] ) and is perhaps justified if depression and psychosis ( taken here as examples ) are considered as being complex syndromes with heterogeneous etiologies , but common pathogenesis , more than specific entities . however , this approach does not identify possible differences between specific psychiatric entities , which could in turn mask differences in therapeutic responses and , therefore , therapeutic outcome . this is compounded by the current disharmony between the nosological classification of diseases , drug development , clinical research , and therapeutic uses of psychotropic drugs . functional pharmacology targeting abnormal behavioral traits could represent an avenue for future research and treatment . objective . the objective was to investigate factors that affect the efficient management of solid waste produced by commercial businesses operating in the city of pretoria , south africa . methods . data was gathered from 1,034 businesses . efficiency in solid waste management was assessed by using a structural time - based model designed for evaluating efficiency as a function of the length of time required to manage waste . data analysis was performed using statistical procedures such as frequency tables , pearson 's chi - square tests of association , and binary logistic regression analysis . odds ratios estimated from logistic regression analysis were used for identifying key factors that affect efficiency in the proper disposal of waste . results . the study showed that 857 of the 1,034 businesses selected for the study ( 83% ) were found to be efficient enough with regards to the proper collection and disposal of solid waste . based on odds ratios estimated from binary logistic regression analysis , efficiency in the proper management of solid waste was significantly influenced by 4 predictor variables . these 4 influential predictor variables are lack of adherence to waste management regulations , wrong perception , failure to provide customers with enough trash cans , and operation of businesses by employed managers , in a decreasing order of importance .

parkinson 's disease ( pd ) is a neurodegenerative condition characterised clinically as a movement disorder consisting of bradykinesia , rigidity and rest tremor , as a consequence of striatal dopamine depletion secondary to degeneration of the dopaminergic neurons of the substantia nigra . in addition , pathology in other regions results in cognitive deficits and a dementia in some cases , neuropsychiatric manifestations including depression and anxiety , sleep disturbances including rapid - eye movement sleep behavior disorder , and autonomic features ( kalia and lang , 2015 ) . the motor disorder of pd responds well to dopamine - replacement therapies such as levodopa or dopamine agonists . however , systemic administration of dopaminergic agents results in non - physiological release of dopamine in the striatum resulting over time in disabling motor dyskinesias , and off - target effects resulting in neuropsychiatric complications , including impulse control behaviours , which can be very severe in some patients . there remains therefore , a currently unmet need for a means of introducing dopamine to the striatum in a more physiological and targeted manner , and as such there is much interest in emerging cell - based regenerative treatments for pd ( jenner , 2003 ; barker et al . , 2015 ; kalia and lang , 2015 ) . a variety of sources of dopaminergic cells have been investigated for treating pd in this way , and in this review we discuss the basis of these , as well as their prospects for use in clinical practice . dopaminergic neurons can be obtained from several sources to serve as the basis for potential cell - based therapies . any neural tissue grafted must have the ability to produce dopamine , demonstrate axonal outgrowth , and form connections with the host striatum , without widespread migration of grafted cells or malignant transformation . fetal ventral mesencephalon ( fvm ) tissue contains progenitors for dopamine - producing neurons , and has been transplanted into the striatum of pd patients with good results in a number of patients ( kefalopoulou et al . , 2014 ; barker et al . , clinical efficacy and safety has been highly variable in trials of human fvm grafting , partly due to suboptimal patient selection , grafting technique , immunosuppressive regimes and trial design ( freed et al . , 2001 ; however , in appropriately selected patients ( using criteria including age less than 65 years and minimal pre - existing dyskinesia ) , this strategy seems to be effective and at the very least has shown an important proof - of - concept that dopaminergic neuron replacement for pd is a therapeutic option ( barker et al . , 2015 ) . although fetal dopaminergic cells have shown an important proof - of - principle , there are major logistical and ethical problems linked to their use which has led to the search for a better source of cells for grafting in pd . the generation of induced pluripotent stem cells ( ipscs ) through forced expression of pluripotency factors in adult human somatic cells led to much interest in these as a potential cell - based treatment for pd ( takahashi et al . , 2007 ) . nigral dopaminergic neurons can be generated from ipscs by exposure to specific factors that induce neuronal differentiation and midbrain patterning ( soldner et al . this approach potentially would mean that a patient - specific therapy could be generated for each individual ( autologous grafting ) , circumventing the need for the immunosuppression that is required when using allografts . comparison of the major approaches to cell - based therapies in parkinson 's disease an alternative approach to the use of ipsc - derived neurons is to generate dopaminergic neurons from embryonic stem ( es ) cells which could be used for allogeneic grafting ( thomson et al . , 1998 ) . like ipscs , these can be differentiated into neurons and directed to a midbrain lineage to produce authentic a9 dopaminergic neurons ( kriks et al . , 2011 ) . it has recently emerged that the markers that have been traditionally used to signify nigral dopaminergic neuronal fate fail to predict positive graft outcomes . this has now been achieved showing that midbrain patterning alone is not sufficient to produce high numbers of dopaminergic neurons , but rather neural progenitors must be specifically patterned to the caudal midbrain in order to achieve high yields of nigral dopaminergic neurons ( kirkeby et al . , 2017 ) . this finding reiterates the importance of protocol optimization prior to commencement of clinical trials of cell - based therapies that involve cellular reprogramming . a more recent development has been the direct conversion of adult somatic cells into induced neurons ( ins ) using viral vectors to transfer proneural transcription factors , bypassing the stem cell intermediate stage ( vierbuchen et al . , this approach could theoretically be used to generate patient - specific dopaminergic neuronal autografts , and human ins have been shown to survive transplantation into rodents , and maintain their phenotype ( pereira et al . , 2014 ) . however , the lack of highly efficient , consistent protocols for deriving functional dopamine - producing ins at the present time means that their utility is likely to be limited to in vitro