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hypertension is common in hemolytic uremic syndrome ( hus ) , and often difficult to control in an acute stage . malignant hypertension associated with hus leads to reversible posterior encephalopathy syndrome , seizures , heart failure , and other adverse consequences which increase the morbidity and mortality of the disease . renin - mediated mechanism is believed to be the main factor responsible for hypertension seen in these cases . drugs that act by blocking renin - angiotensin axis ( ras ) are thus ideal for such cases , however , due to concern of progression of renal failure and lack of experience of these agents in children , these are not preferred or used commonly in acute stages . we hereby report two cases of hus with severe refractory malignant hypertension in which we targeted ras by using intravenous ( iv ) enalaprilat , oral aliskiren , and oral enalapril with quick and dramatic response of blood pressure ( bp ) . a 6-year - old male was admitted with a history of vomiting , fever since 2 weeks , hematuria and decreased urine output since 1 week . on evaluation by his local practitioner , he was found to have anemia ( hemoglobin [ hb ] 6.3 g / dl ) , thrombocytopenia ( platelet 72,000/mm ) , active urine sediment ( red blood cell [ rbc ] 4060/hpf , albumin 3 + ) , and azotemia ( blood urea 200 mg / dl , creatinine 4.2 mg / dl ) . he had an episode of seizure ( due to accelerated hypertension ) , hence was brought to our hospital for further management . on evaluation , he was hypertensive ( bp 150/100 mmhg ) with generalized edema , oliguria , and a normal systemic examination . investigations were suggestive of hus ( hb 4.8 g / dl , white blood cell [ wbc ] 11,190 cmm , platelet 1.84/mm , peripheral smear : schistocytes positive , reticulocyte count 6.8% , lactate dehydrogenase [ ldh ] 4300 u / l , direct coombs test and indirect coombs tests were negative , urea 67 mg / dl , creatinine 2.6 mg / dl ) . septic work up , dengue serology , and malarial antigen were negative , and he became afebrile on the 4 day of admission . his antinuclear antibodies ( ana ) and antineutrophil cytoplasmic antibody ( anca ) were negative . he was started on empiric antibiotics ( injection ceftriaxone ) and daily plasmapheresis for hus . detailed complement regulator assay showed very high anti - factor h antibody ( 41,000 iu ) . c3 , c4 , antigenic levels of factor h , factor i , factor b , and cd46 were normal [ table 1 ] . he was given a blood transfusion and initiated on hemodialysis and daily plasma exchanges in view of oligo - anuric acute kidney injury ( aki ) . complement assay in cases * for the child 's height percentile , the bp percentiles were : 90 percentile : 113/72 mmhg and 95 percentile 117/76 mmhg ( blood pressure references used were as per the fourth report ) . for arterial hypertension [ figure 1 ] , he was started on sustained release nifedepine , clonidine , and metoprolol and subsequently prazosin with a gradual increase in dosage . however , arterial bp remained persistently high ( > 99 centile ; up to 170/120 mmhg ) , and he developed blurring of vision , with abdominal pain and vomiting on the 3 day of admission necessitating need for iv nitroglycerine ( up to 5 mcg / kg / min ) and subsequently labetolol infusion ( up to 2 mg / kg / h ) for refractory hypertension . child had persistent arterial hypertension ( > 99 centile for his age ) , despite vigorous fluid removal in hemodialysis sessions . response to antihypertensive medications in case 1 oral enalapril and minoxidil were also added and dosage of other oral antihypertensives optimized to the maximal doses [ figure 1 ] but arterial bp remained high and was difficult to control . but since within 48 h of adding oral enalapril and oral minoxidil , child went into hypertensive emergency ( bp 180/120 mmhg ) , with hallucinations and visual blurring , iv enalaprilat was added . hypertension showed a significant improvement after addition of iv enalaprilat ( 10 g / kg / dose q 8 hourly ) on the 5 day of admission . there was a consistent fall of bp within hours of giving individual iv enalaprilat boluses ( average fall in mean bp 9.5 mmhg ) . arterial bp decreased to 110/78 mmhg ; patient became asymptomatic , and nitroglycerine and labetolol infusions were tapered off successfully . he developed neutropenia a week following enalaprilat therapy ( wbc 1500 cmm , 50% neutrophils ) , which could not be attributed to any other cause ; hence , it was stopped followed by a rebound in hypertension ( arterial bp 190/136 mmhg ) . he was not dialysis dependent at this stage with a good urine output , and serum creatinine had fallen to 0.6 mg / dl , and his hemodialysis catheter was removed . aliskiren ( 2 mg / kg / dose ) was then added with good response ( arterial bp decreased to 150/110 mmhg over 24 h , and 138/110 mmhg over 48 h ) ; however , it was withdrawn after 4 days due to hyperkalemia ( serum potassium 6.5 mmol / l ) . after improvement of neutopenia , oral enalapril was reintroduced along with telmisartan . bp control improved within 48 h on this angiotensin - converting enzyme inhibitor - angiotensin receptor blockade ( acei - arb ) combination ( bp < 90 percentile for age ) along with other oral antihypertensive agents . he received seven daily plasmapheresis sessions till active hemolysis subsided followed by alternate day sessions . he received prednisolone ( 1 mg / kg / day ) and iv immunoglobulin 2 g / day ( day 10 of admission ) , iv cyclophosphamide during his hospital stay . he received total six doses of iv cyclophosphamide followed by maintenance azathioprine . at his 1 year of follow - up , he is doing well , normotensive ( bp < 90 percentile for age ) and no proteinuria ( urine protein / creatinine ratio < 0.2 ) and a normal urine examination on angiotensin - converting enzyme ( ace ) and arb combination . a 7-month - old male was admitted with a history of vomiting , fever since 5 days and anuria since 2 days . , he was hypertensive ( bp 130/60 mmhg ) , with pallor , facial puffiness , and normal systemic examination . investigations were suggestive of atypical hus - microangiopathic hemolytic anemia with aki ( hb 7 g / dl , wbc 13,280/mm , platelets 100,000/mm , peripheral smear : schistocytes , elliptocytes , reticulocyte count 5.6% , ldh 4300 u / l ) and active urine sediment ( rbc 1020/hpf , albumin 2 + ) . the child had no evidence of pneumonia or sepsis , on clinical evaluation , and all cultures were sterile . the child had advanced azotemia ( urea 216 mg / dl , creatinine 7.2 mg / dl ) with severe hyperkalemia and metabolic acidosis . for hus with evidence of ongoing hemolysis and dialysis dependence , he was started on daily plasmapheresis . a detailed complement assay ( including c3 , c4 , antigenic levels of factor h , factor i , factor b , cd46 , and autoantibodies to factor h ) were normal [ table 1 ] . bp was observed to be high since admission and increased up to 160/110 mmhg on serial monitoring , although patient remained asymptomatic . for his arterial hypertension ( > 99 centile for age ) [ figure 2 ] , he was started on amlodipine and prazosin initially ; dosage was increased to the maximal dose , and clonidine and oral enalapril were added on the 3 and 4 day of admission , respectively . despite the addition of multiple antihypertensive agents and dosage optimization and aggressive ultrafiltration in hemodialysis sessions , arterial bp showed only marginal decrease and mean arterial bp remained high ( > 99 centile for age ; 100110 mmhg ) . intravenous enalaprilat ( 10 g / kg / dose q 8 hourly ) was added on day 6 of admission . the mean arterial bp improved ( 80 mmhg ) within 12 h of addition of enalaprilat . once arterial bp was controlled with no further increase , dose of oral enalapril was maximized , while tapering off iv enalaprilat and other antihypertensive medications were continued . response to antihypertensive medications in case 2 daily hemolytic parameters and renal function were monitored . he was discharged in 2 weeks time in a stable condition , with adequate urine output and bp controlled on oral antihypertensive therapy . at a follow - up of 1 year , currently the infant is doing well , with serum creatinine 0.7 mg / dl , urine protein / creatinine ratio 1.5 , and normal bp ( bp < 90 percentile for age ) on oral enalapril 0.4 mg / kg / day . a 6-year - old male was admitted with a history of vomiting , fever since 2 weeks , hematuria and decreased urine output since 1 week . on evaluation by his local practitioner , he was found to have anemia ( hemoglobin [ hb ] 6.3 g / dl ) , thrombocytopenia ( platelet 72,000/mm ) , active urine sediment ( red blood cell [ rbc ] 4060/hpf , albumin 3 + ) , and azotemia ( blood urea 200 mg / dl , creatinine 4.2 mg / dl ) . he had an episode of seizure ( due to accelerated hypertension ) , hence was brought to our hospital for further management . on evaluation , he was hypertensive ( bp 150/100 mmhg ) with generalized edema , oliguria , and a normal systemic examination . investigations were suggestive of hus ( hb 4.8 g / dl , white blood cell [ wbc ] 11,190 cmm , platelet 1.84/mm , peripheral smear : schistocytes positive , reticulocyte count 6.8% , lactate dehydrogenase [ ldh ] 4300 u / l , direct coombs test and indirect coombs tests were negative , urea 67 mg / dl , creatinine 2.6 mg / dl ) . septic work up , dengue serology , and malarial antigen were negative , and he became afebrile on the 4 day of admission . his antinuclear antibodies ( ana ) and antineutrophil cytoplasmic antibody ( anca ) were negative . he was started on empiric antibiotics ( injection ceftriaxone ) and daily plasmapheresis for hus . detailed complement regulator assay showed very high anti - factor h antibody ( 41,000 iu ) . c3 , c4 , antigenic levels of factor h , factor i , factor b , and cd46 were normal [ table 1 ] . he was given a blood transfusion and initiated on hemodialysis and daily plasma exchanges in view of oligo - anuric acute kidney injury ( aki ) . complement assay in cases * for the child 's height percentile , the bp percentiles were : 90 percentile : 113/72 mmhg and 95 percentile 117/76 mmhg ( blood pressure references used were as per the fourth report ) . for arterial hypertension [ figure 1 ] , he was started on sustained release nifedepine , clonidine , and metoprolol and subsequently prazosin with a gradual increase in dosage . however , arterial bp remained persistently high ( > 99 centile ; up to 170/120 mmhg ) , and he developed blurring of vision , with abdominal pain and vomiting on the 3 day of admission necessitating need for iv nitroglycerine ( up to 5 mcg / kg / min ) and subsequently labetolol infusion ( up to 2 mg / kg / h ) for refractory hypertension . child had persistent arterial hypertension ( > 99 centile for his age ) , despite vigorous fluid removal in hemodialysis sessions . response to antihypertensive medications in case 1 oral enalapril and minoxidil were also added and dosage of other oral antihypertensives optimized to the maximal doses [ figure 1 ] but arterial bp remained high and was difficult to control . but since within 48 h of adding oral enalapril and oral minoxidil , child went into hypertensive emergency ( bp 180/120 mmhg ) , with hallucinations and visual blurring , iv enalaprilat was added . hypertension showed a significant improvement after addition of iv enalaprilat ( 10 g / kg / dose q 8 hourly ) on the 5 day of admission . there was a consistent fall of bp within hours of giving individual iv enalaprilat boluses ( average fall in mean bp 9.5 mmhg ) . arterial bp decreased to 110/78 mmhg ; patient became asymptomatic , and nitroglycerine and labetolol infusions were tapered off successfully . he developed neutropenia a week following enalaprilat therapy ( wbc 1500 cmm , 50% neutrophils ) , which could not be attributed to any other cause ; hence , it was stopped followed by a rebound in hypertension ( arterial bp 190/136 mmhg ) . he was not dialysis dependent at this stage with a good urine output , and serum creatinine had fallen to 0.6 mg / dl , and his hemodialysis catheter was removed . aliskiren ( 2 mg / kg / dose ) was then added with good response ( arterial bp decreased to 150/110 mmhg over 24 h , and 138/110 mmhg over 48 h ) ; however , it was withdrawn after 4 days due to hyperkalemia ( serum potassium 6.5 mmol / l ) . after improvement of neutopenia , oral enalapril was reintroduced along with telmisartan . bp control improved within 48 h on this angiotensin - converting enzyme inhibitor - angiotensin receptor blockade ( acei - arb ) combination ( bp < 90 percentile for age ) along with other oral antihypertensive agents . he received seven daily plasmapheresis sessions till active hemolysis subsided followed by alternate day sessions . he received prednisolone ( 1 mg / kg / day ) and iv immunoglobulin 2 g / day ( day 10 of admission ) , iv cyclophosphamide during his hospital stay . he received total six doses of iv cyclophosphamide followed by maintenance azathioprine . at his 1 year of follow - up , he is doing well , normotensive ( bp < 90 percentile for age ) and no proteinuria ( urine protein / creatinine ratio < 0.2 ) and a normal urine examination on angiotensin - converting enzyme ( ace ) and arb combination . a 7-month - old male was admitted with a history of vomiting , fever since 5 days and anuria since 2 days . there was no history of diarrhea or dysentery in the past . on admission , he was hypertensive ( bp 130/60 mmhg ) , with pallor , facial puffiness , and normal systemic examination . investigations were suggestive of atypical hus - microangiopathic hemolytic anemia with aki ( hb 7 g / dl , wbc 13,280/mm , platelets 100,000/mm , peripheral smear : schistocytes , elliptocytes , reticulocyte count 5.6% , ldh 4300 u / l ) and active urine sediment ( rbc 1020/hpf , albumin 2 + ) . the child had no evidence of pneumonia or sepsis , on clinical evaluation , and all cultures were sterile . the child had advanced azotemia ( urea 216 mg / dl , creatinine 7.2 mg / dl ) with severe hyperkalemia and metabolic acidosis . for hus with evidence of ongoing hemolysis and dialysis dependence , he was started on daily plasmapheresis . a detailed complement assay ( including c3 , c4 , antigenic levels of factor h , factor i , factor b , cd46 , and autoantibodies to factor h ) were normal [ table 1 ] . bp was observed to be high since admission and increased up to 160/110 mmhg on serial monitoring , although patient remained asymptomatic . echocardiography and fundus were normal . for his arterial hypertension ( > 99 centile for age ) [ figure 2 ] , he was started on amlodipine and prazosin initially ; dosage was increased to the maximal dose , and clonidine and oral enalapril were added on the 3 and 4 day of admission , respectively . despite the addition of multiple antihypertensive agents and dosage optimization and aggressive ultrafiltration in hemodialysis sessions , arterial bp showed only marginal decrease and mean arterial bp remained high ( > 99 centile for age ; 100110 mmhg ) . intravenous enalaprilat ( 10 g / kg / dose q 8 hourly ) was added on day 6 of admission . the mean arterial bp improved ( 80 mmhg ) within 12 h of addition of enalaprilat . once arterial bp was controlled with no further increase , dose of oral enalapril was maximized , while tapering off iv enalaprilat and other antihypertensive medications were continued . response to antihypertensive medications in case 2 daily hemolytic parameters and renal function were monitored . he was discharged in 2 weeks time in a stable condition , with adequate urine output and bp controlled on oral antihypertensive therapy . at a follow - up of 1 year , currently the infant is doing well , with serum creatinine 0.7 mg / dl , urine protein / creatinine ratio 1.5 , and normal bp ( bp < 90 percentile for age ) on oral enalapril 0.4 mg / kg / day . the extent of renal microangiopathic involvement appears to be responsible for the development of hypertension and renal failure in atypical hus . renal ischemia is triggered which leads to maximal activation of ras resulting in accelerated hypertension which is often very severe and resistant to antihypertensive therapy . local ras activation is believed to be an important key factor in the thrombotic microangiopathy in hus leading to intractable hypertension . this has been demonstrated to occur via enhanced tissue factor expression on glomerular endothelial cells which is enhanced by angiotension ii . on the other hand two studies have shown elevated plasma renin levels in children with hus , irrespective of systemic hypertension . severe hypertension that ensues is a clinician 's nightmare as it is hard to control despite use of multiple drug combinations and careful drug titration . in extreme cases , bilateral nephrectomy is ultimately required as a life - saving measure for achieving control of bp , thus again underlining the pivotal role of hyperreninemia in the development of hypertension in hus . although there is plenty of evidence in favor of renin - mediated mechanism in the pathogenesis of hypertension in hus , in practice there is hesitation to use ras inhibitors or their combinations in the acute stage for hypertension . this is due to fear of worsening of renal failure and lack of pediatric experience with newer ras inhibitors . oral aceis ( enalapril , captopril ) have been shown to be renoprotective and of benefit for long - term bp control and reduction in proteinuria in patients with persistent disease . however , they are not preferred in the acute stage of disease , used with great caution and generally initiated after improvement of renal function . moreover , aceis including enalaprilat are seldom used in hypertensive emergencies due to concerns regarding slow onset of action and variable effectiveness , especially in children . we used iv enalaprilat in both patients for acute hypertension who showed significant and consistent decline in bp . it helped in successful reversal of hypertensive urgency in the older child whose bp remained high and was extremely difficult to control despite multiple drugs including iv nitroglycerine and labetolol . the onset of action begins in 15 min , but the peak effect may take 14 h. the duration of action is usually 46 h. the half life of the drug is usually 11.1 h in infants and children . there is one case series reporting the neonatal use of enalaprilat that reported that doses even at the lower end of what was used in this cohort may lead to significant , prolonged hypotension and oliguric acute renal failure . if it is used in the newborn or children , it should be used with caution with ongoing monitoring of bp , serum potassium , and renal function . the adverse effects of enalaprilat are hyperkalemia , hypotension , cough , diarrhea , angioedema , and leucopenia ( agranulocytosis ) . we encountered neutropenia in one of our patients a week after initiation of iv enalaprilat which reversed quickly on drug withdrawal . however , severe rebound hypertension also occurred on stopping enalaprilat , while other drugs were continued , which reiterates its effectiveness in the management of the hypertensive urgency . studied the bp response to iv enalaprilat in 35 patients with hypertensive crisis , and found that the extent of systolic and diastolic bp reduction correlated well with the pretreatment plasma renin and angiotensin ii levels . thus , it appears that the status of ras determines the efficacy of iv enalaprilat , and hence it was successful in both our patients with hus - induced hypertension . therapeutic enalaprilat levels can probably be achieved with 1/4 total cumulative dose of enalapril , administered as 6 hourly enalaprilat : recommended pediatric dosing is 510 mcg / kg / dose . we used a dose of 10 mcg / kg / dose q 8 hourly in both of our cases based on the previous report . we used aliskiren in the older child which too was very effective in lowering bp . aliskiren is the first direct renin inhibitor available in an oral form approved for adults by the us food and drug administration in 2007 . as renin is the first and rate - limiting step in angiotensin ii synthesis in ras , its direct inhibition is theoretically more advantageous as compared to ace inhibition / arb . as it is a nonpeptide molecule , it has better bioavailability and a long half - life and can , therefore , lower bp effectively . once administered orally , the effect of the drug peaks in 13 h , achieves its steady state in 57 days and has a half - life of 40 h. aliskiren produces dose - dependent bp reduction and its potency has been shown to be equivalent or better than aceis , arbs in various trials . moreover , when administered as a combination with acei , it helps to block an increase in plasma renin activity induced by acei monotherapy . combination therapy ( aliskiren + acei ) has been demonstrated to have greater bp - lowering potential as compared to either alone . dual ras blockade however must be cautiously monitored as there is a higher chance of adverse effects . a case series of children with chronic kidney disease receiving combination aliskiren / acei showed > 45% proteinuria reduction , however side effects in the form of hyperkalemia , worsening of renal function , and hypotension were seen . mild hyperkalemia was seen in our patient but was asymptomatic , and potassium normalized quickly on stopping the drug . other adverse effects including nausea , angioedema , diarrhea , abdominal pain , and headache may be seen with aliskiren but are usually mild and not dose related . adult studies have shown aliskiren to be a safe and effective antihypertensive drug ; however , its use in children has been restricted so far due to the paucity of data . a recent prospective , randomized controlled in children between ages 6 and 17 years concluded that aliskiren in once daily doses of 2 mg / kg or 6 mg / kg was well tolerated and safe . we used a dose of 2 mg / kg in case 1 . in the above two patients , we used two unconventional drugs : aliskiren and iv enalaprilat , both of which were very quick and effective in controlling high bp refractory to multiple antihypertensive medications and aggressive ultrafiltration during dialysis sessions . the limitation of the report is small number , multiple antihypertensives in these patients , and simultaneous use of plasma exchanges and immunosuppression in anti - factor h antibody positive case 1 to help resolution of illness , which might make interpretation difficult . these appear to be promising alternatives in the treatment of severe atypical hus - induced hypertension and hypertensive emergency , and there is a need to have more trials targeting renin in these cases . in 2009 , a 52-year - old woman presented with a single lesion on her nose , which started as a papule , referred to sedighe tahereh clinic , isfahan , iran . the lesion had existed for a period of 14 months and was slowly increasing in size , enlarging to a plaque . the diagnosis of leishmaniasis was confirmed with a positive smear of the lesion showing leishmania bodies about 1 year before . all five members of her family had had a history of proven leishmaniasis . in the past medical history , the patient was a renal failure case since 11 years before and received a renal transplant 4 years after the diagnosis of renal failure . she was receiving oral mycophenolate mofetil ( 2 g daily ) and cyclosporine ( 100 mg daily ) . a 33 cm indurated ulcer with elevated borders was present on the tip of her nose ( figure 1 ) . her therapeutic plan was intralesional glucantime injection ( approximately 1 ml of 1.5 g vial per week , intralesional injection ) . after completing a therapeutic course of 20 sessions receiving intralesional glucantime injections , she was considered as glucantime therapy resistant . the occurrence of malignant neoplasms in sites of scars is an infrequent but well - known phenomenon.5 although the coexistence of cutaneous leishmaniasis and bcc may have been coincidental , some studies suggest that an association between these two entities does exist.6 leishmaniasis can directly or indirectly alter the diagnosis and course of different malignancies.7 there are reports of bcc in chronic leg ulcers.8 cases of bcc developing in a leishmania scar have also been documented,9 but to our knowledge , cases of both leishmaniasis and bcc in the same site and the same lesion are rare.10 however , in this case , solid organ transplantation and long term immuno suppressive therapy should be considered as risk factors for malignancy . advances in effective immuno suppression after organ transplantation have led to increased risk of malignancies , particularly skin cancers11 including squamous cell carcinoma , basal bcc and malignant melanoma.12 thus , malignancies should be considered in the differential diagnosis of leishmaniasis lesions difficult to treat . the possible role of cutaneous leishmaniasis , as a predisposing factor for skin cancer , should also be kept in mind . aa was the main therapeutic physician and helped write the manuscript . i m and pk contributed in writing the manuscript .

hypertension is common in hemolytic uremic syndrome ( hus ) and often difficult to control . local renin - angiotensin activation is believed to be an important part of thrombotic microangiopathy , leading to a vicious cycle of progressive renal injury and intractable hypertension . this has been demonstrated in vitro via enhanced tissue factor expression on glomerular endothelial cells which is enhanced by angiotensin ii . we report two pediatric cases of atypical hus with severe refractory malignant hypertension , in which we targeted the renin - angiotensin system by using intravenous ( iv ) enalaprilat , oral aliskiren , and oral enalapril with quick and dramatic response of blood pressure . both drugs , aliskiren and iv enalaprilat , were effective in controlling hypertension refractory to multiple antihypertensive medications . these appear to be promising alternatives in the treatment of severe atypical hus - induced hypertension and hypertensive emergency . leishmaniasis is a protozoan infection due to organisms of the genus leishmania . the differential diagnosis of cutaneous leishmaniasis includes arthropod bites , basal cell carcinoma ( bcc ) and other malignancies . bcc is the most common form of skin cancer . we present a case of cutaneous leishmaniasis resistant to standard intralesional glucantime injection in an immunocompromised patient , which was proved to be bcc after surgical excision .

a 60-year - old man was admitted because of visual disturbances and ocular pain following penetrating keratoplasty in the right eye for a corneal chemical burn . on slit lamp examination , we observed a feather - like corneal opacity , corneal infiltrations , and epithelial defects . subconjuctival amphotericin b ( 1 mg ) , topical amphotericin b 0.125% , and levofloxaxin 0.5% were subsequently administered every hour for 16 weeks . the patient was restarted on and maintained on a topical amphotericin b treatment ( 0.125% twice daily ) for 1 year . in addition , levofloxaxin 0.5% ( three times daily ) and fluorometholone 0.1% ( twice daily ) were administered for 1 year . the lesion did not progress , but there was no change in corneal infiltration . on examination one year later , an elevated corneal lesion and increased corneal infiltration with a yellow color were observed despite the topical amphotericin b treatment . seven days later anterior chamber hypopyon and vitreous opacity on a b - scan were observed ( fig . we immediately performed an intravitreal injection of amphotericin b 5 g/0.1ml and vancomycin 1 mg/0.1ml . intravitreal voriconazole 80 g/0.1ml and ceftazidime 2 mg/0.1ml were injected and topical voriconazole 1% was administered every 2 hours . the patient discontinued the oral voriconazole because of abdominal pain , dry mouth , and scaling of the oral mucosa . the patient was treated topically for 6 weeks , after which the corneal lesion and endophthalmitis had improved . the patient subsequently underwent another penetrating keratoplasty and demonstrated a visual acuity of 0.1 at 10 months follow - up with no signs of recurrence . a 60-year - old man was referred to us because of a lack of improvement after a month of empiric treatment with topical amphotericin b for fungal keratitis . on our initial examination after 10 days the culture revealed no growth and the corneal epithelium appeared healed ; however , there was no change in corneal infiltration ( fig . because of a lack of improvement in the corneal lesion a month later , a new treatment regimen was initiated with topical voriconazole 1% administered every hour . six weeks later we observed a decreasing density of the infiltrate and healing of the corneal epithelium ; the topical voriconazole was decreased to twice daily . following completion of the therapy , complete healing of the corneal epithelium and resolution of the corneal infiltrate were observed ; however , the corneal opacity persisted ( fig . 2c ) voriconazole , a derivative of fluconazole , is a new triazole antifungal agent.4 like other triazoles , this voriconazole inhibits cytochrome p450 demethylase , which is essential for the synthesis of ergosterol . it is hypothesized that this adversely affects the permeability of the fungal cell membrane.7 voriconazole has excellent oral bioavailability and a broad spectrum of activity . therapeutic aqueous and vitreous levels are achieved after oral administration of voriconazole.8 voriconazole showed lower mics compared to other antifungal agents when tested against five corneal isolates of scedosporium apiospermum,2 and it had the best in vitro susceptibility profile for 34 common fungal pathogens compared to other antifungal agents.9 gao et al.10 reported that direct intravitreal voriconazole injections of 25 g / ml ( equivalent to 100 mg per injection in a human eye ) caused no electroretinographic or histopathologic abnormalities in rodent retinas . kramer et al.11 reported that intravitreal injection of voriconazole ( 100 g/0.1ml ) with pars plana vitrectomy was an effective therapy for aspergillus endophthalmitis . lee et al.7 reported a case of drug - resistant penicillium endophthalmitis that was treated with intravitreal voriconazole injection ( 50 g/ 0.1ml ) . in these cases , voriconazole has been shown to be highly effective against filamentous organisms and is more potent in invasive aspergillosis than amphotericin b.12,13 according to a report by jang et al.,5 voriconazole is also effective against candida chorioretinitis . ozbek et al.14 reported one case of alternaria keratitis that showed improvement with 1% topical voriconazole . in contrast , giaconi et al.15 reported two cases of fungal keratitis caused by fusarium oxysporum and colletotrichum dematium that did not respond to treatment with 1% topical voriconazole . our report indicates that 1% voriconazole is an effective treatment for candida spp . and unknown fungal keratitis . in conclusion , voriconazole is a new , promising therapy for fungal keratitis refractory to standard antifungal agents . nevertheless , more clinical trials will be necessary to investigate the effectiveness of systemic voriconazole and corneal transplantation . liver plays a pivotal role in detoxification of xenobiotics , environmental pollutants , and chemotherapeutic agents . for this reason , this organ acetaminophen ( apap ) is a widely used antipyretic and analgesic which is currently the most frequent cause of drug - induced hepatic failure in the united states with intentional and unintended overdose . cytochrome p450 enzymes convert a relatively minor portion of apap to the highly reactive intermediate metabolite n - acetyl - p - benzoquinone - imine ( napqi ) . under normal physiological conditions , apap overdose increased napqi formation which exceeds the rate of detoxification by gsh . in recent times , much attention was attracted to herbal medicines as alternative medicines . quite a few plant remedies have been examined for the treatment of liver disorders for centuries prosopis farcta ( pf ) [ figure 1 ] from leguminosae and sub - family mimosoideae has some medicinal properties such as antiinflammatory effects , treating gastric ulcers , fetus abortion , dysentery , arthritis , larynx inflammation , heart pains , and asthma . this plant is the indigenous to dry and semi - dry areas of america , asia , and africa some of the compounds existing in prosopis plant are : quercetin ( flavonoids ) , tryptamine , apigenin 5-hydroxytryptamine ( alkaloids ) , l - arabinose , and lectin . moreover , a number of phenolic compounds with strong antioxidant activity has been identified in extracts of this plant such as vicenin-2 , apigenin c - glycoside , iso - orientin , vitexin , luteolin 7-o - glucoside , isovitexin , quercetin 3-o - glucoside , rutin , kaempferol 3-o - rutonoside , caffeic acid derivative , and luteolin . antioxidants play an important role in inhibiting and scavenging free radicals and thus , providing protection against infections and degenerative diseases . there is no in vitro or in vivo report to show an academic research to evaluate the protective effect of this plant so far . since pfe has some antioxidant property and may alleviate hepatotoxicity , this study evaluated the possible preventive hepatoprotective effect of pfe on liver injury induced by apap in rat . thirty - six male rats of wister strain , weighing 220 30 g were randomly assigned to six groups of six . all animals were housed individually with a 12 h alternating light - dark cycle and had free access to food and water . ambient temperature in the animal facility was kept at 22c 2c with humidity between 30% and 50% . pf beans were collected from outskirts of qom in iran and were coded with the university of birjand , iran herbarium ( herbarium code 1952 ) . the fruit of pf was crushed to moderately coarse powder and for the extraction , 50 g of powder dissolved in 1000 ml ethanol 80% and placed on a shaker for 24 h. after 24 h , the solution is passed through a filter paper . to remove the solvent , the solution was filtered and placed in the oven for 1 - 2 days at 40c . after evaporation of the solvent , the samples maintained at 20c . a stock solution of 1 g / ml in 0.9% ( w / v ) normal saline after 2 weeks of adaptation , animals were orally administered pfe ( 50 or 75 mg / kg ) or saline once daily for 7 consecutive days . to induce hepatotoxicity , 1-h after the last pretreatment by pfe 5 - 6 ml of blood was obtained by cardiac puncture 24 h after induction of hepatotoxicity and plasma samples were separated by the heparinized syringe and analyzed for various biochemical parameters . total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , total protein , albumin , alanine aminotransferase ( alt ) , and aspartate aminotransferase ( ast ) concentrations were measured using standard techniques and commercial kits ( pars azmoon co. , iran ) with an auto analyzer ( gcsan chem 2000 , spain ) . very low density lipoprotein ( vldl ) was calculated by deduction of the sum of the cholesterol fractions from the tc plasma concentration as described by salau et al . statistical significance between two groups of parametric data was analyzed using one - way analysis of variance followed by tukey 's multiple comparisons test . the experiment was approved by the animal welfare committee of the agriculture faculty of birjand university ( research project number : 2131336 ) . tables 1 and 2 reveal that plasma ast and alt levels were increased in rats administered with apap when compared with controls indicating reliable induction of hepatotoxicity by apap ( p < 0.01 ) . while the groups received pfe at each dose alone showed no significant changes in any of the biochemical parameters . treatment of rats with pfe before apap administration , dose - dependently prevented the increase in ast and alt . furthermore , administration of apap significantly decreased plasma total protein and albumin levels ( p < 0.05 ) as well . as shown , pretreatments with different doses of the extract attenuated the reduction in plasma levels of total protein and albumin to some extent . moreover , plasma cholesterol , tg , ldl , and vldl levels markedly increased in rats administered with apap when compared with controls ( p < 0.05 ) . pretreatment with pfe , especially at 75 mg / kg , provided marked protective effect and comparable improvement on these biochemical indices . effect of prosopis farcta on liver enzymes in rats effect of prosopis farcta on liver enzymes and total protein , albumin and lipid profile in rats the present study for the first time brings about the potential hepatoprotective activity of pfe against apap - induced hepatotoxcity . the results of the study indicated that the pfe significantly reversed the enhancement of ast and alt levels . necrosis or membrane damage releases the intracellular enzymes into circulation and hence , it can be measured in the plasma . elevated levels of plasma enzymes are indicative of cellular leakage and loss of functional integrity of the cell membrane in liver prior to other indicators of liver function tests . the most important enzymes are alt and ast that are present in the liver parenchyma . alt predominantly found in liver unlike ast which is also abundantly present in other organs namely , cardiac muscle and kidneys . for this reason , alt is more specific indicator of liver inflammation than ast , though the parallel increases in alt and ast often observe . in the present study , this enhancement may be due to disruption of hepatic cell as a result of necrosis or a consequence of altered membrane permeability . pretreatment with oral doses of p. farca extract attenuated the enzyme activities which are indications of the protective activities of the pfe against apap hepatotoxicities . the most critical assessment if an intervention is used as a pretreatment is the evaluation of metabolic activation ( gsh depletion and protein adduct formation ) which has not been assessed in this study . metabolism of paracetamol produces a highly toxic metabolite , napqi via the cytochrome p450 pathway which induces the process of lipid peroxidation . although , accumulation of napqi occurs when the rate of formation exceeds the rate of detoxification by gsh or the depletion of gsh stores . in addition , covalent bindings of napqi to hepatocyte macromolecules cause necrosis of liver cell and consequent release of the cytosolic liver enzymes particularly the aminotransferases that are regarded as signs of hepatocellular injury . given the large number of compounds present in the extract , it is highly likely that several p450 inhibitors are present . quercetin , a flavonoid antioxidant , is one of the compounds found in prosopis with an excellent radical scavenger activity which attenuated oxidative stress induces by apap , as well as other chemical . it is possible that presence of polyphenols and flavonoids in pf might be responsible for its protective effect on apap - induced liver damage in rats . concerning plasma lipid profile , oral administration of apap increased plasma cholesterol , tg and ldl with no significant alteration in plasma hdl level . lipid deposition in the liver may be as a result of excessive supply of lipids to the liver or interference with lipid deposition . formation of phenoxyl radicals in the presence of peroxidases act as an ldl prooxidant is responsible of ldl elevation in apap animals . it is documented that lipid - lowering drugs have antioxidant properties which prevent ldl peroxidation . pf with antioxidant property is capable of preventing ldl peroxidation and inhibiting elevation of ldl by apap . three molecules of acetyl coa combined and produce 3-hydroxy-3-methyl - glutaryl - coa , which it affected by different enzymes converted to malonic acid , and during different reactions , convoluted and converted to cholesterol . hdl - c is responsible for reverse transport of cholesterol from peripheral cells to the liver cells . cholesterol is transformed to bile acids , which are excreted into the intestine via the biliary tract . the findings of omidi et al . , while preliminary , suggests that the efficacy of pf beans powder as a beneficial agent to decrease ldl - c and increase hdl - c concentration . it produces proteins for its own cellular needs , as well as secretory proteins that are released into the circulation . one of the most important of these secretory proteins is albumin . in this study , the decreased total protein and albumin levels by apap were significantly improved in pfe - treated groups indicating of its hepatoprotective effect . based on the results of the present study , it can be concluded that the aqueous extracts of pf beans exhibit hepatoprotective activity against apap . however , the mechanism by which pfe exhibited protection is not clear by the present study . further investigations on cellular and molecular mechanism of the plant may throw more light on the use of pf for hepatoprotective activity .

we describe two patients with fungal keratitis refractory to standard antifungal therapy whose conditions were managed with voriconazole.the first case is a patient with endophthalmitis and corneal ulcer due to candida parapsilosis after receiving a corneal transplant . the patient was treated with amphotericin but showed no signs of improvement . topical voriconazole , oral voriconazole , and intravitreal voriconazole yielded signs of improvement . the second case is a 63-year - old male who underwent a month of empiric treatment with 0.2% topical amphotericin for fungal keratitis but showed no signs of improvement . treatment was then provided with 1% voriconazole . both cases showed effective treatment with voriconazole.voriconazole may be considered as a new method to treat fungal keratitis refractory to standard antifungal therapy . background : hepatotoxicity by acetaminophen is the most frequent cause of acute liver failure in many countries . prosopis farcta beans extract ( pfe ) has some antioxidant property and may alleviate hepatotoxicity . therefore , the aim of this study was to evaluate effects of pfe against acetaminophen - induced hepatotoxicity.methods:thirty-six male wistar albino rats weighing 220 30 g were distributed into six groups . two groups were pretreated with pfe ( 50 and 75 mg / kg ) for 7 days before administration of acetaminophen ( 600 mg / kg ) . two were given acetaminophen or pfe ( 50 and 75 mg / kg ) alone , and the control received normal saline . one day after acetaminophen , administration blood samples were collected by cardiac puncture to determine liver function enzymes markers ; aspartate aminotransferase and alanine aminotransferase ( ast and alt ) , cholesterol , triglyceride ( tg ) , high , low , and very low density lipoproteins ( ldl and vldl).results : in acetaminophen - treated rat plasma ast ( 314 18.54 vs. 126.37 4.13 ) , alt ( 304 49.24 vs. 187.33 3.71 ) , cholesterol , tg , ldl , and vldl were increased by 149 , 160 , 37 , 92 , 60 , and 94% , respectively . pfe at both doses significantly ( p < 0.05 ) attenuated the above biochemical indices to near normal.conclusions:prosopis farcta beans extract ( 50 and 75 mg / kg ) exhibited hepatoprotective activity against apap .