disease - modelling , at least for the foreseeable future . in order for any of the above approaches to be adopted in a clinical setting , they need to not only be effective and capable of restoring striatal dopaminergic tone , but also commercially viable and ethically acceptable . furthermore , any cell - based therapy must be able to compete with current treatments such as pharmacological dopamine replacement and deep brain stimulation , and also emerging pharmaceutical treatments . as work towards disease - modifying therapies progresses , it may be that the optimal treatment approach involves a combination of pharmacological agents ( e.g. , neurotrophic factors or antioxidants ) or immunotherapies targeting alpha - synuclein to limit disease progression , and cell - based therapies to restore the dopaminergic motor function that has already been lost . clinical trials investigating these approaches must be initiated only on the basis of sound pre - clinical data , with flawed , premature trials likely to be detrimental to the whole field , especially in the case of cell - based therapies . furthermore , these treatments must be targeted to the population of patients that are most likely to benefit from them . there is clearly heterogeneity within the pd spectrum , with some patients experiencing a predominantly motor disorder , and others at increased risk of early cognitive impairment and dementia ( williams - gray et al . , 2007 ) . it is the former group that is likely to derive the most benefit from dopamine cell - based treatments , which do not target the cortical and subcortical cholinergic pathology , which is presumably more prominent in the latter group . in view of the discordance seen between the promising early trials of human fvm grafting and the two negative randomized - controlled trials , further investigation is necessary to determine which factors are critical for successful transplant outcomes ( freed et al . , 2001 ; olanow et al . , 2003 ) . an ongoing clinical trial for human fvm grafting ( transeuro ) , in which refined patient selection and an optimized grafting and immunosuppression protocol has been adopted ( based on an analysis of all available data from prior trials ) , is expected to be published in 2020 . in this trial , at least 3 fvms are grafted per side , with tissue being sited stereotactically via five to seven tracts into the posterior putamen . patients receive 12 months of standard triple agent immunotherapy , in the form of cyclosporine a , azathioprine and prednisolone an approach that bears a close resemblance to the early trials undertaken in sweden in the late 1980s/1990s . one of these patients grafted with human fvm during these early trials in sweden has recently come to post - mortem . histological analysis demonstrated survival of the graft , albeit with some intragraft lewy body pathology , 24 years post - transplantation ( li et al . , 2016 ) . whilst fvm grafts are clearly effective in a population of pd patients , the ethical considerations surrounding the use of fetal tissue , and the logistical challenge of accessing adequate tissue ( at least three fetal midbrains are required per grafted side of brain ) means that this approach will not be suitable for widespread clinical use . nevertheless , if the transeuro trial demonstrates clinical benefit , this will serve as an important stepping - stone for clinical trials using stem cell products . the important remaining question will then be whether to pursue autologous ipsc - derived grafts or allogeneic ipsc or es cell - derived grafts ( barker et al . , 2015 ) . ipscs are a means of generating patient - specific autologous grafts , theoretically meaning that they have a low risk of rejection , circumventing the need for immunosuppression . this is often cited as the major advantage of this approach over the use of allogeneic tissue , such as grafts derived from es cells ( barker et al . , 2015 ; stoker and barker , 2016 ) . however , generation of patient - specific grafts from ipscs on a large scale for use in healthcare brings about significant technical , regulatory and financial challenges . variability in the response of each individual cell line to differentiation protocols means that the product will differ between patients , meaning that each graft generated will necessarily be subject to regulatory approval , and the associated costs . additionally , it may not be possible to generate specific target cell types for every pluripotent cell line without time - consuming and expensive optimisation of the differentiation protocol for that particular cell line . the cost of generating autologous grafts via ipscs is therefore likely to be prohibitively high , meaning that this approach is unlikely to be practicable for treatment of common diseases for the foreseeable future ( table 1 ) . one potential avenue to reduce the regulatory and economic barriers to the use of ipsc - derived autografts , is the development of haplobanks consisting of graft products derived from a relatively small number of individuals with specific hla types , allowing hla - matched grafts to be available for the majority of the population . it has been estimated that hla - matched grafts could be generated for almost 95% of the uk population by obtaining cell lines from 150 individuals ( taylor et al . , 2012 ) . this is an appealing prospect , but in order to keep the required number of donor cell lines to relatively low levels , mismatch of up to two hla loci must be accepted . in this circumstance , a period of immunosuppression may therefore still be necessary , nullifying the main advantage of ipsc - derived grafts over allogeneic grafts . indeed , as the intended site of these grafts ( the central nervous system ) is a relatively immunologically privileged site , long courses of immunosuppression are unlikely to be necessary , regardless of whether grafts are autologous or allogeneic . six to twelve months of standard immunotherapy has been employed in fvm grafts to date , which seems sufficient to prevent graft rejection ( barker et al . , 2015 ) supported by the survival of the aforementioned fvm graft at 24 years ( though it must be noted that this patient had received 64 months of immunosuppression ) ( li et al . , 2016 ) . similarly , ins are unlikely to offer a commercially viable alternative even if efficient protocols for producing functional dopaminergic neurons are developed . in addition to the above challenges , another disadvantage of autologous grafts is the fact that , given that pd pathogenesis is increasingly understood to involve several genetic susceptibility factors , they will retain the individual 's inherent propensity to develop recurrent pd pathology . taking these considerations into account , es cells currently present the most promising approach for a commercially viable cell - based treatment for pd , especially also given some concerns over the stability of ipsc reprogramming . although a period of immunosuppression will be necessary with this approach , this is an acceptable cost to allow development of a product that is both clinically effective and commercially viable . furthermore , as mentioned above , this course of immunosuppression is likely to be limited to a period of months , restricting the burden of adverse effects . the ability to generate a standardized product from the most efficient es cell line means that neurons can be cryogenically stored , avoiding the need to navigate the regulatory environment for each individual , as would be the case with autologous grafts ( table 1 ) . clinical trials of es cell - derived grafts will be commencing in the next few years , with the american nystem trial due to commence in 2018/2019 , and the stem - pd trial in the united kingdom and sweden likely to begin in 2019/2020 . of course , ethical objections to the use of embryonic tissue will remain a potential barrier to use in some circumstances , but taking into account logistical and economic factors , es cell - derived treatments appear to be the most likely to fulfil the criteria for widespread clinical use . development of cell - based treatments for pd has been a long process , with progress being iterative . prematurely conducted trials in the past have cast doubt over this approach , but robustly designed studies are currently ongoing or are planned , to determine whether these treatments can offer a clinically useful therapy . human fvm grafting has served as a stepping stone to stem cell - based treatments , offering important proof - of - concept data . the significant economic and logistical challenges of using autologous tissue relating to variation in the original cell sample , response to differentiation protocol , and the final product , means that es cells probably offer the greatest hope of a useful therapeutic approach , at least in the short to medium term . clinical trials in pd of midbrain dopaminergic neurons derived from es cells ( usa , united kingdom / sweden ) and from ipscs ( japan / usa ) are planned to commence in the next few years , the results of these will be crucial in determining the future direction for the field of regenerative medicine in pd . panoramic radiography is a cost - effective , low - dose method used in dental radiology for evaluating oral health status in routine dental practice.1 this technique allows examination of the maxillary and mandibular arches and their supporting structures on a single image.23 the advantages of panoramic radiography are time - saving , broad anatomic coverage , and high patient acceptability.34 abnormalities such as root fragments , impacted teeth , neoplasms , and foreign bodies are often overlooked when they do not cause symptoms or clinical signs.5 panoramic radiography is often used in routine examinations of edentulous jaws to detect asymptomatic conditions such as root fragments , retained teeth , radiolucent lesions , and foreign bodies.67 thus it is a valuable diagnostic tool in prosthetic treatment planning . in addition , they provide the clinician with information about the sinus floor position in edentulous regions for implant placement . several studies have been carried out including the occurrence rate of these asymptomatic pathologies.2467 however , only a few studies have documented the rate of these radiographic findings requiring treatment.4567 consequently , the aim of this study was to report the frequency of significant radiographic findings , to discuss utility of panoramic radiographs and to obtain the rate of the conditions which treatment is necessary before conventional or implant supported prosthetic rehabilitation in edentulous patients . this retrospective study investigated 743 patients who applied to istanbul university department of dentomaxillofacial radiology between 2009 and 2011 . a retrospective analysis was carried out of using panoramic radiographs taken either due to patient complaints or prior to prosthetic denture treatment , using a panoramic machine ( kodak 8000 digital panoramic machine , carestream health , inc . , rochester , ny , usa ) with 60 - 85 kvp and 10 ma , with total filtration of 2.5 mm aluminum . the radiographs were evaluated by four oral radiology specialists and one oral surgery specialist for impacted teeth , retained root fragments , radiolucencies , radiopacities , foreign bodies , proximity of the maxillary sinus to the crest of the residual alveolar ridge , soft tissue calcifications , and mucous retention cysts . the maxilla and mandible were divided into three areasright and left posterior ( includes bilateral premolar and molar teeth regions ) and anterior ( includes incisors and canines region)-for evaluation of root fragments and radiolucent - radiopaque areas . the maxilla was divided into two areas , left and right , for evaluation of mucous retention cysts and proximity of the maxillary sinus to the crest of the residual alveolar ridge . the patients whose subantral residual bone height is 1 - 2 mm on panoramic radiography ( precise indication for lateral maxillary sinus lift procedures ) were included in the study . soft tissue calcifications ( stc ) the area number 1 ( stc1 ) indicates possible tonsilloliths , parotid calcifications , possible tonsilloliths and parotid calcifications ; the area number 2 ( stc2 ) indicates possible submandibular calcifications and lymph node calcifications ; and area number 3 ( stc3 ) indicates possible carotid calcifications . then , two different options of prosthetic rehabilitation were planned for each subject including conventional removable prosthesis and implant supported fixed prosthesis . the patients who required surgical treatment for each treatment modality were determined based on radiographic findings . out of 743 patients , 428 ( 57.6% ) were female and 315 ( 42.4% ) were male . the mean age of the patients was 59.42 ; minimum age was 16 and maximum age was 88 . a total of 331 significant radiographic findings were detected in 256 patients . among these 256 patients , the frequency of radiographic findings and the findings which required treatment before conventional or implant supported prosthetic treatment are summarized in table 1 . seventy - four patients had proximity of the maxillary sinus to the crest of the residual alveolar ridge . the frequency of the subjects with posterior atrophic maxilla , soft tissue calcifications and mucus retention cysts are shown in table 2 . seventy - one root fragments were detected in 60 ( 8% ) of the 743 patients . of the 60 patients who had root fragments , 50 ( 83% ) had one root fragment , nine ( 15% ) had two , and only one ( 2% ) patient had three root fragments . one were ( 8% ) residual cyst , 11 ( 92% ) were idiopathic bone cavity . nine ( 82% ) radiopacities had well - defined borders and two ( 18% ) had diffuse borders . three ( 19% ) were extruded root canal materials , 13 ( 81% ) were other foreign bodies including eleven ( 69% ) retained amalgam fragments and two ( 12.5% ) gunshot fragments . the distribution of the foreign bodies , root fragments and radiopaque - radiolucent conditions are shown in table 3 . a total of 36 ( 5% ) impacted teeth were found in 27 different patients . nineteen patients ( 70% ) had only one impacted tooth , seven ( 26% ) had two impacted teeth , and one ( 4% ) had three impacted teeth . one of the most important goals of panoramic imaging is to detect any underlying pathology of both maxilla and mandible before prosthetic treatment.8 moreover , many reports have concluded that due to the high percentage of significant radiographic findings , radiographic screening should be required in all edentulous patients.67 similarly , our study showed that most of significant findings are