melamine ( 2,4,6-triamino-1,3,5-triazine , cas # 108 - 78 - 1 ) was first prepared and described by liebig in 1834 ( 1 ) and has since become an increasingly important chemical commodity . most of the melamine production is used in the fabrication of melamineformaldehyde resins ( 2 ) . the first analytical methods related to food were therefore developed to detect melamine migration from resins used in food contact materials ( 3 ) . recently , melamine has become infamous as being one of the most effective adulterants used to increase the nitrogen content in foods and feeds . melamine contains about 66.6% nitrogen , and the addition of 1% melamine to protein leads to a false increase in the kjeldahl protein content by 4.16% ( 6 ) . the first cases of melamine adulteration were detected in fish meals from italy in the late 1970s ( 7,8 ) , and methods to detect melamine in potato proteins were developed in switzerland in the 1980s ( 6 ) . since then , melamine adulteration cases have not been reported in the literature until 2004 and 2007 , when melamine was found in pet foods , causing renal failure in dogs and cats ( 9,10 ) . nephrotoxicity also appears to be the major toxic effect in humans ( 11,12 ) . with the intentional adulteration of human foods , including baby foods , with melamine , the economically motivated fraud in the food chain has reached a new dimension , for which food control systems were unprepared . the problem first became evident in china as an increase in urinary tract stone formation in infants beginning in the spring of 2008 ( 13 ) . more than 294,000 children have reportedly been affected by adulterated formula , with over 50,000 hospitalized , and at least 6 deaths ( 14 ) . twenty - two dairy companies were implicated in the melamine fraud , with the sanlu company being identified as the one that had most seriously violated the law ( 13 ) . apparently , the adulterations occurred at the raw milk collection stations , for which no systematic state surveillance had been implemented ( 13 ) . the adulterators ostensibly used relatively sophisticated techniques such as special protocols for premixes and were said to have provided training regarding the use of those premixes after diluting the milk with water ( 15 ) . because of globalization and the worldwide food trade , the melamine contaminated foods were detected in a large number of countries , including the united states ( 14 ) and the european union ( 16 ) . some commentators expect similar cases in the future with even improved techniques ( 15 ) . this crisis has highlighted not only the hazards of an increasingly globalized food chain but also the weaknesses in the control systems ( 17 ) . we think that in order to improve consumer protection in the future , completely new strategies for food surveillance must be developed . the current food control system is generally target - oriented , meaning that quantitative analyses of certain health - relevant compounds such as heavy metals , pesticides , or carcinogenic contaminants are conducted ( 18 ) . while the current system has not paid great attention to intentional adulterations ( 17 ) , testing laboratories are also faced with the problem of choosing parameters for spot checks , as systematic full analyses are not possible for economical reasons ( 19 ) . therefore , analysis parameters are usually chosen by purely arbitrary choices ( 19 ) or by a risk - oriented approach ( 20 ) , both of which most certainly would not include novel high - tech adulterants ( 15 ) chosen specifically with the intent of evading the food control system . for all of these reasons , we propose that future food control must implement what we call a nontargeted approach . this is an approach that must be able to detect deviations in food composition even by compounds that are not specifically being searched out or ones that are unknown to the analyst . this would enable food control to work proactively , rather than being one step behind the adulterators , as is currently the case . the most preferred form of nontargeted analysis would be a screening technique that would allow measurement of a large number of samples in a short time frame . nuclear magnetic resonance ( nmr ) spectroscopy has been proposed as probably the best nontargeted technique for use in screening food extracts ( 21 ) . in a comparison of spectroscopic screening techniques , much richer information was provided using nmr in comparison to near - infrared or fourier transform infrared spectroscopy , and selective and sensitive qualitative and quantitative information could be gathered from the spectra ( 22 ) . even though 400 mhz nmr machines are still expensive , compared with the cost of other analytical systems , the cost per sample for nmr can be very low , depending on the turnover to be achieved . the successful application of nontargeted screening approaches for food analysis has been previously demonstrated for a number of food matrices ( 21 ) . however , the focus was always placed on authenticity control and not on the detection of health - relevant compounds . in this study , on the basis of our experience with beer ( 23 ) and fruit juice screening ( 19,24 ) , we demonstrate for the first time that nmr is useful for nontargeted screening of baby food formula for health - relevant compounds such as melamine and also for labeling controls ( e.g. , lactose - free products ) . additionally , we provide information that the 400 mhz nmr spectra of liquid sample extracts can also be used for quantification of melamine . using 700 mhz high - resolution magic angle spinning ( hrmas ) nmr , we demonstrate that melamine can be quantitatively detected in complicated food matrices without any sample preparation . authentic chinese infant formulas ( n = 9 ) , including one contaminated sanlu infant formula , were purchased in beijing ( china ) in september 2008 during the melamine crisis . german infant formulas ( n = 13 ) were sampled in the context of official food control by governmental food inspectors in the german federal state baden - wrttemberg in december , 2008 . melamine - positive chinese candy from the german market was also included in the study . all nmr measurements were performed on a bruker avance 400 spectrometer ( bruker biospin , rheinstetten , germany ) equipped with a 5-mm bbi inverse probe with z - gradient coils , using bruker automatic sample changer ( b - acs 60 ) . all spectra were acquired at 300.0 k. h spectra were acquired using one - dimensional ( 1d ) nuclear overhauser enhancement spectroscopy ( noesy ) pulse sequence implemented with a low - power continuous wave presaturation during the relaxation delay and during the mixing time . the relaxation delay and mixing time the data were acquired automatically under the control of icon - nmr ( bruker biospin , rheinstetten , germany ) , requiring about 12 min per sample . the proton hrmas experiments were carried out using a 700 mhz av iii nmr spectrometer ( bruker biospin , rheinstetten , germany ) with a 4 mm hrmas standard bore probe ( h , c , p , and h - lock ) and gradient . the temperature was controlled at 300.0 k using a bcu05 precooling unit for the n2 bearing gas flow and a temperature control unit ( bvt300 mas ) . the rotor spin rate was 7000 hz ; therefore , the spectral window was free from spinning side bands . a single pulse proton experiment with a 30 flip angle was executed with 4 s acquisition time , a relaxation delay of 4 s , and a sweep width of 20 ppm . the number of scans for the standard experiments was ns = 64 ( experimental time 9:15 min ) . the processing was done with zero - filling to 128k data points and an exponential multiplication with lb = 1 . the chemometrical analyses of the spectra were performed using matlab ( the mathworks , version r2008a ) routines developed in - house . for the quantitation of the melamine concentrations , the signal of the nh2-groups at about 5.93 ppm ( figure 1 ) was investigated . its intensity was derived by fitting a theoretical signal on the spectrum , which minimized the sum of squares of the residues and therefore optimally explained the melamine influence on the nmr spectrum . the integral value of this fitted signal was linearly dependent on the melamine concentration and did not require a calibration for each sample when fixed acquisition and preparation parameters were used . the fitting was done in a fully automated mode using a nonlinear bound optimization algorithm , which optimized parameters such as line shape , position , height , and signal - underground ( baseline ) . comparison of melamine - contaminated sanlu sample with the reference distribution of a collection of infant formulas ( measurement in dmso - d6 at 400 mhz ) . the nontargeted analysis of the data was performed using simple robust techniques to describe the univariate distributions of the nmr spectra at each ppm value . figure 1 illustrates this method which uses the quantiles of the distributions of the reference samples as an estimate of the medians and the corresponding variances at each chemical shift . since this procedure yields a robust description , a few atypical samples in the reference group the melamine signal of the sanlu infant formula was easily detected ( manually and in automation ) since its intensity translates into a z - score larger than 56 ( medianreference + z stdreference ; a z - score of about 3 would cover 99% of all samples under normal distribution assumption ) . this approach emphasizes deviating spectral regions and signals , which can then be investigated with sophisticated nmr experiments or other conventional analyses . nondeviating samples will pass this screening , and adulterations up to certain limit can be ruled out . in the first stage , for nontargeted analysis , 20 0.4 mg of infant powder was weighed and dissolved in 2 ml of waterbuffer solvent ( 90% distilled water and 10% buffer ; 1.5 m kh2po4/d2o and 0.1% tsp at ph 7.0 ) to obtain a final concentration of 10 0.2 this solution was shaken for 1 min on a vortex mixer and 3 min in an ultrasonic homogenizer . then 600 l of homogenized solution was transferred to a standard 5 mm nmr tube for analysis . when it became evident that the melamine peak itself could not be measured in water , the sample preparation was repeated in 99.8% dmso - d6 ( deutero gmbh ) , while 20 0.4 mg of infant powder was dissolved in 2 ml of dmso - d6 . for calibration , melamine standards in dmso - d6 at a concentration range between 2 and 200 mg / l were measured . to determine the limit of detection , a separate calibration curve in the range between 0.2 and 2 was filled into the 50 l hr - mas rotor , and 45 l of dmso - d6 was added and stirred . the rotor was prepared with a 50 l upper spacer placed at a 3.0 mm depth and sealed with a teflon screw . the material from the white rabbit candy was scraped off and powdered in a mortar before analysis . for hrmas calibration , a blank matrix ( nestl student sweetened milk powder , nestl china , absence of melamine was confirmed by reference analysis ) was filled in the rotor , and 45 l of melamine standard solution in dmso - d6 was added . the calibration range was 9 to 900 mg / kg , and the second curve for the determination of limit of detection was in the range between 0.9 and 9 mg / kg . for infant formula , 1 g of sample was added to a centrifuge tube , and 10 ml of acetonitrile was added . ten milliliters of deionized water , a small amount ( covering the tip of a spatula ) of ammonium acetate , and 0.2 ml of glacial acetic acid were then added to the tube . the solution was again vortexed for 1 min and centrifuged for 10 min at 4000 g . a 2.5 ml aliquot of the supernatant was placed into a conditioned solid - phase extraction ( spe ) tube ( strata - x - c 200 mg/6 ml tubes , phenomenex , aschaffenburg , germany ) . for candy , a different sample preparation had to be conducted because they were not soluble in cold solvents . therefore , 1 g of candy sample was dissolved in 50 ml of hot water after the addition of 1 ml of acetic acid . after cooling , 30 ml of acetonitrile was added , and the solution was ultrasonicated for 15 min . after adjustment with acetonitrile to 100 ml , an aliquot of the extract was centrifuged and transferred to an spe tube as described above . the spe tubes were conditioned using 15 ml of methanol followed by 15 ml of water . cleanup was conducted using 6 ml of hcl ( 0.1 m ) and 3 ml of methanol . after each elution step , the extract was dried at 5060 c using a nitrogen stream . then , 500 l of acetonitrile / water ( 1:1 ) was added , and the solution was membrane filtered ( 0.2 m ) and used for chromatography ( injection volume 20 l ) . the lc / ms / ms system consisted of an agilent ( waldbronn , germany ) 1100 hplc system ( binary pump , degasser , and autosampler ) coupled with a thermo fisher scientific ( formerly thermo finnigan ) ( dreieich , germany ) tsq 7000 mass spectrometer . , 3 m , 125 , c18 column ( phenomenex , aschaffenburg , germany ) at 30 c , using mobile phase a ( acetic acid , 0.1% ) and mobile phase b ( acetic acid in methanol , 0.1% ) in the following gradient program . flow rate : 0.2 ml / min , 05 min , 90% a ; 55.5 min , 90% a to 20% a ; 5.57.5 min , 20% a ; 7.58 min , 20% a to 90% ; 813 min , 90% a. atmospheric pressure chemical ionization ( apci ) used a capillary temperature of 270 c , a vaporizer temperature of 400 c , and a corona discharge current of 2.0 a . the sheath gas was nitrogen at 50 psi , and argon was used as the collision gas . the collision cell of the triple quadrupole was operated with a collision energy of 35 ev for all transitions . for quantitative analysis , the following fragmentations were monitored in the selected reaction monitoring ( srm ) mode : m / z 127 85 , m / z 127 68 , and m / z 127 43 for melamine . in the calibration range between 5 to 1000 ng / ml , melamine exhibited good linearity , with regression coefficients greater than 0.99 . the limit of detection was 10 ng / ml and the limit of quantitation was 30 ng / ml . the recovery was 105 5% in spiked baby formula ( n = 8) , 81 13% in spiked ready - to - eat baby purees ( n = 6 ) , 98 3% in different spiked egg powders ( n = 4 ) , or 92 6% in candy ( n = 11 ) . the precision ( expressed as coefficient of variation ) was 7.5% ( n = 6 , bakery products ) , 5.1% ( n = 8 , baby formula ) , or 6.4% ( n = 11 , candy ) . at this point in time , only one study in the literature has presented h nmr data on melamine in the context of a kinetic study ( 25 ) . the signal assignment ( dmso - d6 , 6.02 , s , nh2 ) reported previously corresponds excellently with the findings in our study . the identification of the singlet peak of nh2 at 5.93 ppm in dmso - d6 for melamine was confirmed using measurements of pure standards as well as standard addition to positive samples . other studies have used hrmas nmr to study the structure of a cyanuric acidmelamine system ( 26 ) or the formation of melamine condensation products ( 27 ) ; however , no h data were presented in either study that could be used as a comparison with our results . table 1 shows a comparison between the sensitivities of the different methodologies under investigation . from the methods we evaluated , the reference lc / ms / ms procedure was the most sensitive , followed by 700 mhz hrmas , and then 400 mhz liquid nmr . this was not unexpected , on the basis of the physical characteristics of the methods . the results clearly show that nmr can be used for screening purposes at levels down to the lower mg / kg range . for example , the who melamine recommendations of 1 mg / kg in powdered infant formula and 2.5 mg / kg in other foods ( 28 ) can be reached by hrmas . the sensitivity of the 400 mhz procedure could be increased if an optimized sample extraction or sample preconcentration step was included ( e.g. , a spe procedure similar to that conducted prior to lc / ms / ms ) ; however , it was our intention to leave the sample preparation as simple as possible . in contrast to the previous belief that nmr is only suitable for structure verification , elucidation , and purity analysis ( 24 ) , our results clearly demonstrate that nmr can be used for quantitative analysis of foods for health - relevant compounds . of course , the power of nmr resides in the nontargeted approach ( see below ) , but as we also had previously demonstrated in beer and fruit juice analysis ( 19,23,24 ) , the same spectra can be used to acquire quantitative data along with the results from multivariate analysis . determined according to din 32645 using a separate calibration curve in the range of lod . measuring solution is the final sample extract filled into the nmr tubes or injected into the lc / ms / ms system . calculated for a sample weight of 10 mg in 1 ml of dmso - d6 . determined using a hrmas rotor filled with 10 mg of authentic matrix . calculated for a sample weight of 1 g reconstituted in 500 l after spe cleanup . regarding the precision of the methods , the 400 mhz method showed a coefficient of variation of 3.2% ( n = 9 ; replicated measurement of authentic contaminated sanlu sample ) , while the coefficient of variation of the hrmas 700 mhz method was 2.6% ( measurement of spiked infant formula , n = 11 ) . the precision of nmr was judged to be sufficient for the purposes of food analysis . the lc / ms / ms procedure , in comparison , also had precisions of this order of magnitude or above ( due to the complicated sample preparation procedure ) . regarding accuracy , the nmr results were compared to the lc / ms / ms reference procedure . for the contaminated sanlu formula , the 400 mhz nmr , the 700 mhz nmr , and the lc / ms / ms melamine results were 412 mg / kg , 460 mg / kg , and 470 mg / kg , respectively , and for the contaminated candy , the melamine results were 47 mg / kg , 74 mg / kg , and 68 mg / kg , respectively . the 400 mhz procedure showed lower results than either of the other procedures , which was judged to be due to an incomplete extraction during sample preparation . this exemplifies the advantages of the hrmas procedure , which gave results very similar to those of the reference procedure . nevertheless , we also judge the 400 mhz procedure in the current form as suitable for food screening because any amount of melamine is of course not admissible so that even a qualitative result would be sufficient ( e.g. , in the context of raw product screening in industry ) . our study is the first to apply hrmas nmr to quantitatively determine melamine in authentic food matrices . hrmas appears to be eminently suitable for analyzing complicated matrices , such as solid or semisolid foods , that are difficult to extract by conventional means . many of these foods form emulsions or are not completely soluble in organic solvents , as in the case of the candy . hrmas is a form of gel - phase nmr , where the sample is mixed with a solvent that swells the matrix . using rotation of the sample , we can obtain high resolution h spectra in a routine manner ( 29 ) . a major application of hrmas appears to be the detection of tumor tissues ( 3033 ) . so far , relatively few applications in the field of food control have been presented , and these have involved the authentication of cereal foods ( 34,35 ) , cheese ( 3638 ) , or beef ( 39 ) . lc / ms / ms with spe extraction is clearly the preferred method for melamine quantification in all kinds of foods ( 4043 ) . according to our own experience the advantage of nmr and especially hrmas is the minimal time for sample preparation compared to that in the tedious lc / ms / ms procedure . unfortunately , melamine contamination is not the first instance of unsafe milk formula in china . in 2003 , 12 children died of malnutrition in anhui province as a result of being fed infant formula of poor quality . in this incident , 55 different infant formulas , 40 corporations , and 10 provinces were involved , which exposed key public health gaps in food safety and public protection ( 44 ) . this incident might even have indirectly led to the melamine crisis because government directives against diluted preparations were implemented . the fact that melamine could increase the apparent protein content and , furthermore , could make the product look milky may have been irresistible to those who would adulterate milk formulas ( 14 ) . as we have detailed in , consumer safety can not be ensured only by targeted analysis of compounds such as melamine because the adulterators are always one step ahead . in our research to establish a quantitative method for nmr analysis of melamine , it became quickly evident that even by a simple visual inspection of the nmr spectra , the sanlu sample showed differences . these were observed in several spectral ranges , even in the analysis of the aqueous solutions , in which the melamine peak at 5.93 ppm is not visible because of rapid proton exchange on the nh2 groups with the solvent protons . for example , the sanlu sample showed deviations in the range between 3.6 and 4.2 ppm . a search in our library of nmr spectra of pure compounds showed that this spectral pattern can be assigned to sucrose ( figure 2 ) . upper panel : comparison of melamine - contaminated sanlu sample with the reference distribution of a collection of infant formulas ( measurement in water at 400 mhz ) . lower panel : reference measurement of sucrose from the spectral library . in the same fashion , we found that one of the german infant formula samples diverged from all other samples . in this case this finding shows that the nmr method can also allow one to check recipes and labeling claims ( such as lactose - free , hypoallergenic , etc . ) . confirming these factors is comparably important to ensure the health of those subgroups of consumers with special nutritional needs . we therefore conclude that the routine application of nmr in the screening of baby food products would provide a considerable improvement in consumer protection . similar to what we have shown in the screening of fruit juices or beer , our chemometric approach allows the establishment of a standard model of a typical infant formula . this model can then be used without manual intervention to provide a judgment about a sample , regarding whether it does or does not correspond to the typical composition . in the case of noncompliance , the large depth of spectral information of nmr then allows one to make the assignment of compounds using databases ( as in the case of lactose or sucrose ) . in the case of compounds not in databases ( e.g. , such as melamine when we started our study ) , the option for nontargeted screening , along with the structural information inside the spectra , is certainly the major advantage of nmr over other techniques that have been recently proposed . for example , surface desorption mass spectrometry ( 4547 ) or enzyme - linked immunosorbent assays ( 48 ) again have the disadvantage that they are focused on sensitive target analysis . these will certainly detect melamine but will miss novel adulterants that might be used in its place in the future . the only other proposals in the literature that might be usable for a nontargeted approach are several infrared spectroscopy methods ( 4951 ) . multivariate regression analysis was able to derive models to quantify melamine from raman , mid , or near - infrared spectra . so far , only nmr can be seen as a holistic approach for detecting adulteration in infant formula . fatigue is a transitional state between awake and sleep which manifests itself as lack of alertness and deteriorated mental or physical performance and often associated with drowsiness . road accidents due to fatigue are often much more severe than other crashes , since the driver reaction time increases ( 1 ) . driver fatigue is deemed to account for up to 40% of road accidents ( 2 ) . it is conjectured that 1030% of road deaths are related to driver fatigue ( 3 ) . mental fatigue causes reactions become prolonged , more fluctuable , and more error tending ( 4 , 5 ) . grandjean defined fatigue as a state with decreased efficiency and lack of general willingness to work ( 6 , 7 ) . these can be classified as subjective , psychological , performance and physiological methods . in subjective methods , standard questionnaires such as f - vas and moreover , the use of behavioral and psychological techniques in mental fatigue investigation has been adopted in several preceding studies ( 1417 ) . among these studies , a set of video recordings of facial expressions , mannerisms and personality traits questionnaires were common methodologies with high reliability and validity ( 17 ) . in addition , some fatigue studies on driving simulator exploited performance features such as steering wheel angle and lane departure ( 1820 ) . some other researchers have focused on driver s physiological changes , such as the measurement of eye activity , heart rate , skin electrical potential and specially eeg activity as a means to detect cognitive states ( 21 , 22 ) . although several physiological indices available for assessing the alertness level , eeg signal may be one of the safest and most predictive , ( 2325 ) , since it immediately reflects brain activity . driving involves several tasks such as motion , reasoning , visual and auditory processing , decision - making , perception and cognition . driving is also under the influence of emotion , anxiety and many other psychological factors ( 24 ) . the brain electrical activity rhythms are classified according to frequency bands including delta , theta , alpha and beta waves ( 26 ) . alpha rhythm has the frequency range from 813 hz , which occurs during wakefulness , especially in the occipital cortex area of the brain . it typically appears during eye closure and reduced when eyes open and attenuates severely during attention tasks ( 27 ) . in previous researches , it is known that increase in delta power during internal processing is associated with mental task performance ( 28 ) . other researchers have reported that an increase in eeg theta power depends on decreased performance in monotonous tasks ( 29 ) . although some definite trends were observed in the delta , theta and alpha power frequency bands during fatigue , the results of different studies may be influenced by inter - individual and intra - individual variations in eeg data ( 30 ) . changes in eeg power spectra are associated with fluctuations in the alertness state ( 31 ) . monitoring physiological signals while driving can provide the possibility to detect and warn fatigue ( 32 ) . most investigations revealed that changes in delta and theta activity are related to the transition to fatigue . therefore , eeg monitoring during driving may be a promising variable for using in fatigue countermeasure devices ( 24 ) . it has been suggested that during driving at night , delta band varies significantly with the degree of fatigue ( 33 ) . some researchers have presented an eeg - based cerebral workload index based on the increase of eeg power spectra in the theta band over prefrontal areas and the immediate decrease of eeg power spectra over parietal areas in alpha band during driving ( 34 ) . while physiological mental fatigue level increases , the relative power of theta , alpha and beta rhythms decrease , but the relative power in delta rhythm increases ( 35 ) . the relative power of alpha increased while the attention level of the driver decreased ( 36 ) . however , research on the physiological links specially eeg frequency bands to driver fatigue is still exploratory and is an important area that needs further investigation . therefore , this study attempted to detect the driver mental fatigue through changes in eeg alpha power activity . twelve healthy male volunteer car drivers ( ranging 20 to 30 years old ) participated in an overnight study from 2 to 6 a.m. in virtual reality laboratory of khaje nasir toosi university of technology in 2013 . all the subjects held valid driving license with at least 2 years driving experience and had no history of prior brain injuries . the epworth sleepiness scale was used to measure the participants for any trait sleepiness . after taking informed written consent , the subjects were requested to stay awake 18 h before the experiments and refrain from caffeinated drinks or any other stimulant as well as cigarette smoking for 12 h prior to the experiments . the drivers should have regular sleep pattern ( i.e. sleeping not later than 1 a.m. and awaking not later than 9 a.m. ) and not get used to daily napping which was studied through sleep diary for one week before the experiment . this research employed a fixed - based car driving virtual reality simulator ( ci004 semi ) in a calm controlled room with fixed temperature and illumination conditions . 1 shows a snapshot of the car - driving simulator developed in mechatronics department of k.n . snapshot of the car - driving simulator used for the implementation of the proposed protocol a set of 110 km road sceneries had been designed and simulated . the first 90 km of the road was monotonous and straight with minimal side components to induce more fatigue and the last 20 km was winding mountainous road ( see fig . 2 ) . a scenery of the road captured from the car simulator lcd the designed road was from the most dangerous parts of real road pattern of iran , so called haram to haram road , photographed through google maps using autodesk autocad civil 3d ( version 2013 ) and autodesk 3ds max ( version 2012 ) softwares . a portable g.usb amp bio - signal amplifier with 16 channels was used with 256 hz sampling rate and 24 bit quantification with active electrodes for signal acquisition . during driving on the simulator , eeg and the electrode positions on different areas of the scalp were based on the 1020 international electrodes placement guideline . the main eeg signal channels were o1 , o2 , p3 , p4 , p7 , p8 , oz , fp1 , fp2 , cz , fz , t7 and t8 . the eeg data analysis involved pre - processing , artifact removal , and features extraction . the eeg signal was first processed executing a band pass filter between 0.5 and 60 hz . eeglab ( version 10.2.5.6a ) was utilized to remove muscular and ocular artifacts by visual inspection . all the signals were sequenced to epochs each lasting two seconds ( 1 epoch consisting of 512 samples = 2 seconds ) . this research exploits power spectrum density and fast fourier transform ( fft ) analyzing technique to determine the absolute and relative powers of alpha frequency band . relative alpha power was computed as the ratio between absolute alpha power and the total spectral power of the signal . we compared the absolute and relative alpha power variations in the first and last 10 minutes of driving with the f - vas scores . for further certainty , this study employed the double check validity method by comparing the extracted features from eeg signals with the video rating scores . before resuming the experiments , then , eeg and eog signals were recorded from the subjects in relaxed sitting posture for 3 minutes with closed eyes ( imagining driving on a highway ) and 3 minutes with open eyes ( looking at a road picture on the screen ) as a baseline for alertness . the subjects were asked to drive the monotonous road at a constant speed of 90 km / h . at the same time , a continuous eeg and eog records were taken during the driving on the car simulator . meanwhile , the drivers faces and behaviors were monitored via video recordings in lateral and front views . these videos were used to rate the fatigue level of drivers on a four - point scale from 1(alert ) to 4 ( very tired ) by two trained observers . moreover , the self - rating of fatigue was performed during the driving using fvas scored from 0 ( no fatigue or energetic ) to 10 ( worst possible fatigue ) in every 10 minute interval . the descriptive statistics such as central and scattered indices were computed in order to describe the variables . pearson and spearman correlation coefficients were employed to explore the associations between f - vas scores and video ratings , fvas and absolute and relative alpha powers . paired - sampled t - test was utilized to compare the means of f - vas scores , video ratings , absolute and relative alpha powers in the initial and final 10 minutes of driving . twelve healthy male volunteer car drivers ( ranging 20 to 30 years old ) participated in an overnight study from 2 to 6 a.m. in virtual reality laboratory of khaje nasir toosi university of technology in 2013 . all the subjects held valid driving license with at least 2 years driving experience and had no history of prior brain injuries . the epworth sleepiness scale was used to measure the participants for any trait sleepiness . after taking informed written consent , the subjects were requested to stay awake 18 h before the experiments and refrain from caffeinated drinks or any other stimulant as well as cigarette smoking for 12 h prior to the experiments . the drivers should have regular sleep pattern ( i.e. sleeping not later than 1 a.m. and awaking not later than 9 a.m. ) and not get used to daily napping which was studied through sleep diary for one week before the experiment . this research employed a fixed - based car driving virtual reality simulator ( ci004 semi ) in a calm controlled room with fixed temperature and illumination conditions . 1 shows a snapshot of the car - driving simulator developed in mechatronics department of k.n . snapshot of the car - driving simulator used for the implementation of the proposed protocol a set of 110 km road sceneries had been designed and simulated . the first 90 km of the road was monotonous and straight with minimal side components to induce more fatigue and the last 20 km was winding mountainous road ( see fig . 2 ) . a scenery of the road captured from the car simulator lcd the designed road was from the most dangerous parts of real road pattern of iran , so called haram to haram road , photographed through google maps using autodesk autocad civil 3d ( version 2013 ) and autodesk 3ds max ( version 2012 ) softwares . a portable g.usb amp bio - signal amplifier with 16 channels was used with 256 hz sampling rate and 24 bit quantification with active electrodes for signal acquisition . during driving on the simulator , eeg and the electrode positions on different areas of the scalp were based on the 1020 international electrodes placement guideline . the main eeg signal channels were o1 , o2 , p3 , p4 , p7 , p8 , oz , fp1 , fp2 , cz , fz , t7 and t8 . the eeg data analysis involved pre - processing , artifact removal , and features extraction . the eeg signal was first processed executing a band pass filter between 0.5 and 60 hz . eeglab ( version 10.2.5.6a ) was utilized to remove muscular and ocular artifacts by visual inspection . all the signals were sequenced to epochs each lasting two seconds ( 1 epoch consisting of 512 samples = 2 seconds ) . this research exploits power spectrum density and fast fourier transform ( fft ) analyzing technique to determine the absolute and relative powers of alpha frequency band . relative alpha power was computed as the ratio between absolute alpha power and the total spectral power of the signal . we compared the absolute and relative alpha power variations in the first and last 10 minutes of driving with the f - vas scores . for further certainty , this study employed the double check validity method by comparing the extracted features from eeg signals with the video rating scores . before resuming the experiments , the car driver s sleepiness propensity was measured by the epworth sleepiness scale . then , eeg and eog signals were recorded from the subjects in relaxed sitting posture for 3 minutes with closed eyes ( imagining driving on a highway ) and 3 minutes with open eyes ( looking at a road picture on the screen ) as a baseline for alertness . the subjects were asked to drive the monotonous road at a constant speed of 90 km / h . at the same time , a continuous eeg and eog records were taken during the driving on the car simulator . meanwhile , the drivers faces and behaviors were monitored via video recordings in lateral and front views . these videos were used to rate the fatigue level of drivers on a four - point scale from 1(alert ) to 4 ( very tired ) by two trained observers . moreover , the self - rating of fatigue was performed during the driving using fvas scored from 0 ( no fatigue or energetic ) to 10 ( worst possible fatigue ) in every 10 minute interval . the descriptive statistics such as central and scattered indices were computed in order to describe the variables . pearson and spearman correlation coefficients were employed to explore the associations between f - vas scores and video ratings , fvas and absolute and relative alpha powers . paired - sampled t - test was utilized to compare the means of f - vas scores , video ratings , absolute and relative alpha powers in the initial and final 10 minutes of driving . the drivers mean age was 23.8 years ( sd = 1.44 years ) with mean body mass index ( bmi ) of 21.57 ( sd = 2.01 ) . other demographic characteristics and work - related information of the drivers drivers demographic and background characteristics data as shown in table 2 the self - report scale measured by means of f - vas suggested that the participants had not been fatigued during the first 10 minutes of driving and moderately to extremely fatigue during the final section of driving . compared with the initial section , the f - vas scores increase significantly ( p = 0.001 ) during the final section of driving task , which demonstrates that continuous monotonous driving may predispose the subjects to cognitive fatigue . different fatigue measures in i10d and f10d i10d : the initial 10 min of driving ; f10d : the final 10 min of driving furthermore , table 2 presents the video scores rated by trained observers in the initial and final 10 minutes of driving . paired - sampled t test suggests significant increase during the final 10 minutes of driving ( p = 0.001 ) . the results showed a mild , but significant correlation between f - vas and video rating scores in the initial 10 minutes of driving ( r = 0.404 , p = 0.001 ) . meanwhile , spearman s correlation test showed a relatively strong and significant association between f - vas and video rating scores in the final 10 minutes of driving ( r = 0.62 , p = 0.001 ) . table 2 also indicates the absolute and relative alpha powers in the initial and final 10 minutes of driving . there was a significant increase in the absolute alpha power during the final 10 minutes of driving ( p=0.006 ) . moreover , our findings showed no differences in absolute alpha power in different areas of the scalp in the initial and final 10 minutes of driving , except in the parietal area of the brain ( p=0.005 ) . paired - sampled t test suggests statistically significant difference in p4 site between absolute alpha powers in the initial and final 10 minutes of driving ( p = 0.026 ) . this study attempted to present a simple and reliable method for early detection of driver mental fatigue based on eeg alpha power in healthy sleep deprived drivers . in our experiment , different fatigue outcome measures including subjective self - report of fatigue ( f - vas ) , video ratings as well as eeg alpha powers were employed to evaluate the change in fatigue for drivers during the initial and final 10 minutes of driving . the experimental results suggested a significant increase in the subjective self - report of fatigue ( fvas scores ) during the final 10 minutes of driving . the same results were obtained from zhang ( 2008 ) study which found that the level of both subjective sleepiness and fatigue increase significantly from pre - task to post - task ( 37 ) . they studied the impacts of visual display terminal ( vdt ) task on autonomic nervous system and central nervous system through subjective self - report of sleepiness and some physiological measures such as power spectral indices of hrv and wavelet packet parameters of eeg . it should be regarded that our findings from fvas scores showed lowered variability ( all participants reached the state of fatigue and extremely fatigue ) in the final section of driving . this may be because a subject who is fatigue will be less likely to evaluate his / her state sufficiently . in other words , mental fatigue may impact the drivers judgment of their existing state . the findings of this preliminary research indicated a significant increase in absolute alpha power in the final section of driving . this is consistent with the known aspects of eeg alpha rhythm ( 38 , 39 ) . the increase in alpha rhythm in the final 10 minutes of driving depicted the decrease in the level of alertness and attention and the commencement of fatigue and drowsiness . the findings of the present study are well be in line with the findings of researchers in which they found significant difference between the alpha frequency band in the first and fifth section of driving ( 40 ) . in another study , delta and theta activity increased in the final 3 hours of 7 truck drivers during night driving ( 41 ) . nevertheless , our results revealed no significant difference between the relative alpha powers in the initial and final 10 minutes of driving . the reason for this may be due to the increase in delta and theta rhythms , which causes less relative alpha variability in the final section of driving . in our study , most changes occurred in the right parietal region ( p4 ) which was in contrast with the schier ( 2000 ) study that showed the greatest changes in the right frontal region ( f4 ) of the scalp ( 36 ) . however , this is consistent with the reported role of the right hemisphere via blood flow measurement in attention demanding tasks ( 42 ) . since the subjective self - report of fatigue was measured in the initial and final 10 minutes of driving , sudden variations can not be detected using f - vas technique . another limitation to employing this scale is that drivers verbal expression of their level of fatigue may alert them , thereby decreasing their fatigue level . meanwhile , it is almost unfeasible to obtain fatigue feedback from a driver in real driving conditions unless significantly distracting the driver attention from the driving task . our study benefited from using video rating of facial expressions and driver behaviors by trained observers that might not be able to see what goes on behind the facial behavior and expression . placing the electrodes on the scalp to obtain signals is an intrusive technique that should be addressed as another limitation of researches using wired biological signals . therefore , future projects should make the use of less - intrusive systems such as wireless electrodes and benefit the strengths of the various fatigue monitoring methodologies into a hybrid system to develop an efficient fatigue detection device in real driving situations . the present study specifically deals with eeg alpha power in the initial and final section of driving in partially sleep - deprived drivers - a very prominent aspect of driver fatigue analysis . the results of this study may serve as a baseline for the development of anin - vehicle device preventing fatigue related crashes on monotonous roads and greatly improving the transport industry in terms of socio - economic benefits and safety . this study suggests that alpha brain wave rhythm can be a good indicator for early prediction of driver fatigue . meanwhile , image processing of facial expressions may be a complementary method for road accident prevention . these indicators can be incorporated to develop a fatigue countermeasure device to prevent road accidents and reduce fatigue related costs . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

the recent melamine crisis in china has pointed out a serious deficiency in current food control systems , namely , they specifically focus on selected known compounds . this targeted approach allowed the presence of melamine in milk products to be overlooked for a considerable time . to avoid such crises in the future , we propose that nontargeted screening methods need to be developed and applied . to this end , nmr has an extraordinary potential that just started to be recognized and exploited . our research shows that , from the very same set of spectra , 1h nmr at 400 mhz can distinguish between melamine - contaminated and melamine - free infant formulas and can provide quantitative information by integration of individual lines after identification . for contaminated chinese infant formulas or candy , identical results were obtained when comparing nmr with spe - lc / ms / ms . nmr was found to be suitable for routine nontargeted and targeted analyses of foods , and its use will significantly increase food safety . background : driver fatigue is one of the major implications in transportation safety and accounted for up to 40% of road accidents . this study aimed to analyze the eeg alpha power changes in partially sleep - deprived drivers while performing a simulated driving task.methods:twelve healthy male car drivers participated in an overnight study . continuous eeg and eog records were taken during driving on a virtual reality simulator on a monotonous road . simultaneously , video recordings from the driver face and behavior were performed in lateral and front views and rated by two trained observers . moreover , the subjective self - assessment of fatigue was implemented in every 10-min interval during the driving using fatigue visual analog scale ( f - vas ) . power spectrum density and fast fourier transform ( fft ) were used to determine the absolute and relative alpha powers in the initial and final 10 minutes of driving.results:the findings showed a significant increase in the absolute alpha power ( p = 0.006 ) as well as f - vas scores during the final section of driving ( p = 0.001 ) . meanwhile , video ratings were consistent with subjective self - assessment of fatigue.conclusion:the increase in alpha power in the final section of driving indicates the decrease in the level of alertness and attention and the onset of fatigue , which was consistent with f - vas and video ratings . the study suggested that variations in alpha power could be a good indicator for driver mental fatigue , but for using as a countermeasure device needed further investigations .

there are many published reports of malfunction of the inner tube of the bain co - axial circuit , with potentially lethal complications for the patient . the following case report describes a case where profound hypercapnia occurred consequent to avulsion of the inner tube of the co - axial circuit at the machine end of the circuit , which was neither apparent nor visible to the anaesthesiologist . this report therefore emphasizes the need for testing the co - axial circuit for any circuit malfunction before each use . a 30 year - old male presented as an emergency for repair of a crush injury of the left hand . induction was done with thiopentone sodium 5 mg / kg and fentanyl citrate 1.5 mg / kg . after achieving muscle relaxation with succinylcholine 1 mg / kg , anaesthesia was maintained with oxygen , nitrous oxide , isoflurane , and intermittent doses of vecuronium bromide . the vital signs monitored were blood pressure , oxygen saturation , electrocardiogram , temperature , and end - tidal carbon dioxide ( etco2 ) . the gas flows , airway pressure , temperature , chest expansion and bilateral air entry in to lungs were all checked and found to be normal . we changed the d - fend of our side - stream capnograph for a new one but the etco2 continued to rise . the heart rate rose from the baseline of 80 per minute to 110 per minute and blood pressure rose from a baseline of 110/70 mmhg to 140/94 mmhg ; however , the oxygen saturation was 100% . not finding any cause for the rise of etco2 , we decided to change the circuit . following this , the etco2 curve began to fall and reached the normal value of 40 mmhg over the next couple of minutes and the inspiratory carbon dioxide baseline returned to zero . close examination of the original circuit revealed that the inner tubing of the co - axial tube had become disconnected from its seat at the machine end of the circuit [ figure 1 ] . this was a previously unused bain circuit and we had not checked the integrity of the inner tubing before inducing the patient . the inner coloured tube carries the inspiratory gases . if this tube should become disconnected or develop any breach in its integrity there will be a huge increase in dead space , with consequent hypercapnia and its complications . four main causes for development of hypercapnia during anaesthesia have been described : ( 1)reduced alveolar ventilation , with rise in partial pressure of co2(2)inhalation of exhaled co2 ( as has been noted in association with a defective fresh gas flow tube of a bain circuit)(3)inhalation of exogenous co2(4)increased metabolic rate ( as in malignant hyperthermia ) reduced alveolar ventilation , with rise in partial pressure of co2 inhalation of exhaled co2 ( as has been noted in association with a defective fresh gas flow tube of a bain circuit ) inhalation of exogenous co2 increased metabolic rate ( as in malignant hyperthermia ) hypercapnia leads to sympathetic system stimulation , with tachycardia , hypertension , arrhythmias , excessive sweating , and peripheral vasodilatation , which may lead to excessive intraoperative blood loss . our patient had significant increase in heart rate and blood pressure , which was initially erroneously attributed to the light plane of anaesthesia . a number of tests have been described to assess any co - axial circuit malfunction ; these include : ( 1)visual inspection of tubing for any obvious disruption or obstruction.(2)pethick test : this tests the low - pressure system and the integrity of the inner tube . collapse of the reservoir bag due to the creation of venturi effect in the outer tube is an indication that the inner tube is intact . this test will not detect a system in which inner tube is omitted or does not extend to the patient port or one that has holes at the patient end of the inner tube.(3)foex - crampton - smith manoeuvre : this manoeuvre assesses the gas flow line from the flowmeters of the machine to the patient end of the circuit . with an oxygen flow at 2 litres / minute , the patient end of the inner tube is occluded briefly for 23 seconds using the forefinger . a positive test is indicated by descent of the rotameter bobbin due to back pressure ; with removal of finger , the bobbin ascends to its original position . ghani suggested the use of the plunger of a 3-ml syringe to occlude the inner tube more precisely . pethick test : this tests the low - pressure system and the integrity of the inner tube . collapse of the reservoir bag due to the creation of venturi effect in the outer tube is an indication that the inner tube is intact . this test will not detect a system in which inner tube is omitted or does not extend to the patient port or one that has holes at the patient end of the inner tube . foex - crampton - smith manoeuvre : this manoeuvre assesses the gas flow line from the flowmeters of the machine to the patient end of the circuit . with an oxygen flow at 2 litres / minute , the patient end of the inner tube is occluded briefly for 23 seconds using the forefinger . a positive test is indicated by descent of the rotameter bobbin due to back pressure ; with removal of finger , the bobbin ascends to its original position . ghani suggested the use of the plunger of a 3-ml syringe to occlude the inner tube more precisely . this case report highlights the possibility of severe hypercapnia due to dead - space rebreathing as a result of disconnection of the inner tube of the co - axial circuit . we present this as a warning and reminder that the integrity of the co - axial circuit must be always checked visually as well as mechanically . in this regard , the foex - crampton - smith manoeuvre with the ghani modification appears to be most satisfactory method of assessing the integrity of the gas line from the flowmeter up to the patient end of the bain circuit . we attempted to estimate the perceived degree of urgency of the visit and to identify reasons for seeking non - urgent care in the ped by patients and parents . a prospective survey was completed by parents ( for children 17 and younger ) and patients ( 18 - 21 ) presenting to a suburban academic ped that sees approximately 15,000 patients per year . three hundred and five of 334 surveys were completed ( 91% response rate ) over a 3-month period . twenty - four percent of the chief complaints were perceived by those surveyed as emergent or possibly life - threatening , 23% were felt to be very urgent , and 52% were deemed somewhat urgent or minor . twenty - five percent of those with minor or somewhat urgent complaints arrived by ambulance . overall , 79% of those surveyed identified a primary care provider ( pcp ) for themselves or their child . of those , 54% had attempted to contact the pcp prior to coming to the ped . six percent of those who attempted to reach their primary care providers were able to contact them and 52% were told to come to the ped . more than half of patients and parents presenting to the ped believed they had minor or somewhat urgent complaints . while the majority of patients have a regular provider , limited access to timely primary care and convenience may make the ped a more attractive care option than primary care for many parents and patients .

the bain co - axial circuit is fully established in general anaesthesia practice . a major concern is the potential malfunctioning of the circuit due to avulsion of the inner fresh gas delivery tube at the machine end of the circuit . the following case report presents a case in which a patient connected to the bain circuit developed severe hypercapnia in the early intraoperative period due to the above mentioned defect . objectives : pediatric emergency department ( ped ) patients often present with non - urgent complaints . we attempted to estimate the perceived degree of urgency of the visit and to identify reasons for seeking non - urgent care in the ped by patients and parents.methods:a prospective survey was completed by parents ( for children 17 and younger ) and patients ( 18 - 21 ) presenting to a suburban academic ped that sees approximately 15,000 patients per year . a convenience sample of participants was enrolled.results:three hundred and five of 334 surveys were completed ( 91% response rate ) over a 3-month period . twenty - four percent of the chief complaints were perceived by those surveyed as emergent or possibly life - threatening , 23% were felt to be very urgent , and 52% were deemed somewhat urgent or minor . twenty - five percent of those with minor or somewhat urgent complaints arrived by ambulance . weekend visits and minority race correlated with a lower degree of perceived urgency . overall , 79% of those surveyed identified a primary care provider ( pcp ) for themselves or their child . of those , 54% had attempted to contact the pcp prior to coming to the ped . six percent of those who attempted to reach their primary care providers were able to contact them and 52% were told to come to the ped.conclusions:more than half of patients and parents presenting to the ped believed they had minor or somewhat urgent complaints . while the majority of patients have a regular provider , limited access to timely primary care and convenience may make the ped a more attractive care option than primary care for many parents and patients .