easily detected on the panoramic radiographs of edentulous jaws . the most important limitation of this study is that the treatment planning was performed by using radiographic findings . in fact , both clinical and radiological correlation is very important to assess effective treatment planning and 3-d evaluation by using cone - beam computed tomography ( cbct ) can be necessary for specific conditions . thus , it is reported that almost all of the findings on panoramic radiographs coincide with clinical findings.9 in the present study , our observations were in accordance with this hypothesis . additionally , we did not achieve cbct views due to comprehensive treatment planning for implant placement . moreover , several clinical conditions which require surgical treatment before application of conventional removable prosthesis in edentulous patients such as epulis fissuratum or alveolar ridge disruption were excluded . bohay et al.10 reported 68.3% range of one or more significant radiographic findings in 375 edentulous patients . in addition , they determined 8.3% of these patients required treatment before treatment with removable dentures . similarly , masood et al.4 suggested a few ( 3.8% ) of the positive radiographic findings required treatment before denture fabrication . our results revealed an important part of these findings did not require surgical intervention before conventional removable prosthodontic treatment . on the other hand , a significant amount of the radiographic findings in edentulous patients require treatment before implant supported prosthetic treatment . lyman and boucher11 reported only one impacted tooth which required extraction among 300 edentulous patients . by this conclusion , they have not suggested routine panoramic examination for every edentulous patient to avoid cumulative effects of radiation exposure . however , today 's implant supported prosthetic rehabilitation becomes the most preferred treatment option for edentulous patients.13 hence , radiographic examination should be based on the concept that the edentulous patient is a candidate for implant placement . retained root fragments and impacted teeth are the most frequent significant radiographic findings in edentulous patients.4712 previous research has shown that most root fragments are localized in the molar region of the maxilla.4714 in our study , retained root fragments represented the second most frequent pathology . the majority of these root fragments were localized in the premolar - molar region of the maxilla . the reasons for this finding could be morphology and number of roots , as they were located posteriorly , where it is difficult to perform an operation . in addition , extraction of these roots poses several risks of complications , such as nerve injury ( inferior alveolar , lingual , and mental nerves ) and displacement of the roots into the maxillary sinus.1516 in particular , dental surgery in older patients carries a high risk of these complications . impacted teeth are , of course , critically important in preoperative planning for dental prostheses and implants in edentulous jaws , and they affect patients ' oral health and function.17 as such , patients with impacted teeth have a variety of complaints , such as carious lesions , dentigerous cysts , tooth eruption abnormalities , pain , and infections . stathopoulos et al.18 retrospectively investigated 7782 impacted third molars in 6182 patients and reported that the pathologic conditions related to these teeth were lower than 2.77% . sumer et al.6 reported teeth impaction in 3.1% of 676 edentulous patients . in our study , we found 36 impacted teeth , representing a frequency of 3.6% . this result may be related to elective procedures recommended for impacted teeth in edentulous patients by specialists . today , recent studies suggested an implant placement protocol encroaching upon residual roots and impacted teeth.1920 this unconventional method has been proposed to assess minimal invasive surgical procedures in implant dentistry . panoramic radiographs have been used frequently for preoperative assessments of the maxillary sinus for implant placement . these assessments include the vertical dimension of the alveolar crest to the maxillary sinus.2122 in completely edentulous patients , the upper alveolar ridge should be related to the floor of the maxillary sinus because of bone resorption . in these circumstances , panoramic radiographs simply allow an evaluation of this relation by using a lower effective dose.23 in this radiographic study , we determined that 22.4% of all radiographic findings were in relation of the floor of their maxillary sinuses with alveolar ridge . therefore , open maxillary sinus augmentation was required before implant surgery for all subjects . when maxillary sinuses are imaged , some maxillary sinus pathologies such as mucosal cysts can be detected with panoramic radiography . sinus mucosal cysts were another frequent significant finding in our study , observed in 6.3% of all patients . as the prevalence of mucous cysts in radiographic studies has been reported as 2 - 13% , our finding was in accordance with the literature.624 it has been reported that a mucus retention cyst of the maxillary sinus is not a contraindication for sinus membrane elevation.25 feng et al.26 retrospectively evaluated the survival rate of 21 endosseous implants placed into the elevated maxillary sinus area in the presence of mucus retention cysts . they reported that all of the implants were functionally stable during the 27-months follow up period . nevertheless , other maxillary sinus pathologies ( acute or chronic infections ) accompany to positive radiological findings of maxillary sinuses should be evaluated carefully for implant surgery in posterior maxillary area.27 carotid area calcifications can be detected in panoramic radiographies , and its prevalence has been reported as 3 - 5% in the general dental population . however , in a study conducted with a younger population ( with a mean age of 32 - 35 ) , the incidence was found to be very low , in a range of 0.4 - 0.8%.28 in previous studies , the frequency of radiopaque findings which might be due to the inclusion of soft tissue calcifications was reported as 9.3 - 9.9% ; thus , our findings seem relatively low by comparison.67 in our study , calcifications were detected in the area considered as tonsillolith in 23 patients ( 3.1% ) , in the submandibular area and radiopaque findings considered as lymph node calcification in 30 patients ( 4% ) , and in the carotid area in 11 patients ( 1.5% ) . these additional panoramic findings did not affect the treatment planning of implant placement or prosthetic rehabilitation . nevertheless , it seems that panoramic radiographs may include critical important findings in the head and neck region . in conclusion , to achieve successful results in prosthetic dentistry , preprosthetic - presurgical phase of treatment planning should be made carefully . it is our opinion that due to the high frequency of significant radiographic findings , panoramic radiography should be analyzed , even in the absence of clinical symptoms . within the limitation of this study , patients candidates for implant placement can more frequently require additional surgical procedures to eliminate pathological conditions of edentulous jaws .

parkinson 's disease ( pd ) is a neurodegenerative condition which causes a characteristic movement disorder secondary to loss of dopaminergic neurons in the substanitia nigra . the motor disorder responds well to dopamine - replacement therapies , though these result in significant adverse effects due to non - physiological release of dopamine in the striatum , and off - target effects . cell - based regenerative treatments offer a potential means for targeted replacement of dopamine , in a physiological manner . dopaminergic neurons for cell - based therapies can be obtained from several sources . fetal ventral mesencephalon tissue contains dopaminergic neuron progenitors , and has been transplanted into the striatum of pd patients with good results in a number of cases . however , the ethical implications and logistical challenges of using fetal tissue mean that fetal ventral mesencephalon is unlikely to be used in a widespread clinical setting . induced pluripotent stem cells can be used to generate dopaminergic neurons for transplantation , providing a source of autologous tissue for grafting . this approach means that challenges associated with allografts , such as the potential for immune rejection , can be circumvented . however , the associated cost and difficulty in producing a standardized product from different cell lines means that , at present , this approach is not commercially viable as a cell - based therapy . dopaminergic neurons derived from embryonic stem cells offer the most promising basis for a cell - based therapy for parkinson 's disease , with trials due to commence in the next few years . though there are ethical considerations to take into account when using embryonic tissue , the possibility of producing a standardized , optimized cell product means that this approach can be both effective , and commercially viable . purposethe aim of this study was to determine the frequency of significant panoramic radiographic findings and eventual treatment requirements before conventional or implant supported prosthetic treatment in asymptomatic edentulous patients.materials and methodsa total of 743 asymptomatic edentulous patients were retrospectively evaluated using a digital panoramic system . we analyzed the radiographic findings , including impacted teeth , retained root fragments , foreign bodies , severe atrophy of the posterior maxillary alveolar bone , mucous retention cysts , soft tissue calcifications and radiopaque - radiolucent conditions.resultsfour-hundred-eighty-seven ( 65.6% ) patients had no radiographic finding . a total of 331 radiographic findings were detected in 256 ( 34% ) patients . in 52.9% ( n=175 ) of these conditions , surgical treatment was required before application of implant - supported fixed prosthesis . however , before application of conventional removable prosthesis surgical treatment was required for 6% ( n=20 ) of these conditions.conclusionthe edentulous patients who will have implant placement for implant - supported fixed prosthesis can frequently require additional surgical procedures to eliminate pathological conditions .

spinal epidural lipomatosis ( sel ) is a rare disease that is characterized by pathologic overgrowth of lumbar or thoracic epidural fat tissue . although the etiology of sel is not known , sel has been frequently diagnosed in obese patient groups and exogenous steroid administration groups , so steroid metabolism is presumed to be related with sel . excessive epidural fat deposition in the spinal canal can contribute to various neurologic symptoms , such as back pain , radiculopathy , myelopathy , claudication and even cauda equina syndrome . in this case , we present a sel patient who had taken steroid hormone medicine for 20 years , after a suprasellar tumor resection . she presented with leg weakness and radiating pain on both buttock , posterior thigh & calf for 1 year . because pain and weakness in both legs was gradually aggravated during 4 months , she could not walk or stand when she was admitted . prior to admission , she underwent an operation for a suprasellar tumor resection in our hospital when she was 14 years old . at that time , the pathologic diagnosis was pilocytic astrocytoma . after the operation , panhypopituitarism occurred , so steroid hormone replacement therapy was started in 1993 . for a period of 19 years , exogenous steroid maintenance had been administrated ( median dose of prednisolon 7.5 mg ) . the neurologic examination revealed both legs weakness ( motor grade iv / iv ) and radiating pain on the l5 and s1 dermatomes . the straight leg raising test was negative , but the bilateral knee jerk and ankle jerk were slightly hyperreflexive . a magnetic resonance imaging ( mri ) study showed severe compression of the lumbar spinal cord by epidural fat tissue . at the dorsum of the spinal cord from the l1 to l5 levels , high signal lesions on t1- and t2-weighted images were checked . the longest length from the dorsum of the spinal cord to the ligamentum flavum was 11.4 mm on the l3 level and the median length was 9.2 mm at the l1-l5 levels . the engorged epidural vessels were positioned beneath the nerve root exit site , bilaterally ( fig . 2 ) . considering the long - term exogenous steroid administration , her neurologic symptoms and mr images , we could make the diagnosis of spinal epidural lipomatosis . because she complained of radiating pain on the bilateral l5 and s1 dermatomes , she had an operation to decompress the l5 and s1 nerve roots . in the operation field , fat tissue compressed the dorsum of the thecal sac and engorged epidural vessels were found on nerve root exit sites ( fig . we removed the epidural fat tissue and coagulated the engorged epidural vessels for nerve root decompression . the radiating pain in both legs was dramatically relieved and motor power was slightly improved . after 1 month , we did a follow - up mri . on the follow - up mri , the epidural fat tissue did not compress the thecal sac and the courses of nerve roots were not interrupted . spinal epidural lipomatosisis a rare disease characterized by overgrowth of epidural fat tissue , causing compression of the spinal neural components . after the first report , subsequent reports about this disease have been published , but the pathological mechanism of sel still remains unclear16 ) . there are many case reports about sel patients who were under long - term steroid therapy for curing ulcerative colitis , asthma , sarcoidosis , rheumatoid arthritis or renal disease3,8,10,12,15 ) . al - khawaja et al.2 ) reported that sel is classified in the idiopathic group and secondary group . in this review , they studied 111 patients , and both idiopathic and secondary groups are strongly male - predominant . in the idiopathic group , lumbar segments are more involved than thoracic segments and leg pain and back pain are the most frequent symptoms . relatively , in the secondary group ( steroid administration or endocrinopathy group ) , thoracic segments are more involved than lumbar segments and myelopathy is the main symptom . the symptoms of sel are dependent on the level of spinal canal compromise2,4 ) . in reported cases , the symptoms are gradual onset and similar to degenerative spinal stenosis . and the severity of symptoms are various from painful back pain to paraplegia . the diagnostic criterion of sel is greater than 6 mm thickness of epidural fat tissue . possible differential diagnoses include encapsulated spinal lipomas , spinal stenosis , and degenerative joint disease , which can also cause spinal compression and present with similar symptoms17 ) . conservative managements such as exogenous steroid tapering and weight loss are recommended in early sel patients while patients with severe symptoms or progressive neurologic deficits are indicated for operation1,7,9,13 ) . in a study by fogel et al.7 ) , seventy - seven percent of patients of the steroid group who underwent on decompressive laminectomy had symptom improvements . but in a study by fassett et al.6 ) , the mortality rate in these patients within 1 year after surgical decompression was 22% , because of concomitant medical problems and comorbidities . consequentially , we must make a treatment plan that considers various factors , such as neurologic severity , general health , steroid therapy and lifestyle . in patients who underwent an operation for suprasellar lesions such as pituitary adenoma , suprasellar meningioma , craniopharyngioma , and so forth , we can occasionally observe complications from corticosteroid hormone deficiency ; in which case , we start corticosteroid hormone replacement therapy without worrying about the sel . in our case , we reviewed the studies showing that long - term corticosteroid replacement therapy after neurosurgery can lead to spinal epidural lipomatosis . increased long - term survival following effective treatment of childhood lymphomas has placed greater emphasis on residual sequelae of therapy for lymphoma and the impact of these sequelae as the cause of further morbidity and mortality . following treatment of childhood hodgkin 's disease , cardiac pathology is second only to neoplasm as the cause of death in initial survivors . as the number of cancer survivors increases , so does the number of patients exposed to therapies toxic to the heart and lung , including radiation and chemotherapeutic modalities that employ anthracyclines . the literature is replete regarding effects of anthracyclines and radiation on the heart ; however , there is still debate regarding the detection of cardiac sequelae , their significance , and the relation between different treatment modalities and the development of cardiac sequelae . we hypothesized that protocols employing anthracyclines and radiation would adversely affect long - term cardiac function and that these effects would differ between survivors who received different treatment modalities . we therefore prospectively evaluated long term cardiac function in 45 survivors of childhood lymphomas , using clinical parameters , electrocardiography , and echocardiography and compared these parameters between lymphoma subtypes . the study included survivors of histology - diagnosed childhood lymphomas who had at least 5 years of followup from termination of therapy , and who were less than 18 years of age at treatment initiation . these criteria identified 45 survivors of 108 children treated for lymphoma at the pediatric hematology and oncology department of a tertiary care center during a nineteen - year period . all participants underwent evaluation which included an interview to assess functional class ( new york heart association ) , physical examination , 12-lead electrocardiography ( ecg ) , and echocardiography . data were taken from a single assessment at the last available follow - up . statistical analysis was performed using a commercially available package ( sigmastat 5.0 jandel scientific , san rafael , california ) . comparisons between the different lymphoma subgroups were made using one way anova or the tukey correction of this test where normality failed , and where relevant by the student 's t - test or the mann - whitney rank sum test where normality failed . characteristics of the study population including treatment duration and mean follow - up duration are presented in table 1 . the bl subgroup was divided between those treated in earlier years with protocols ( comp ) that did not include anthracyclines ( 7/12 patients ) , and those that were treated more recently ( 5/12 patients ) with protocols ( lmb , nci ) containing anthracyclines . these two subgroups did not differ significantly with regard to age at diagnoses ( 6.7 3.7 ; 8.30 5.1 years , resp . , p = .52 ) or age at follow - up ( 21.2 4.2 ; 16.1 5.8 years , p = .1 ) . the mean dose of doxorubicin ( adriamycin ) , for the group as a whole was 25 mg / m per treatment , leading to a cumulative dose of 150 mg / m or a total cumulative dose of 225250 mg . all participants underwent evaluation which included an interview to assess functional class ( new york heart association ( nyha ) ) , physical examination , 12-lead ecg , and echocardiography . cardiovascular physical examination , ecg , and echocardiography were performed by 2 senior staff pediatric cardiologists ( a. lorber and y. braver ) . analyses of ecg and echocardiography images were then undertaken in a blinded manner by different investigators ( m. friedberg and i. solt ) . stroke volume was calculated by multiplying the aortic valve area , measured from the 2-dimensional echocardiographic long axis view of the internal aortic diameter at valvular level , with the velocity time integral ( vti ) of aortic flow . cardiac index ( ci ) was then calculated by multiplying this value by heart rate , and dividing the product by body surface area . mitral regurgitation was graded according to the routine clinical gradation used in the lab as none , trivial , mild , moderate or severe . normal values were defined as being within 2 standard deviations of the mean normal value : for lv ejection fraction 66 4% , and lv fractional shortening 36 4% , lv mass index 70.4 gram / m for males ( 1095 percentile 48.5103 gram / m ) and 60.7 gram / m for females ( 1095 percentile 35.681 gram / m ) . early diastolic mitral inflow velocity ( e wave ) to late diastolic mitral inflow velocity ( a wave ) ratio is 1.7 0.4 to 2.5 0.9 . cardiac dimensions were defined as being within 2 standard deviations of the normal mean for body surface area . the nyha functional class was class i in 43 patients ( 96% ) and class ii in 2 patients ( 4% ) . these 2 patients did not have other comorbidities or notable toxic effects on follow - up . no events of near - syncope , syncope , or sudden death were noted . clinical features of the cohort