suicide of which the literal meaning is to kill oneself is an act of intentionally causing one 's own death . the reasons are mostly related with some mental disorders such as depression , bipolar disorder , schizophrenia , alcoholism , or sometime drug abuse . the triggering factor is stress which is many times related with either financial difficulties or troubles within their personal relationships . it is 10 leading cause of death worldwide in which around 800,000 to a million people die every year and an estimated 10 - 20 million nonfatal attempted suicides occur each year . in india , most suicides are under - reported and very little data is available about attempted suicides . indian union health ministry estimated that around 1.2 lakh people commit suicide , and over 4 lakhs people attempt suicide in india . unfortunately , 11% of these cases are reported from andhra pradesh state of india . national crime record bureau of india reported that in last three and half decades an increase in 175% of suicidal rate is observed and one suicide occurs in every 5 min in india although the suicidal act is made illegal in the country under the section 309 and 306 indian penal code ( ipc ) and even attempt to commit suicide is part of criminal activity under section 309 of ipc . number of suicidal incidences is proportional to attempted suicide cases hence if attempt cases reduced number of suicidal death can also be decreased and for that purpose risk factors should be identified and reduced . there are number of risk factors for suicide which varies according to country , culture , religion , gender , age , and social values . hence , the present study is planned to identify risk factors among lower socioeconomic rural population of both genders of surrounding areas of hyderabad region of india as little data is available . moreover , hyderabad is a very important densely populated metropolitan city of south india . surrounding rural population including rural population of adjoining districts therefore , it is important to properly understand the risk factors for attempted suicide cases of little studied lower socioeconomic class rural population so that suitable policies and programs can be initiated for prevention . this was a prospective study in which all the suicide attempt cases reported at bhaskar medical college and general hospital were included . it was conducted during the period of january 2013 to july 2013 . studied population belongs to surrounding rural areas of hyderabad region of india . all the cases were diagnosed and managed by emergency and general medicine department of the institute and as per the routine standard protocol , were referred to psychiatric outpatient department for further evaluation and intervention , once the patients were medically became fit for the psychiatric interview . all the patients clearly making an attempt on their life by themselves or assisted by someone were included , and accidental cases of poisoning were excluded from the study . these patients have undergone a detailed psychiatric interview including their demographic details and complete suicide risk assessment was done using beck 's suicide intent scale . the end point of the psychiatric management was counseling and treatment in the form of medication and follow - up for further management . in this study , a total of 36 patients were studied in which 20 were females , while 16 were males . mean age was 25 years for female , and 34 years for males , the age difference was statistically significant ( p < 0.05 ) in them . minimum age of the female was 16 years and the maximum was 40 years , while in the male it was 18 years and 70 years respectively . in both the groups , most of the cases were in between the age group of 21 and 30 years [ table 1 ] . age group distribution female ( n=20 ) , male ( n=16 ) more than 50% subjects were uneducated in both groups . in females majority of the subjects ( 75% ) were in the category of uneducated to secondary education while in males same trend is seen , but the numbers were much higher ( 93.75% ) . moreover , in the female group , substantial number of subjects ( 20% ) was intermediate passed , while in males , no intermediate qualified subject was seen . as far as a profession is concerned the subjects were housewife , laborer , students , and low income self - employed or were farmers . about 25% male subjects were farmers and 50% were laborer , while in the female group 35% were housewife and 45% were laborer [ table 2 ] . their married life ranged in between < 1 and > 10 years of marriage . in the male group almost 40% subjects majority of females ( 75% ) and males ( 56.25% ) subjects had dependent children between 1 and 3 kids [ table 3 ] . marital status and children majority of subjects attempted suicide 1 time although 10% female and 6.25% male attempted 2 time . most of the females ( 75% ) were free from any kind of substance use , and only 25% of them occasionally took toddy . in case of males , most of them ( 62.5% ) were consuming alcohol regularly , although 25% of males are nonalcoholics and the rest ( 12.5% ) were occasional users . apart from alcohol and toddy , subjects in both groups were not involved in any kind of drug or substance use . at the time of attempt , attempts , substance use , substance influence most of the males took organophosphorus ( 87.5% ) for suicide attempt followed by calotropis ( 12.5% ) while the majority of females also took organophosphorus ( 45% ) followed by rat poison ( 15% ) , known / unknown tablets ( 15% ) , phenyl ( 10% ) , animal / plant energy mix ( 10% ) and calotropis ( 5% ) [ table 5 ] . methods / substances for suicide attempt among the study population , 10 individuals met the criteria for alcohol dependence syndrome and 14 had depression . most of them had depressive disorder ( 38.9% ) , followed by alcohol dependence syndrome ( 27.8% ) . both of them together were present in 13.8% of the individuals [ table 6 ] . prevalence of psychiatric disorders on the suicide assessment scale in isolation parameter , score 2 was predominantly observed in females ( 70% ) , while in males scores 2 and 3 was equal ( 43.8% each ) . on timing parameter , score 2 was mainly seen in both the genders ( 65% females and 56.3% males ) , while in precautions against discovery / intervention parameter , score 1 was predominant in both the genders ( 70% females and 68.8% males ) . score 2 was also common in acting to get help during / after attempt parameters in both the groups [ tables 7 and 11 ] . scores on suicide assessment scale score 1 was mainly observed in final acts in anticipation of death ( female 95% , male 93.8% ) , active preparation for attempt ( female 90% , male 56.3% ) and a suicide note ( 100% in both groups ) parameters although 31.3% males scored 2 on active preparation for attempt parameter . most of the female and male subjects score 2 on overt communication of intent before attempt parameter ( female 80% , male 75% ) . on alleged purpose or intent parameter , score 3 was common in females ( 70% ) while in males score 3 was observed in 50% subjects and score 2 in 43.8% individuals [ tables 8 and 11 ] . scores on suicide assessment scale on expectations of fatality parameter , score 3 was more common in females ( 60% ) while in males score 2 was common ( 68.8% ) . on the conception of method 's lethality parameter , score 2 was common in both the genders ( females 65% , males 68.8% ) and also in seriousness of attempt parameter score 3 was more common in both the genders ( females 60% , males 68.8% ) . we observed score 3 predominantly in females ( 60% ) and males ( 60% ) on the attitude toward living / dying parameter although score 2 was also seen in the high number of subjects ( females 40% , males 43.8% ) . score 2 was the predominant in both the genders ( females 80% , males 68.6% ) although the number of female subjects were higher in comparison to their male counterparts on the parameter of conception of medical rescuability . most of the subjects of both the genders ( females 75% , males 68.6% ) scored 1 on the degree of premeditation parameter [ tables 9 and 11 ] . scores on suicide assessment scale total score on suicide assessment scale scores on suicide assessment scale when we assessed total score of all the parameters of suicide assessment scale it was found that in the female group equal number of ( 50% each ) cases were in medium ( total score between 20 and 28 ) and high risk ( > 28 ) categories . in the male group , number of males with medium risk were slightly more ( 56.3% ) in comparison to high risk group ( 43.8% ) . in both groups , not a single case was found to be in low risk category [ tables 10 and 12 ] . this prospective study was conducted to explore the sociodemographic variables in individuals with suicidal attempt in which females ( n = 20 ) have outnumbered males ( n = 16 ) . kessler et al . , also found female in dominance in their study , but das et al . , have reported a higher incidence of suicide attempts in males when compared to females in indian population , which is contrast to our finding in south india . das et al . , suggested that the trend might be because of higher responsibility of males in the family financial issues that contributes to increased exposure to stressful events . bashir et al . , and kiran et al . , also observed male dominance in their respective studies in tribal and urban regions of india . but in our study , this difference could be because of the study population considered . most of our study population is from the rural background and both husband and wife work as daily laborers , sharing equal amount of stress at work . besides this females have additional family responsibilities at home which in fact make them more prone for stress than males . joseph et al . , also found female dominance in rural regions of india . with increasing age , there was a decline in the number of the individuals who attempted suicide . people in 21 - 30 years age group had more suicidal rate than those belonging to other age groups . with the increasing age , individuals develop more adaptability to the stressful conditions , helping them to encounter challenging situations in a better way , thus contributing to lesser incidence of suicides . , on incidence rates of suicide in west bengal , which concluded that the vulnerable age group being those between the ages of 18 and 30 years . high proportion ( 87.5% ) of the individuals who committed suicide was married and out of total married females 15% were pregnant at the time of the attempt . marital conflicts , family stress and financial issues would have contributed to the increased number of suicides in married group . ramdurg et al . , also found predominance of married individuals in their study of which was conducted to find out sociodemographic profile clinical factors and mode of attempt of suicide attempt . , observed that most of people of developing countries live in rural regions and they are involved in agriculture and farm small areas of land . these farmers keep all their agriculture materials including pesticides , commonly within , or close to , the household . the easy availability of these pesticides when the individual is impulsive would have contributed to the high number of them attempting by this method . most of the individuals ( 62.5% ) had regular intake of alcohol and 31.25% of the study population attempted suicide under the influence of alcohol . this could be probably because of the impulsivity , lack of ability to think logically during the intoxicated state . hufford , in his study found that alcohol intoxication increases suicide risk up to 90 times , in comparison with abstinence . , also reported that , constructs related to aggression and impulsivity confers additional risk for suicidal behavior among persons with alcohol dependence . this is in accordance with the study conducted by potukuchi and rao in which there was a high prevalence of alcohol intake in the females of rural population of this region . in this study , diverse precipitating factors for suicide attempt were observed . including quarrel with close relatives , husbands or fathers were alcoholic and abusive , over all family disputes stand as a major reason for the suicidal attempt . this finding is in harmony with the findings of siwach and gupta , who reported that marital disharmony , economic hard ships and disagreement with other family members as the major precipitating factors for suicide . depressive disorders were diagnosed in 38.9% of individuals and stands as the most common associated disorder . parkar et al . , in mumbai region of indian also observed similar trend , as in their studied population 40% of individuals with intentional self - harm were suffering from depression and significant number of them also had alcohol use related disorders . increased risk of suicide with alcohol intake the individuals included in the study were referred from other departments of the institute . therefore , this may not represent all the individuals who had attempted suicide as there is a chance of people not consulting psychiatry department . a few individuals were referred to other health care institutes or expired during the medical management were not included in the study . we conclude that in the studied population risk of suicide attempt is almost equal in terms of medium and high category of suicide assessment scale in both the genders . females in between the age group of 20 and 30 years , uneducated , married and daily laborers by occupation are involved more than males and thus such category of females have greater risk for suicide attempt . depressive disorders are most common associated psychiatric disorders in both the genders , followed by alcohol use related problems . we suggest that all the individuals with alcohol related disorders must be screened for suicidal ideation , so that appropriate methods can be adopted to reduce the risk . single - cell microorganisms ( scm ) constitute an emerging alternative to source high - value lipids for a series of growing markets demanding low - cost , high - quality alternatives . scm as a broad class display a series of advantages when compared to plants and animals as lipid sources . in addition to being more genetically accessible , scm are capable of producing greater diversity and storing higher percentages of lipids . therefore , their productivity per volume and energy input can be up to 5 or 6 times that of plants and even more when compared to animal sources . additionally , single - cell microorganism architecture and circuitry tend to be more straightforward than those in plant or animal cells . for example , there are fewer organelle compartments in fungal cells than in plant cells , and some microalgal metabolic pathways ( e.g. , fatty acid synthesis ) are encoded by single - copy genes , whereas plants have multiple genes encoding proteins with redundant enzymatic activities . scm can be readily grown under controlled conditions , and the key metabolic and biochemical questions can be answered more simply and quickly than in complex multicellular systems . in principle , scm can achieve greater sustainability to alleviate the increasing problem of sourcing oils for both the fuel and human consumption markets , thus mitigating the continuous increase in commodity oil prices . microbial lipids can also become sources of safe and clean biomaterials ( e.g. , biosurfactants ) at reduced costs and continuous availability . a complete mechanistic understanding of cellular machinery is the key to moving from principle to practice in lipid production . the purpose of this paper is to provide an overview of the current knowledge of the different cellular and biochemical mechanisms involved in neutral and polymeric lipid accumulation within four main single - cell microbial groups : yeasts , microalgae , bacteria , and archaea . special emphasis is given to the production of triacylglycerols ( tag ) and polyhydroxyalkanoates ( pha ) as the hallmark lipids accumulated in eukaryotes and prokaryotes , respectively . insights are provided on the mechanistic differences existing in each group during the transformation of substrates ( e.g. , acetyl coenzyme a ) , formation of intermediate pools ( e.g. , fatty acyl chains ) , allocation to intracellular lipid pools ( e.g. , polar lipid pools or neutral lipid pools ) , and the architecture of lipid accumulation storage ( e.g. , lipid droplets ) . in microorganisms , different types of neutral lipids are accumulated by different types of scm . for example , in oleaginous yeast , tag and/or sterol esters ( se ) are the primary neutral lipids accumulated inside the cell , whereas in specific types of bacteria and archaea polyhydroxyalkanoates ( pha ) , and to a lesser extent tag and wax esters , are preferentially stored . these lipids are currently viewed as valuable potential biofuel sources , high - value compounds in the food and pharmaceutic industries , building blocks for biomaterials , potential tools to treat chronic diseases , and natural alternatives for the production of oleochemicals , among many other uses . neutral lipids follow distinct cellular accumulation strategies compared to polar lipids , such as phospholipids ( pl ) , galactolipids , and sulfolipids among others . however , in most cases , both classes share key biosynthetic steps . depending on cell needs , the cell is capable of transforming polar lipids into neutral lipids and vice versa due to the different pools present in strategic cellular locations . for instance , polar lipids are pooled and may be mobilized from the different cell membranes . key membranes involved in the accumulation of lipids include the plasma membrane in both eukaryotes and prokaryotes , and in the case of eukaryotes the endoplasmic reticulum ( er ) , peroxisome , mitochondrial , plastidial ( and their variants , such as apicoplastidial ) , or even thylakoid membranes , depending on the organism . key intermediates , such as acetyl coenzyme a ( ac - coa ) , malonyl coenzyme a ( mal - coa ) , acyl coenzyme a ( acyl - coa ) , and glycerol-3-phosphate ( g3p ) are also pooled in strategic cellular locations such as the cytosol , plastidial stroma , and mitochondrial intermembrane space . the distribution of the different biosynthetic enzymes plays a key role in achieving the correct lipid traffic toward accumulation . the accumulation of intracellular neutral lipid is a highly reductive process , requiring more nadph and atp than the storage of glucose and glucose derivatives . accumulation of lipids is a biological process that fulfills several roles in microorganisms . storage of lipids contributes to cell growth , cell division , stress response , and as energy storage for survival . in some cases , some scm have the capacity to accumulate lipids > 20% of their weight when exposed to an environmental stress , such as the lack of a key nutrient . when growing on limiting concentrations of a key nutrient ( typically nitrogen ) and with sufficient or excess carbon sources , oscm will stop their replication processes and utilize the available carbon to synthesize and store it in the way of reduced lipids . in the case of tag and se , oscm shift their metabolic machinery to generate a pool of ac - coa , as well as nadph , which is the reducing power driving fatty acid ( fa ) synthesis forward the strategies vary among different classes of oscm , and details will be discussed in the following sections . in the second stage , ac - coa is carboxylated to produce mal - coa , transferred to acyl carrier protein ( acp ) , and further transformed into an acyl acp by sequential turns within the fatty acid synthase ( fas ) . there are two types of fas : type i is an integrated enzyme complex ( common in eukaryotic cytoplasm ) , and type ii is a dissociated version in which all subunits are independent ( common in prokaryotes and in organelles of prokaryotic origin in primary and secondary endosymbionts , such as plastids and mitochondria ) . type i can be subdivided in type ia , present in fungi ( 66 complex in the cytoplasm ) , and type ib , present in animals ( as cytoplasmic 2 dimers ) . for reviews describing type i fas and type ii fas , the reader is referred to the works of schweizer and lu et al . , depending on the cell s needs and the type of scm , the acyl chain might end in a specific type of lipid pool , such as membrane lipid ( coupling the acyl chain to g3p , ac - coa , or other glycerol - based backbone to form pl or other types of polar lipid ) or can eventually be incorporated into a neutral lipid droplet core ( via the construction of tag or se molecules ) . the nature , location , and dynamics of this stage vary among species and are explained more in detail in the following sections . in this stage , further elongation and desaturation of the acyl chains may also occur , and these phenomena vary throughout the different domains of life . finally , the fourth stage involves the formation of intracellular lipid droplets ( ld ) . variations occur depending on the organism and the environmental conditions . in the case of prokaryotes , accumulation of tag or se is less common . in some members of the gram - positive actinomycete group , such as mycobacterium , rhodococcus , nocardia , dietzia , and streptomyces , tag interestingly , bacterial tag biosynthesis occurs frequently in the environment because actinomycetes are the most abundant microorganisms in soil . accumulation of tag in gram - negative bacteria has been reported only in species from the genus acinetobacter , but only as a minor component of the neutral accumulated lipid . wax esters are the main compound stored in these species . for the rest of the gram - negative bacteria , however , several studies have successfully transformed model bacteria such as escherichia coli by inserting the required genes to become oleaginous , with remarkable success . in addition , tag have been reported as major components of the neutral lipid of the cyanobacterium nostoc commune , although no indications were obtained for the storage of lipid inclusions in the cells . in the case of archaea , it is more common in prokaryotes to accumulate pha , comprising specialized lipids such as poly(3-hydroxybutyrate ) ( p3hb ) , polyhydroxyvalerate ( phv ) , or copolymers such as poly(3-hydroxybutyrate - co-3-hydroxyvalerate ) ( phbv ) . pha possess thermoplastic and elastomeric properties and are recyclable materials that can be easily degraded into carbon dioxide and water . the most promising are as film formation , paper coating , foils and diaphragms , and multiple - use packages , because the melted polymers have low viscosity , permitting the injection molding of objects with thin walls . the end product is very hard and can be used at temperatures from 30 to 120 c . chemical structures of three different types of pha , synthesized and accumulated in certain prokaryotes . the first stage involves the generation of a suitable cytoplasmic ac - coa pool . the second one involves the synthesis of the hydroxyalkanoate ( ha ) monomer or monomers , which can change depending on the species and substrates . the third stage consists of polymerizing or copolymerizing the monomers into pha chains , and the fourth one involves the formation of the intracellular pha ld . in both prokaryotes and eukaryotes , accumulation of neutral lipids results in the formation of intracellular fat bodies typically called lipid droplets ( ld ) . ld are also called lipid bodies , fat bodies , or adiposomes , and in the case of plants they have also been termed oleosomes , spherosomes , or plastoglobules , the latter if their location is inside the plastid . for the present discussion , the term lipid droplet will be used . in most of the eukaryotes ld are intracellular sphere - like particles composed of a core of neutral lipids , such as tag or se or both , coated by a monolayer of polar lipids , mainly pl , and decorated with a series of proteins . the polar heads face the cytoplasm , and the nonpolar tails face the inside , where the neutral lipids reside . the inserted proteins within the polar lipid monolayer play functional , structural , and regulatory roles in the life cycle of ld . in the case of polyhydroxyalkanoate - accumulating prokaryotes , they form ld , which are also called lipid granules or carbonosomes . in some higher organisms ( e.g. , humans ) , lipid droplet interaction with other cellular organelles , and their dynamics is closely related to progression of metabolic diseases , such as obesity , fatty liver , type 2 diabetes mellitus , and atherosclerosis . therefore , it has become apparent that ld are not just a static storage compartment in the cell , but rather a dynamic organelle that interacts with other organelles within the cell and actively participates in the intricate cellular processing symphony , to the point that some authors consider ld as specialized organelles in cells . as described in later sections , strategies for lipid accumulation in scm vary among the different types of scm , and it is important to understand these differences to improve and/or create productive strategies that can become economically as well as technologically feasible for the generation of biofuels as well as nutritional lipid metabolites such as long - chain polyunsaturated fatty acids ( lc - pufa ) . thus , they are capable of metabolizing carbon from simple sugars or from other simple compounds such as glycerol . when oleaginous yeasts encounter an environmental stress , such as a limiting nutrient ( e.g. , nitrogen ) , they shift their metabolic machinery to stop synthesizing proteins and nucleic acids and , thus , begin to allocate the available carbon in the form of reduced lipids . other examples of limiting nutrients can be phosphorus or magnesium . in practice to date , nitrogen has been extensively used because it seems to yield higher lipid accumulation when compared to other nutrients . the carbon to nitrogen ratio is a useful tool to construct nitrogen - rich or nitrogen - depleted media . for example , it has been shown that for yeasts , poor lipid accumulation occurs in media in which the carbon to nitrogen ( c : n ) ratio is < 20 , whereas ideal lipid production occurs in a c : n ratio range of 3080 . the optimal c : n ratio for lipid accumulation varies greatly with the microbial species , strain , and carbon sources present in the growth medium . in yeasts , oleaginicity depends on the ability to produce ac - coa , the necessary precursor of fatty acids , in an effective manner . the conversion of 1 mol of ac - coa to fatty acids requires the formation of 2 mol of nadph , which constitutes the reducing power necessary to drive the reaction forward . the metabolic steps for lipid accumulation in yeast can be divided into four main stages , as mentioned before ( see figure 2 ) . the diagram also includes the connection between fatty acid synthesis , triacylglycerol synthesis , and lipid droplet formation . abbreviations : me , malic enzyme ( cytosolic ) ; me 2 , malic enzyme ( er membrane , responsible for fatty acid desaturation ) ; acl , atp : citrate lyase ; i d , isocitrate dehydrogenase ; nld , nascent lipid droplet ; ld , lipid droplet ; er , endoplasmic reticulum ; lpa , lysophosphatidic acid ; pa , phosphatidic acid , fas i , fatty acid synthase i ; dag , diacylglycerol ; tag , triacylglycerol ; ampd , amp deaminase . the cell responds by activating amp deaminase and inducing an acute decrease of cellular amp content . in mitochondria of oleaginous species , the decrease in cellular amp levels causes isocitrate dehydrogenase ( idh ) activity to decrease or even stop . as a consequence , the production of -ketoglurarate drops and the tricarboxylic cycle ( tca ) is dramatically reduced or even stopped . to reverse this situation , aconitase transforms isocitrate back to citrate ( cit ) , leading to an accumulation of cit in mitochondria . cit is transported by an antiport protein , known as citrate / malate translocase ( cmt ) , from the mitochondria to the cytoplasm . in the cytoplasm , cit is cleaved to form oxaloacetate and ac - coa , by atp : citrate lyase ( acl ) . cytosolic malate is converted to pyruvate by malic enzyme ( me ) , encoded by me1 . this reaction generates nadph and is coupled to a series of parallel reactions as well . the first is the carboxylation of pyruvate to form oxaloacetate by pyruvate carboxylase inside mitochondria . this nadph is the reducing power required to convert ac - coa into fatty acids . thus , pyruvate carboxylase , md , and me are known as the transhydrogenation machinery . the resulting pyruvate completes the cycle and goes inside the mitochondria ( figure 2 ) . me is present in all fungi , but its role in supplying nadph for de novo lipogenesis is preponderant in oleaginous species . it has been found that , upon nitrogen limitation , me changes from isoform d to isoform e , which supplies nadph for de novo lipogenesis . ac - coa is converted by acetyl - coa carboxylase ( acc ) to mal - coa , which is then used to synthesize fatty acids . in yeast , cytosolic acc the mitochondrial version closely resembles in both its molecular mass and amino acid sequence the cytoplasmic one . acc uses biotin as a cofactor to transfer co2 to ac - coa in a two - step process . it is a trifunctional enzyme , harboring a biotin carboxyl carrier protein domain , a biotin carboxylase domain ( where co2 binds to biotin ) , and a carboxyl - transferase domain ( where co2 is transferred to ac - coa to yield mal - coa ) . the fatty acyl chain is built in a type i cytosolic fatty acid synthase ( fas i ) . it is an 66 complex encoded by two genes , fas1 ( subunit ) and fas2 ( subunit ) . it is suggested to contain 6 equivalent sites of fa synthesis with a total of 42 catalytic domains , organized in a ring - like structure . however , variations to this architecture exist and probably may affect the performance in de novo lipogenesis . for example , ac - coa is always the basis of fa biosynthesis and is the substrate of -ketoacyl acp synthase ( ksa ) in bacteria . the sequence of reactions in yeast fas ii is condensation of ac - coa with mal - coa ( via ks ) , reduction ( via ketoacyl reductase , kr ) , dehydration ( via a dehydratase , dh ) and further reduction ( via enoyl reductase ear ) in a repetitive manner until palmitoyl - acp is formed . release of the acyl moiety is a key step that differs from species and is currently a subject of intense research . yeasts employ malonyl - palmitoyl transacylase ( mpt ) to transfer the acyl chain from acyl acp to acyl - coa . in algae , the acyl chain can be released in three ways , depending on the cell s needs . first , it can be hydrolyzed from acp as a free fatty acid by means of a thioesterase ( te ) . second , it can be transferred either to g3p or monoacylglycerol-3-phosphate ( mag3p ) through an acyltransferase ( at ) in the chloroplast . lastly , if the acyl chain is destined to leave the plastid , it can also be released in the acyl - coa form by acyl - coenzyme a synthetases ( acs ) . it has been found that acs play a key role in regulating in each compartment the internal acyl - coa pools by esterification of fa to coa . the localization of the pools is maintained due to acyl - coa s not being able to cross the intracellular membranes . the final fatty acid composition of different types of microalgae is in great part dependent on the activity of these enzymes . for example , microalgal chain length specific te have been reported , which can release acyl moieties of specific length ( e.g. , c 12:0 or even c 8:0 ) . this type of fatty acid is more suitable for the production of gasoline and jet fuel . in contrast , gram - positive bacteria first form acyl - phosphate from acyl - coa ( via plsx ) , which is then transferred onto g3p by plsy . gram - negative species use plsb acyltransferase only to load an acyl group directly onto g3p from acyl acp . in yeast , acyl - coas released from cytosolic fas i system there , a series of esterifications to a g3p backbone occur , also known as the kennedy pathway . the first step consists of esterifying the acyl moiety from acyl - coa to g3p or dihydroxyacetone 3-phosphate via glycerol-3-phosphate acyl transferase ( g3pat ) , on the sn-1 position . g3pat is encoded in yeast by gat1 and ayr1 , when the acceptor is g3p or dihydroxyacetone 3-phosphate , respectively . gat1 does not exhibit a particular preference for acyl - coa , whereas ayr1 prefers palmitoyl - coa , thus defining fa composition . a second acyl moiety can be attached on the sn-2 position to generate phosphatidate ( pa ) , catalyzed by acyl - coa : lysophosphatidic acyltransferase ( lpaat ) . pa plays a key role in the regulation of acc1 , fas1 , and fas2 genes , as it is involved in an autoregulatory loop directed by the concentrations of inositol and choline in the cytosol and pa in the er membrane . expression of upstream activating sequence ( uasino)-operated genes changes in parallel to pa , inositol , and choline concentrations . in the case when pa is going to be transformed into tag first , the phosphate group can be directly hydrolyzed by a phosphatidate phosphatase ( pap ) to create dag . the second one involves synthesizing cytidine diphosphate dag ( cdp - dag ) from pa , catalyzed by cdp - dag kinase , encoded by cds1 . cdp - dag is the precursor of the different pl in the er membrane . it thus stays as either phosphatidylcholine ( pc ) , phosphatidylethanolamine ( pe ) , or another type of pl . eventually , dag is generated by cleavage of the pl via phospholipase c. both alternatives are in metabolic interlock with each other . this creates a positive feedback system that channels pa into the tag pathway if cdp - dag concentration is higher than dag concentration or directs pa to the membrane pool in the opposite situation . lastly , dag is transformed into tag by esterifying an acyl moiety from acyl coa catalyzed by an acyl coa : dag acyltransferase ( adat ) , encoded by either dga1 or lro1 , depending on whether dag is generated directly from pa via pap or it comes from the er membrane pl pool , respectively . another me , different from the cytosolic malic enzyme , catalyzes the conversion of malate to pyruvate and the consequent reduction of nadp to nadph . nadph then couples to a series of electron transfer reactions involving cytochrome b5 reductase and further activating the desaturase , which adds a double bond on the fatty acid chain at the expense of converting 1 mol of oxygen into a mole of water . , ergosterol is the major sterol present in yeast . for a review in the biosynthesis of ergosterol backbone yeast se are synthesized via transesterification of ergosterol with acyl - coa . in yeast , two acyl - coa cholesterol acyltransferase ( acat ) related enzymes , are1p and are2p ( encoded by are1 and are2 ) , catalyze the reaction . microscopic localization of green fluorescent protein hybrids and enzyme measurements showed that both are1p and are2p are localized to the er . are1p esterifies ergosterol and its precursors with nearly equal efficiency with a slight preference for lanosterol , whereas are2p uses ergosterol as a preferred substrate . an acyl - coa - independent pathway for the formation of ses has not been identified in yeast . lipid droplet biogenesis is an area of intensive current research , and there is emerging scientific evidence showing that in yeast it takes place between the two membrane leaflets of the er . by mechanisms still not clearly understood there are spots in the er membrane where there is a concentration of adats dga1 and lro1 . the first one is responsible for tag synthesis in the outer leaflet of the endoplasmic reticulum , whereas the second is responsible for tag synthesis in the inner leaflet of the er . the synthesized tag begin to accumulate , generating a lens - like protrusion and promoting the recruitment of structural proteins . in yeast , pat proteins ( from the initials of perilipin , adipocyte differentiation - related protein , and tip47 ) accumulate in the outer leaflet ( see figure 3 ) , whereas in plants this role is played by oleosins . when there is enough accumulation of tag between the leaflets , the outer buds off and the lipid droplet is formed ( figure 3 ) . model of lipid droplet formation in the er membrane . adapted from ref ( 11 ) . there seems to be a functional relationship between the lipid droplet membrane and the er membrane , because several studies have shown that certain functional proteins can migrate between the lipid droplet and er membranes by mechanisms that do not require energy expenditure . in yeast , for example , experiments using yeast mutants unable to synthesize tag revealed that ld were not formed . however , the proteins that were present in the wild forms in ld were also present in the er of the yeast mutants , suggesting a relationship between er proteins and lipid droplet proteins . in some cases , lipid droplet - localized proteins can relocate back to the er , indicating that some continuity between the two organelles is maintained , even if only transiently , in a way that allows the two - way partitioning of proteins between the two compartments . in describing the functions of proteins embedded in the droplet monolayers , two mechanisms have been proposed . examples of this strategy are the multifunctional caveolin protein , which has been localized to both the plasma membrane and ld , and dga1 , the major enzyme catalyzing triacylglycerol synthesis . their long internal hydrophobic stretch may enable them to be embedded in either bilayers or monolayers . the second mechanism is best represented by the previously mentioned pat protein family . they display a four - helix bundle with great similarity to the n - terminal domain of apolipoprotein e. upon binding to lipids , the apoe four - helix bundle opens to expose amphipathic helices that can bind the monolayer surfaces of lipoproteins . in an analogous manner , pat proteins may bind to lipid droplets by embedding hydrophobic helices into the droplet surface . pat proteins also share a common structural element : n - terminal 11-mer repeats that have an amphipathic helical structure . it is still unclear why or how they do it . in the specific case of perilipins ( members of the pat family ) , there are five groups , and their abundance has been correlated with the abundance of tag in the lipid droplet as well . it is important to mention that perilipin - like proteins are not found in plants . as was already mentioned , functionally similar oleosins and caleosins are present in plants instead . the accumulation of lipids that promote concavity decreases the energy burden required for budding . it has been shown that lipid droplet pl contain more lysophospholipids and less sphingomyelin and pa compared to the total membrane . in silico studies proposed a packing parameter s to quantify the degree of convexity / concavity in pl . lysophosphatidylcholine and phosphatidylinositol ( pi ) promote a convex shape where their lipid footprint areas are much smaller than their headgroup areas . lipids with s values < 1 adopt a convex surface , favoring lipid droplet formation . configurations adopted by different phospholipids , which can affect the curvature during lipid droplet formation . eukaryotic microalgae are classified into nine divisions : glaucophyta , rhodophyta ( red algae ) , heterokontophyta , haptophyta , cryptophyta , dinophyta ( dinoflagellates ) , euglenophyta , chlorarachniophyta , and chlorophyta ( green algae ) all of these divisions include single - cell strains , which can be either motile , nonmotile , or both . in terms of their nutritional strategies , microalgae can be divided into obligate heterotrophs , obligate photoautotrophs , facultative mixotrophs , and obligate mixotrophs . most algal divisions contain colorless heterotrophic species that can obtain organic carbon from the external environment either by taking up dissolved substances ( osmotrophy ) or by engulfing bacteria and other cells as particulate prey ( phagotrophy ) . in most cases , lipid accumulation strategies in microalgae and some single - cell protists ( both primary and secondary endosymbionts ) follow the four - stage lipid accumulation strategy described for yeast . , further studies are demonstrating that lipid biosynthesis pathways in microalgae are not a simple mirror image of what happens in higher plants , which have been more thoroughly studied . it seems likely that regulation of triacylglycerol synthesis and breakdown in microalgae tends to obey a stress response phenomenon , whereas in plants it follows a developmental phenomenon . for a review comparing lipid metabolism between microalgae and plants , see liu . microalgae differ from yeast in the location of the acetyl - coa pools within the cell . microalgae display plastidial and cytosolic acetyl - coa pools , which are key for lipid accumulation . in contrast , yeast s main acetyl - coa pool used for lipid accumulation is located in the cytosol . there is an additional acetyl - coa pool present in mitochondria as well as a mitochondrial lipid biosynthesis pathway employing a type ii fas ( different from plastidial fas ii ) both in algae and in yeasts . they play important roles in different cell processes , such as rna processing , mitochondrial lipoic acid synthesis , and protein lipoylation . however , mitochondrial lipid synthesis is not the main avenue for lipid accumulation and will not be covered in the present work . for a review of mitochondrial lipid biosynthesis and its relevance in cell function , plastids present in algae play a key role in de novo lipid biosynthesis ( for a review of plastid evolution and diversification , see the work of keeling ) . in photoautotrophic microalgae , photosynthesis provides an endogenous source of plastidial acetyl - coa , although more than one pathway may contribute to maintaining the acetyl - coa pool . for instance , in photoautotrophic microalgae plastidial pyruvate can be sourced via transformation of photosynthesis - derived glyceraldehyde-3-phosphate to phosphoenolpyruvate ( pep ) . pep is irreversibly converted to pyruvate ( see figure 5 ) by pyruvate kinase ( pk ) . finally , the plastidial pyruvate dehydrogenase complex ( pdh ) catalyzes the oxidative decarboxylation of pyruvate to produce plastidial ac - coa , co2 , and nadh . pdh contains three components : e1 ( pyruvate dehydrogenase , composed of e1 and e1 subunits ) , e2 ( dihydrolipoyl acyltransferase ) , and e3 ( dihydrolipoamide dehydrogenase ) . it is possible that photosynthesis - derived pyruvate is the major contributor to plastidial ac - coa for de novo fatty acid synthesis . however , in mixotrophic grown cultures of heterokonts such as nannochloropsis sp . , incorporation of acetate directly into lipids occurs . the acetyl - coa synthetase ( acsin ) converts acetate to plastidial ac - coa . additionally , a study showed that under nitrogen deprivation chlamydomonas is capable of changing its metabolism from converting acetate to glucose to a more direct incorporation of acetate into fatty acids by down - regulating glyoxylate cycle activity and gluconeogenesis . abbreviations : er , endoplasmic reticulum ; accase , acetyl - coa carboxylase ; acp , acyl carrier protein ; dagat , diacylglycerol acyltransferase ; dhap , dihydroxyacetone phosphate ; enr , enoyl - acp reductase ; fat , fatty acyl - acp thioesterase ; g3pdh , glycerol-3-phosphate dehydrogenase ; gpat , glycerol-3-phosphate acyltransferase ; hd , 3-hydroxyacyl - acp dehydratase ; kar , 3-ketoacyl - acp reductase ; kas , 3-ketoacyl - acp synthase ; lpaat , lysophosphatidic acid acyltransferase ; lpat , lysophosphatidylcholine acyltransferase ; mat , malonyl - coa : acp transacylase ; pdh , pyruvate dehydrogenase complex . in microalgae , the cytosolic ac - coa pool is mainly fueled by the release of mitochondrial cit to the cytosol and further cleaved into oxaloacetate and ac - coa by acl . cytosolic ac - coa is the building block used for lc - pufa elongation in the er . concomitant production of nadph by me generates the reductive power necessary to drive plastidial de novo fatty acid synthesis forward . availability of nadph can increase the reaction velocity of acc 2 ( see stage 2 ) and acl . fatty acid synthesis is an energy - demanding process due to the activity of elongases and desaturases . for instance , the formation of a c18 fa requires 54 nadph from oxygenic photosynthesis . other functions of malic enzyme in algae may include delivery of co2 from the tca for the plastidial ribulose-1,5-bisphosphate carboxylase ( rubisco ) . studies also suggest the existence of a plastidial me ( absent in yeast ) , which can provide electrons for plastidial fa synthesis . for a review about some microalgae are capable of accumulating intracellular starch , such as the thoroughly studied microalgal model chlamydomonas . starch synthesis is an example of how carbon partitioning might play a key role in lipid accumulation . one study showed that in wild - type chlamydomonas , tag accumulated only after the maximum amount of starch was reached , whereas starchless chlamydomonas mutants initiated tag accumulation earlier and reached a higher level than wild - type strain . therefore , it is possible to engineer high tag algal strains by eliminating competing carbon utilization pathways such as starch synthesis to maximize lipid biosynthesis . in algae , the committed step in plastidial fatty acid synthesis is the conversion of plastidial ac - coa to mal - coa by acc1 . the three domains of the homomeric acc1 are located on a multifunctional polypeptide encoded by a nuclear gene . others , such as t. pseudonana and p. tricornutum , contain two homomeric accases , acc1 ( described above ) and a cytosolic acc ( acc2 ) , which uses cytosolic ac - coa to generate mal - coa . the latter plays a role in lc - pufa elongation in the er membrane . fas are synthesized in microalgae s plastid via a dissociated type ii fas , containing discrete , monofunctional enzymes encoded by distinct genes . in plastidial fas ii mal - coa is loaded to acp via malonyl - coa : acp transacylase encoded by fabd . malonyl - acp is used in the cyclic condensation reactions to extend the acyl group to palmitoyl acp or stearoyl acp . the acyl acp can be released from fas ii in several ways : it can be hydrolyzed by a fatty acyl - acp thioesterase located in the chloroplast envelope , forming a free fatty acid , or it can be transesterified from acp to coa via ( acs ) , or it can even be coupled to either g3p or mag3p through an at in the chloroplast . there are some heterotrophic microalgal species that contain a cytosolic type i fas , synthesized from one or two polypeptides , different from plastidial type ii fas . for example , aurantiochytrium(100 ) contains a type i fas , which synthesizes saturated c14:0 and c16:0 . the synthesized free fas and the absence of genes homologous to a type ii te may indicate integration of te activity into the synthase . several attempts have been made to overexpress specific enzymes in the lipid biosynthetic pathways . in the cases of acc and ks ( ks iii ) , overexpression failed to increase lipid accumulation . the released acyl moieties as free fatty acids destined to stay within the plastid may be further desaturated ( typically to hexadecatrienoic acid hdt , c16:3n-4 ) and coupled to a monogalactosyldiacylglycerol ( mgdg ) backbone . mgdg , along with other types of galactosylglycerides ( gg ) , is a major component of photosynthetic membranes in microalgae and in some cases may be even more abundant than pl . in plants , the fa combination of gg can be traced back to their biosynthetic pathways ; the so - called eukaryotic molecular species ( c18/c18 ) of gg are synthesized outside the chloroplast in the eukaryotic pathway , and the prokaryotic molecular species ( c18/c16 ) are synthesized in the plastid via the prokaryotic pathway . microalgae differ from plants in that most c20 fas are synthesized outside the chloroplast and are present in both the eukaryotic - like ( c20/c20 , c18/c18 ) and prokaryotic - like ( c18/c16 , c20/c16 ) molecular species . c20/mlc ( where mlc means medium to long chain ) rather than eukaryotic- and prokaryotic - like gg . when acyl acp s are esterified to either g3p or mag3p , they can join the plastidial pl pool . in plants two ats the second one resides on the inner chloroplast envelope membrane and preferentially selects palmitoyl - acp . in some algal species , such as c. reinhardtii and p. lutheri , pc is absent in both plastidial and extraplastidial phospholipid pools . instead , they contain the non - phosphorus betaine lipid diacylglyceryl - n , n , n - trimethylhomoserine ( dgts ) , which has similar physicochemical properties , as a major membrane component . it has been suggested that dgts may have a role in lipid droplet formation similar to that of pc in higher plants . however , they contain other types of phospholipids , such as pa , pe , and pi . acyl acps synthesized in plastidial type ii fas can be transesterified with coa via acs . thus , stearoyl acp can leave the fas ii complex via transesterification by a series of acs responsible for maintaining both intraplastidial and cytosolic acyl - coa pools . in both cases , acyl - coa is destined to leave the plastid and enter the er membrane for further oxygen - dependent elongation and desaturation , to become long - chain polyunsaturated fatty acids ( lc - pufa ) , such as eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) . the first are front - end desaturases containing an n - terminal cytochrome b5 domain and insert the new double bond between the fa carboxyl group and a possible existing double bond . one example is the high substrate specific plastidial - located 12 desaturase , identified in p. tricornutum , which desaturates palmitoleic acid c16:1n-7 to hexadecadienoic acid 16:2n-4 . the second group comprises the less common 6/3 desaturases capable of inserting a new double bond between the fa methyl end and a pre - existing double bond . stearoyl coa can be desaturated by an er membrane - bound 9 desaturase to oleyl - coa and then linked to a glycerol backbone for further processing . some microalgae contain a plastidial 9 desaturase , capable of direct desaturation of stearoyl acp to be then transesterified to coa and sent to the er for elongation and desaturation . in most microalgae , n-3 lc - pufa are more abundant than n-6 lc - pufa , whereas in filamentous fungi , such as mortierella and mucor , n-6 lc - pufa are more common . in microalgae the most common pathways for epa and dha synthesis are the n-3 and n-6 pathways , but variations of the theme occur . in the n-3 pathway , oleic acid already linked to a glycerol backbone in the er membrane is desaturated to linoleic acid ( la ) , via a 12 desaturase ( encoded by fad2 ) . a third double bond inserted by an n-3 15 desaturase gives -linolenic acid ( lna ) , which is further desaturated to produce stearidonic acid ( sa ) by a 6 desaturase . sa is then elongated to c20:4 n-3 ( eicosatetraenoic acid , ea ) and finally desaturated by a 5 desaturase to produce epa . in most dha - producing microalgae this is a different , less complicated strategy for dha synthesis from epa , compared to the sprecher pathway , present in mammals , which involves an additional elongation and a -oxidation . in contrast to the n-3 pathway , some microalgae synthesize epa via the n-6 pathway , following 6 desaturation of la to -linolenic acid ( gla ) , elongation to dihomo--linolenic acid ( dgla ) , creation of a fourth double bond by 5 desaturase to produce arachidonic acid ( ara ) , and a final desaturation to create epa via a n-3 17 desaturase . some microalgae express 9 elongase and 8 desaturase , allowing them to synthesize epa in a different way compared to the n-3 and n-6 pathways . in a variation of the n-3 pathway , lna is elongated by a 9 elongase to eicosatrienoic acid ( eta ) and desaturated by the 8 desaturase , creating ea . on the other hand , a variation of the n-6 pathway using this pair of enzymes involves 9 elongation of la to eicosadienoic acid ( eda ) and 8 desaturation to dgla . thus , microalgae can switch from n-6 fatty acids to n-3 fatty acids via the n-3 15 desaturase and the n-3 17 desaturase . in some species of the thraustochytrids , it is also possible to switch from n-6 docosapentaenoic acid ( dpa ) to dha , via an n-3 4 desaturase . in addition to these pathways , there is a different n-3 lc pufa biosynthetic pathway present in one of the three genera of heterotrophic thraustochytrids , namely auranthiochytrium , based on an anaerobic polyketide synthase pathway ( pks ) . a large multifunctional enzyme complex carries out the multitude of individual reactions , utilizing mal - coa and producing free n-3 lc - pufas . the major free fas dpa ( 22:5n-6 ) and dha ( 22:6n-3 ) are then activated to acyl - coa and incorporated into tags . because pks does not require aerobic desaturation , the pathway is energetically favorable compared to the membrane - bound desaturases and elongases . tag synthesis in microalgae follows the kennedy pathway in the er in a similar fashion as yeast ( see figure 5 ) . however , an acyl - coa - independent mechanism for triacylglycerol synthesis in some plants and yeast has been reported . this pathway uses pl as acyl donors and dag as the acceptor , and the reaction is catalyzed by the enzyme phospholipid : dag acyltransferase . there are two diacylglycerol acyltransferase families identified in chlamydomonas , involved in the final step of triacylglycerol synthesis : type one ( dgat ) , encoded by dgat1 , and type two ( dgtt ) , encoded by five dgtt genes , which do not share sequence similarity . two independent studies showed the expression levels of dgat1 and dgtt1 increased considerably following nitrogen deprivation . however , it remains unclear which of these dgats are primarily responsible for the accumulation of tags under this condition or whether individual isoforms have specific roles . triacylglycerol synthesis can also occur within the plastid , through a series of acyl - acp esterifications to plastidial g3p , catalyzed by plastidial ats similar to the kennedy pathway in the er . the involvement of gg ( the major polar lipid family in plastidial membranes in several microalgae ) in this process remains to be elucidated . during light dark cycles , many microalgae initiate triacylglycerol storage during the day and deplete those stores at night to support cellular atp demands and/or cell division . this cycling has to be taken into account when scaling up processes for production of lipids from algae . this variable may be key to the overall success of an open pond process or a closed photoreactor process . they can grow facing the cytosol or toward the inside of the plastid , facing the stroma , which is the major aqueous fluid surrounding the thylakoids inside the chloroplast . the mechanisms underlying the orientation of lipid droplet growth in plastid membranes are not well understood . when ld grow toward the inside of the plastid ( facing the stroma ) , they can also be called plastoglobules ( ptg ) . ptg can be considered to be functionally equivalent to cytosolic ld , but differ in three major respects . second , they can assume several different forms including rods , fibers , and globules ; and third , they are bound by a specific family of proteins , variously termed plastoglobulins , plastid lipid - associated proteins , and fibrillins . oleosins ( the major lipid droplet proteins present in plants ) are not present in green algae , but a major lipid droplet protein was identified by applying proteomics in chlamydomonas and shown to modulate lipid droplet size . a wide variety of parameters affect the abundance of ld in chlamydomonas , and there is ample evidence that turnover of ld plays crucial roles in cellular lipid or carbon homeostasis . in bacteria , the most frequent types of neutral and polymeric lipids synthesized and accumulated are pha , tag , wax esters ( we ) , and , to a lesser extent , se . however , a special situation occurs in bacteria such as rhodococcus ruber , and other related bacteria , capable of accumulating both types of lipids from unrelated carbon sources such as glucose . the mechanisms of wax ester accumulation have been reviewed elsewhere . in a nutshell , an acyl - coa is transformed to an aldehyde via an nadph - dependent acyl - coa reductase ( encoded by acr1 ) , which is then reduced to an alcohol via an also nadph - dependent fatty aldehyde reductase . the alcohol is then transesterified to an acyl - coa via an enzyme displaying both wax ester synthase ( ws ) and acyl - coa : diacylglycerol acyltransferase ( dgat ) activities ( abbreviated ws / dgat ) . the only report of the presence of an sterol ester - synthesizing enzyme in prokaryotes was provided by thornton et al . to date , triacylglycerol biosynthesis has been detected only in aerobic heterotrophic bacteria and in cyanobacteria . in most bacteria , accumulation of tag and other neutral lipids , such as we , is stimulated by a carbon source present in excess , together with limited nitrogen in the medium . structurally , fluorescence staining experiments showed that lipid biosynthesis starts at peripheral lipid domains close to the cytoplasm membrane . biochemically , fatty acid biosynthesis begins in bacteria with acc , a heterotetrameric enzyme encoded by four genes , acca , accb , accc , and accd . ac - coa is converted to mal - coa and transferred to acp by malonyl - coa : acp transacylase ( fabd in bacteria ) to form malonyl - acp . a first condensation of malonyl - acp with acetyl - coa by -ketoacyl - acp synthase iii ( fabh ) to form -ketobutyryl - acp and co2 initiates a cycle that can elongate the fatty acyl - acp by two carbon units for each cycle until a saturated fatty acid of 16 or 18 carbons is made . ksi ( fabb ) and ksii ( fabf ) are responsible for the subsequent elongation cycles of the growing acyl - acp chain . to balance chain initiation with growth and utilization in bacterial fasii , ksiii , enoyl - acp reductase ( fabi in bacteria ) , and -ketoacyl - acp reductase ( fab g in bacteria ) are under negative feedback control by long - chain acyl - acps . long - chain acyl - acps directly control reductase activities ; consequently , fasii is biased to catalyze forward if these products are withdrawn from the system by any conversion , including phospholipid or triacylglycerol synthesis . in some bacteria , fas ii yields unsaturated fatty acids , which play a key role in bacterial membrane fluidity and function . unlike er desaturases present in microalgae or yeast , which introduce double bonds into the completed fatty acid chains at the expense of oxygen , the bacterial fas ii system can also desaturate fatty acids anaerobically , because it does not require molecular oxygen . faba introduces the double bond at the 10-carbon intermediate , forming cis-2-decenoyl - acp . it additionally isomerizes it into trans-3-decenoyl - acp , which is further elongated by fabb . two genes , faba and fabb , are the key players in this pathway and occur together in bacteria that produce unsaturated fatty acids . some rhodococcus and nocardia bacteria are capable of incorporating branched or phenylic groups into their intracellular tag if the corresponding substrate is fed to the medium . for example , nocardia globerula strain 432 accumulated tag containing the branched fatty acid 4,8,12-trimethyltridecanoic acid after cells were fed pristane ( a branched alkane ) , and tag with a subfraction containing phenyldecanoic acid residues were detected in cells of r. opacus pd630 after phenyldecane was fed as sole carbon source . the enzymes involved in the esterification of the glycerol moiety probably act via sequential acylation of the sn-1 , -2 , and -3 positions of g3p , with the removal of the phosphate group occurring before the final acylation step . the first esterification is catalyzed by a g3pat using either acyl - coa or acyl acp to form lysophosphatidic acid . a second acylation to lysophosphatidic acid gives pa , which is the first branchpoint for the synthesis of tag and pl , because it can be converted to cdp - dag , the precursor of the different pl species in bacterial membranes . dag itself is also at a metabolic branchpoint that divides phospholipid and triacylglycerol formations , because it acts as a precursor for tag , pc , and pe biosynthesis . in addition , dag can also be derived from pl by the action of phospholipase c the distribution of acyl groups on the hydroxyl groups of the glycerol backbone is nonrandom , as has been demonstrated for r. opacus pd630 . the shorter and saturated fatty acids were esterified to the hydroxyl group at position 2 , whereas unsaturated fatty acids were preferentially found at position 3 . this distribution in bacterial tag is different from the tag of mammals and plants , where the longer unsaturated fatty acids are found at position sn-2 . the final step in wax ester and triacylglycerol biosyntheses in bacteria is catalyzed by ws / dgat . ws / dgat is encoded by atfa and is not related to any known at involved in the formation of tag and we in eukaryotes . it has also been shown that ws / dgat is localized at the bacterial cytoplasmic membrane , presumably attached to the inner leaflet of the membrane , probably via ionic interactions . for a detailed review about ws / dgat , the reader is referred to the work of waltermann et al . alternative pathways for triacylglycerol synthesis that do not involve dag in bacteria have also been reported and may involve an enzyme similar to that reported by dalquist in yeast , which catalyzes the formation of tag from the transesterification of an acyl donor ( e.g. , acyl - coa ) to pl . this follows observations that following a double knockout of ws / dgat genes in a. borkumensis , cells were still capable of substantial triacylglycerol accumulation in ld . triacylglycerol / wax ester lipid droplet biogenesis presumably begins by allocation of newly formed tag in a hydrophobic zone within ws / dgat . as time goes by , more ws / dgat attaches to the membrane , and cumulative synthesis advances , presumably leading to the formation of very small triacylglycerol agglomerates , depicted by wltermann et al . as small lipid droplets ( sld ) . sld apparently recruit pl from the membrane by a mechanism that is still not well understood , forming a layer toward the cytoplasmic side ( see figure 6 ) . the agglomeration of phospholipid - coated sld appears in microscopy as an oleaginous layer just parallel to the membrane . then , accumulation of sld in a given point gives birth to ld , which are coated by pl and detach from the oleaginous layer . it has been suggested that acquisition of additional tag is by merging with other ld freshly synthesized from the oleaginous layer via coalescence and/or through a protein identified as tada . an interesting study by ding and co - workers examined the proteome of rhodococcus sp . rha 1 ( a gram - positive bacteria capable of accumulating tag ) grown both in nitrogen - abundant ( not favoring lipid accumulation ) and nitrogen - depleted conditions ( favoring lipid accumulation ) . using a combination of techniques , including lc - ms , sds - page , and immunoblot assays , they reported 228 lipid droplet - associated proteins , which clustered primarily into metabolism - related enzymes , transcriptional regulators , ribosome proteins , and cell division - related proteins . interestingly , they identified two major proteins , ro02104 and pspa , which constituted about 15% of the total lipid droplet protein . according to their findings , the structure predicted for ro02104 resembles that of apolipoproteins , the structural proteins of plasma lipoproteins in mammals . suggested mechanisms of neutral ld formation in bacteria . adapted from ref pha comprise a complex class of storage polyesters , with almost 150 different hydroxyalkanoic acids known as constituents . a wide variety of gram - positive as well as gram - negative bacteria synthesize pha . examples include pseudomonas , bacillus , ralstonia , aeromonas , and rhodobacter , among others . pha are divided into two groups on the basis of the number of constituent carbon atoms in their monomer units : short chain length phas ( scl pha ) and medium chain length phas ( mcl pha ) . monomers of scl pha are 35 carbon atoms long , compared to 614 carbon atoms in mcl pha . in addition , scl pha are stiff and brittle with a high degree of crystallinity , whereas mcl pha are flexible and have low crystallinity , tensile strength , and melting point . accumulation of pha starts with the creation of hydroxyalkanoate monomers from three different biosynthetic pathways . the first one involves incorporation of two acetyl - coas to form acetoacetyl - coa , by the enzyme -ketothiolase . this pathway creates hydroxybutyrate ( hb ) monomers exclusively and is used by bacteria such as cupriavidus necator and azotobacter beijerinckii ( see figure 7 ) . on a second pathway involving de novo fatty acid biosynthesis , monomers of different lengths can be formed by transesterifying 3-hydroxyacyl - acp , an intermediate of fas ii to 3-hydroxyacyl - coa , presumably by the enzyme acyl - acp - coa transacylase , encoded by phag . this enzyme is the key link between de novo fatty acid synthesis and polyhydroxyalkanoate biosynthesis . this pathway is of biotechnological interest because it helps generate monomers for polyhydroxyalkanoate synthesis from structurally unrelated and simple , inexpensive carbon sources such as glucose or related simple sugars . on a third embodiment , monomers of different lengths can also be sourced from fatty acid -oxidation pathway either by conversion of 2-enoyl - coa by an r - specific enoyl - coa hydratase , encoded by phaj , or by reduction of 3-ketoacyl - coa , presumably by fabg or phab . in this case the ability of microorganisms to synthesize a particular form of polyhydroxyalkanoate is mainly due to the substrate specificity of polyhydroxyalkanoate synthases . these enzymes are divided into four classes , depending on their structure and specificity . class i enyzmes utilize coa thioesters of 3-hydroxyalkanoates ( 3-has ) , 4-has , and 5-has comprising three to five carbon atoms . members of class ii display major specificity for monomers ranging from 6 to 14 carbon atoms . enzymes from both classes consist of a single subunit of an average size of 6070 kda and are encoded by phac . in contrast , class iii and class iv synthases are encoded by two genes , phac / phae , and phac / phr respectively , and consist of two subunits . class iii members are capable of polymerizing preferably monomers ranging from three to five carbons , yet can utilize monomers from six to eight carbons as well . species such as allochromatium vinosum contain class iii synthases , whereas class iv has been reported only in bacillus sp . all polyhydroxyalkanoate synthases share a conserved cysteine as a catalytic site to which the growing polyhydroxyalkanoate chain is covalently attached . the active - site cysteine , histidine , and aspartate constitute a catalytic triad similar to esterases . two models currently exist that may explain the formation of in vivo polyhydroxyalkanoate ld ( which are also called granules or carbonosomes ) : the micellar and the budding models . the first one is based on the assumption that the polyhydroxyalkanoate synthase is present in the cell as a soluble enzyme , distributed throughout the cytoplasm . once polymerization of substrate molecules ( coa - thioesters of suitable hydroxyalkanoic acids ) starts , the nascent polyester chain converts the initially soluble enzyme into an amphipathic molecule and the increasingly hydrophobic polyhydroxyalkanoate chains aggregate into a micelle - like structure . polyhydroxyalkanoate synthase remains attached to the surface of the granule and therefore becomes insoluble ( see figure 8) . in this model , pl and proteins of the surrounding layer would gradually become incorporated as the self - assembled polyhydroxyalkanoate inclusion increases in size . this model requires the polyhydroxyalkanoate granule to be localized in the cytoplasm at all stages of formation . in contrast , the budding model assumes that polyhydroxyalkanoate synthase is associated with the inner face of the cytoplasmic membrane , either inherently or as soon as a polyhydroxyalkanoate chain emerges from the enzyme . in this case , biosynthesis of the polyester would be directed into the intermembrane space where the extending chains would accumulate until eventually the granules detach from the membrane and polyhydroxyalkanoate - specific surface proteins can be attached to the growing granules . although the micelle model is supported by the fact that polyhydroxyalkanoate granules can be produced in vivo in the absence of membranes , most of the recently emerging evidence is in favor of the budding model . phas may constitute approximately 5% ( w / w ) of total cellular proteins , and they play a main structural role in preventing polyhydroxyalkanoate granules from aggregating and in preventing the nonspecific attachment of other proteins to polyhydroxyalkanoate granules . in addition , phasins are presumably involved in the regulation of polyhydroxyalkanoate synthesis , polyhydroxyalkanoate degradation , polyhydroxyalkanoate granule size control , the formation of networks on the polyhydroxyalkanoate granule surface , and the distribution of polyhydroxyalkanoate granules during cell division . another important group of proteins present in the external granule layer is the depolymerases , which are responsible of catalyzing polyhydroxyalkanoate breakdown . this is a relevant step in the role of polyhydroxyalkanoate accumulation as a survival mechanism in the absence of suitable energy / carbon sources , as was demonstrated many years ago in r. eutropha . phazs have been investigated much less than extracellular depolymerases , and the mechanism by which intracellular native polyhydroxyalkanoate granules can be reutilized is still not well understood . interestingly , the gene coding for phazs is located between two copies of phac1 and phac2 ( both polyhydroxyalkanoate synthase genes ) in all investigated bacteria that accumulate mcl pha . in contrast , secreted depolymerases are used by most bacteria to assimilate pha present in the environment from , for example , other nonliving cells . proposed mechanism of pha containing lipid droplet formation in bacteria ( micelle model ) . adapted from ref ( 22 ) . polyhydroxyalkanoate granule synthesis and phasin production are tightly regulated by the effectiveness of the transcriptional regulator phar . genes encoding proteins homologous to phar are widely distributed among scl pha producing bacteria , indicating an important role in the regulation of scl pha biosynthesis . the discovery of the archaea domain in 1977 revealed a novel class of microorganisms encountered in exceptional ecological niches such as high ( thermophiles and hyperthermophiles ) or low ( psychrophiles ) temperatures , acidic media ( acidophiles an thermoacidophiles ) , anaerobic atmosphere ( methanogens ) , and high salinity ( halophiles ) . the unique chemical structure of their core membrane lipids is in part responsible for their adaptation to such hostile environments . archaeal membrane lipids , in contrast to those of bacteria and eukaryotes , are made up of saturated chains containing methyl branches , attached to glycerol by ether linkages with a stereochemistry in the 2-position of the glycerol opposite that of conventional mesophilic lipids . ( for a review on archaeal ether lipid structures , the reader is referred to the work of jacquemet et al . ) moreover , archaea do not synthesize fatty acyl esters , which are the most common constituents of ld ; instead , their lipids are based on isoprenoid chains . therefore , no accumulation of tag has been reported yet in archaea . despite these differences , however , evidence of polyhydroxyalkanoate accumulation was first reported in haloarchaea back in 1972 . the strains were called at that time halobacterium sp . from the dead sea , but later identified as haloarcula marismortui . since then , strains of several other haloarchaeal genera , including haloferax , halobiforma , and haloquadratum , have been found to accumulate pha . as in bacteria , archaea produce pha under conditions of nutrient limitation but where carbon is available in excess . the mechanisms of polyhydroxyalkanoate accumulation within archaea are beginning to be understood , and work is underway to elucidate the type of proteins involved in archaeal polyhydroxyalkanoate accumulation . genes involved in polyhydroxyalkanoate biosynthesis in haloarchaea were not recognized until recently , when the polyhydroxyalkanoate synthase genes were identified and characterized for haloarcula marismortui and haloferax mediterranei . these archaeal polyhydroxyalkanoate synthases are all composed of two subunits , phae and phac , and they are homologous to class iii bacterial polyhydroxyalkanoate synthases but have a longer c - terminal extension in the phac subunit . the close similarity of archaeal and bacterial type iii polyhydroxyalkanoate synthase genes and the lack of other polyhydroxyalkanoate gene types in archaea suggest that archaeal pha originated from the horizontal transfer of an ancestral type iii gene from a bacterium . some authors suggest this transfer to have occurred already before permian times . genome - wide analysis of h. marismortui atcc 43049 revealed eight paralogues of a short - chain dehydrogenase / reductase , responsible for reduction of acetoacetyl - coa to ( r)-3-hydroxybutyryl - coa ( 3-hb coa ) , a monomer used by polyhydroxyalkanoate synthase to produce polyhydroxybutyrate ( phb ) . another study demonstrated that a similar paralogue in h. hispanica , namely fabg1 , encodes a pha - specific acetoacetyl - coa reductase responsible for providing 3-hb - coa for polyhydroxyalkanoate biosynthesis in haloarcula species . the authors concluded that the polyhydroxyalkanoate biosynthesis pathway from ac - coa , catalyzed by -ketoacyl thiolase , acetoacetyl - coa reductase , and polyhydroxyalkanoate synthase , as distributed in bacteria , likely also exists in the domain of archaea . nonetheless , they pointed out that for phb - accumulating haloarchaeal natrialba strain 56 , no enzyme activity of acetoacetyl - coa reductase or -ketoacyl thiolase was detected in the crude extract , indicating that a different metabolic route toward production of phb might be employed . polyhydroxyalkanoate synthase , putative enoyl - coa hydratase , and two structural phasin - like proteins have been identified in haloarchaeal polyhydroxyalkanoate granule surfaces . the phasin - like proteins in haloarchaea share some structural features with bacterial phas , such as the presence of hydrophobic domains and a high -helix content . after a genome - wide investigation into the 12 haloarchaea that harbored the phap gene , most of these archaea were found to possess a similar pha cluster , with five genes , namely , maoc - gap12-phap - phae - phac , having the same organization as that in h. mediterranei . the extensive existence of this pha gene cluster was suggested as an indication of an evolutionarily conserved pha gene cluster unique to haloarchaea . a promising approach is to develop archaeal species as industrial scale polyhydroxyalkanoate producers . in particular , several halophilic archaea have the advantages of utilizing much cheaper carbon sources ( including waste materials ) , as well as having less strict sterilization requirements , plus easier and more efficient methods for polyhydroxyalkanoate extraction . microorganisms provide an exciting platform for the development of lipid technologies . in the course of evolution , they have developed elegant pathways to synthesize a wide array of lipids , providing a versatile and cost - effective approach for sourcing lipids to virtually all sectors of industry . the understanding of the biochemical and cellular mechanisms of lipid production , accumulation , and secretion will provide valuable insights on innovations to overcome the hurdles in microbial lipid utilization . ongoing studies using different omics approaches will provide a holistic view of flows and interactions between the different metabolic pathways involved in lipid accumulation . tools such as next - generation sequencing , transcriptome analysis , proteomics , and mass spectrometry are already clearing out the missing links in single - cell lipid biosynthesis . this information will permeate in the creation and scaleup of more efficient processes . it is thus important to project the potential applications and envision the frontiers to which this valuable toolset can lead the diverse fields of lipid technology . for example , in the field of biotherapeutics , microbial - based approaches have the flexibility to construct platforms to manufacture personalized lipid therapies starting as simply as lc pufa combinations , appropriate to the metabolic phenotype of each individual and eventually becoming as complex and selective as personalized cancer interventions . this approach of personal medicine will carry improved benefits in the treatment of a range of conditions : in immunological diseases from infections to autoimmunities ; in metabolic conditions from diabetes to cardiometabolic diseases ; and in microbiota dysbiosis from ibs to ibd . microbial technologies can also provide an alternative pathway for sourcing promising lipids and fatty acids that are unavailable in the market at present . for example , oil - producing microbes can be considered as appropriate vehicles in which foreign ( plant ) genes could be cloned for the production of commercially attractive fatty acids such as nervonic acid ( c24:1 15 ) obtained from honesty ( lunaria ) , which is used in small amounts in the treatment of particular neuropathies . another example is sterculic acid { -(2n - octylcycloprop-1-enyl)-octanoic acid } , which has been considered as a treatment for certain cancers of the bowel . one more promising example is represented by the non - methylene - interrupted fatty acids ( e.g. , c20:3 5 , 11 , 14 ) , which can be obtained from juniperus chinensis seed oil and is known to reduce the amount of arachidonic acid in certain phospholipid pools and , thus , act to alter eicosanoid signaling . in the field of energy and biofuels , understanding the mechanisms by which each group of microorganisms generates highly combustible lipids will foster the production of cost - effective fuels through sustainable processing and with improved performance . it is yet to be tested whether the biochemical mechanisms for amphipathic lipid secretion present in some microorganisms would work for the secretion of neutral lipids . this concept , if brought to practice , will significantly reduce the processing costs and render high - quality combustible lipids for the production of biofuels . in the case of the food industry , a microbial - based approach will allow food companies to expand their core businesses by creating new product portfolios out of their current byproducts . two promising examples are the production of cocoa butter analogues and the production of high - value oils out of spent agricultural materials . in the field of biomaterials , microbial lipids are already being used for producing biosurfactants and bioplastics , replacing synthetic analogues due to their improved biodegradability and reduced cost . products such as metabolix pha and biomer are examples on how microbial - derived plastics are gaining momentum in the market . their specific modes of action , low toxicity , relative ease of preparation , and widespread applicability are increasing their use in applications such as emulsifiers , wetting and foaming agents , functional food ingredients , detergents in petroleum , petrochemicals , environmental management , agrochemicals , cosmetics and pharmaceuticals , commercial laundry detergents , mining and metallurgical industries ( for an overview of microbial surfactant applications , see mukherjee ) . the scope of applications goes to such an extent that the petroleum extractive industry is already researching the use of microbial lipid - based biosurfactants for increasing oil recovery yields from subterranean depots . the great era of chemistry culminating in the restructuring of the human condition in the 20th century has been defined by the principles of reductionism and simplicity . in contrast , the 21st century heralds the era of complexity for which the inherent biological diversity and information content of microorganisms will be key to adding value to all industrial chains . a search performed on july 6 , 2013 , with the terms microbial lipids in the u.s . patent publication since 2008 , showing the increasing interest in the generation of intellectual property around this area of research . the estimated patents by the end of 2013 according to the current trend will be around 1420 , almost doubling the number registered in 2009 . the next step in microbial lipid technology is the integration of processes and applications to generate comprehensive , sustainable solutions . the immediate challenge is to combine desired metabolic pathways present in different species or strains to generate superior microbial species . benefits will include improved yields , targeted lipidomic profiles , and structural features to synthesize in a sustainable and cost - effective way the lipid structures necessary to satisfy the breadth of needs of 21st century industry . for example , an appropriate integration of microbial - based lipid technologies will allow delivery of smart biofuels and even personalized edible oils derived from microorganisms and reporter - equipped biodegradable containers also derived from microorganisms . currently the most used packaging material for commercial edible oils is polyethylene terephthalate , which is virtually a nondegradable plastic . with the appropriate use of microbial pha , it could be possible to see in the same facility the production of high - value microbial oils ( rich in a combination of lc pufa ) , along with the production of polyhydroxyalkanoate - based bottles for its commercialization . furthermore , additional microbial biotechnology platforms will be integrated to transform the byproducts either into biomass nutrients to make the process sustainable or even into another portfolio of high - value products , as a way to increase the business profitability ( figure 10 ) . conceptual flowchart for an integral , sustainable microbial - based edible oil process . microbial lipid technologies are a valuable toolset available for the future generation of scientists , which will help them push the boundaries of production solutions ultimately toward a more sustainable society .

background : suicide is an act of intentionally causing one 's own death . number of suicidal incidences is proportional to attempted suicide cases hence if attempt cases are reduced , number of suicidal death can also be decreased and for that purpose risk factors should be identified and reduced . therefore , this study is planned to identify risk factors among lower socioeconomic rural population of surrounding areas of hyderabad in india.materials and methods : this was a prospective study in which all the suicide attempt cases reported at bhaskar medical college and general hospital were included . the study period was from january 2013 to july 2013 . they were undergone a detailed psychiatric interview , including their demographic details , and complete suicide risk assessment was done using beck 's suicide intent scale.results:it was found that females in the age group of 20 - 30 years , uneducated , married and daily laborers by occupation had higher incidence of suicidal attempts . depressive disorder is the most common associated psychiatric disorder in both the genders , followed by alcohol use related problems . family disputes are the other major risk factors . most common mode for attempt was organophosphorous poisoning followed by ingestion of calotropis.conclusion:risk of suicide attempt is almost equal in terms of medium and high category of suicide assessment scale in both genders . we suggest that all individuals with alcohol related disorders must be screened for suicidal ideation so that appropriate methods can be adopted to reduce the risk . in recent years attention has been focused on the utilization of microorganisms as alternatives for industrial and nutritional applications . considerable research has been devoted to techniques for growth , extraction , and purification of high - value lipids for their use as biofuels and biosurfactants as well as high - value metabolites for nutrition and health . these successes argue that the elucidation of the mechanisms underlying the microbial biosynthesis of such molecules , which are far from being completely understood , now will yield spectacular opportunities for industrial scale biomolecular production . there are important additional questions to be solved to optimize the processing strategies to take advantage of the assets of microbial lipids . the present review describes the current state of knowledge regarding lipid biosynthesis , accumulation , and transport mechanisms present in single - cell organisms , specifically yeasts , microalgae , bacteria , and archaea . similarities and differences in biochemical pathways and strategies of different microorganisms provide a diverse toolset to the expansion of biotechnologies for lipid production . this paper is intended to inspire a generation of lipid scientists to insights that will drive the biotechnologies of microbial production as uniquely enabling players of lipid biotherapeutics , biofuels , biomaterials , and other opportunity areas into the 21st century .

large elastic arteries in the central region and medium - sized muscular arteries have two functions , i.e. , they act as low resistance conduits and as flow pulsation buffers ( 1 ) . moreover , a reduction in buffering capacity may increase systolic blood pressure ( bp ) , left ventricular afterload , and pulsatile flow in capillary beds and reduce the diastolic contribution to blood flow in the coronary artery ( 2 ) . arterial stiffness is determined by the properties of the arterial wall matrix and by vascular smooth muscle tone , and may be changed immediately by an alteration in vascular smooth muscle tone caused by exercise ( 3 ) . exercise training - induced alterations in arterial stiffness would be of great benefit to those with coronary artery disease ( cad ) , and would potentially reduce myocardial oxygen demand and ischemic symptoms ( 4 ) . in the present study , we investigated the effect of short - duration exercise on arterial stiffness in patients with coronary artery disease , by repeatedly measuring brachial - ankle ( ba ) pulse wave velocity ( pwv ) ; an established non - invasive means of assessing arterial stiffness . fifty patients that underwent percutaneous coronary intervention ( cad group ) and 50 patients without a history of cardiovascular disease ( control group ) who were referred for treadmill testing by physicians mostly due to atypical chest pain , were prospectively enrolled . patients who were positive for myocardial ischemia on treadmill tests or those with comorbid conditions that limited exercise were excluded to ensure adequate exercise duration . thus , patients with residual ischemia after pci was excluded from cad group and patients with overt clinical coronary artery disease was excluded from control group . brachial - ankle pwv was measured using an automatic pwv measurement system ( form - pwv / abi , colin , komaki , japan ) in both brachia and ankles before treadmill exercise testing . this instrument simultaneously records bapwv , and brachial and ankle blood pressures on left and right sides , and provides an electrocardiogram and heart sounds . after baseline measurements , each subject performed symptom limited treadmill exercise testing according to the bruce protocol . at 10 min after the completion of exercise , bapwv was remeasured . heart rate and blood pressure ( bp ) were continuously monitored during exercise . for the analysis , statistical analysis was performed using sas ( sas system for windows 9.00 , cary , nc , u.s.a . ) . the chi - square test and the unpaired t - test the paired t - test was used to compare pre- and post - exercise results , and the independent t - test was used to compare the effect of exercise in both groups . multiple linear regression analysis was used to evaluate associations between bapwv changes and independent variables , and stepwise regression was used to select independent variables . fifty patients that underwent percutaneous coronary intervention ( cad group ) and 50 patients without a history of cardiovascular disease ( control group ) who were referred for treadmill testing by physicians mostly due to atypical chest pain , were prospectively enrolled . patients who were positive for myocardial ischemia on treadmill tests or those with comorbid conditions that limited exercise were excluded to ensure adequate exercise duration . thus , patients with residual ischemia after pci was excluded from cad group and patients with overt clinical coronary artery disease was excluded from control group . brachial - ankle pwv was measured using an automatic pwv measurement system ( form - pwv / abi , colin , komaki , japan ) in both brachia and ankles before treadmill exercise testing . this instrument simultaneously records bapwv , and brachial and ankle blood pressures on left and right sides , and provides an electrocardiogram and heart sounds . after baseline measurements , each subject performed symptom limited treadmill exercise testing according to the bruce protocol . at 10 min after the completion of exercise , bapwv was remeasured . heart rate and blood pressure ( bp ) were continuously monitored during exercise . for the analysis , statistical analysis was performed using sas ( sas system for windows 9.00 , cary , nc , u.s.a . ) . the chi - square test and the unpaired t - test the paired t - test was used to compare pre- and post - exercise results , and the independent t - test was used to compare the effect of exercise in both groups . multiple linear regression analysis was used to evaluate associations between bapwv changes and independent variables , and stepwise regression was used to select independent variables . the clinical characteristics and laboratory findings of the study subjects are shown in table 1 . mean age was higher in the cad group , and the cad group contained more male patients and hypertensive patients than the control group . the cad group had a lower mean left ventricular ejection fraction , a lower mean ldl cholesterol , a higher bapwv , and a shorter treadmill exercise duration than the control group . the patients with cad took more medicines , such as aspirin , -blockers , renin - angiotensin system inhibitors , and hmg - coa reductase inhibitors . baseline bapwv values were found to correlate significantly with age , systolic bp ( sbp ) , mean arterial pressure ( map ) , and diastolic bp ( dbp ) ( table 2 ) . brachial - ankle pwv values were significantly lower at 10 min after exercise than at baseline in both groups . however , this decrease was significantly larger in the cad group , thus bapwv in the cad group was initially higher than in the control group but became similar after exercise . in the control group , sbp and map were significantly lower at 10 min after exercise than at baseline , but dbp was not . in the cad group , map was significantly lower at 10 min after exercise than at baseline ( table 3 ) , whereas sbp was marginally lower , and dbp was not significantly different . heart rates were higher at 10 min after exercise than at baseline in both groups ( table 3 ) . by multivariate analysis , the cad group showed a larger decrease in bapwv after exercise than the control group after adjusting for age , bmi , sbp , map , map reduction , and baseline bapwv ( table 4 ) . arterial stiffness increases left ventricular afterload and alters coronary perfusion ( 5 ) , and has been independently associated with target organ damage and increased cardiovascular morbidity and mortality ( 6 ) . brachial - ankle pwv is a simple marker of arterial stiffness ( 7 , 8) and mainly reflects large artery stiffness , although it has also been reported to reflect endothelium - dependent peripheral vasodilation . changes in vascular wall distensibility may be induced by changes in the quality and quantity of vascular fibrous matrix ( e.g. , elastic fibers and collagen fibers in media : an organic factor ) and by changes in smooth muscle tone ( a functional factor ) . elastic fiber is the primary determinant of vascular distensibility under physiologic conditions ( 9 , 10 ) . moreover , the elastin - collagen compositions of arterial walls represent a more chronic component of arterial stiffness and changes only over years , thus it is unlikely that short - duration aerobic exercise changes these structural components ( 11 ) . instead , arterial compliance is probably altered in the short terms , or even acutely , via the modulation of the sympathetic - adrenergic tone of smooth muscle cells in arterial walls ( 12 ) , which is affected by autonomic nervous activity and vasoactive agents derived from vascular endothelial cells , e.g. , nitric oxide ( no ) , prostacyclin , and endothelium - derived hyperpolarizing factor ( 13 ) . in particular , the production of no is important , because it is a potent endothelium - dependent vasodilator and reduces vasoconstrictor response to -adrenergic receptor stimulation ( 14 ) . moreover , pulsatile flow in the aorta associated with exercise training might evoke the acute release of no , upregulate no production , and increase the productions of other vasodilatory factors ( 15 - 17 ) . in cad patients , endothelial dysfunction develops secondary to reduced no production and early reactivation by reactive oxygen species ( 18 ) . in the present study , bapwv , which was higher in the cad group at baseline , was found to be reduced significantly at 10 min after exercise in both groups , and because this decrease was larger in the cad group , no difference in bapwv was observed between the two groups after exercise . these observations suggest that short - duration exercise affects arterial stiffness even in patients with cad . we speculate that the mechanism involved may be related to the restoration of an equilibrium between no production and inactivation by reactive oxygen species , which also appears to be the primary mechanism underlying exercise training - mediated perfusion improvements in cad patients ( 18 ) . bp and age have been reported to be important determinants of bapwv in healthy individuals ( 7 , 19 ) , which is consistent with the findings of the present study . exercise duration is significantly different between the two groups which might be a confounding factor . however , exercise duration is longer and the proportional change of bapwv is smaller in control group , and moreover exercise duration is not correlated to both absolute and relative change of bapwv ( data not shown ) . thus it is not probable that different exercise duration is a significant confounding factor . because , as mentioned above , cad group members were taking more medications , it is unclear whether our findings suggest that patients with stable cad have the potential to reverse arterial stiffness with exercise despite the presence of disease or whether they reflect an effect of the medications taken , such as aspirin , -blocker , renin - angiotensin system inhibitor , and hmg - coa reductase inhibitor . the lower level of ldl - cholesterol in the cad group was probably due to hmg - coa reductase inhibitors . however , considering the higher baseline bapwv value in the cad group , it appears that medications do not completely normalize arterial stiffness in cad patients and that short - duration exercise seems to independently improve arterial stiffness immediately . because majority of patients in cad group and only a small number of persons in control group was taking one or more of the drugs which influence endothelial functions , statistical adjustment is not feasible with our data . complete exclusion of the effects of medication probably can only be performed by experimental design in which normal control group is given the same medications . still , it is rather puzzling that cad patients showed more prominent reduction in bapwv than control group and resulted in similar level of bapwv after exercise . this apparent ' reversibility ' of arterial stiffness might be related to the duration and extent of atherosclerosis . as discussed above we speculate that those in relatively early course of cad may have more reversible component related to endothelial dysfunction and less irreversible component such as structural change of vascular wall . this group of patients has higher baseline bapwv due to the endothelial dysfunction but much of this functional abnormality might be reversible by some intervention , such as short - duration exercise in this study . patients in the cad group had undergone percutaneous coronary intervention and those with findings of residual ischemia were excluded by exercise test . this exclusion may have resulted in selection of patients with lower risk and less extensive coronary artery disease . however , as a limitation of this study , we do not have enough information on the duration and extent of cad in the patient group . also , it is not possible to investigate this speculation further by measuring biomarkers of no production and oxidative stress . if markers of central blood pressure ( 20 , 21 ) had been measured , different findings might have been found . in previous studies , aerobic exercise improved peripheral arterial stiffness but not central arterial stiffness ( 22 ) , and central arterial stiffness was a better prognostic factor than peripheral arterial stiffness ( 23 ) . however , this does not mean that peripheral arterial stiffness is meaningless , which has been shown to have prognostic value in another study ( 24 ) , and also shown to be correlated to central arterial stiffness ( 8) . it is likely that central arterial stiffness is a better index but peripheral arterial stiffness measured by bapwv is a very convenient alternative . another weakness of the study is that we do not have data on the heart rate and blood pressure at the time of pwv measurement . because these variables acutely influence bapwv , this can be a potential source of confounding . however , we assume that heart rate and blood pressure was probably not different from the baseline at 10 min post - exercise . though our study showed immediate short - term response to exercise , evidence is scarce on whether repeated short - duration exercise may result in a persistent and long - term improvement of arterial stiffness . further study is needed on this question , considering that frequent short - bout exercise is being discussed as a practical alternative to conventional long - duration exercise ( 25 , 26 ) . in conclusion , in the present study , we prospectively investigated the effects of short - duration exercise on arterial stiffness by measuring bapwv in patients with or without cad . a significant reduction in bapwv was observed at 10 min after short - term aerobic exercise in both groups , and in the cad group this decrease was more prominent . these observations suggest that short - duration exercise training may be effective at improving arterial stiffness even in patients with coronary artery disease , at least in short - term . clinical study is needed to see whether repeated short - duration exercise will improve arterial stiffness in cad patients in long - term . with cataract surgery regarded as the most widely performed surgical procedure , a demand exists for continued innovation and technology . the latest advances evolved through application of well - defined principles to current surgical goals and patient expectations . for example , femtosecond laser technology emerged after fifty years of employing laser technology in ophthalmology . theodor scheimpflug described the principle of scheimpflug images in 1904 , but he was actually an austrian army captain who spent his life 's work dedicated to designing methods and tools to create maps depicting aerial photography . application of these principles to ophthalmology in the last few years has advanced our understanding of corneal biomechanics . the latest highlights in technology include advances in preoperative and intraoperative diagnostics , femtosecond laser - assisted cataract surgery ( flacs ) , and a new generation of intraocular lenses ( iols ) . more than ever , patients have the desire to reduce their dependence on spectacles after cataract surgery . physicians now have access to advanced diagnostics that can better quantify conditions such as dry eye , light scatter , and posterior corneal astigmatism . mcdonald recently reported that the postoperative prevalence of dry eye related symptoms is approximately 88% . analysis and optimization of dry eye preoperatively and postoperatively has a beneficial impact on visual outcomes after cataract surgery . therefore , increased interest among ophthalmologists to utilize objective measurements to assess the ocular surface exists . the acutarget hd ( visiometrics sl , spain ) assesses the objective scatter index , which can objectively evaluate dry eye disease severity using the degradation of image quality over time ( figure 1 ) . the keratograph ( oculus , germany ) noninvasively measures tear break up time , tear meniscus height , and meibography , providing a functional and qualitative analysis of the corneal surface and tear film [ 8 , 9 ] . the tearlab osmolarity system ( tearlab corporation , san diego , california ) uses a small tear sample to measure tear osmolarity using a microelectrode . compared to other commonly used diagnostic tests for dry eye disease , test results were better at predicting dry eye severity . the lipiflow ( tearscience , morrisville , north carolina ) combines heat and eyelid pressure to treat dry eye disease due to meibomian gland dysfunction . recent studies showed consistent improvement in meibomian gland function up to 12 months after the treatment . evaluation of optical quality also aids in decision making between corneal or lens - based procedures . the c - quant ( oculus , germany , optikgenrate gmbh ) assesses straylight subjectively by utilizing a compensation comparison method . the acutarget hd ( visiometrics sl , spain ) uses a double pass system to measure point spread function ( psf ) , modulation transfer function ( mtf ) , strehl ratio , and intraocular scattering of the light . these data allow clinicians to evaluate the quality of a patient 's optical system objectively . another objective functional diagnostic is the salzburg reading desk ( srd vision , vienna , austria ) , which allows measurement of the variable read print sizes and distances with differences in contrast sensitivity and luminance . both anterior and posterior corneal astigmatism should be taken into account in iol planning , particularly in patients desiring astigmatic correction . inaccuracies arise when posterior corneal astigmatism is measured based on the assumption of a fixed - ratio relationship with the anterior curvature . the cassini corneal shape analyzer ( i - optics bv , the hague , the netherlands ) is a new topographer that uses led ray tracing technology with 700 diode lights to measure anterior and posterior corneal astigmatism . these advances in cylinder and axis measurement precision can be useful for preoperative planning of toric iol implants and in postrefractive surgery patients [ 1517 ] . patients with a history of corneal refractive surgery expect reduced dependence on spectacles after cataract surgery . the optiwave refractive analysis ( ora , alcon , fort worth , tx ) uses wavefront interferometry to produce a fringe pattern , and distortions in this pattern are translated into refractive values and aphakic and pseudophakic readings ( figure 2 ) . studied mean postoperative residual refractive astigmatism in patients receiving toric iols with power selection aided by intraoperative aberrometry . surgeons altered cylindrical power 24% of the time and spherical power 35% of the time . patients were 2.4 times more likely to have less than 0.50 d of residual refractive astigmatism when intraoperative aberrometry was used . in contrast , huelle et al . published a study where aphakic spherical equivalent- ( se- ) based iol formulas were generated from repeated intraoperative wavefront aphakic measurements of se . the agreement of repeated aphakic se readings ranged from 0.69 diopters to + 0.66 diopters . the authors concluded that measurement precision is limiting reliability of intraoperative aberrometry and application to routine cataract surgery . however , it may be useful in guiding limbal relaxing incision enhancements and has resulted in the need for fewer subsequent laser enhancements . this technology is particularly useful in postrefractive patients and those with astigmatism uncertainty or other corneal pathology . other intraoperative inconsistencies include cyclotorsion , variable anterior chamber depth and intraocular pressure , variability in wound hydration , and use of viscoelastic device versus balanced salt solution . although limitations may exist in quality and measurement precision , the future of this technology is promising . the use of the electroretinogram ( erg ) has been well described and may have a novel application for refractive cataract surgery . richard mackool described the use of flash erg testing with office - based electroretinography ( diopsys , pine brook , new jersey ) in preoperative cataract evaluation . it can provide an objective evaluation of macular function and could be useful in influencing lens selection for patients with conditions such as epiretinal membrane , diabetic retinopathy , and age - related macular degeneration . more studies evaluating ergs in preoperative cataract assessment need to be done to further assess its value and implications . noninferiority has been established relative to manual cataract surgery , and some reports have suggested superiority relative to manual methods . potential advantages include customized corneal incisions and capsulotomy position , precision in shape and size of capsulotomy , custom lens fragmentation patterns , endothelial cell loss reduction , and better refractive stability and predictability . after the food and drug administration ( fda ) approval of laser - assisted capsulotomy and lens fragmentation in 2010 , five platforms have been released : lensx by alcon ( fort worth , tx ) ; the lensar by lensar ( orlando , fl ) ; the catalys by abbott / optimedica ( north chicago , il ) ; the victus by bausch and lomb ( rochester , ny ) ; and the ldv z8 by ziemer ( port , switzerland ) ( figure 3 ) . the docking process using the femtosecond laser - eye interface uses a suction ring to stabilize the eye , thereby allowing imaging and laser delivery through a clear optical pathway . considerations for docking include complete coupling , patient comfort , intraocular pressure elevation , and minimal distortion of anatomy to avoid disruption of the beam path . in a study using alcon 's lensx platform to compare curved direct contact and modified soft interfaces ( softfit by alcon ) , mayer et al . showed that redocking was unnecessary when a modified soft interface was used , even though some cases resulted in incomplete incisions requiring manual opening . schultz et al . found significantly fewer intraocular pressure elevations after docking using a liquid interface ( liquid optics interface , catalys precision laser system ) in comparison to flat and curved interfaces . while docking is a necessary step with femtosecond laser technology , laser incisions are optional in flacs . comparatively analyzed femtosecond laser incisions and manual incisions and cited better tunnel morphology with flacs incisions . flacs theoretically decreases endothelial cell loss relative to manual techniques by reducing the use of ultrasound energy . however , krarup et al . compared endothelial cell loss rates between phacoemulsification and flacs and showed there were no differences between both modalities . published similar findings but did cite a difference in favor of flacs that was limited to the early postoperative period . they also showed that laser corneal incisions themselves may influence endothelial cells , as there may be a disturbance in the postoperative inflammatory response after laser application [ 3032 ] . new surgical techniques , in combination with more advanced lens fragmentation patterns , will allow the lens to be extracted through an aspiration mechanism that may reduce endothelial cell loss . the size , shape , and position of a capsulotomy should theoretically lead to a more predictable lens position by enhancing uniform capsule - optic overlap , thereby reducing the incidence of lens tilt and leading to an overall better effective lens position and visual outcome ( figure 4 ) . recently , toto and colleagues found no difference in prediction error when comparing traditional phacoemulsification with flacs but did find higher refractive stability and iol centration with flacs . this similarity in prediction error may be a consequence of unexplored potential with iol calculations and algorithms . ma approached the prediction of true lens position using an algorithm based on oct anterior segment 3-d reconstruction . this prediction model could have great potential once there is consistent alliance of oct measurements with flacs to provide more precise outcomes . this is particularly relevant with premium iols , as there is a lower tolerance threshold for minor unanticipated miscalculation and decentration . okulix ( tedics peric & joher gbr , dortmund , germany ) is an innovative software program that calculates iol power using ray tracing combined with corneal topography . evaluated its accuracy in post - lasik eyes in comparison with camellin - calossi , shamas - pl , haigis - l formulas and double - k srk - t method . they reported that this technology provides sufficient predictability outcomes in postrefractive myopic lasik , even though a small hyperopic shift tendency was noted in the study . g6 lens professional ( ziemer , port , switzerland ) is an optical biometer that integrates placido rings with a dual rotating scheimpflug camera as well as an optical coherence tomography based a - scan in a single device . shin et al . compared its accuracy with the lenstar ls 900 ( haag - streit , koeniz , switzerland ) , for intraocular lens ( iol ) power calculation . they noted that axial length , lens thickness ( lt ) , and white - to - white ( wtw ) values were statistically different . thus , even though high repeatability was present , and the iol powers were not statistically different between the two devices , the values provided by the galilei g6 were not interchangeable with the lenstar in the clinical setting . the goal of appropriate iol selection is to provide the best visual outcome that meets a patient 's individualized goals and expectations . variability in materials , optical properties , and designs are important factors to consider in the patient - specific selection of an iol . advancements in iol technology aim to improve visual functionality by creating customized iols or modifying optical power postoperatively . the concept of adjustable iols involves the correction of residual refractive error postoperatively or customization after lens implantation . this new paradigm in iol manufacturing may be subdivided into two major categories : a modular multicomponent category requiring a separate intraocular procedure and another category where the optic is adjusted postoperatively with a secondary device . the first category includes multicomponent iols ( clarvista harmoni modular iol system , clarvista medical , aliso viejo , ca ; and omega lens , omega ophthalmics , lexington , ky ) ; infinite vision iol ( infinite vision optics , france ) ; and mechanically adjustable iols ( acritec ar-1 pc / iol , acri.tec , hennigsdorf , germany ) . the second category includes magnetically adjustable iols ( university of missouri - rolla , rolla , and eggleston adjustable lens , st . louis , mo ) , light adjustable iols ( calhoun vision , pasadena , ca ) , and the perfect lens ( perfect lens , llc , irvine , ca ) . the latter is a novel platform , which can be adjusted with the femtosecond laser based on the concept of refractive index shaping . its mechanism involves the use of infrared light to polymerize photosensitive silicone macromers , which results in changes in lens morphology and optical properties . a new generation of optics with extended depth of focus , multifocal rotational symmetry and asymmetry , and accommodating capabilities offers promising strategies to advance functional vision in refractive cataract surgery . the tecnis symfony ( amo ) is an extended depth of focus iol that works by correcting chromatic aberration using diffractive optics and reduces glare and halo symptoms classically associated with conventional multifocal iols . multifocal lenses with bifocal and trifocal designs include the rotationally asymmetric lentis mplus ( topcon europe medical bv , the netherlands ) and the rotationally symmetric finevision ( physiol , liege , belgium ) and at lisa ( zeiss , oberkochen , germany ) . found that trifocal lenses provide satisfactory intermediate vision without compromising near and far distance visual acuity . the fluidvision iol ( powervision , belmont , ca ) has silicone oil inside the lens that moves in response to ciliary contraction forces . an electroactive iol with a liquid crystal that is sensitive to electric current is also in development ( sapphire autofocal iol , elenza , roanoke , va ) . dual accommodating iols designed for sulcus placement include the dynacurve iol ( nulens , israel ) and the lumina iol ( akkolens , the netherlands ) . the injectable polymer smartiol ( medennium , irvine , ca ) , which is a thermodynamic , pliable capsule - filling iol , is the only bag filling technology in development . the future of refractive cataract surgery is exciting ; in time , these new technologies may be the standard of care . with refinements of the latest technology , flacs and other parallel advances will provide surgeons with the potential to perform an even safer , predictable , and effective surgery .

arterial stiffness is an important contributor to the development of cardiovascular disease . we investigated the effect of short duration exercise using the treadmill test on arterial stiffness in the presence of coronary artery disease . we enrolled patients with and without coronary artery diseases ( cad and control group , 50 patients each ) referred for treadmill testing . brachial - ankle pulse wave velocity ( bapwv ) were measured before and after treadmill testing . values of bapwv were significantly reduced at 10 min after exercise in both groups , more in the cad group than in the control group ( baseline bapwv and post - exercise change [ cm / sec ] : 1,527245 and -132155 in the cad group , 1,439202 and -7793 in the control group , respectively , p for change in each group < 0.001 , p for difference in changes between the two groups < 0.001 ) . these findings persisted after adjusting for age , body mass index , systolic blood pressure , mean arterial pressure ( map ) , map decreases , and baseline bapwv . significant post - exercise bapwv reductions were observed in both groups , and more prominently in the cad group . this finding suggests that short - duration exercise may effectively improve arterial stiffness even in patients with stable coronary artery disease . technology in cataract surgery is constantly evolving to meet the goals of both surgeons and patients . recent major advances in refractive cataract surgery include innovations in preoperative and intraoperative diagnostics , femtosecond laser - assisted cataract surgery ( flacs ) , and a new generation of intraocular lenses ( iols ) . this paper presents the latest technologies in each of these major categories and discusses how these contributions serve to improve cataract surgery outcomes in a safe , effective , and predictable manner .