as a whole and of the hodgkin 's disease subgroup are shown in table 3 . the percentage of survivors with a prolonged qtc interval by lymphoma subgroup is shown in figure 1(a ) . a prolonged qtc interval was not associated with sinoatrial or atrioventricular node dysfunction - conduction anomalies . incomplete right bundle branch block was recorded in 4 patients ( 9% , all diagnoses ) ; complete right bundle branch block was recorded in 2 patients . cardiac systolic and diastolic function was normal or low - normal for the group as a whole without significant differences between the subgroups ( table 3 ) . lv mass and shortening fraction did not correlate with mantle irradiation dose , qtc interval , or length of follow - up . mean lv mass was within normal limits ( 97 40 grams / m , mean s.d . ) . lv mass was significantly higher for males as compared to females ( all subjects , 112 grams versus 76 grams , resp . ; p < .01 ) . left ventricular end diastolic internal dimensions did not increase following relatively smaller shortening fraction values . cardiac index and mass by lymphoma subgroup are presented in figures 1(b ) and 1(c ) , respectively . the percentage of survivors with mitral regurgitation by subgroup is shown in figure 1(d ) . pulmonary valve insufficiency , more than the accepted physiological norm , was found in 1 survivor of nhnb . asymptomatic small pericardial effusions were found in 3 of the 45 patients ( 7% ) . no larger effusions or pericardial thickening was noted in any of the patients . a mitral inflow pattern consistent with constrictive or restrictive physiology ( elevated e wave velocity , elevated e / a ratio , and short deceleration time ) was not noted in any of the patients . selected electrocardiographic and echocardiographic parameters for the subset of bl survivors treated with anthracyclines versus those not treated with anthracyclines are presented in table 4 . cardiac abnormalities described after treatment for childhood lymphomas can involve almost any aspect of cardiac function and include pericarditis , pericardial effusions , pericardial fibrosis , pancarditis , myocardial fibrosis with functional impairment , valvular disease , conduction defects , and coronary artery disease . although sequelae may develop at variable time intervals following treatment , emphasis is now placed on the long - term sequelae of lymphoma therapy [ 7 , 8 ] , which are caused mainly by anthracyclines and radiation . in our study population , previous study has shown that an average cumulative dose of 450 mg / m of doxorubicin causes a 23% abnormality rate in echocardiographic findings during a 7-year follow - up period and that sequelae are rare at doses under 300 mg / m [ 10 , 11 ] . our study population received an average of 150 mg / m of doxorubicin , leading to a total dose of 225250 mg , which is less than the average toxic dose . the average follow - up duration for our population was 10.9 years , which is longer than that of many other studies , with relatively few cardiac sequelae from anthracyclines . however , these patients still warrant long - term cardiac follow - up , as the risk for cardiomyopathy continues to increase over time [ 12 , 13 ] . this vigilance is further justified by previous findings that patients may develop cardiac complications from anthracyclines , even when lower doses are used . studies performed 2 to 3 decades ago identified factors that augment the cardiotoxicity of anthracyclines . these include radiation of the mediastinum [ 14 , 15 ] , previous cardiac abnormalities , including involvement of the heart by the tumor [ 16 , 17 ] , uncontrolled hypertension [ 11 , 12 ] , and exposure to other chemotherapy modalities such as cyclophosphamide , dactinomycin , mitomycin , vincristine , bleomycin and methotrexate [ 13 , 18 , 19 ] . demographic factors include young age , female sex , black race and also trisomy 21 . among these , our study population was exposed to radiation of up to 4400 cgy ( but in most instances less than this ) and to polychemotherapy , including vincristine and bleomycin , as dictated by the various treatment protocols . more recently a pathophysiological role for the tyrosine kinase receptor , erbb2 , has been implicated in cardiomyocyte susceptibility to anthracyclines . again , the total dose of doxorubicin was probably the most important factor in preventing significant cardio toxicity among our population . these findings are consistent with other studies that found that modern protocol therapies using mopp / abvd and radiation hold low risk for cardiac toxicity [ 21 , 22 ] . . suggested that cardiac status following anthracyclines is described by a pattern consistent with a thin - walled , compliant left ventricle with reduced muscle mass performing under above - normal levels of wall stress . our results are consistent with this description and show left ventricular dimensions at the lower level of normal , with good compliance . it is surprising that no statistical differences in systolic function and left ventricular mass and thickness were found between the lymphoma type subgroups , in the light of the substantial differences in therapy that these groups received . although differences in left ventricular mass between bl patients who received anthracyclines and those who did not reach statistical significance , our data may correlate with the aforementioned findings that anthracyclines reduce ventricular mass and reduce left ventricular wall thickness . however , no differences were found between the various lymphoma subgroups , among whom hd patients were exposed both to anthracyclines as well as to radiation . indeed , these patients exhibited increased cardiac output in comparison to the other lymphoma subgroups . more importantly , cardiac index was at the low range of normal , or slightly below normal , for all groups . differences between males and females were significant regarding lv mass , a factor that contributes to the greater susceptibility of females to anthracyclines , and the greater prevalence of prolonged qtc interval among females . minimal mitral insufficiency , in the absence of valve deformity , is usually of little clinical significance . . detected an increase in the prevalence of mitral regurgitation among children who had received anthracyclines , 12% ( 4 of 34 ) of whom later developed left ventricular systolic dysfunction , concluding that mitral regurgitation might be an early marker of anthracycline - related cardiac dysfunction . our findings correlate with those of previous studies , where both qt and qt dispersion were prolonged following treatment with anthracyclines [ 27 , 28 ] . a prolonged qt interval may result from myocardial cell damage but does not correlate with decreased contractile function [ 29 , 30 ] . as dysrhythmias may occur years after termination of treatment and these are potentially life - threatening , patients should be evaluated at regular followup with a 12-lead ecg [ 8 , 31 ] . although no sudden death events were observed in our population , following our findings of a prolonged q - t interval in a substantial percentage of the population , it may be suggested that a 24-hour holter - ecg recording be part of the routine follow - up of these patients . coronary artery disease has been reported after radiation therapy to the mediastinum , but not after anthracycline therapy [ 1 , 32 ] . our results did not show resting ecg changes , and there was no premature mortality among our study group on a longer follow - up period . in the light of findings from other studies , we would suggest periodic ergometry in the long - term follow - up of patients who have undergone radiation therapy . additional well - described sequelae of radiation therapy to the mediastinum are pericardial effusion and constrictive pericarditis [ 34 , 35].we did not note these pathologies to any significant degree in our study population . the use of possible cardioprotective agents such as dexrazoxane may provide additional protection against long - term cardiac sequelae of anthracyclines , especially in girls [ 36 , 37 ] . these agents were not used in our study , and we can not comment further on their effects . . we did not compare the study group with a control group , as our primary objective was definition of cardiac status among survivors . however , we used well - established norms that are commonly and widely used as the basis for this definition . further comparisons between the different lymphoma subgroups , provided a comparative basis for the different treatment modalities , and specific comparison between burkitt lymphoma survivors who had been exposed to anthracyclines and those who had not strengthened this comparison . echocardiographic parameters such as ejection fraction and fractional shortening are limited by load dependency and constitute basic indicators of global ventricular function . although other echocardiographic methods are available for assessment of cardiac function , each has its specific limitations and in daily clinical practice many oncologists are more familiar with the traditional parameters used in this study . this study focused on long - term follow - up with data collected at the last available assessment , and we did not collect data on acute or subacute toxicity.in summary , in this cohort , relatively few long - term cardiac complications were seen in most patients following low - dose anthracycline therapy used in modern treatment protocols for childhood lymphoma . however , anthracycline cardio toxicity has previously been well established and in the current study , abnormalities were found in mitral valve regurgitation abnormalities , qt prolongation , low - normal cardiac index , and in 2 survivors functional capacity reduction leading to a compromised quality of life . these findings necessitate comprehensive and long - term cardiac follow - up of these patients . the study group is relatively small and the number of patients in subgroups is small . we did not compare the study group with a control group , as our primary objective was definition of cardiac status among survivors . however , we used well - established norms that are commonly and widely used as the basis for this definition . further comparisons between the different lymphoma subgroups , provided a comparative basis for the different treatment modalities , and specific comparison between burkitt lymphoma survivors who had been exposed to anthracyclines and those who had not strengthened this comparison . echocardiographic parameters such as ejection fraction and fractional shortening are limited by load dependency and constitute basic indicators of global ventricular function . although other echocardiographic methods are available for assessment of cardiac function , each has its specific limitations and in daily clinical practice many oncologists are more familiar with the traditional parameters used in this study . this study focused on long - term follow - up with data collected at the last available assessment , and we did not collect data on acute or subacute toxicity . in summary , in this cohort , relatively few long - term cardiac complications were seen in most patients following low - dose anthracycline therapy used in modern treatment protocols for childhood lymphoma . however , anthracycline cardio toxicity has previously been well established and in the current study , abnormalities were found in mitral valve regurgitation abnormalities , qt prolongation , low - normal cardiac index , and in 2 survivors functional capacity reduction leading to a compromised quality of life . these findings necessitate comprehensive and long - term cardiac follow - up of these patients .

spinal epidural lipomatosis ( sel ) is an abnormal localized accumulation of fat tissues in the epidural space . it is strongly related with steroid administration . the symptoms of sel are various and range from back pain to paraplegia . in severe cases , decompressive laminectomy is the choice of treatment . a 32-year - old woman who had been under long - term steroid administration after suprasellar tumor resection was admitted for both leg radiating pain and weakness . she was diagnosed with sel and had a decompressive laminectomy . during the operation , we found the nerve roots were compressed by epidural fat tissues and engorged vessels . after the operation , her radiating pain was relieved and motor weakness was improved . objectives . we studied long - term effects of therapy for childhood lymphoma on cardiac function . design and patients . we prospectively evaluated 45 survivors of childhood lymphoma , using clinical parameters , electrocardiography and echocardiography . further comparisons were made between lymphoma subgroups and between males and females . results . mean age at diagnosis was 9.1 years . mean followup duration was 10.9 years . the nyha functional class was i in 43 patients and ii in 2 patients . a prolonged qtc interval ( > 0.44 msec ) was found in 8 patients . left ventricular ( lv ) systolic function and compliance were normal ( lv shortening fraction 40 5.6% ; cardiac index 2.84 1.13 l / min / m2 ; e / a wave ratio 2.5 1.3 ; mean s.d . ) , lv mass was normal ( 97 40 grams / m2 , mean s.d . ) . mitral regurgitation was observed in 7/45 patients ( 16% ) . asymptomatic pericardial effusions were found in 3/45 ( 7% ) patients . conclusions . long - term follow - up shows that most parameters of cardiac function are normal in survivors of childhood lymphoma . this is likely due to relatively low doses of anthracyclines in modern protocol modalities . abnormalities in mitral valve flow , qtc prolongation and in a small proportion of survivors , and functional capacity necessitate long - term cardiac follow - up of these patients .