epilepsy is the most common serious neurological disorder of young people affecting nearly 3.4 million individuals in europe . societal costs are considerable as individuals with medically intractable seizures make up a third of the epilepsy population . more than eu15 billion is spent annually on the treatment of epilepsy in europe , a financial burden comparable to that of lung and breast cancer combined [ 2 , 3 ] . a recent irish prevalence study estimated that up to 40,000 children and adults in ireland have the disorder , which gives a point prevalence of about 0.9% ; in line with other industrialized nations . studies in the last decade have shown that the majority of patients with epilepsy will be urgently admitted to secondary and tertiary care institutions ( mostly through the ed ) at some point in the history of their illness and a significant proportion will require multiple visits . furthermore , symptomatic seizures , secondary to acute medical or surgical illness , alcohol and drug intoxication , brain trauma and stroke , add to the burden of seizure pathology in the ed . finally , a range of mimic disorders from psychogenic nonepileptic seizures ( pnes ) to blackouts caused by impaired vascular responsiveness contribute to the diagnostic and therapeutic challenges . despite the heavy burden of seizures in the ed , international studies suggest that the majority of patients are referred unnecessarily for admission and that the acute treatment of seizures is often ineffective indicating that seizure admissions are a cause of unnecessary medical intervention , delayed diagnosis , and prolonged length of stay [ 5 , 6 ] . beginning in 2006 we first sought to determine the impact of emergency seizure admissions on the resources of a large irish teaching hospital . on the basis of two baseline audits we identified a number of areas where we could improve the quality of service to patients with seizures by the employment of an evidence - based seizure care pathway in the emergency department ( ed ) and acute medical admissions unit ( amau ) . this is a report of preliminary quality and safety metrics accrued by this intervention over a 12-month period ( november 2008 - 2009 ) . the study consisted of three parts : retrospective audit of admissions with seizures through the emergency department in 2004 . measurement of quality and safety metrics after the implementation of an evidence - based seizure care pathway from november 2008 to november 2009 . the first study group was restricted to in - patients discharged in 2004 with a diagnosis of a seizure or convulsion . these patients were identified through the hospital in - patient enquiry ( hipe ) system ( a national coding system for hospital discharges in ireland ) , which identified 341 patients with epilepsy or seizure as the primary reason for admission . particular attention was paid to investigations , diagnosis , specialist referral , and follow - up . the charts were also examined for what we designated as necessity of admission . an admission was deemed medically necessary if there was a history of prolonged ( > 5 minutes ) or clustered ( > 2 ) events , a history of status epilepticus ( > 30 minutes of seizure activity ) , or an abnormal neurological exam 90 minutes after arrival to the ed . hipe system was used to determine if any of these patients was readmitted or died over a one - year period following admission . the second study group represented all patients who attended the ed with an event that was deemed likely to have been a seizure or its aftermath over one calendar month in 2006 . patients who presented with a complaint of seizure , weakness , confusion , head injury , dizziness , and collapse of unknown cause were examined for possible inclusion in the study . in a similar vein to part 1 of the study , attention was paid to investigations , diagnosis , referral , and follow - up in all patients . after the baseline audits , a seizure care pathway was designed and implemented by the neurology service with the cooperation of the ed staff . the pathway required early rapid access ambulatory follow - up for patients fit for discharge from the ed . a rapid access clinic ( rac ) was established and run by an existing epilepsy nurse specialist ( ens ) . in addition to patients discharged from wards and the ed , this clinic also reviewed new referrals from gps and patients with an established diagnosis of epilepsy who had exacerbations of their illness , all of which were designed to avoid ed referrals . education sessions were provided by the ens who also provided a phone help - line and e - mail service to facilitate follow - up care . patients presenting to the ed or admitted to the amau overnight with seizures were seen by the seizure service fellow . patients were managed according to a locally designed , evidence - based , seizure care pathway ( figure 1 ) . the pathway clearly laid out the criteria for : admission , discharge and follow - up . the pathway was designed with reference to published international guidelines of care ( scottish intercollegiate guideline network ( sign ) 2003 ( 8) and national institute of clinical excellence ( nice ) 2005 ( 9 ) ) . all patients seen by the seizure service were provided with printed cards with full details for phone and e - mail contact . length of stay and time to ct , mri and eeg were analysed using kruskal wallis test for nonparametric data . the rate of representation was analysed using pearson chi - square test . times to follow - up the reduction in admission rates was analysed using chi - square test with yates correction . during 2004 , hipe data identified 341 admissions with a specific diagnosis of epilepsy or seizure out of a total of 11,721 admissions from all causes through the ed . in the 50 charts randomly selected out of this group , 34% of patients had a previously documented diagnosis of epilepsy at presentation . investigations performed included ct brain ( 84% ) , mri brain ( 28% ) , and eeg ( 56% ) . median delay to ct , mri , and eeg were 2 days , 5 days , and 5 days , respectively . ambulatory follow - up was evenly divided between neurology ( 28% ) , general medicine ( 28% ) , and general practitioner ( 20% ) , with a further 24% having no follow - up whatsoever . 23/50 patients ( 46% ) represented to the emergency department with further seizures over the next 12 months . using our criteria for necessity of admission , we concluded that 36% could have been discharged earlier or from the ed , had appropriate investigations and neurological opinion been available in a timely manner . of the 341 patients with a diagnosis of epilepsy , 10 died over the subsequent year but only one of those who presented with status epilepticus died as a direct result of their epilepsy . 20 ( 19% ) of the 102 patients included in the study had a previously established diagnosis of epilepsy . of the special investigations required for epilepsy , ct brain was the only one conducted on the day of admission and in only 5.8% of cases . neither mri nor eeg 's were performed on any patient on the day of presentation . ultimately of the 34 admitted , 14 ( 41% ) had an eeg , 21 ( 61% ) patients had a ct brain , and 4 ( 12% ) had an mri brain . median delay for eeg was 2 days ; ct brain was 1 day , and mri was 2.5 days . no data was collected on mortality or follow - up as it was designed primarily to gather data on patients presenting to the ed . during 2009 , there were 276 admissions with a primary diagnosis of epilepsy out of 12 , 607 admissions from all causes through the ed . 350 patients who presented to ed between november 2008 and november 2009 with seizures and other forms of collapse were referred to the seizure team for assessment and had the seizure care pathway applied . 97 patients had an established history of either generalised or focal epilepsy and 72 patients had epilepsy associated with significant medical and surgical comorbidities . 34 patients were referred with undefined collapse , 12 patients were referred with confusion , and 4 patients with myoclonic jerks . of the 181 eegs requested during the intervention study period , 99 ( 55% ) were done on the same day . 66 ( 36% ) were done within 1 to 3 days , and 16 ( 9% ) were done as outpatients within 4 weeks . 150 patients had ct brain requested and 140 ( 93% ) were performed on the same day and median delay for ct brain was again zero days . in 2008 - 2009 , 68 ( 19% ) of the total cohort of 350 , had mr imaging of the brain requested . same day mr brain acquisition however went up from 0% in 2004 to 7.2% in 2008 - 2009 and another 8.8% cases were done within 1 to 3 days . of the 57 patients with nonepileptic collapse , 12 had an eeg , 18 patients had ct brain and only one patient had an mri brain performed . 110 patients ( 31.4% ) were seen in the rapid access clinic ( rac ) . 64 patients ( 18% ) were seen in the subspecialty epilepsy clinic and median follow - up time for this more stable group was 8 weeks . 6 patients ( 1.71% ) were followed up in other hospitals 31 ( 8.9% ) of the total study group were readmitted in the 12-month follow - up period . of the patients seen during the study period , 19 ( 5.4% ) the neurological causes of death were herpes encephalitis , obstructive hydrocephalus , nonconvulsive status epilepticus and subdural haematoma . three patients died of direct seizure - related causes , two with nonconvulsive status epilepticus , and one with convulsive status epilepticus . during the intervention study period no patient who was discharged from the ed or within 2 days of admission died . the number of admissions with epilepsy or seizure dropped significantly from 341 out of 11,721 ( 2.9% ) in 2004 to 276 out of 12 , 607 ( 2.2% ) in 2009 ( p = 0.0006 ) . there was a significant reduction in median length of stay between the first 2 audits and the intervention study ( p < 0.001 ) . figure 2 summarises the median length of stay over the 3 studies there was a significant improvement in time to diagnostic investigations such as ct brain , mri brain , and electroencephalography between the first two audits and the intervention study ( p 0.001 , p 0.048 , p 0.001 ) . figure 3 summarizes the median delay to investigations on admitted patients across the three studies . there was a significant reduction in follow - up times from a median of 16 weeks to 5 weeks ( p < 0.001 ) . figure 4 shows median times to follow - up in the baseline audit in 2004 and in the intervention study in 2008 - 2009 . there was a significant reduction in readmission rates from 45.1% to 8.9% ( p 0.001 ) . figure 5 shows the change in readmission rates between 2004 and the intervention study in 2008 - 2009 the use of care pathways in modern healthcare delivery has been somewhat controversial since the expected gains are not always forthcoming . for instance a cochrane review of the implementation of a care pathway in stroke rehabilitation did not endorse any benefit to patient care . nevertheless , given the highly variable care delivered in the ed in relation to seizure care , we felt that a care pathway could provide much needed improvement . the aim of this study was to demonstrate improvements in for patients presenting to the ed with seizures and related disorders , without compromising safety by the use of an evidence - based seizure care pathway . the main quality indicators measured were requirement for admission , median length of stay , time to diagnostic tests , specialist follow - up , readmission rates , and mortality . the main findings of the study are that through the utilization of the seizure care pathway the ed and amau can reduce unnecessary admissions and safely discharge patients for early follow - up , which has a very significant impact on reducing representation rates . timely decision support has the effect of significantly reducing time to diagnostic tests , particularly eeg , and thus reducing median length of stay by up to 3 days . all of these outcomes were significant statistically and support the use of care pathways for patients presenting to the ed with seizures without any increase in mortality . it has been suggested that admission of seizure patients is only warranted in patients who are at high risk of further events , remain drowsy or comatose following a period in the ed , or in whom the neurological exam reveals signs indicative of an underlying lesion or treatable infective cause . however , international studies suggest that the majority of patients are referred unnecessarily to the in - house medical or neurological services for admission [ 5 , 6 , 9 , 10 ] . in our original retrospective audit ( part 1 ) , using a set of criteria based on the above indications for admission , we determined that 36% could have avoided admission . the intervention study showed the stability of this figure with 31% of patients actually being discharged from the ed and a further 8.5% within 24 hours of admission . comparison of hipe data between 2004 with that of 2009 following implementation of the seizure care pathway shows a reduction in the number of admissions with a specific diagnosis of epilepsy from 2.9% ( 341 ) of total hospital admissions to 2.2% ( 276 ) . this is despite an increase in overall admission rates from ed of 7.56% from 11,721 in 2004 to 12,607 in 2009 . had admissions continued at the rate of 2.9% with no seizure care pathway in place , it would have resulted in 365 epilepsy - related admissions in 2009 , suggesting that 89 epilepsy specific admissions were avoided due to implementation of the seizure care pathway in 2008 to 2009 . if we consider the median los to be 4 days ( without the seizure care pathway being applied ) this would have resulted in 356 bed days saved . combining this figure with the 478 bed days saved by an overall median reduction in los of 2 days , a total of 834 is the projected bed days saved in one 12-month period as a direct result of the implementation of the seizure care pathway . the reduction in median length of stay from 4 days in 2004 and 5 days in 2006 to 2 days during the intervention study in 2008 - 2009 was made possible by an emphasis upon early safe discharge in the pathway with an eye to reduce bed occupancy days . establishment of a separate rapid access follow - up clinic made routine and even unscheduled early follow - up possible , which increased the safety and ease of early discharge . reductions in length of stay secondary to implementation of care pathways have been reported in other areas of heath care , but not in the case of patients with seizures [ 1113 ] . of the discharged patients , the readmission rates show a significant drop from 47% to 8.9% between 2004 and 2008 - 2009 . it is possible that some unobserved bias meant that patients more likely to return were seen in the 2004 audit . it may be due to a combination of more timely and effective inpatient management including the delivery of inpatient ens education , the provision of phone and e - mail advice services , the use of rapid access to ambulatory clinics for exacerbations of existing epilepsy , and improved , timely communication with primary care teams . the reduction of median follow - up time from 16 weeks during 2004 to 5 weeks in 2008 - 09 may also have helped in reducing re - admissions to the ed . in relation to the overall safety of the seizure service with its emphasis on reducing admissions and length of stay , we found that only three of the 19 deaths in the study group were directly attributable to epilepsy . in 2004 , only one death was attributable to epilepsy . while there is a slight increase in epilepsy - related deaths , there was no excess mortality in any patients discharged from the ed or within the 2-day median length of stay window . accurate diagnosis and classification of seizure type are essential to the provision of quality patient care and good control . eeg is described as an important aid in the evaluation of seizure patients . in relation to the baseline audit the median waiting time to eeg was 5 days , demonstrating that most patients were not being tested soon after the seizure period . such waiting times observed in the baseline audits suggested that eeg contributed significantly to the length of stay of individual patients . during the intervention study the involvement of early specialist opinion allowed for the streamlining of those who required eeg which occurred on the day of admission in approximately 54% of the study group and in total over 90% had the test done in 3 days or less . neuroimaging is essential to identify structural lesions , which may result in the development of a seizure disorder . despite strong consensus within the literature for performing mri over ct especially in focal seizures [ 5 , 15 , 16 ] ct was the preferred modality for neuroimaging within all our study groups . this is largely to do with the ease of access to ct over mri at our institution , which has not changed significantly since 2004 . 84% of the retrospective sample had a ct performed , whilst only 28% had an mri . in the intervention study only 42% of patients had a ct requested reflecting the fact that in a number of cases of either established epilepsy or indeed eeg proven primary generalized epilepsy , the pathway was able to obviate the need for ct . an improvement in time to brain imaging was demonstrated in the intervention study with 93% of patients having their ct on the same day of request . unusually , the mean wait time for mri brain increased in 2009 compared to 2004 . this , we believe , was due to the steadily increasing demand for mri brain in acute presentations of seizure over those years coupled with limited availability of our mri resource ( one magnet open only during office hours ) . this has been ameliorated lately by the addition of a second scanner . as outlined by the sign and nice guidelines , adults with epilepsy should have a specialist expert opinion in an ambulatory setting including regular structured annual review . in the retrospective 2004 audit , mean time to neurology clinic follow - up was the lack of decision support and or expert neurological opinion to two - thirds of the 2004 study group may have contributed to the longer length of stay in this group as there may have been a delay in diagnosis and pursuing appropriate investigations . only 28% of that group were followed up by the neurology service in outpatient clinics , which we speculate may have contributed to return ed presentations and readmission rates . the intervention study suggests that the decision support embodied in the seizure care pathway and early follow - up contributed significantly to a reduction in readmission rates . retrospective chart reviews , which formed the basis of initial baseline data , are hazardous for deciding on service provision due to the unreliability and potential bias in the data . furthermore , the initial analysis was on a relatively small number of charts . in this study we complimented the retrospective audit with a short prospective audit , which validated some of the retrospective audit and independently verified characteristics of patients admitted with epilepsy and their course in hospital . the intervention study was large enough to draw conclusions but its comparison to the two prior audits must be done with caution as the patient characteristics may have been biased in the smaller studies . this was exemplified by the significant differences in readmission rates between the two audits and the intervention study . while some of the difference were undoubtedly due to service improvements finally , for system - wide change the decisions that contributed to the improved quality metrics would have ideally been made by ed and acute medical staff and specialist nurses applying the principles in the seizure care pathway . in this study the pathway was implemented by a specialist service and thus the generalizability of the results in unclear . however , the lack of widespread use of pathways for seizure presentations requited that a proof of principle study was required . future study should now focus on the use of an integrated care pathway ( icp ) without resource necessarily to a specialist at the ed / amau interface . it appears that a large proportion of seizure - related presentations are referred to the in - house medical or neurological teams for admission , due in large part to the lack of access to appropriate algorithms for admission and decision support for early treatment and diagnostic investigation and the difficulty in obtaining outpatient investigations and specialist epilepsy follow - up in a reasonable length of time . this study conducted over a 12 month period using baseline data collected between 2004 and 2006 shows that using an evidence - based care pathway with early specialist advice and follow - up , along with directed patients education and a range of communication tools to aid in self - management such as telephone and e - mail advice , can contribute significantly to quality and value improvements in epilepsy care without compromising safety . we recommend further study of this programme and we have embedded a continuous improvement cycle into prospective audit . after getting the university ethics committee 's approval , a prospective case - control study was conducted . all patients who admitted to the boo - ali hospital , zahedan , southeastern iran , with a clinical suspicion of pulmonary tuberculosis or non tuberculous pulmonary infections from september 2006 to august 2007 were included for comparison . one hundred and seventy eight patients were included in the study in which 67 were documented cases of ptb and 111 were patients with pulmonary infections other than tuberculosis as control group ( mostly acute pneumonia and acute exacerbation of chronic bronchitis ) ( figure 1 ) . multiple sputum samples are usually collected from a single patient in the process of tb diagnosis . patho - tb test , ziehl - neelsen staining and culture for mycobacterium tuberculosis were done on all specimens . polymerase chain reaction based on detecting a 123-bp dna segment belonging to insertion sequence is6110 specific for mtb was just done in 42 sputum smear negative samples . analysis of data was performed in confirmed ptb patients ( 63 culture - positive and 4 pcr - positive cases ) . patients with pulmonary infections with negative acid fast bacilli ( afb ) smear and culture results were included in the control group . forty three patients in the tb group were sputum smear positive and the rest were sputum smear negative . we used the patho - tb kits ( anda - rt mycobacteria patho - tb , anda biologicals , strasbourg , france ) which were developed in collaboration with the pasteur institute of iran . the patho - tb test was performed as the manufacturer brochure 's following stages : 1- sample decontamination and neutralization : the sputum sample was treated for decontamination by the kubica method in which 2 ml of sample was put in a 50 ml conical centrifuge tube . an equal volume of solublization / decontamination solution was added and mixed well by vortexing and incubating for 20 minutes on an orbital or turning agitator . at least 0.2 ml aliquot of the re - suspended pellet was kept to perform the rapid patho - tb test and the rest was used to seed culture and perform other required diagnostic test . 2- re - suspended pellet treatment : the re - suspended 0.2 ml aliquot was diluted in 1 to 5 volumes of dns . subsequently , an equal volume of negative control and rehydrated positive control were transferred to two other boiling resistant microtubes . from dissolving solution , the rapid patho - tb test was performed with those treated samples following the procedure presented in the following section . 3- rapid test on the cartridge : only one pre - filter was placed in the center of each cartridge ( filtering unit ) . this pre - filter was essential to retain large particulate matter that would clog the filter . a funnel was made on the pre - filter by snapping it securely in place on the cartridge without damaging the pre - filter . thereafter , the funnel and the pre - filter were removed and a second wash was done with 2 drops of washing solution . then 3 drops of antibody solution was added . a red - pink color at the filter center , as mentioned earlier , indicated the presence of tuberculous bacilli in the processed sample . 4- result interpretation : the color intensity is proportional to the number of antigens in the sample . it is classified into three categories : ( + ) a visible central pinkish color , lighter than the control ; ( + + ) a pronounced central pinkish red color , similar to the control ; and ( + + + ) a central purple - red color , darker than the control . when the result of a test was compatible with the first category , the test should be repeated . the appearance of a red or pink ring with or without a red - pink central area is an artifact that does not influence the results and should be considered negative . data were analyzed using the spss software for windows , version 11.5 ( spss inc , chicago , il , usa ) . the diagnostic performances of the test characteristics including sensitivity , specificity and positive and negative predictive values were measured . a two - sided significance level of 0.05 was used . the patho - tb test was performed as the manufacturer brochure 's following stages : 1- sample decontamination and neutralization : the sputum sample was treated for decontamination by the kubica method in which 2 ml of sample was put in a 50 ml conical centrifuge tube . an equal volume of solublization / decontamination solution was added and mixed well by vortexing and incubating for 20 minutes on an orbital or turning agitator . at least 0.2 ml aliquot of the re - suspended pellet was kept to perform the rapid patho - tb test and the rest was used to seed culture and perform other required diagnostic test . 2- re - suspended pellet treatment : the re - suspended 0.2 ml aliquot was diluted in 1 to 5 volumes of dns . subsequently , an equal volume of negative control and rehydrated positive control were transferred to two other boiling resistant microtubes . from dissolving solution the rapid patho - tb test was performed with those treated samples following the procedure presented in the following section . 3- rapid test on the cartridge : only one pre - filter was placed in the center of each cartridge ( filtering unit ) . this pre - filter was essential to retain large particulate matter that would clog the filter . a funnel was made on the pre - filter by snapping it securely in place on the cartridge without damaging the pre - filter . thereafter , the funnel and the pre - filter were removed and a second wash was done with 2 drops of washing solution . then 3 drops of antibody solution was added . a red - pink color at the filter center , as mentioned earlier , indicated the presence of tuberculous bacilli in the processed sample . 4- result interpretation : the color intensity is proportional to the number of antigens in the sample . it is classified into three categories : ( + ) a visible central pinkish color , lighter than the control ; ( + + ) a pronounced central pinkish red color , similar to the control ; and ( + + + ) a central purple - red color , darker than the control . when the result of a test was compatible with the first category , the test should be repeated . the appearance of a red or pink ring with or without a red - pink central area is an artifact that does not influence the results and should be considered negative . data were analyzed using the spss software for windows , version 11.5 ( spss inc , chicago , il , usa ) . the diagnostic performances of the test characteristics including sensitivity , specificity and positive and negative predictive values were measured . the patho - tb test was performed as the manufacturer brochure 's following stages : 1- sample decontamination and neutralization : the sputum sample was treated for decontamination by the kubica method in which 2 ml of sample was put in a 50 ml conical centrifuge tube . an equal volume of solublization / decontamination solution was added and mixed well by vortexing and incubating for 20 minutes on an orbital or turning agitator . at least 0.2 ml aliquot of the re - suspended pellet was kept to perform the rapid patho - tb test and the rest was used to seed culture and perform other required diagnostic test . 2- re - suspended pellet treatment : the re - suspended 0.2 ml aliquot was diluted in 1 to 5 volumes of dns . subsequently , an equal volume of negative control and rehydrated positive control were transferred to two other boiling resistant microtubes . from dissolving solution the rapid patho - tb test was performed with those treated samples following the procedure presented in the following section . 3- rapid test on the cartridge : only one pre - filter was placed in the center of each cartridge ( filtering unit ) . this pre - filter was essential to retain large particulate matter that would clog the filter . a funnel was made on the pre - filter by snapping it securely in place on the cartridge without damaging the pre - filter . thereafter , the funnel and the pre - filter were removed and a second wash was done with 2 drops of washing solution . then 3 drops of antibody solution was added . a red - pink color at the filter center , as mentioned earlier , indicated the presence of tuberculous bacilli in the processed sample . 4- result interpretation : the color intensity is proportional to the number of antigens in the sample . it is classified into three categories : ( + ) a visible central pinkish color , lighter than the control ; ( + + ) a pronounced central pinkish red color , similar to the control ; and ( + + + ) a central purple - red color , darker than the control . when the result of a test was compatible with the first category , the test should be repeated . the appearance of a red or pink ring with or without a red - pink central area is an artifact that does not influence the results and should be considered negative . data were analyzed using the spss software for windows , version 11.5 ( spss inc , chicago , il , usa ) . the diagnostic performances of the test characteristics including sensitivity , specificity and positive and negative predictive values were measured . a two - sided significance level of 0.05 was used . one hundred and seventy eight patients were enrolled in the study who were finally classified into two groups ; a group of documented pulmonary tuberculosis ( n = 67 ) and a group of non - tuberculous pulmonary infection ( n = 111 ) . the mean age of the tb patients was 58 17 years and 42% of them ( 28 patients ) were men . the mean age in non tb patients was 60 15 and 46% of them ( 51 patients ) were men . twenty ( 11% ) of the patients were from afghanistan and the rest were iranian . anorexia , weight loss , hemoptysis and malaise were significantly higher in the tb group but fever was more presented in the non - tb group ( table 1 ) . positive reactions to ppd skin test ( more than 10 mm ) and presence of bcg scar were seen in 62% and 37% of ptb group comparing to 54% and 45% in non - tb group , respectively ( p > 0.05 ) ( table 1 ) . the sensitivity , specificity , ppv and npv of ppd test versus culture were 63% , 46% , 41% and 67% , respectively . as it was shown in table 1 , 43 out of 67 ptb patients had positive direct smear examination . the sensitivity of sputum smear examination was 64% versus 90% in patho - tb test but the specificity was 100% versus 70% . the most common clinical findings of pulmonary tuberculosis and non - tuberculous patients the results of patho - tb test were positive in 40 of smear positive and 20 of smear negative tuberculous patients and 33 cases of non - tuberculous control group . four specimens in control group were positive which were retested and only one specimen was reported positive at the end . the sensitivity of patho - tb test in sputum smear positive and negative ptb was 93% and 83% , respectively . specificity of the test was 70% and positive predictive value ranged from 38% in sputum smear negative ptb to 65% in the total number of ptb patients . the negative predictive value was more than 90% in each group ( table 2 ) . among communicable diseases , tuberculosis ( tb ) is the second leading cause of death worldwide , killing nearly 2 million people each year . about one - third of the world population is infected with tb in which 10% progress toward active disease . most of these patients are living in underdeveloped countries of the world.10 the incidence of total ptb and sputum smear positive ptb were estimated about 53.4 and 22.4 per 100,000 respectively in the study region during 2006.11 the definite diagnosis of pulmonary tuberculosis is difficult because of low sensitivity of sputum direct examination . despite the low cost and easy performance of direct sputum smear microscopy , it has a sensitivity of 61.3 - 63.4% and specificity of 97.3 - 97.4% and is being used as the primary diagnostic test for the diagnosis of ptb.12 culture requires several weeks to provide results and other complex tests such as pcr are expensive and rarely available in low income countries . world health organization targets 70% of case detections and 85% of successful treatments and they are not likely to be achieved with the existing methods of diagnosis.13 attempts to develop a rapid and inexpensive diagnostic method with high sensitivity and specificity are still highly desirable for the diagnosis of ptb . different methods by using new reagents or purified antibodies try to provide high sensitivity and specificity for the tests comparable to direct smear microscopy or afb culture.14 there are many different free mycobacterial antigens which can be detected in different types of body fluid . the most commonly used antigens are lipoarabinomannan ( lam , mycodot serologic test ) , antigen 5 ( 38 kda , pathozyme - tb elisa test and ict diagnostics ) , lam and 38 kda ( pathozyme - myco elisa test ) , a-60 antigen ( antigen a-60 elisa test ) , 45/47 kda antigen , kp90 antigen , 30 kda antigen , p32 antigen and some other antigens.the standard methods used for those different antigen assays include sandwich elisa , inhibition elisa , latex agglutination and reverse passive hemagglutination tests.1416 dipstick method had a sensitivity and specificity of 93% and 95% , respectively.17 recently it was reported that patho - tb test has a reasonable sensitivity and specificity . among 310 tb patients , the sensitivity of patho - tb was 91.1% comparing to direct examination that was 91.8% and the specificity was 85.5% versus 100%.18 direct examination in that study was done by auramine staining which was confirmed by ziehl - neelsen in positive specimens but in the present study only ziehl - neelsen staining for direct examination was performed . sensitivities in both of these studies revealed approximately similar results but the specificity was lower in the present study . in the present study , the sensitivity of patho - tb test was 90% in contrast with 64.1% for the direct smear method . the higher sensitivity , coupled with prompt response , low cost and easy performance , will enable the identification of a sizeable number of pulmonary tuberculosis . another advantage of the test is using just a small amount of sputum ( 0.2 ml ) . in order to have good sensitivity , the antibodies used in this test are polyclonal . these antibodies are raised and purified against tb bacilli , especially 35 , 65 and 85 kda mycobacterial antigens , but can also react with other mycobacterial species . thus it is necessary to analyze results together with the clinical data of each patient to obtain a complete diagnosis . concerning the high number of false positive , using a confirmatory diagnostic procedure like culture in association with clinical features is mandatory . none of the patients in this study were hiv positive so a study in immune compromised patients with several opportunistic infections is recommended . as mentioned earlier , performance of anti - mycobacterial antibody test revealed contradictory results , regardless of type of tuberculosis . the value of detection of specific igg or igm antibodies against antigenic particles of mycobacterium tuberculosis revealed different results.1923 rapid immunochromatographic test ( ict ) for detection of antibodies directed against mtb antigens and mtb - specific enzyme - linked immunospot assay , with early antigenic target-6 and culture filtrate protein-10 peptides are two other different new diagnostic methods for tb.2425 despite all these different types of rapid diagnostic tests , newer and more accurate methods of diagnosis are still needed . although clinical manifestations in table 1 such as malaise , anorexia , weight loss and hemoptysis showed a significant difference between ptb and control groups , they are not characteristic for tb . positive tuberculin skin test in our study also did not show any significant difference and the results of diagnostic performance characteristics were much lower than patho - tb test results . although sputum smear examination was highly specific ( 100% ) , it was just 64% sensitive . higher sensitivity of this new rapid flow - through method comparing to direct smear microscopy of afb , might enable using patho - tb test for rapid diagnosis of ptb as a screening test along with traditional diagnostic tests . positive predictive values of the test are low therefore patients with positive patho - tb test need a confirmatory test such as sputum culture to confirm the diagnosis of ptb . concerning the high percentage of npvs , it would be possible to rely on them for patients with a negative patho - tb test and clinical manifestations which are not suggestive of ptb . further studies on larger number of sputum smear negative ptb patients are required to clear the characteristic performance of patho - tb test . ran was the main researcher , wrote the manuscript and coordinated in most of the experiments .

aim . to evaluate the utility of a seizure care pathway for seizure presentations to the emergency department ( ed ) in order to safely avoid unnecessary admission and to provide early diagnostic and therapeutic guidance and minimize length of stay in those admitted . methods . 3 studies were conducted , 2 baseline audits and a 12-month intervention study and prospective data was collected over a 12-month period ( nov 2008 - 09 ) . results . use of the pathway resulted in a reduction in the number of epilepsy related admissions from 341 in 2004 to 276 in 2009 ( p = 0.0006 ) ; a reduction in the median length of stay of those admittedfrom 4 - 5 days in the baseline audits to 2 days in the intervention study ( p 0.001 ) ; an improvement in time to diagnostic investigations such as ct brain , mri brain and electroencephalography ( p 0.001 , p 0.048 , p 0.001 ) ; a reduction in readmission rates from 45.1% to 8.9% ( p 0.001 ) ; and an improvement in follow - up times from a median of 16 weeks to 5 weeks ( p < 0.001 ) . from a safety perspective there were no deaths in the early discharged group after 12 months follow - up . conclusion . the burden of seizure related admissions through the ed can be improved in a safe and effective manner by the provision of a seizure care pathway . background : despite recent technologic improvements in identifying mycobacterium tuberculosis , we are still facing problems in rapid diagnosis of tuberculosis . the objective of this study is to determine the diagnostic value of a new rapid screening test ( patho - tb ) for diagnosis of pulmonary tuberculosis.methods:between september 2006 to august 2007 , 178 patients were enrolled in the study who were finally classified into two groups ; a group of documented pulmonary tuberculosis ( n = 67 ) and a group of non - tuberculous pulmonary infection ( n = 111 ) . patho - tb test , ziehl - neelsen staining and culture were done on all specimens.results:of all , 43 patients with pulmonary tuberculosis were sputum smear positive for acid fast bacilli and the rest were smear negative . mean age of the patients was 59.8 16.1 years and 44% of them were men . the results of patho - tb test were positive in 40 of smear positive and 20 of smear negative tuberculous patients and 33 cases of non - tuberculous control group . the sensitivity , specificity , positive and negative predictive values and accuracy of patho - tb test were estimated 89.5% , 70.2% , 64.5% , 91.7% and 77.5% , respectively.conclusions:according to the present study it would be suggested that patho - tb test could be a rapid and inexpensive method for diagnosis of pulmonary tuberculosis , given by its high sensitivity and negative predictive value . concerning the high number of false positive results , using a confirmatory diagnostic procedure is mandatory .

a 14-year - old , domestic shorthair cat was treated for transient diabetes mellitus for 3 months with glargine insulin , which was discontinued when the diabetes mellitus resolved . approximately 36 months later the diabetes mellitus recurred and glargine insulin was restarted . within 23 mins of the first injection the cat collapsed , developed profuse vomiting and diarrhea , as well as facial swelling and diffuse erythema . a hypersensitivity reaction was suspected and the cat was treated with antihistamines , aggressive fluid therapy and gastrointestinal support . six months later the cat re - presented for relapse of its diabetes mellitus and an intradermal skin challenge with 1:20 diluted insulin was performed confirming a hypersensitivity to glargine . the cat continues to be well regulated on porcine zinc insulin without any hypersensitivity reactions noted . this is the first reported case of a hypersensitivity reaction secondary to glargine insulin in a cat . clinicians should be aware of this potential complication , particularly in animals with a previous history of insulin administration and the potential to utilize intradermal testing with insulin . hypersensitivity reactions ( hrs ) most commonly occur after an allergen binds to ige on the surface of mast cells and basophils precipitating release of vasoactive substances such as histamine , chymase , tryptase and other chemokines . it is usually associated with prior exposure to the antigen causing sensitization of the immune system . clinical manifestations can range from facial pruritis and urticaria , to cardiovascular collapse and death . previous reports of suspected hrs to pharmaceuticals in veterinary medicine include dexamethasone , ophthalmic medication and human albumin among others . the purpose of this case report is to describe the clinical manifestation and management of a cat with an hr to subcutaneous glargine insulin ( gi ) administration . a 14-year - old , female , spayed , domestic shorthair cat weighing 5.68 kg presented on referral to our hospital for evaluation of acute collapse . the cat had a history of diabetes mellitus ( dm ) , which was first treated with gi in 2013 . the cat was treated for diabetes , which became transient after 3 months of treatment . one week prior to presentation at our institution , the dm had recurred and re - initiation of gi was recommended . the bottle had not been opened prior to use and it was stored in a refrigerator . the evening of presentation the cat had ingested all of its meal ( which had not been changed in over a year ) , according to the owner , and received 1 unit of gi subcutaneously for the first time since 2013 . approximately 23 mins after administration , the cat collapsed and started panting . the cat also demonstrated projectile vomiting and diarrhea . on presentation to the primary veterinarian , the cat was noted to be laterally recumbent and was exhibiting horizontal nystagmus . a blood glucose ( bg ) was not obtained , but the cat was given dextrose submucosally as there were concerns about a hypoglycemic crisis . the cat was then referred to our facility . on presentation to our institution , the cat was noted to be obtunded , was tachycardic ( heart rate of 220 beats per minute ) and tachypneic ( respiration of 50 breaths per minute ) . the respiratory effort was increased and infrequent stridor noted . the face and muzzle were swollen and edematous . the cat again produced a large amount of vomiting and diarrhea ; at this time hematochezia was also noted . though mentation was dull , the nystagmus had resolved and no cranial nerve deficits were noted . bg was measured on presentation with a handheld glucometer ( alphatrak ; abbott animal health ) and was noted to be 517 mg / dl ( reference interval [ ri ] 75116 mg / dl ) ; this was approximately 2 h after the gi had been administered . the cat had no measurable systolic blood pressure on doppler measurement ( parks doppler machine ) and a 10 ml / kg bolus of crystalloids was administered . owing to the concerns about an hr to the insulin , diphenhydramine ( 2 mg / kg ) was administered intramuscularly . within an hour , the muzzle swelling and erythema had improved after antihistamine administration . the cat continued to experience recurrent systemic hypotension in the subsequent hours and after receiving a 3 ml / kg synthetic colloid bolus , was started on a continuous crystalloid ( 75 ml / kg / day ) and colloid infusion ( 8 ml / kg / day ) and the systemic hypotension resolved . blood work performed on admission revealed hyperglycemia ( 666 mg / dl ; ri 75116 mg / dl ) , hypokalemia ( 3.36 meq / l ; ri 3.624.60 treatment for her gastrointestinal signs consisted of maropitant ( 1 mg / kg sc q24h ) , dolestron ( 0.6 mg / kg iv q24h ) and ampicillin ( 20 mg / kg iv q8h).the cat continued to experience a large volume of diarrhea and hematochezia over the first 24 h after admission , but the vomiting resolved . an abdominal ultrasound was performed 1 day after presentation and revealed an enlarged and hyperechoic liver with few small hypoechoic nodules throughout the parenchyma , assessed as consistent with a diabetic hepatopathy . both kidneys had a mild decrease in corticomedullary definition , and the left and right adrenal glands were noted to have normal shape and size . blood work was also repeated at this time and showed normalization of the lactate and potassium levels . during hospitalization , the bg continued to be monitored and ranged from 107355 mg / dl ; the cat s appetite remained poor . no insulin was administered while hospitalized , owing to the poor appetite , persistent diarrhea and the concerns about an hr to gi . the cat was discharged 3 days after presentation on metronidazole ( 10 mg / kg po q12h ) and famotidine ( 1 mg / kg po q24h ) . the owners continued to monitor the cat s bg at home with a handheld glucometer and it ranged between 88 and 380 mg / dl . the owners tended to measure the bg if they felt the cat appeared lethargic ; they did not have a set protocol of when and how frequently to measure its bg . at this time the cat was still not consuming its full caloric needs and we recommended that the owners continue to monitor the cat s bg at home to determine when it would require insulin again . the owners infrequently measured the cat s bg 12 times weekly and it was never greater than 200 mg / dl . six months later , the cat re - presented for recurrence of polyuria / polydipsia and being lethargic at home . the owners were continuing to measure the cat s bg at this time and were intermittently obtaining values greater than 250 mg / dl . the cat was sedated with ketamine ( 0.5 mg / kg iv ) and valium ( 0.3 mg / kg iv ) , and the fur over the lateral left thorax was clipped . one unit of gi and porcine zinc insulin ( pzi ) were each diluted into 20 units/0.2 ml of 0.9% nacl and from each of these mixtures , 0.05 ml/5 units was injected intradermally . as a negative control , within 30 mins of the procedure , a palpable swelling was noted over the gi site . the cat also developed erythema and swelling of its muzzle ( figure 1 ) and vomited ; diphenhydramine ( 2 mg / kg i m ) was administered and the clinical signs resolved . two hours after the procedure , the cat s bg was 410 mg / dl ( ri 90180 mg / dl ) and it was elected to administer 0.5 units pzi subcutaneously , owing to the hyperglycemia . the cat was kept overnight for monitoring and was noted to be painful and intermittently pruritic over the injection site . the cat was discharged the following day and continues to be on subcutaneous pzi every 12 h and has not exhibited any recurrent signs of systemic or cutaneous hr . evidence of cutaneous anaphylaxis after the administration of glargine insulin ; note the generalized erythema and swelling over the muzzle and periocular regions type i hrs , as previously noted , can vary in severity from urticaria to anaphylaxis . this can occur as a biphasic reaction , with immediate itching and burning at the injection site , followed by a more sustained generalized reaction 48 h later with a combination of cutaneous , gastrointestinal and/or respiratory symptoms reported . this presents as small , localized tenderness and painful non - erythematous nodules and central hematomas at injection sites , occurring around 68 h after insulin injection and lasting for 48 h. clinical examples of type iii hrs include serum sickness , adenopathy and insulin resistance . the cutaneous nodules associated with a type iv hr can be distinguished from a type iii hr as the former usually occurs 24 h or more after the insulin injection and last for 47 days . in humans , type iv hrs it appears , given the acute presentation and history of prior exposure to insulin , the cat in this case demonstrated a type i hr , but this was not conclusively proven . severe anaphylactic reactions in cats can cause a multitude of signs , but dyspnea is most common as the lungs are believed to be the shock organ of cats . in the case previous case reports of anaphylactic reactions in cats have also shown that some will also exhibit simultaneous gastrointestinal and respiratory signs . while a gastrointestinal infection or toxin can not be fully ruled out , the cat s diet was strictly controlled by its owners , owing to its dm , and an infection also seemed less likely as it was kept indoors only and there were no other pets in the house . also , the association of the signs with the administration of the gi is also indicative of an hr . confirmation of a cutaneous hr to insulin administration is performed by intradermal testing using a 1:20 dilution of insulin preparation . appearance of a cutaneous lesion within 60 mins of injection indicates a type i hr , as exhibited by the cat in this case , whereas a type iii or iv hr would be suggested by a response seen between 2 and 24 h. human protocols were used as intradermal testing with insulin had never been performed in a cat prior to this . there was subjectively more swelling and discomfort noted at the gi injection site ; the gi site also appeared more erythematous . the increased swelling and erythema over the gi site also provides more evidence that the cat was more sensitive to the gi than the pzi . the cat in this case exhibited more severe cutaneous and gastrointestinal signs than the typical human patients who undergo intradermal testing . should this protocol be utilized in the future in small animal patients , the insulin may need to be further diluted to reduce the severity of adverse effects . other diagnostic modalities for hrs reported in dogs include identification of an elevated alanine transaminase and increased gallbladder wall thickness and a striated wall pattern on abdominal ultrasound . it is unknown if such abnormalities are identified in cases of feline insulin hr ; the abdominal ultrasound performed in the cat reported here failed to identify any gall bladder abnormalities . management of insulin hr involves exclusion of poor injection technique , use of alternative forms of insulin , splitting the dose and injection into separate sites , and addition of antihistamines . if these methods fail , a local injection of dexamethasone in combination with insulin is sometimes used . steroid therapy was not pursued in this cat as we were concerned it would further complicate the management of the dm . similarly , we also did not attempt to divide the dose or inject it in separate sites as the cat had exhibited such severe clincial signs of hypersensitivity initially . poor injection technique or improper storage of insulin was not considered likely as the owners had successfully managed the cat s dm for 3 years . desensitization , using gradually increasing dilutions of insulin intradermally in a controlled setting , can be successful in type i hr , but can require long periods of time to be beneficial . desensitization in people appears to be less successful in patients with the biphasic type i hr . use of newer insulins such as aspart or gi has also been recommended , but hrs to these insulins have also been recently recognized in human medicine . in the cat corticosteroids and epinephrine have been recommended for hrs in veterinary patients but were considered potentially contraindicated given the pre - existing dm , severe hyperglycemia and potential to cause ketosis . the cat appears to be well managed on pzi without any reported signs of an hr . insulin hrs were much more common in human patients prior to the use of human insulin analogues when bovine and porcine insulin were more commonly administered . it is believed that humans that have an hr to insulin are reacting to the type of protein in the insulin given that there are molecular differences between insulin from different species . we elected to use pzi , a protamine - containing recombinant human insulin , in this cat rather than gi , a recombinant human insulin analogue , as this contains a different form of protein . both gi and pzi also contain different adjuvants , with pzi mostly containing zinc and gi mainly containing metacresol . as such , it is unclear if the hr developed secondary to the adjuvants rather than the different insulin protein molecules . in human medicine it is much more common to react to the insulin molecule rather than the adjuvants . furthermore , while we can not completely rule out improper storage or poor administration , the bottle of gi was newly purchased , stored in a refrigerator and had never been used prior to the suspect hr event . the owners had also successfully managed the cat s dm for 3 years and were well versed in subcutaneous administration of insulin , so poor technique does not seem likely . we can not rule out that cats do not naturally develop pruritis and erythema from intradermal gi injections and further studies should evaluate intradermal gi injections in healthy cats . clinicians should be aware of the possibility of hrs to insulin administration in cats , particularly in cats with a previous history of insulin administration . the incidence of hrs to insulin and use of intradermal insulin testing for confirmation in veterinary medicine requires further evaluation . furthermore , it is the most common cause of antenatal hospital admission.1 the incidence of preterm birth rate in us was 12% in 2011.2,3 at siriraj hospital , a tertiary care in central part of thailand , the preterm birth rate was 9.4% in 2004 and 13.7% in 2010.4 srinagarind hospital is another tertiary care center in northeastern part of thailand ; the preterm birth rate was as high as 11.5% in 2013 ( unpublished data ) . the complications of preterm births arise from immature organ systems that are not yet ready to support life in the extrauterine environment . for instance , respiratory distress syndrome is caused by a lack of surfactants in the lung epithelium . antenatal administration of corticosteroids to women who were expected to give preterm birth was associated with a significant reduction of neonatal morbidity and mortality , respiratory distress syndrome , intraventricular hemorrhage , and necrotizing enterocolitis of preterm infants . national institute of health and the american college of obstetrician and gynecologists recommended a single course of corticosteroids for pregnant women between 24 and 34 weeks of gestation who are at risk of preterm delivery within 7 days.5,6 the use of tocolytic agents to suppress preterm uterine contraction can delay delivery for at least 48 hours , and this prolongation enable the complete administration of corticosteroids to obtain their maximum effect for gestational age of 2434 weeks.6 despite of the available modalities to prevent preterm birth and its consequences , the proportions of preterm birth are not reduced.7 therefore , we planned to evaluate the success rate of preterm uterine contraction inhibition with tocolytic agents and factors associated with inhibition failure to aware the possibility of preterm delivery . this observational retrospective study was approved by the khon kaen university ethics committee for human research based on the declaration of helsinki and the ich good clinical practice guidelines ( he571337 ) . after reviewing the data collection forms from the medical records of the project , the ethics committee deemed patient written informed consent was not required , as this was a retrospective study . we have reviewed the medical records of all singleton pregnant women between 24 and 33 weeks of gestation admitted to the labor room of srinagarind hospital , khon kaen university , thailand , between january 2013 and july 2014 , and selected the patients who received uterine contraction inhibition with tocolytic drugs under diagnosis of preterm labor or threatened preterm labor . multiple pregnancies , women with diagnosis of dead fetus in utero , and lethal fetal anomalies were excluded . preterm labor was defined as regular uterine contractions accompanied by a change in cervical dilatation , effacement , or both.6 threatened preterm labor was defined as regular uterine contractions occurring at the frequency of at least once in every 10 minutes during 30 minutes of monitoring , with no dilatation and effacement of cervix.4 the high risk of preterm delivery was defined as group of women with any history of obstetric , medical , or surgical complication during pregnancy . the gestational age determination was based on the certain date of the last menstrual period or first trimester ultrasound examination . their medical records were assessed for the baseline characteristics including age , weight , gravidity , parity , gestational age on admission , numbers of antenatal care visit , characteristics of uterine contraction , and cervical status to classify studied women to preterm labor or threatened preterm labor . the types , modes , duration of administration , and the adverse effects of the tocolytic agents used for uterine contraction inhibition were also retrieved . the pregnancy prolongation was measured in terms of the duration ( days ) and the gestational age ( complete weeks ) whether it was beyond 34 and 37 weeks or not . additionally , we also reviewed all of neonatal outcomes including gestational age , route of delivery , appearance , pulse , grimace , activity , respiration ( apgar ) scores , and birth weight . the tocolytic agents commonly used in srinagarind hospital are adalat ( nifedipine ) , bricanyl ( terbutaline ) , and ventolin ( salbutamol ) . the course of oral nifedipine ( 10 mg ) started with the initial load every 15 minutes with the maximum dose of 40 mg until no uterine contraction was observed , followed by the maintenance dose of 10 mg every 6 hours for 24 hours , an then tapered every 8 hours for 24 hours , every 12 hours for 24 hours , and once daily , and discontinue if there was no more contraction . for terbutaline , 4 ampules ( 2 mg ) were added to 5% d / w 100 ml and intravenously dripped beginning with the rate of 5 microdrops / min , then increased 5 microdrops / min every 30 minutes until there was no uterine contraction or getting the maximum rate of 45 microdrops / min . if there was no uterine contraction for 24 hours , the dripping rate would be reduced to 5 microdrops / min until off . in case of salbutamol , 1020 mg of salbutamol if the preterm pregnant women still have uterine contraction while using these drug regimens or have some intolerable side effects , they were classified as inhibition failure and the attending physicians would change to other drugs or none . doses of these drugs could be modified in some cases , depending on the associated maternal or fetal conditions and the judgment of attending physicians . since in our setting , only preterm pregnant women of 24 to 33 weeks of gestation would receive a course of antenatal corticosteroid administration for accelerate fetal lung maturation . so the success of preterm uterine contraction inhibition was assessed as the delay in delivery for at least 48 hours after tocolytics administration between 24 and 33 week of gestation while the inhibition failure was defined as the delay in delivery for < 48 hours . the risk factors of the inhibition failure were also analyzed in this study . the sample size calculation based on the objective to assess the success rate of preterm uterine contraction inhibition with the maximal proportion of defined women was 0.5% and 10% of allowance error . the study required at least 96 singleton pregnant women who admitted to the labor room with preterm uterine contraction and received tocolytic agents for inhibition . for statistical analysis , mean , standard deviation ( sd ) , number , and percentage were calculated . the chi - square and fisher s exact test were used to evaluate the risk factors of labor inhibition failure . these variables were then included if p - value < 0.20 in a stepwise logistic regression analysis to determine which factors , if any , were independently associated with the risk of labor inhibition failure . adjusted odds ratio with a 95% confidence interval ( ci ) , which did not include unity were considered statistically significant . during the studied period , a total of 424 pregnant women met the diagnosis of preterm labor or threatened preterm labor . after exclusion of 39 multiple pregnancies , a women with dead fetus in utero , 2 women with lethal fetal anomalies , 3 women whose medical records were lost , 262 women between 34 and 36 weeks of gestation and also 14 women between 24 and 33 weeks of gestation whose uterine contractions were not inhibited , 103 singleton pregnant women at 24 to 33 weeks of gestation who received tocolytic agents were reviewed . around three - fourth of them ( 77.7% ) met the clinical criteria of threatened preterm labor , while 22.3% met the diagnosis of preterm labor . there were histories of obstetrical , medical , and/or surgical complications during pregnancy among studied women . anemia was the most frequently associated condition ( 34% ) , followed by previous abortion ( 29.1% ) , preterm pre - labor rupture membranes ( 17.5% ) , urinary tract infection or asymptomatic bacteriuria ( 15.5% ) , and history of preterm labor / delivery ( 11.7% ) and vaginal bleeding ( 9.7% ) . few cases of other complications were autoimmune diseases ( 3 cases ) , genital tract infection ( 3 cases ) , infective diarrhea / food poisoning ( 3 cases ) , polyhydramnios ( 3 cases ) , and 1 case each of chorioamnionitis , sepsis , uterine leiomyoma , abdominal surgery during pregnancy , and uterine cervical surgery . in table 2 , the overall success rate of preterm uterine contraction inhibition for prolongation of pregnancy at least for 48 hours was 86.4% . the success rate among who were diagnosed as threatened preterm labor on admission was high ( 93.8% ) , while the success rate among who were diagnosed as preterm labor on admission was low ( 60.9% ) . approximately half of the studied women ( 55.3% ) had prolonged pregnancy beyond 34 weeks . one - third of them ( 38.8% ) gave birth after 37 weeks of gestation . regarding the type of tocolytic agents , terbutaline was the most frequently selected tocolytic agent , followed by nifedipine . intolerable side effects of tocolytic agents were observed in 13 cases , whose medication was discontinued . among these 13 cases , 5 cases developed hypotension after nifedipine treatment and 8 cases showed maternal tachycardia / dyspnea and fetal tachycardia after terbutaline treatment . duration of tocolytic agent administration for labor inhibition was 25 days ( median = 4 days ) . mean baby birth weight was 2,370.9 g ( sd : 833.6 ) , and their mean gestational age at birth was 34.4 weeks ( sd : 3.5 ) . almost half of them ( 49.5% ) were low birth weight ( < 2,500 g ) babies . there were 4 cases of severe birth asphyxia whose apgar score was < 4 at 5 minutes after birth . in the univariate analysis , risk factors including preeclampsia with severe feature , history of abortion , more than 24 hours of uterine contraction , and history of cervical surgery were not significantly associated with inhibition failure ( p - value > 0.20 ) . when using multivariate logistic regression analysis , only preterm labor had statistical significant associated with inhibition failure . estimated worldwide incidence of preterm birth was 9.6% in 2005 , mostly occurred in africa and asia.8 in 2010 , low birth weight babies worldwide were estimated to be around 18 million , and 59% of them were small - for - gestational age term and 41% were preterm . two - third of small - for - gestational age infants were born in asia.9 the understanding of the causes of premature delivery , low birth weight babies and improvement of the effectiveness of available modality of intervention are needed to improve birth outcomes . the results of this study from a tertiary care hospital in northeastern thailand showed the same tendency of worldwide burden of preterm pregnancy complications . women with uterine contraction before 34 weeks of gestation were admitted to the hospital to prolong pregnancy with tocolytic agents . the majority of them ( 86.4% ) could continue pregnancy at least 48 hours after receiving the tocolytic agents and receive completed corticosteroids course to accelerate fetal lung maturity . however , 77.7% of these studied women presented with threatened preterm labor , which is defined as uterine contraction without cervical dilatation . the significant risk factor of inhibition failure was preterm uterine contraction with cervical dilatation ( preterm labor ) . similar to the authors present results , motazedian et al reported that a 48-hour prolongation of pregnancy was achieved in 87% patients receiving terbutaline and 84% patients receiving salbutamol.10 likewise , the study at siriraj hospital , bangkok , thailand , revealed that the success rate in threatened preterm labor inhibition for 12 hours with nifedipine was about 80%.11 however , in cases of women with advanced cervical dilatation ( 48 cm ) who received one or more tocolytics ( magnesium sulfate , indomethacin , or nifedipine ) , only 23% could prolong pregnancy at least 48 hours after admission.12 similarly , in cases of women with the diagnosis of preterm labor based on the presence of uterine contraction with documented cervical change , success rate by nifedipine was 70%.13 in these two studies with the lower success rates , the patients presented as preterm labor with apparent cervical changes . we noticed that , regardless of number and the type of tocolysis , the success rate was higher when uterine contraction inhibition was applied to women with preterm uterine contraction without cervical changes . in the present study , the success rate to prolong pregnancy at least 48 hours was 93.8% in threatened preterm labor group and that of preterm labor group was 60.9% . furthermore , the authors data revealed that preterm labor ( uterine contraction with cervical change ) was the significant risk factor of labor inhibition failure . the main rationale of tocolysis for preterm labor are to delay delivery for at least 48 hours to allow completed course of corticosteroids to accelerate fetal lung surfactant production and allow to transfer mother to a tertiary facility . betamimetics and calcium channel blockers reduce the number of preterm labor women who will give birth within 48 hours . nevertheless , tocolysis does not appear to significantly lengthen the gestational age beyond 7 days.14 it is known that nifedipine is easy to administer and have fewer side effects relative to betamimetics.15 the authors results also support this notion as intolerable side effects occurred more in cases of betamimetics treatment . in spite of the success rate of preterm uterine contraction inhibition was as high as 86.4% in this study , for example , the mean birth weight of babies was approximately 2,300 g and a half of them had low birth weight ( < 2,500 g ) . severe birth asphyxia was seen in 3.9% and more than half ( 58.3% ) of the delivery required surgical intervention . thus , we should provide advanced neonatal care and prepare for complicated delivery for the pregnancy complicated with preterm uterine contraction with or without cervical dilatation . limitations of this study were those inherent to any retrospective study which made it difficult to gain the complete data . fortunately , most of the outcomes of interest were available in the retrieved medical records . another was the limited number of studied women , in that the majority of women enrolled in this study were those with threatened preterm labor that would lead to overestimation of the overall success rate of uterine contraction inhibition . however , we tried to minimize such effects by separating the patients into threatened preterm and preterm labor groups for outcome analysis . regarding the evaluation of pregnancy outcomes of uterine contraction inhibition with tocolytic agents , further research on threatened preterm labor with or without tocolysis would be necessary . also , whether preterm uterine contraction with cervical change should get high success rate as preterm uterine contraction without cervical change if the tocolytics could attenuate uterine contraction should be explored in future . the success rate of preterm uterine contraction inhibition with tocolytic agents to delay delivery for at least 48 hours was high in threatened preterm labor and low in preterm labor . a significant risk factor for inhibition failure was the preterm uterine contraction with cervical change .

case summarya 14-year - old , domestic shorthair cat was treated for transient diabetes mellitus for 3 months with glargine insulin , which was discontinued when the diabetes mellitus resolved . approximately 36 months later the diabetes mellitus recurred and glargine insulin was restarted . within 23 mins of the first injection the cat collapsed , developed profuse vomiting and diarrhea , as well as facial swelling and diffuse erythema . a hypersensitivity reaction was suspected and the cat was treated with antihistamines , aggressive fluid therapy and gastrointestinal support . the cat made a full recovery and was discharged 3 days later . six months later the cat re - presented for relapse of its diabetes mellitus and an intradermal skin challenge with 1:20 diluted insulin was performed confirming a hypersensitivity to glargine . the cat continues to be well regulated on porcine zinc insulin without any hypersensitivity reactions noted.relevance and novel informationhypersensitivity reactions to insulin administration are rarely described in human medicine . this is the first reported case of a hypersensitivity reaction secondary to glargine insulin in a cat . clinicians should be aware of this potential complication , particularly in animals with a previous history of insulin administration and the potential to utilize intradermal testing with insulin . objectivethis study aims to assess the success rate of inhibiting preterm uterine contraction with tocolytic agents to delay delivery for at least 48 hours and risk factors of failure inhibition.materials and methodsbetween january 2013 and july 2014 , medical records of all singleton pregnant women between 24 0/7 and 33 6/7 weeks of gestation with the diagnosis of preterm labor ( with cervical dilatation ) or threatened preterm labor ( without cervical dilatation ) who received tocolytic agents were reviewed . the success rate of preterm uterine contraction inhibition was accounted in patients with 48 hours delayed delivery . the risk factors of the inhibition failure and neonatal outcomes were also investigated in this study.resultsamong 424 pregnant women diagnosed of preterm labor or threatened preterm labor , 103 singleton pregnant women met the study criteria . overall success rate of preterm uterine contraction inhibition to prolong pregnancy for at least 48 hours was 86.4% ( 95% confidence interval [ ci ] : 78.3 , 92.3 ) . however , the success rate among the threatened preterm labor group was 93.8% ( 95% ci : 88.3 , 99.1 ) while the preterm labor group was 60.9% ( 95% ci : 39.3 , 82.4 ) . the significant factor associated with inhibition failure was preterm labor ( adjusted odds ratio 7.22 ; 95% ci : 1.99 , 26.20).conclusionthe success rate of preterm uterine contraction inhibition with tocolytic agents to delay delivery for at least 48 hours was high in threatened preterm labor and low in preterm labor . a significant risk factor for inhibition failure was the preterm uterine contraction with cervical change .

achalasia is a primary esophageal motor disorder of unknown etiology characterized by selective loss of inhibitory neurons in the esophageal wall , resulting in insufficient relaxation of the lower esophageal sphincter and loss of esophageal peristalsis.1,2 jackhammer esophagus is a newly introduced term to describe patients with at least 20% of the swallows with distal contractile integral ( dci ) 8000 mmhg.sec.cm.3 several reports have described patients with diffuse esophageal spasm ( des ) , non - specific esophageal motor disorder ( nsemd ) , nutcracker esophagus , and gastroesophageal reflux disease ( gerd ) progressing to achalasia.49 although no causal relationship has been identified , these reports suggest that the different esophageal motor disorders represent a spectrum rather than unique and stable disorders . we describe , for the first time , a case of a patient who progressed from jackhammer esophagus to type ii achalasia . a 66 year - old woman with a history of atrial fibrillation , hypertension , gastritis , and hypothyroidism presented to our clinic in july 2014 with progressive dysphagia for solids and liquids over 18 months . her symptoms occurred daily and resulted in an 11-kg weight loss over this period of time . she denied heartburn , regurgitation , choking or coughing during eating , chest pain , vomiting , abdominal pain , or change in bowel habits . the barium swallow showed a mildly dilated esophagus with tertiary contractions , delayed emptying of the esophagus , and a narrowed gastroesophageal junction ( fig . 1 ) . the upper endoscopy demonstrated mild - moderate antral gastritis , normal appearing esophageal mucosa , and it was noted that the patient might have a tight gastroesophageal junction . unfortunately , after completing these tests she missed 3 follow - up appointments and was seen in our clinic only one year later . given her progressive dysphagia symptoms and weight loss , a high resolution esophageal manometry ( hrem ) was performed ( fig . the study showed a normal median integrated residual pressure of 8 mmhg , normal mean resting pressure of 21 mmhg , normal mean residual pressure of 7 mmhg , and mean dci of 10 770 mmhg sec cm . of the 10 swallows , 30% were hypercontractile ( > 8000 mmhg sec cm ) , 50% were normal and 20% simultaneous . overall , the findings were consistent with jackhammer esophagus based on the chicago classification of motility disorders.3 pantoprazole was increased to 40 mg twice daily with plan to add a pain modulator if her symptoms recurred . unfortunately , she was lost to follow - up again until she was seen in our clinic a year later in june 2015 . she continued to have worsening dysphagia , lost an additional 7 kg , and reported new onset of occasional post - prandial chest tightness . at this point , 3 ) . this study demonstrated diffuse esophageal dilatation with retained secretions and barium with only 020% change in esophageal volume after 5 minutes . in addition , tertiary peristaltic waves within the distal esophagus , limited emptying of the contrast into the stomach and an air - fluid level were also noted . 4 ) revealing a median integrated residual pressure of 71.5 mmhg , mean resting pressure of 97 mmhg , and mean residual pressure of 90 mmhg . there was panesophageal pressurization with every swallow and 80% of the swallows demonstrated prolonged pan - pressurization , all consistent with type ii achalasia . therapeutic options were discussed with the patient , and she elected to undergo heller myotomy . the patient was referred to general surgery but presented urgently to gi clinic 2 weeks later for worsening dysphagia , dehydration , weakness , inability to tolerate oral intake , and an additional 7-kg weight loss . the patient was admitted to the hospital and was noted to develop acute kidney injury . in addition , a nasogastric tube was placed and she was started on tube feeds to optimize her nutritional status . a week after discharge the patient underwent heller myotomy with dor fundoplication . on a follow - up visit with her general surgeon , she reported that she was tolerating liquids without any difficulties . to our knowledge , this is the first case that describes progression of jackhammer esophagus to achalasia over a period of about one year . smart et al5 reported 5 patients with gerd who subsequently developed achalasia over a period of 210 years . robson et al4 reported a patient with gerd who developed des one year after initial diagnosis and achalasia one year later . several reports describe the progression of des to achalasia.68 khatami et al10 conducted the first prospective cohort study with 12 patients . they observed the progression of des to achalasia in only one patient after 10.6 years . they also noted that low esophageal body amplitude contractions was a predictor of this progression . fontes et al11 conducted the largest prospective study to date to assess the progression of des to achalasia . patients with gerd confirmed by ph monitoring or systemic diseases that may affect the esophagus were excluded . five ( 14% ) of the patients progressed to achalasia at a mean follow - up of 2.1 years . although demographic characteristics were not predictive of the transition to achalasia , the authors observed that amplitude contractions of less than 50 mmhg was a predictive factor ( p = 0.002 ) . anggianash et al9 described the progression of nutcracker esophagus to achalasia in one patient after 3 years . vantrappen et al13 described six patients with nsemd who progressed to achalasia . while our patient presented with symptoms that could be considered classic for achalasia , her initial conventional manometry was consistent with nsemd . only the second hrem as well as the timed barium esophagram were consistent with achalasia . this progression within one year of an esophageal motor disorder was faster than what has been documented in previous reports . however , this case suggests that all esophageal motility disorders represent a spectrum of disorders , where patients may progress over time from one to another . this case also demonstrates the importance of following patients who were diagnosed with non - achalasia motility disorders with a repeat hrem in one year to assess for progression or a shift to a different esophageal motility disorder . because most of the studies documenting progression of a motility disorder to achalasia were case reports using conventional manometry , long - term studies evaluating this progression with hrem are needed . perhaps with the current availability of hrem , researchers may be able to identify predictive factors associated with progression of certain esophageal motility disorders to achalasia . the international cancer genome consortium ( icgc ) is a multidisciplinary , multi - institutional collaborative effort aiming to systematically and comprehensively characterize somatic mutations in 50 different cancer types and subtypes ( 1 ) . five hundred tumor genomes , as well as matched normal control genomes for each cancer type , will be analyzed using high - throughput next - generation sequencing technologies to detect a wide range of somatic mutations , including single nucleotide mutations , small insertions / deletions , copy number alterations , translocations and other chromosomal structural rearrangements . genome - wide methylation state analysis and whole - transcriptome sequencing have also been planned to provide additional molecular - level characterizations . to make the effort more scalable each member institution specializes in generating data for a particular tumor type . at the time of writing , figure 1.international cancer genome consortium ( icgc ) projects ( march 2011 ) . international cancer genome consortium ( icgc ) projects ( march 2011 ) . one of the major goals of icgc is to rapidly bring these data to the cancer research community in order to accelerate studies on the discovery of cancer causes , to enhance the accuracy of diagnoses and to improve treatments . in order to achieve this task , the data generated by the consortium members have to be managed efficiently . amongst the most important data management challenges faced by the consortium is the high complexity and heterogeneity of the data types involved , the necessity to link different data types , and the need to protect controlled data . furthermore , the high volume of data and the distributed nature of the sources make traditional centralized approaches to data management impractical . consequently , the icgc has adopted federated data architecture to address their data management needs . the scalability of the system is improved by having each member institution store and process data locally ; the data federation software then presents these separate sources as a single access point for remote data access . biomart , an open source data federation system ( 2 ) , has been chosen as the icgc data management platform . biomart s flexible data model makes it generally applicable to a wide range of biological data types and built - in query optimizations make it suitable for large data sets . in addition , biomart supports an industry - standard security framework that is needed to provide secure access to the controlled data . finally , a large number of existing expert - maintained public annotation databases can be readily federated , adding value to the interpretation of the icgc experimental data . the architecture of the icgc data management system has been modeled on the biomart central portal ( 3 , 4 ) . for a number of years , the portal has been successfully providing a single point of access to a large number of biological databases distributed across the world . each biomart database federated in this portal is maintained independently , released and updated on its own schedule . similarly , each icgc member maintains their local biomart database where clinical annotations and the data produced from genomic analyses are deposited . however , in order to make these data comparable across different cancer projects and create a unified icgc data set , a mechanism for enforcing uniformity is required . thus , the same set of data models , controlled vocabularies , ontologies and reference data sets are used in all of the icgc member databases . furthermore , a gene - centric data model adopted from ensembl mart ( 5 ) is used to reference experimental data to the same set of annotated genes . in order to link individual biomart databases into a unified system , these servers can each access data in three ways : they can access their own local databases directly ; they can communicate with the other icgc biomart servers , in order to retrieve data from remote databases ; and they can communicate with non - icgc biomart servers , such as cosmic ( 6 ) and reactome ( 7 ) , to retrieve publicly - available annotation data . in this manner , the biomart server maintained by each icgc member acts as a fully featured icgc data portal , providing unified access to all the consortium data . the users select a combination of different data sets and specify query criteria using a variety of graphical user interfaces and analysis tools that are available from the portal . application programming interfaces ( apis ) for java , rest , soap and sparql are also available for programmatic access . behind the scenes , biomart software breaks down this query into smaller parts that are distributed to the remote data sources . the results are collected and compiled into a single unified results set that is presented to the user . some of the data in the portal has access restrictions in order to preserve patient privacy . to protect this sensitive data , communication between biomart servers when a user logs into an icgc data portal server using his / her openid , the server will consult the central registry to ensure that the user is authorized to access the data ( figure 2 ) . at present the portal contains data from 24 cancer projects , consisting of 3478 genomes and 13 cancer types and subtypes . this includes the data generated from seven studies performed by five icgc participating institutions located in four countries . the data portal also hosts data from other large - scale cancer genome projects including the cancer genome atlas ( tcga ) ( 8 , 9 ) , tumor sequencing project ( tsp)(10 ) and johns hopkins university ( 1114 ) . open access data sets include ( i ) simple somatic mutations , ( ii ) copy number alterations , ( iii ) structural rearrangements , ( iv ) gene expression , ( v ) mirna , ( vi ) dna methylation and ( vii ) exon junctions . secure access to controlled data sets , such as germline variations , is available to authorized users . a summary of data currently available on the data portal is shown in table 1 . in addition to cancer genomic data , the data portal also federates several public databases ( table 2 ) . currently this includes ensembl genome database ( 5 ) , kyoto encyclopedia of genes and genomes ( kegg ) ( 1517 ) , reactome ( 7 ) , cosmic ( 6 ) , pancreatic expression database ( 18 ) and the breast cancer campaign tissue bank ( 19 ) , and the number of resources is continuously growing . table 1.the summary of data available on the icgc data portal divided by cancer projectsourcecancer projectdata setsimple mutationscopy number alterationsstructural rearrangementsgene expressionmirna expressionexon junctionsdna methylationgermline variationsicgcbreast carcinoma ( wtsi , uk)liver cancer ( ncc , jp)liver cancer ( riken , jp)malignant melanoma ( wtsi , uk)pancreatic cancer ( oicr , ca)pancreatic cancer ( qcmg , au)small cell lung carcinoma ( wtsi , uk)tcgaacute myeloid leukemiabreast invasive carcinomacolon adenocarcinomaglioblastomamultiformekidney renal clear cell carcinomakidney renal papillary cell carcinomalung adenocarcinomalung squamous cell carcinomaovarian serous cystadenocarcinomarectum adenocarcinomastomach adenocarcinomauterine corpus endometrioid carcinomaotherbreast cancer ( jhu , us)colorectal cancer ( jhu , us)glioblastomamultiforme ( jhu , us)lung adenocarcinoma ( tsp , us)pancreatic cancer ( jhu , us) table 2.public databases federated with the icgc data portalsourceurldescription of contentsensemblwww.ensembl.orggenome annotationreactomewww.reactome.orgpathway annotationkeggwww.genome.jp/keggpathway annotationcosmicwww.sanger.ac.uk/genetics/cgp/cosmic/somatic mutations in cancerpancreatic expression databasewww.pancreasexpression.orgpancreatic cancer expression databreast cancer campaign tissue bankwww.breastcancertissuebank.org/bio-informatics.phpbreast cancer expression data the summary of data available on the icgc data portal divided by cancer project public databases federated with the icgc data portal the data portal provides three major interactive entry points : identifier search , analysis and database search ( figure 3 ) . identifier search ( figure 3a ) lets users input identifiers that are commonly used in public annotation databases ( e.g. hgnc gene symbol , ensembl i d , refseq i d , uniprot i d and other accessions ) and returns links to the corresponding gene report page , which displays basic gene description , pathway annotation , mutations found in the cosmic database , as well as pancreas and breast cancer expression data ( figure 4 ) . in addition , the gene report displays a summary of mutation frequencies in the selected gene across all cancer projects ( figure 4d ) . records in the gene report ( i.e. genomic coordinates , pathway name , publication ) are linked to appropriate resources when available , allowing users to easily retrieve additional information on their data of interest . three main entry points are available : ( a ) identifier search , ( b ) analysis and ( c ) database search . figure 4.gene report for kras includes : ( a ) gene annotation data from ensembl , pathway annotation from ( b ) kegg and ( c ) reactome , ( d ) summary of mutation frequencies in each cancer project , ( e ) mutation data from cosmic , expression data from ( f ) pancreatic expression database and ( g ) breast cancer campaign tissue bank ( bcctb ) . different sections of this page come from federated biomart sources . three main entry points are available : ( a ) identifier search , ( b ) analysis and ( c ) database search . gene report for kras includes : ( a ) gene annotation data from ensembl , pathway annotation from ( b ) kegg and ( c ) reactome , ( d ) summary of mutation frequencies in each cancer project , ( e ) mutation data from cosmic , expression data from ( f ) pancreatic expression database and ( g ) breast cancer campaign tissue bank ( bcctb ) . different sections of this page come from federated biomart sources . to help with the interpretation of cancer data , gene and pathway analysis tools are available in the analysis section of the data portal ( figure 3b ) . these tools enable users to view the most commonly affected genes or pathways in one or more cancer projects . results are presented in an easy - to - follow chart that can be exported as an image file , and the numerical data can also be downloaded for further processing ( figure 5 ) . ( a ) affected pathways are shown in a chart , with bars representing the number of affected genes in each pathway . ( b ) by clicking on the bar , users are able to view and download the genes that were mutated in each pathway . ( a ) affected pathways are shown in a chart , with bars representing the number of affected genes in each pathway . ( b ) by clicking on the bar , users are able to view and download the genes that were mutated in each pathway . using the database search entry point ( figure 3c ) users can interactively query the database by several data types : genes , samples , simple mutations , copy number alterations , structural rearrangements , gene expression , mirna , dna methylation and exon junctions . the quick interface contains a pre - selected set of the most commonly used filters such as gene type , mutation type and chromosome , and outputs a fixed set of attributes . the flexible interface contains additional filters and a selection of attributes , allowing the user to choose which data are displayed in the output . the advanced interface contains the complete set of filters and attributes , including technological platforms used for sequencing , and clinical parameters such as patient gender and tumor histopathology . to demonstrate the utility of the icgc data portal we present several queries that can be performed using different query interfaces . search for genes affected by copy number loss and also detected as deletion from structural rearrangement analysis. ( figure 6 ) data setscancer projectsfiltersattributesgenespancreatic cancer ( qcmg , au)rearrangement type : deletionensembl gene idcopy number alteration type : lossgene symbolgene description figure 6.a screenshot of the quickquery interface . query # 2 ( flexible search ) : retrieve clinical staging data for colorectal cancer patients with non - synonymous simple mutations in genes that are involved in wnt signaling pathway. ( figure 7 ) data setscancer projectsfiltersattributesgenescolorectal cancer ( jhu , us)pathway : signaling by wnttumor sample idconsequence type : non_synonymous _ codingmutation idmutation typedonor iddiagnosis idclinical staging ( who)ensembl gene idgene symbol figure 7.a screenshot of the flexiblequery interface . a screenshot of the flexiblequery interface . query # 3 ( advanced search ) : in pancreatic cancer data set , retrieve all rna - seq expression data for genes that are affected by copy number gains. ( figure 8) data setscancer projectsfiltersattributesgenespancreatic cancer ( qcmg , au)copy number alteration type : gainensembl gene idplatform : solid sequencinggene expression : sample idnormalized read countraw read count figure 8.a screenshot of the advancedquery interface . the icgc data portal is the first project to successfully federate large amounts of cancer genomics data and rich annotation data in a single access point . it presents a scalable approach , not only in the traditional sense of parallelizing data processing and storage , but also in a more general sense of outsourcing the external annotation expertise , by federating annotations from independently maintained databases . this approach has proved to be successful in addressing icgc data management needs and can be useful for similar , large - scale collaborative projects . the icgc data portal will continue to expand by adding more data , both from within the project in the form of new cancer genomics data from icgc members , and by integrating other public annotation databases to further the depth of analysis possible through the data portal . additionally , new tools will be developed to increase the flexibility and utility of the system . this includes tools for icgc deployers , to further streamline the data processing , transformation and loading processes , and tools for users , to add new methods of data visualization and analysis to the portal .

it has been suggested that patients with certain motility disorders may progress overtime to develop achalasia . we describe a 66 year - old woman who presented with dysphagia for solids and liquids for a period of 18 months . her initial workup showed normal endoscopy and non - specific esophageal motility disorder on conventional manometry . six months later , due to persistence of symptoms , the patient underwent a high resolution esophageal manometry ( hrem ) demonstrating jackhammer esophagus . the patient was treated with a high dose proton pump inhibitor but without resolution of her symptoms . during the last year , the patient reported repeated episodes of food regurgitation and a significant weight loss . a repeat hrem revealed type ii achalasia . multiple case reports , and only a few prospective studies have demonstrated progression from certain esophageal motility disorders to achalasia . however , this report is the first to describe a case of jackhammer esophagus progressing to type ii achalasia . the international cancer genome consortium ( icgc ) is a collaborative effort to characterize genomic abnormalities in 50 different cancer types . to make this data available , the icgc has created the icgc data portal . powered by the biomart software , the data portal allows each icgc member institution to manage and maintain its own databases locally , while seamlessly presenting all the data in a single access point for users . the data portal currently contains data from 24 cancer projects , including icgc , the cancer genome atlas ( tcga ) , johns hopkins university , and the tumor sequencing project . it consists of 3478 genomes and 13 cancer types and subtypes . available open access data types include simple somatic mutations , copy number alterations , structural rearrangements , gene expression , micrornas , dna methylation and exon junctions . additionally , simple germline variations are available as controlled access data . the data portal uses a web - based graphical user interface ( gui ) to offer researchers multiple ways to quickly and easily search and analyze the available data . the web interface can assist in constructing complicated queries across multiple data sets . several application programming interfaces are also available for programmatic access . here we describe the organization , functionality , and capabilities of the icgc data portal.database url : http://dcc.icgc.org

a 64-year - old male patient who had been on diabetes medication with no other particular medical history visited the clinic for symptoms of chillness , and his temperature was measured to be 38.8. an abdomical ct revealed a 6-cm abscess surrounding a pseudoaneurysm of about 4 cm near the right internal iliac artery , and mycotic aneurysm was diagnosed . although no particular strain was identified from the blood culture test conducted at the time of the clinic visit , based on the week - old abdominal ct results , which clearly showed the growing aspect of the aneurysm , risk of death due to rupture was judged to be high . before conducting a femoral artery bypass , a distal part of the right internal iliac artery was occluded using a 10-mm amplatzer vascular plug . the right common iliac artery and external iliac artery were also occluded in succession using 14-mm and 12-mm amplatzer vascular plugs , respectively . the patient was immediately moved to the or and a left fermoral artery - right femoral artery bypass was performed using a 10-mm ringed gore - tex graft . on the third day post - operation , a pigtail catheter was inserted into the abscess near the mycotic aneurysm of the mycotic aneurysm of the right iliac artery and the surrounding abscess was drained . postoperative abdominal ct showed complete occlusion of the right iliac artery and internal and external iliac arteries . when compared to the preoperative abdominal ct , the mycotic aneurysm and abscess were found to be completely removed ( fig . the patient was discharged after 4 weeks of antibiotic treatment with no symptomatic exacerbation found . currently , he has been off antibiotics for one year , and no complications have been detected in outpatient monitoring . a 56-year - old female visited the clinic with symptoms of fever , coughing , and chillness for 5 days . her temperature at the time of the visit was 38.2. she complained of pain in her left leg . two years and 5 months before her visit to the clinic , the patient had been diagnosed with perforated appendicitis and had received an appendectomy . however , postoperative complications including intraperitoneal abscess and enterocutaneous fistula resulted in treatments including right hemicolectomy , oophorectomy , and colostomy . in addition , the histopathology tests of the patient at the time resulted in the diagnosis of stage iia cecal cancer , and she received adjuvant concurrent chemo - radiotherapy for 12 months thereafter . since then , a pet scan for post - treatment evaluation showed a lump on the soft tissue of the right anterior abdominal wall . upon removal the patient received chemotherapy for an additional 11 months and was discharged . a ct scan to determine the cause of pain in the left lower limb at the time of the visit revealed a deep vein thrombosis . in addition , the part of small intestine creating an enterocutaneous fistula was removed following the previous operation . when detaching a severe adhesion on the abdominal wall , the right external iliac artery was damaged and sutured . on the tenth day post operation , lower right abdominal pain and wound infection were found . in a ct scan conducted during conservative treatment , an 8-cm lump suggestive of acute hematoma in the right external iliac fossa and a false aneurysm connected to the right external iliac fossa were found . an s&g stent graft of 10-mm diameter and 40-mm length was promptly inserted into the right external iliac fossa . although no strains were identified from blood culture testing , a joining of the infected area in the lower right abdomen and the false aneurysm was observed , resulting in the diagnosis of mycotic aneurysm . a 10-mm long amplatzer vascular plug was inserted into the right proximal external iliac artery . the patient was then immediately moved to the or and the right distal external iliac artery was ligated . a 12-mm ringed gore - tex graft was used to perform a left femoral artery - right femoral artery bypass . a postopeative lower extremity vascular ct scan showed complete withdrawal of the mycotic aneurysm previously present at the right external iliac artery ( fig . the patient was discharged 18 days after the operation without specific symptoms , but remote metastasis of the cecal cancer aggravated and she died after 4 months . a 64-year - old male patient who had been on diabetes medication with no other particular medical history visited the clinic for symptoms of chillness , and his temperature was measured to be 38.8. an abdomical ct revealed a 6-cm abscess surrounding a pseudoaneurysm of about 4 cm near the right internal iliac artery , and mycotic aneurysm was diagnosed . although no particular strain was identified from the blood culture test conducted at the time of the clinic visit , based on the week - old abdominal ct results , which clearly showed the growing aspect of the aneurysm , risk of death due to rupture was judged to be high . before conducting a femoral artery bypass , a distal part of the right internal iliac artery was occluded using a 10-mm amplatzer vascular plug . the right common iliac artery and external iliac artery were also occluded in succession using 14-mm and 12-mm amplatzer vascular plugs , respectively . the patient was immediately moved to the or and a left fermoral artery - right femoral artery bypass was performed using a 10-mm ringed gore - tex graft . on the third day post - operation , a pigtail catheter was inserted into the abscess near the mycotic aneurysm of the mycotic aneurysm of the right iliac artery and the surrounding abscess was drained . postoperative abdominal ct showed complete occlusion of the right iliac artery and internal and external iliac arteries . when compared to the preoperative abdominal ct , the mycotic aneurysm and abscess were found to be completely removed ( fig . the patient was discharged after 4 weeks of antibiotic treatment with no symptomatic exacerbation found . currently , he has been off antibiotics for one year , and no complications have been detected in outpatient monitoring . a 56-year - old female visited the clinic with symptoms of fever , coughing , and chillness for 5 days . her temperature at the time of the visit was 38.2. she complained of pain in her left leg . two years and 5 months before her visit to the clinic , the patient had been diagnosed with perforated appendicitis and had received an appendectomy . however , postoperative complications including intraperitoneal abscess and enterocutaneous fistula resulted in treatments including right hemicolectomy , oophorectomy , and colostomy . in addition , the histopathology tests of the patient at the time resulted in the diagnosis of stage iia cecal cancer , and she received adjuvant concurrent chemo - radiotherapy for 12 months thereafter . since then , a pet scan for post - treatment evaluation showed a lump on the soft tissue of the right anterior abdominal wall . upon removal the patient received chemotherapy for an additional 11 months and was discharged . a ct scan to determine the cause of pain in the left lower limb at the time of the visit revealed a deep vein thrombosis . in addition , the part of small intestine creating an enterocutaneous fistula was removed following the previous operation . when detaching a severe adhesion on the abdominal wall , the right external iliac artery was damaged and sutured . on the tenth day post operation , lower right abdominal pain and wound infection were found . in a ct scan conducted during conservative treatment , an 8-cm lump suggestive of acute hematoma in the right external iliac fossa and a false aneurysm connected to the right external iliac fossa were found . an s&g stent graft of 10-mm diameter and 40-mm length was promptly inserted into the right external iliac fossa . thereafter , the false aneurysm was found to have disappeared . however , a profound amount of hematoma was present in the affected area . although no strains were identified from blood culture testing , a joining of the infected area in the lower right abdomen and the false aneurysm was observed , resulting in the diagnosis of mycotic aneurysm . a 10-mm long amplatzer vascular plug was inserted into the right proximal external iliac artery . the patient was then immediately moved to the or and the right distal external iliac artery was ligated . a 12-mm ringed gore - tex graft was used to perform a left femoral artery - right femoral artery bypass . a postopeative lower extremity vascular ct scan showed complete withdrawal of the mycotic aneurysm previously present at the right external iliac artery ( fig . the patient was discharged 18 days after the operation without specific symptoms , but remote metastasis of the cecal cancer aggravated and she died after 4 months . mycotic aneurysm constitutes about 1% of all aneurysms , and its high risk of rupture requires immediate treatment . it tends to occur in a wide range of arteries , including in the lesser curve of the aortic arch and on the opposite side of the visceral branch vessel of the abdominal aorta . although the condition is often thought to be related to structural characteristics of the artery or the turbulent blood flow that occurs in the opposite side of abdominal branching blood vessels , the actual cause of the condition is still unknown . the major bacterial strains known to cause mycotic aneurysms are e. coli , staphylococcus , salmonella , and streptococcal species . these strains are related to atherosclerotic ulcers in the inner arterial membrane , and they act as a nidus for invading a secondarily infected arterial wall , triggering a false aneurysm or rupture of the artery . although the onset rate of mycotic aneurysm is comparatively low , the difficulty of diagnosis often results in late discovery of the disease after serious advancement of the condition . the mortality rate is fairly high , since it often develops in patients with lowered immunity or other underlying diseases . diagnosis of mycotic aneurysm can be done by identifying three typical symptoms : abdominal pain , fever , and a pulsating lump . however , it is unusual to find all the symptoms at once and symptoms are often nonspecific . a ct scan of a subject with a suspected mycotic aneurysm can reveal detailed information of the lesion and diagnosis can be done through blood culture testing . however , only 10~27% of postoperative mycotic aneurysms are found to be benign in tissue culture testing , and there have been no reports of any relationship to prognosis , such as a postoperative infection of the graft . treatment of mycotic aneurysm includes early diagnosis as well as broad removal of the lesion surgically following artificial blood vessel placement and long - term use of antibiotics . although treatment plays a critical role in a patient 's prognosis , several studies have reported high postoperative death rates ( 16~44% ) despite ongoing technical advancement . unlike the general aneurysm , mycotic aneurysm is often located near the upper portion of renal arteries . in that case , in situ graft placement is preferred , since the range of the lesion is broad and reconstruction of blood vessels that reach surrounding organs is required . on the other hand , in cases of mycotic aneurysm in the lower portion of renal arteries , extra - anatomic artificial blood vessel bypass has been recognized as the appropriate treatment . in both cases , if staphylococcus aureus , salmonella species , or other pyogenic infection is grossly suspected , in situ graft placement is forbidden , since the chance of a postoperative graft infection is high . a variety of vascular embolizations have been reported recently for extra - anatomic artificial vessel bypass on mycotic aneurysms in the inferior renal artery . vascular embolization is known to be suitable for the treatment of aneurysms in blood vessels that have below - average size , since the operational approach can be difficult . however , it has been controversial to place a foreign material in the infected artery in treatment of mycotic aneurysm and the risk of post - treatment complications such as rupture can increase due to a weakening of the arterial wall . therefore , these treatments can only be applied in patients with a low chance of continuous exacerbation of infection . in this case report , the authors have planned treatments after comprehensive consideration of the patients ' clinical symptoms as well as the location of lesions . we would like to introduce the bypass surgery in between bilateral femoral arteries following vascular embolization as a successful treatment option for mycotic aneurysm . microarray data is deposited at the ncbi gene expression omnibus ( geo ) database under geo series accession number gse52552 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse52552 ) . to gain insight in the mechanisms of complement resistance in m. catarrhalis , the transcriptional response of m. catarrhalis strain bbh18 , a complement resistant isolate , , upon exposure to 10% pooled normal human serum ( nhs ) was analyzed by microarray expression profiling . this concentration was chosen because test experiments demonstrated that a m. catarrhalis bbh18 gene deletion mutant lacking the key complement resistance factor uspa2h was rapidly killed in 10% nhs ( data not shown ) . to reduce the effect of day to day variation , two fully independent microarray expression profiling experiments were performed , indicated hereafter as experiments a and b ( table 1 ) . m. catarrhalis bbh18 was pre - cultured overnight on brain heart infusion ( bhi ) agar plates at 37 c in an atmosphere containing 5% co2 . bacteria were harvested from plates and resuspended in pbs supplemented with 0.15% gelatin ( pbs - g ) . next , 5 ml bhi medium was inoculated to an od600 nm of ~ 0.075 and cultured until mid - log phase ( od600 nm of ~ 1.0 ) , obtaining cultures with a density of 6 10 cfu / ml . of this culture , 4 ml was harvested by centrifugation , washed in 16 ml pbs - g , and resuspended in 20 ml pbs - g with 0.2 mm mgcl2 and 1 mm cacl2 ( ca and mg ) . 5.5 ml of this suspension was mixed with 5.5 ml of either pbs - g ( ca and mg ) ( control ; experiment a ; n = 3 , experiment b ; n = 2 ) or with 20% pooled nhs ( gti diagnostics ) in pbs - g ( ca and mg ) ( experiment a ; n = 3 , experiment b ; n = 3 ) and incubated at 37 c with agitation ( 200 rpm ) . after 0 , 30 ( experiment a only ) , and 60 min , a 3.5 ml aliquot was taken , harvested by centrifugation , and immediately treated with 1 ml of rnaprotect bacteria reagent ( qiagen ) . total rna was isolated using the rneasy mini kit ( qiagen ) according to manufacturer 's instructions . co - purified genomic dna was removed by a double dnase treatment using the dna - free kit ( ambion ) . five micrograms of total rna obtained from samples that were exposed for 60 min to nhs or control conditions was used for microarray expression profiling . the rna was reverse transcribed and labeled for microarray hybridization according to standard nimblegen gene expression array protocols . two micrograms of cy3-labeled cdna probes was hybridized overnight at 42 c to custom - designed nimblegen m. catarrhalis bbh18 expression arrays ( 4 plex arrays ; 72 k ) and subsequently washed ; all procedures were performed according to manufacturer 's instructions . array images were acquired with a nimblegen ms200 scanner , and images were first processed with nimblescan software . raw expression data ( .pair files ) were processed using arraystar ( dnastar ) and normalized using the quantile rma method yielding an average signal intensity per cds . to identify genes differentially expressed in 10% nhs compared to the control condition , statistical analysis was executed using cybert unpaired two - condition data analysis ( http://cybert.ics.uci.edu/ ) . genes were considered to be differentially expressed when expression was increased or decreased by > 2.5-fold and at a bonferroni corrected p < 0.05 . to eliminate genes with no or very low expression from our analysis , we generated frequency plots of log2 average probe signal intensity per gene ; two distributions could be observed ( data not shown ) . based on these frequency plots we eliminated genes with an average normalized probe signal intensity of log2 < 6 in all tested conditions from further analysis . using these criteria we identified 84 genes with a > 2.5 fold increased expression and 134 genes that showed a > 2.5 fold reduced expression in 10% nhs compared to incubation in pbs - g ( nhs dilution buffer ) . to determine if exposure to 10% nhs resulted in the increased expression of genes grouped in the same functional classes , potential enrichment was analyzed with the fishers exact ( one - tail ) test and corrected for multiple testing according to storey and tibshirani . to validate microarray expression data , real - time quantitative pcr ( rt - qpcr ) analysis was performed for a selection of 18 genes on samples obtained after 60 min exposure to nhs or pbs - g ( experiment b ) . a sub - selection of 11 genes was tested on samples obtained after 30 min ( experiment a ) ( table 1 ) . dna - free total rna ( 500 ng ) was reverse transcribed into cdna using the iscript cdna synthesis kit ( bio - rad ) . rt - qpcr was performed in duplicate for each sample using ssoadvanced sybr green supermix ( bio - rad ) and analyzed on a cfx96 real - time pcr machine ( bio - rad ) . expression fold - changes ( nhs versus pbs - g ) between the different groupings were calculated using the ct method and the gyra gene was used for normalization . correlation between microarray and rt - qpcr data was assessed with a two - tailed pearson correlation test using graphpad prism software . to gain insight in the mechanisms of complement resistance in m. catarrhalis , the transcriptional response of m. catarrhalis strain bbh18 , a complement resistant isolate , , upon exposure to 10% pooled normal human serum ( nhs ) was analyzed by microarray expression profiling . this concentration was chosen because test experiments demonstrated that a m. catarrhalis bbh18 gene deletion mutant lacking the key complement resistance factor uspa2h was rapidly killed in 10% nhs ( data not shown ) . to reduce the effect of day to day variation , two fully independent microarray expression profiling experiments were performed , indicated hereafter as experiments a and b ( table 1 ) . m. catarrhalis bbh18 was pre - cultured overnight on brain heart infusion ( bhi ) agar plates at 37 c in an atmosphere containing 5% co2 . bacteria were harvested from plates and resuspended in pbs supplemented with 0.15% gelatin ( pbs - g ) . next , 5 ml bhi medium was inoculated to an od600 nm of ~ 0.075 and cultured until mid - log phase ( od600 nm of ~ 1.0 ) , obtaining cultures with a density of 6 10 cfu / ml . of this culture , 4 ml was harvested by centrifugation , washed in 16 ml pbs - g , and resuspended in 20 ml pbs - g with 0.2 mm mgcl2 and 1 mm cacl2 ( ca and mg ) . 5.5 ml of this suspension was mixed with 5.5 ml of either pbs - g ( ca and mg ) ( control ; experiment a ; n = 3 , experiment b ; n = 2 ) or with 20% pooled nhs ( gti diagnostics ) in pbs - g ( ca and mg ) ( experiment a ; n = 3 , experiment b ; n = 3 ) and incubated at 37 c with agitation ( 200 rpm ) . after 0 , 30 ( experiment a only ) , and 60 min , a 3.5 ml aliquot was taken , harvested by centrifugation , and immediately treated with 1 ml of rnaprotect bacteria reagent ( qiagen ) . total rna was isolated using the rneasy mini kit ( qiagen ) according to manufacturer 's instructions . co - purified genomic dna was removed by a double dnase treatment using the dna - free kit ( ambion ) . five micrograms of total rna obtained from samples that were exposed for 60 min to nhs or control conditions was used for microarray expression profiling . the rna was reverse transcribed and labeled for microarray hybridization according to standard nimblegen gene expression array protocols . two micrograms of cy3-labeled cdna probes was hybridized overnight at 42 c to custom - designed nimblegen m. catarrhalis bbh18 expression arrays ( 4 plex arrays ; 72 k ) and subsequently washed ; all procedures were performed according to manufacturer 's instructions . array images were acquired with a nimblegen ms200 scanner , and images were first processed with nimblescan software . raw expression data ( .pair files ) were processed using arraystar ( dnastar ) and normalized using the quantile rma method yielding an average signal intensity per cds . to identify genes differentially expressed in 10% nhs compared to the control condition , statistical analysis was executed using cybert unpaired two - condition data analysis ( http://cybert.ics.uci.edu/ ) . genes were considered to be differentially expressed when expression was increased or decreased by > 2.5-fold and at a bonferroni corrected p < 0.05 . to eliminate genes with no or very low expression from our analysis , we generated frequency plots of log2 average probe signal intensity per gene ; based on these frequency plots we eliminated genes with an average normalized probe signal intensity of log2 < 6 in all tested conditions from further analysis . using these criteria we identified 84 genes with a > 2.5 fold increased expression and 134 genes that showed a > 2.5 fold reduced expression in 10% nhs compared to incubation in pbs - g ( nhs dilution buffer ) . to determine if exposure to 10% nhs resulted in the increased expression of genes grouped in the same functional classes , potential enrichment was analyzed with the fishers exact ( one - tail ) test and corrected for multiple testing according to storey and tibshirani . to validate microarray expression data , real - time quantitative pcr ( rt - qpcr ) analysis was performed for a selection of 18 genes on samples obtained after 60 min exposure to nhs or pbs - g ( experiment b ) . a sub - selection of 11 genes was tested on samples obtained after 30 min ( experiment a ) ( table 1 ) . dna - free total rna ( 500 ng ) was reverse transcribed into cdna using the iscript cdna synthesis kit ( bio - rad ) . rt - qpcr was performed in duplicate for each sample using ssoadvanced sybr green supermix ( bio - rad ) and analyzed on a cfx96 real - time pcr machine ( bio - rad ) . expression fold - changes ( nhs versus pbs - g ) between the different groupings were calculated using the ct method and the gyra gene was used for normalization . correlation between microarray and rt - qpcr data was assessed with a two - tailed pearson correlation test using graphpad prism software .

mycotic aneurysm is a disease requiring immediate treatment because of the high risk of rupture . a difficult surgical approach , especially in the case of occurrence on the iliac artery , involving endovascular embolization and extra - anatomic bypass grafting , is known to be a suitable treatment . we performed extra - anatomic bypass grafting after endovascular embolization successfully in two patients . the postoperative computed tomography of both patients showed complete exclusion of the mycotic aneurysm . the complement system is an important part of the innate defense against invading pathogens ( blom et al . , 2009 ; [ 1 ] ) . the ability to resist complement - mediated killing is considered to be an important virulence trait for the human - restricted respiratory tract pathogen moraxella catarrhalis , as most disease - associated m. catarrhalis isolates are complement - resistant ( wirth et al . , 2007 ; [ 2 ] ) . here we provide a detailed overview of the experimental methods that we have used to study the molecular basis of m. catarrhalis complement - resistance by transcriptome profiling of the bacterium upon exposure to 10% normal human serum ( nhs ) , associated with the study of de vries et al . published in molecular microbiology in 2014 [ 3 ] .

rectal varices are not an uncommon manifestation in cirrhosis with portal hypertension . however , serious bleeding from rectal varices is uncommon . because of its rarity , several authors have reported the utility of transjugular intrahepatic portosystemic shunt ( tips ) placement to control such bleeding when local therapy , such as endoscopic sclerotherapy or banding , fails [ 2 , 3 , 4 , 5 ] . however , we experienced a case of massive bleeding from large rectal varices that could not be controlled by tips placement , despite normalization of the portosystemic pressure gradient . furthermore , we observed rapid decompensation of the cirrhosis that led to severe encephalopathy and death after the tips placement . we report this case to highlight and discuss potential pitfalls of tips placement when using this technique to treat rectal variceal bleeding . a 59-year - old man with a history of alcoholic cirrhosis was hospitalized at an outside facility for massive hematochezia and fainting . he underwent upper endoscopy and tagged red blood cell scan , both of which were negative for active bleeding . a computed tomography of the abdomen was performed and did not reveal the source of the gastrointestinal bleeding ; a nodular liver with splenomegaly consistent with liver cirrhosis was seen , and a markedly dilated inferior mesenteric vein was also noted . during his 5-day hospitalization , he had approximately 1015 bloody bowel movements and received a total of 6 units of packed red blood cells and 2 units of fresh frozen plasma . the patient was then transferred to our institution for further management of his persistent hematochezia . his admission laboratory tests showed a hemoglobin level of 8.3 g / dl , a serum ammonia level of 45 g / dl , a total bilirubin level of 2.8 mg / dl and a model for end - stage liver disease ( meld ) score of 18 . however , he had another episode of massive hematochezia on the following day , with a decrease in hemoglobin level to 5.0 g / dl . therefore , the decision was made to proceed with emergent tips placement . a tips was successfully created with a 10 90 mm viatorr stent - graft ( gore & associates , flagstaff , ariz . this resulted in a decrease in the portosystemic pressure gradient from 12 to 6 mm hg . after the tips placement , brisk hepatopetal flow through the tips was seen on the portogram . despite the successful normalization of the portosystemic pressure gradient , he had another episode of massive bleeding on the following day . an angiogram of the superior rectal vein was performed , which showed tortuous large rectal varices with brisk hepatofugal flow ( fig . 2 ) . flow into the internal iliac vein branches was observed , confirming portosystemic shunting . embolization of the rectal varices was then performed , which necessitated multiple coils , 1 ml of n - butyl cyanoacrylate glue , 5,000 units of thrombin and several sheets of gelfoam ( fig . 3 ) . after the procedure , the patient did not have any further bleeding episodes , and the serial hemoglobin levels remained stable . the remaining hospital course was significant for rapid decompensation of cirrhosis , with an increase in the total bilirubin level to 8.7 mg / dl , the ammonia level to 121 g / dl and the meld score to 28 . he developed acute hypoxic respiratory failure secondary to multiple causes , including progressive liver failure . this case highlights two potential pitfalls of tips placement when using this technique in the treatment of rectal variceal bleeding . first , tipss may not be always successful in controlling serious bleeding from rectal varices , despite optimal portosystemic pressure reduction . we observed rebleeding after the tips placement despite a 50% reduction in the pressure gradient , with a final pressure gradient of 6 mm hg . some authors have also reported that a tips failed to control bleeding from rectal varices [ 6 , 7 ] . recurrent bleeding from rectal varices after tips placement may be related to the large size of the varices . rectal varices , unlike esophageal varices , are true veins and are likely to have larger diameters . according to laplace 's equation , the tension in the varix wall is proportional to the radius of the vessel for any given transmural pressure . , it is recommended to decrease the portosystemic pressure gradient to < 12 mm hg in esophageal variceal bleeding . however , this targeted pressure gradient may not be adequate for controlling bleeding from rectal varices . some authors advocate embolization of the rectal varices at the time of tips placement , even if normalization of portal hypertension is achieved [ 7 , 10 ] . given the successful control of bleeding following embolization in our case , concomitant variceal embolization at the time of tips placement may be necessary to control massive bleeding , especially when the rectal varices are large . second , tipss can be associated with life - threatening complications that can possibly lead to early mortality . since the first application of a tips in the treatment of recurrent bleeding from anorectal varices in 1993 , a tips alone or a tips combined with variceal embolization has been reported to be useful in controlling such bleeding without significant morbidity or mortality [ 2 , 3 , 4 , 5 , 10 ] . however , tipss are inherently associated with risks of procedure - related complications , hepatic encephalopathy and progressive liver failure . godil and mccracken reported a case of rapid liver function decompensation and encephalopathy following tips placement in a 73-year - old woman with rectal variceal bleeding . although no recurrent bleeding was seen , the patient died 4 weeks after the tips procedure . according to the american association for the study of liver diseases practice guidelines , tips is recommended only in the absence of other options for patients with 30-day predicted mortality ( meld > 1518 or serum bilirubin > 4.0 mg / dl ) . although emergent tips placement can be the last resort to control massive bleeding from rectal varices , even in high - risk patients it must be used cautiously because it can lead to life - threatening complications , as seen in our case . despite its minimally invasive nature , tips placement has been associated with a 30-day mortality rate as high as 36% when it is used to control acute gastroesophageal variceal bleeding . one alternative to tips placement in high - risk patients may be the percutaneous embolization of rectal varices via a transhepatic approach . although reports on the use of this technique for bleeding rectal varices are limited , it could be useful in controlling an initial acute bleeding and in stabilizing a patient without affecting liver function . however , collaterals can redevelop after embolization , and recurrent bleeding requiring repeated intervention has been reported . some authors have reported several cases of obliteration of bleeding large rectal varices with the use of balloon - occluded antegrade transvenous sclerotherapy with or without coil embolization [ 14 , 15 ] . in this technique , a sclerosing agent , such as ethanolamine oleate , was injected into the varices while the superior rectal vein was occluded using a balloon catheter . further research is needed to determine its efficacy . in conclusion , although tipss have been reported to be useful in controlling bleeding from rectal varices , there are potential pitfalls in using this technique to treat rectal variceal bleeding . a tips may not always be successful in controlling massive bleeding from large rectal varices even after normalization of portal hypertension , and concomitant variceal embolization may be necessary . furthermore , tipss can be associated with life - threatening complications that may lead to early mortality . with an ageing population , alzheimer 's disease ( ad ) and related neurodegenerative disorders represent a serious social and economic threat to most societies . the cost of caring for an increasing number of people with dementia continues to rise . at present , there are an estimated 30 million people with dementia worldwide , with numbers expected to further increase to 80 million by 2040 . the countries or regions with the largest numbers of affected individuals are china and the developing western pacific , western europe , and the unites states . the two core histopathological hallmarks of ad are the amyloid- ( a ) plaques and the tau - containing neurofibrillary tangles ( nfts ) , both of which have been described by alois alzheimer more than a century ago . disease - causing mutations in familial cases of ad have been described two decades ago ; yet there is still no cure for this debilitating disease . the current treatment is limited to acetylcholine esterase ( ache ) inhibitors ( donepezil , rivastigmine , and galantamine ) and the nmda receptor antagonist , memantine ; yet neither of these strategies halts the degenerative process that characterizes ad . what are the characteristics of the major component of the two hallmark lesions in ad and what causes the familial cases ? the 3942 amino acid a peptide in the plaques is fibrillar , as is tau , a microtubule - associated protein that becomes hyperphosphorylated in the disease process and eventually aggregates to form nfts , neuropil threads , and the abnormal neurites that are associated with some amyloid- plaques . although tau has been generally perceived as a neuronal protein , with mainly an axonal localization , we have recently challenged this view by demonstrating an essential dendritic function under physiological conditions and in mediating a toxicity [ 5 , 6 ] . in addition to plaques and nfts , the ad brain is characterized by massive neuronal cell and synapse loss . tau pathology , in the absence of overt a plaques , characterizes a series of diseases that are collectively known as tauopathies . this includes ftld - tau ( frontotemporal lobar degeneration with tau inclusions ) with an inherited form known as ftdp-17 ( frontotemporal dementia with parkinsonism linked to chromosome 17 ) . there is a second , major subset of ftld characterized by tau - negative yet ubiquitin - positive lesions . in this subset , the transcription and splicing factor tdp-43 ( tar dna - binding protein 43 ) has been identified as the aggregating protein , and consequently , this form of ftld has been named ftld - tdp . similar to tau , tdp-43 in the aggregates is hyperphosphorylated and fragmented , a process believed to be linked to toxicity [ 811 ] . finally , in a third subset of ftld , ftld - fus , the nuclear protein fus ( fused - in - sarcoma ) has been identified as aggregated protein . while both proteins form also aggregates in subsets of amyotrophic lateral sclerosis ( als ) , the majority of ad cases are sporadic ( sad ) , with familial ( fad ) cases likely accounting for less than 1% . in three genes , autosomal dominant mutations have been identified : amyloid precursor protein ( app ) , presenilin 1 ( psen1 ) as well as presenilin 2 ( psen2 ) . it is the amyloid precursor protein , app , from which the a peptide is derived by proteolytic cleavage . presenilins are components of the multimeric -secretase complex that generates a. in addition to the three fad genes , apolipoprotein e ( apoe ) has been unanimously confirmed as a susceptibility gene in sporadic ad ( sad ) . more recently , additional risk factor genes have been identified , with clu encoding clusterin , picalm the phosphatidylinositol - binding clathrin assembly protein , cr1 the complement component ( 3b/4b ) receptor 1 , and tomm40 a channel - forming subunit of the translocase of the mitochondrial outer membrane ( tom ) complex [ 1517 ] . at present , a total of ten genes , mostly with roles in the immune system and in cholesterol metabolism , are established risk genes for ad . compared to ad , ftld is a more heterogeneous disorder , with variants that are , for example , characterized by language or behavioural problems . ad and ftld both present with a progressive decline of memory function leading to dementia , although in ftld this is often preceded by motor symptoms such as parkinsonism . it was several years after the first mutations had been identified in the app and psen genes in ad , that the first mutations were identified in a subset of ftld - tau , ftdp-17 , where they were found in exonic and intronic sequences of the mapt gene that encodes tau . another subset of familial ftld is characterized by mutations in the pgrn gene that encodes the pleiotropic protein progranulin , in the vcp gene that encodes valosin - containing protein , and also in tardp encoding tdp-43 ; these ftld - tdp cases are characterized by inclusions of tdp-43 . finally , mutations in chmp2b encoding chromatin modifying protein 2b lead to ftld , in the absence of either tau or tdp-43 inclusions . in this paper , we will be discussing a few selected tau transgenic mouse models with memory and motor phenotypes and how one of these , the k369i mutant human tau expressing k3 strain , has been employed in an enu modifier screen , outlining the strategy from breeding to gene identification . as the focus of this review article is the application of enu mutagenesis to one of our mutant tau transgenic mouse strains , k3 , we will be comparing this particular strain with a few other tau transgenic strains we have established in the past . mouse strains that are otherwise available in the field including those characterized by a plaque formation have been discussed by us in detail elsewhere [ 2427 ] . what has the ambition been in developing transgenic mouse strains for the tau pathology of ad in the first place ? the aim has always been to authentically model the human pathology and to gain a deeper understanding of what causes tau pathology , how this is related to neurodegeneration and functional impairment , and ultimately , how pathogenic processes can be prevented or delayed . when we generated the very first tau transgenic mouse model in 1995 by expressing the longest human tau isoform in mice , we reproduced a major aspect of the human pathology in a subset of neurons : somatodendritic accumulation of hyperphosphorylated forms of tau , a feature of pathological tau in ad brains . we failed however to reproduce nft formation , neurodegeneration , and functional impairment . even by using a stronger promoter to drive human tau expression , this goal of the early days was not achieved , although the mice developed a motor phenotype and amyotrophy [ 29 , 30 ] . only with the identification of pathogenic mutations in ftld - tau in the mapt gene encoding tau , by expressing mutant forms such as g272v or p301l tau , we achieved nft formation , as determined by gallyas silver impregnations and electron microscopy [ 31 , 32 ] . while we discontinued the g272v mutant mice , the p301l tau transgenic pr5 strain was employed to provide support for the amyloid cascade hypothesis that places a upstream of tau , to reveal behavioural impairments in amygdala- and hippocampus - dependent tasks [ 34 , 35 ] , to assess the role of site - specific phosphorylation and in particular of protein phosphatase 2a ( pp2a ) in this process [ 36 , 37 ] , to determine the degree of the cholinergic pathology , and to reveal an impaired stress - related unfolded protein response and a mitochondrial dysfunction caused by pathological tau [ 4042 ] . assessing the role of mirnas in ad adds an additional level of complexity [ 43 , 44 ] . while the pr5 mice display memory impairment as a major clinical feature of ad , another feature , parkinsonism , that characterizes a significant subset of ftld cases , has been modelled in k369i mutant tau transgenic k3 mice . we established this strain based on the identification of the k369i mutation of tau in a single patient with pick 's disease ( pid ) , a form of ftld . as is known for pid in general , the tau lesions in the mutation carrier 's brain showed a remarkable feature when analysed histologically : silver impregnation revealed that the tau lesions were bielschowsky - positive and gallyas - negative ; moreover , tau was phosphorylated at many epitopes but not 12e8 ( ser262/ser356 ) . we succeeded in reproducing this distinct pathology fully in the k3 mice that express k369i mutant tau . owing to a unique expression pattern of the k369i transgene that includes the substantia nigra , the k3 transgenic mice also modelled early - onset parkinsonism , that is , resting tremor , bradykinesia , postural instability , and gait anomalies . they showed an increased cataleptic response to haloperidol and an early , but not late , response to l - dopa , indicating that the dopaminergic system is impaired in these mice . in recent years , impaired axonal transport has been identified as a central pathomechanism in ad [ 4651 ] . conceptually , impaired axonal transport is linked to oxidative stress and mitochondrial dysfunction : mitochondria need to be efficiently transported along the long axonal processes for neurons to function ; furthermore they are a major source of reactive oxygen species ( ros ) [ 53 , 54 ] . we found a selectively impaired axonal transport of distinct cargos including mitochondria and th- ( tyrosine hydroxylase- ) containing vesicles . at the molecular level , we found that this impaired transport phenotype is due to trapping of a component of the kinesin motor machinery , the adapter protein jip1 , by elevated levels of phosphorylated tau , preventing jip1 from executing its physiological function in the axon . a pathological interaction between tau and jip1 was further revealed in ad and not , control brain , highlighting the validity of transgenic animal models in dissecting pathomechanisms in ad . as tau only traps jip1 when it is hyperphosphorylated , this study presents inhibition of abnormal hyperphosphorylation of tau as a promising therapeutic strategy in ad . with the pr5 and the k3 mice we do have two complementary models at hand that present themselves for therapeutic intervention : both strains are characterized by hyperphosphorylation and increased insolubility of tau as well as silver - positive inclusions , gallyas - positive nfts in the case of r5 mice , and bielschowsky - positive inclusions predominantly resembling pick bodies in k3 mice . the pr5 mice show an accelerated extinction in the conditioned taste aversion ( cta ) paradigm , while the k3 mice have a memory phenotype as determined by the novel object recognition test , as well as a motor phenotype that can be easily monitored either by scoring of clasping , gait , and tremor ( table 1 ) or on the rota - rod [ 22 , 57 ] . in addition , as the k3 mice age , they lose substantial numbers of neurons both in the substantia nigra ( th - positive neurons ) and in the cerebellum ( basket cells ) [ 22 , 57 ] . experimental diabetes exacerbates the tau pathology as shown for pr5 mice . as therapeutic strategies , different approaches can be envisaged such as the transplantation of cells with the potential to differentiate in situ either into neuronal or glial cell types . this strategy has been successfully applied to mice with a combined tau and a pathology in which neuronal stem cell transplantation has been shown to improve cognition via brain - derived neurotrophic factor ( bdnf ) . we used both active and passive immunization and monitored efficacy of this approach biochemically , histologically , and behaviourally . we have also been testing a small bioavailable compound , sodium selenate that can be supplied with the drinking water . chronic treatment over a period of four months significantly improved the pathology in both young and old k3 mice . this was associated with reduced tau hyperphosphorylation and decreased numbers of tau inclusions including spheroids . in fact , spheroids were completely absent from frontal cortex , suggesting that selenate reverts the functional impairment leading to spheroids . k3 mice are characterized by a substantial , age - dependent degeneration of cerebellar basket neurons , resulting in the absence of pinceau terminals formed by clustered axons surrounding purkinje cells . these findings show that selenate reduces tau phosphorylation and deposition , mitigates pathological spheroid formation , and prevents axonal degeneration of distinct neuronal populations in k3 mice . we also treated 8-month - old pr5 mice with sodium selenate and found that treatment caused a significant impairment in the cta paradigm . as for the k3 mice , selenate both caused reduced hyperphosphorylation and numbers in tau deposits ( nfts ) as well as increased tau solubility . nft numbers in 12-month - old selenate - treated pr5 mice were similar to those in 8-month - old - untreated pr5 mice , suggesting that treatment had halted disease progression . we further showed that the selenate - induced improvements require protein phosphatase 2a ( pp2a ) , by performing in vitro experiments and showing inefficacy of selenate in mice with a combined tau pathology and reductions in pp2a activity [ 66 , 67 ] . while in the ad and ageing field mouse strains are available with traits that arose spontaneously and have been further established by specific breeding programs such as the senescence - accelerated sam mice , the rate at which new models arise spontaneously means that , even with a concerted effort , discovery is on an asymptomatic trajectory towards a phenotype gap . the concept of genomewide mutagenesis is well established in invertebrates such as the fruit fly drosophila melanogaster and in the nematode worm caenorhabditis elegans , with enu mutagenesis mainly producing a to t and t to a transversions . this mutagenesis strategy was used to create the first genetic maps of mendelian inheritance in drosophila , revealing many of the key genes controlling early embryonic patterning and development . what makes it possible to carry out comparable efforts in mice is the extraordinary mutagenic efficiency of enu in mouse spermatogonial stem cells . by expanding the library of alleles with chemical mutagenesis using n - ethyl - n - nitosourea ( enu ) , identification of each of the single nucleotide changes that result in a particular phenotype is feasible in mice and is now providing novel insights into not only immunity , where this approach has been used widely , but also the functioning of the brain . genomewide , random mutagenesis with the ethylating chemical enu provides a way to produce and screen genetic variants in the mouse . intraperitoneal injection of male founder mice ( g0 ) causes random base pair substitutions in the dna of spermatogonial stem cells . informative mutations are identified by screening members of the pedigrees propagated from these founders for phenotypes of interest . introducing point mutations has the advantage that it most often inactivates or alters the function of individual protein domains rather than eliminating the protein altogether ( as is the case for complete knock - outs ) , making it harder for related proteins to compensate , better mimicking the effects of drugs or natural genetic variants , illuminating active sites in proteins , and revealing specific functions of alternatively spliced forms . we have been investigating a phenotype modulation of our k3 mice by crossing enu male founder mice with k3 females as discussed hereinafter . here , and in general , dominant mutations are expected to manifest in the first generation offspring ( g1 ) , while recessive mutations are brought to homozygosity in g3 mice by either g1 . the rate of recessive mutations has been established based on genotype - driven projects using libraries of g1 gametes generated from an enu - treated founder . sequencing of sentinel regions of the genome of g1 sperm provides quantitative and qualitative characteristics of enu mutagenesis and information about the probable yield of mutations from phenotype - driven screens . the consensus is a mutation rate of 0.51.0 mutations / mbp ( 7.5 107 mutations / bp / g0 gamete , or 15003000 mutations / g1 ) from a standard regimen ( 100 mg / kg / week 3 for a c57bl/6 founder ) . assuming a 1600 cm 2.6 gb haploid mouse genome , of which approximately 1.5% is coding , this comes down to approximately 50 functional mutations in each g1 . the efficiency of enu - induced mutation means that the generation of allelic series is highly feasible . based on a conservative estimate of 20 point mutations per g1 gamete , a survey of 17400 g3s would yield a series of 5 functional alleles per gene with > 99% probability . limiting factors of the enu mutagenesis approach are infertility and lethality , with the standard enu regimen resulting in azoospermia ( absence of motile sperm in semen ) for up to 10 weeks , with fertility being resumed when the testes are repopulated by spermatogonia . the presence of mutations that confer lethality before weaning sets the workable upper limit of the enu dose even more stringently . for example , one study found that 1/13 g1 mice contained lethal mutations in the balancer chromosome 11 region that contains 5% of the genome , with the consequence that 18% of the g3 population will not survive because of lethal mutations . for phenotype - driven strategies ( such as a motor phenotype , see the following ) , f1 and f3 mice are examined for dominant - acting and recessive - acting mutations , respectively [ 23 , 82 ] . the recovery rate of dominant mutations is low ( 2% ) ; on the other hand however , costs for identifying recessive mutations are much higher , due to much higher mouse numbers and sample size to be screened and sequenced . in an ideal screen , the phenotypes are qualtitative ( such as having a motor phenotype ) or binomial ( rejection of transplanted tissue or not ) ; they are free of false positives and are sufficiently penetrant to permit meiotic mapping . the latter is typically achieved first by outcrossing and then an f1 intercrossing to yield f2 mice ; 50 f2 mice are required to obtain a 10 mb mapping resolution with a 95% confidence , although haplotype mapping might enable an estimation of the chromosomal location using as few as 10 affected f2 mice . for a more detailed discussion of the likelihood of a confounding mutation cosegregating with the phenotype the reader is referred to two excellent reviews on the topic [ 23 , 69 ] . what makes the approach feasible is a simple read - out , and in this respect , motor phenotypes are much easier to monitor than memory phenotypes . as in our k3 mice , k369i mutant tau expression causes both a motor and memory impairment , changes to the motor phenotype by any intervention although interesting in itself can also serve as a surrogate marker for changes in memory functions . we initiated an enu mutagenesis program in k3 mice that generated transgene - dependent and -independent pedigrees . in the following we will be discussing five pedigrees , enu164 ( partial rescue , transgene - related ) , enu67 ( slow weight gain , transgene - related ) , enu37 ( circling , not transgene - related ) , enu12/30a ( motor phenotype , not transgene - related ) , and enu16/069 ( motor phenotype , not transgene - related ) . to first establish that the k3 phenotype is fully penetrant on the background chosen for enu mutagenesis , we established backcrosses with 20 mice each , using c57bl/10 , c3h / h3h , fvb , c57led , and balb / b mice , all of which are established enu strains at the apf ( australian phenomics facility , canberra ) . we decided to use balb / b as the strain for enu mutagenesis , as the k3 phenotype of parkinsonism is fully penetrant on this background , both with regards to phenotype and age - of - onset . therefore , balb / b males were injected with enu and paired with the k3 mice ( that are on a c57bl/6 background ) , and the f1 offspring was analyzed . the litters born were scored for three read - outs of parkinsonism in mice , tremor ( staging 03 ) , gait anomalies ( staging 03 ) , limb clasping ( staging 05 ) , as well as body weight ( table 1 ) . we had previously phenotyped wild - type balb / b mice for the three parkinsonism characteristics and weight . although none of them displayed abnormal gait or resting tremor , 37% did clasp their hind limbs when stimulated and 5% clasped their hind limbs without stimulation , while their front paws grasped the cage wire . therefore , to ensure accurate identification of the phenotype , the hindlimb clasping alone was not used as sole indicator , but in conjunction with tremor and abnormal gait . phenotyping was done at weaning , and then at one - week intervals until around 8 weeks of age , depending on the scoring outcome . any mice exhibiting all three phenotypes and genotyped k3-tau positive were culled , as were any mice failing to display any of the phenotypes and genotyped k3-tau negative . mice which showed a genotype / phenotype mismatch were genotyped a second time for confirmation and phenotyped for up to 3 months . any mice which did not display the phenotype until after 5 weeks of age were also noted as having delayed onset of the phenotype and were flagged as a putant ( potential mutant ) of interest . this was followed by next generation whole exome sequencing as a means to identifying mutations . we would like to make the comment that while , previously , we have been mapping the enu mutations down to a chromosomal region for most strains these days it is much faster to directly next generation sequence the exomes of one to two affected mice instead . this has greatly accelerated the speed of discovery from years to months and reduced costs of identifying causal mutations . the snps identified with the sequencing were then validated against the phenotype with an amplifluor snp assay for each snp . hereinafter we will be discussing five pedigrees for which we have arrived at different stages of out - crossing and identification of the underlying gene mutations , starting with those pedigrees with which we have the least progressed . enu164 pedigreethis pedigree is characterized by a partial rescue of the parkinsonism phenotype that characterizes the k3 mice , because some transgene - positive mice in the pedigree showed an improvement in our vigorous weekly scorings ; for example , at week 6 they would show a scoring of 1/2/1 for gait / clasp / tremor instead of 3/4/3 as is typical for k3 mice . as mentioned above , consecutive backcrossing onto c57bl/6 reduces the number of functional enu - induced point mutations such that in the 3rd generation ( f3 ) they are reduced to 3 - 4 mutations . as the k3 mice have been generated on a c57bl/6 background , while enu mutagenesis has been on a balb / b inbred genetic background , strain - specific snp detection enabled the genomic mapping of the causative mutation location on the balb / b background . this narrows down the linked mutation - carrying region to less than 20 mb , at which point candidate genes in the region will be sequenced to detect mutant alleles , and eventually , the causative mutation . 6 of these showed the inherited partial rescue phenotype ( ~15% inheritance ) . from f2 # 483 we screened 11 k3-positive mice , with 3 showing partial rescue ( ~27% ) , from f2 # 430 20 k3-positive mice ( ~35% ) . exome ( ngs ) next generation sequencing is ongoing to identify the one single gene mutation that causes the k3 modulating phenotype . this pedigree is characterized by a partial rescue of the parkinsonism phenotype that characterizes the k3 mice , because some transgene - positive mice in the pedigree showed an improvement in our vigorous weekly scorings ; for example , at week 6 they would show a scoring of 1/2/1 for gait / clasp / tremor instead of 3/4/3 as is typical for k3 mice . as mentioned above , consecutive backcrossing onto c57bl/6 reduces the number of functional enu - induced point mutations such that in the 3rd generation ( f3 ) they are reduced to 3 - 4 mutations . as the k3 mice have been generated on a c57bl/6 background , while enu mutagenesis has been on a balb / b inbred genetic background , strain - specific snp detection enabled the genomic mapping of the causative mutation location on the balb / b background . this narrows down the linked mutation - carrying region to less than 20 mb , at which point candidate genes in the region will be sequenced to detect mutant alleles , and eventually , the causative mutation . 6 of these showed the inherited partial rescue phenotype ( ~15% inheritance ) . from f2 # 483 we screened 11 k3-positive mice , with 3 showing partial rescue ( ~27% ) , from f2 # 430 20 k3-positive mice ( ~35% ) . exome ( ngs ) next generation sequencing is ongoing to identify the one single gene mutation that causes the k3 modulating phenotype . enu67 pedigreethis pedigree shows a worsening of the weight phenotype that characterises the k3 mice . for example , while the average weight of transgene - negative mice is 19,0 grams at six weeks of age , this is reduced to 15.8 grams for transgene - positive k3 mice and is even further reduced in the enu67 pedigree ( k3-positive ) that shows an average weight of only 11.7 grams . both k3-positive mice ( # 347 , # 350 ) were identified as slow to gain weight , and we are currently crossing further to determine the underlying gene mutation . for example , while the average weight of transgene - negative mice is 19,0 grams at six weeks of age , this is reduced to 15.8 grams for transgene - positive k3 mice and is even further reduced in the enu67 pedigree ( k3-positive ) that shows an average weight of only 11.7 grams . both k3-positive mice ( # 347 , # 350 ) were identified as slow to gain weight , and we are currently crossing further to determine the underlying gene mutation . the trait shows mendelian inheritance over three generations . following deep sequencing of the exome of 2 affected mice we identified possible causal point mutations in the following genes:5730455p16rik ( riken cdna);tcof1 ( treacher collins franceschetti syndrome 1 ) , responsible for the production of treacle , which plays a role in the formation of tissue in the face . the human disease is also autosomal dominant;tbx1 ( t - box 1 gene ) t - box genes encode transcription factors involved in the regulation of developmental processes . chromosomal deletions in this region have been linked to neural - crest - related defects . a point mutation could potentially cause a mild neural phenotype;senp7 ( sumo1/sentrin specific peptidase 7 gene ) involved in the posttranslational modification of proteins by the addition of ubiquitin - like sumo proteins;ggt7 ( gamma - glutamyltransferase 7 gene ) : this gene is a member of a family that encodes enzymes involved in both the metabolism of glutathione and in the transpeptidation of amino acids ; changes in the activity of -glutamyltransferase may signal preneoplastic or toxic conditions in the liver or kidney in humans;hkdc1 ( hexokinase domain containing 1 gene ) ; chrna4 ( cholinergic receptor , nicotinic , alpha polypeptide 4 gene ) : this gene encodes a nicotinic acetylcholine receptor . polymorphisms in this gene provide protection against nicotine addiction in humans.amplifluor primers were designed to the above snps and affected and unaffected mice genotyped , which led to the confirmation of the causal mutation in the tbx1 gene , with a t to a substitution of bp 857 ( coding position ) resulting in an asn to ile change of amino acid 276 . a complete knock - out of tbx1 is available and is a model of digeorge syndrome ; it is further characterized by perinatal death [ 87 , 88 ] . the digeorge syndrome is caused by a 1.5 to 3.0 mb hemizygous deletion of chromosome 22q11.2 . haploinsufficiency of the tbx1 gene in particular is responsible for most of the physical malformations . there is evidence that point mutations in the tbx1 gene can also cause the disorder . digeorge syndrome is the most frequent microdeletion syndrome in humans , and is characterized by cardiovascular , thymic and parathyroid , and craniofacial anomalies . the underlying cause is disturbed formation of the pharyngeal apparatus , a transient structure present during vertebrate development that gives rise to endocrine glands , craniofacial tissue , and the cardiac outflow tract . the pharyngeal apparatus is composed of derivatives of ectoderm , endoderm , mesoderm , and the neural crest . thus , complex interactions between cell types from different origins have to be orchestrated in the correct spatiotemporal manner to establish proper formation of the pharyngeal system . the analysis of engineered mouse mutants developing a phenotype resembling digeorge syndrome has revealed genes and signalling pathways crucial for this process . intriguingly , these mouse models reveal that interference with either of two distinct phases of pharyngeal apparatus development can contribute to the aetiology of digeorge syndrome . with regards to ad , furthermore , string 9.0 known and predicted protein - protein interactions for tbx1 identify sept5 which is also among 29 genes that are deregulated in pr5 tau transgenic mice ( jg and lim , manuscript submitted ) . the value of the enu37 strain lies in the absence of a perinatal phenotype which allows for studies which are not feasible with the complete knockout . the trait shows mendelian inheritance over three generations . following deep sequencing of the exome of 2 affected mice we identified possible causal point mutations in the following genes:5730455p16rik ( riken cdna);tcof1 ( treacher collins franceschetti syndrome 1 ) , responsible for the production of treacle , which plays a role in the formation of tissue in the face . the human disease is also autosomal dominant;tbx1 ( t - box 1 gene ) t - box genes encode transcription factors involved in the regulation of developmental processes . chromosomal deletions in this region have been linked to neural - crest - related defects . a point mutation could potentially cause a mild neural phenotype;senp7 ( sumo1/sentrin specific peptidase 7 gene ) involved in the posttranslational modification of proteins by the addition of ubiquitin - like sumo proteins;ggt7 ( gamma - glutamyltransferase 7 gene ) : this gene is a member of a family that encodes enzymes involved in both the metabolism of glutathione and in the transpeptidation of amino acids ; changes in the activity of -glutamyltransferase may signal preneoplastic or toxic conditions in the liver or kidney in humans;hkdc1 ( hexokinase domain containing 1 gene ) ; chrna4 ( cholinergic receptor , nicotinic , alpha polypeptide 4 gene ) : this gene encodes a nicotinic acetylcholine receptor . polymorphisms in this gene provide protection against nicotine addiction in humans.amplifluor primers were designed to the above snps and affected and unaffected mice genotyped , which led to the confirmation of the causal mutation in the tbx1 gene , with a t to a substitution of bp 857 ( coding position ) resulting in an asn to ile change of amino acid 276 . a complete knock - out of tbx1 is available and is a model of digeorge syndrome ; it is further characterized by perinatal death [ 87 , 88 ] . the digeorge syndrome is caused by a 1.5 to 3.0 mb hemizygous deletion of chromosome 22q11.2 . haploinsufficiency of the tbx1 gene in particular is responsible for most of the physical malformations . there is evidence that point mutations in the tbx1 gene can also cause the disorder . digeorge syndrome is the most frequent microdeletion syndrome in humans , and is characterized by cardiovascular , thymic and parathyroid , and craniofacial anomalies . the underlying cause is disturbed formation of the pharyngeal apparatus , a transient structure present during vertebrate development that gives rise to endocrine glands , craniofacial tissue , and the cardiac outflow tract . the pharyngeal apparatus is composed of derivatives of ectoderm , endoderm , mesoderm , and the neural crest . thus , complex interactions between cell types from different origins have to be orchestrated in the correct spatiotemporal manner to establish proper formation of the pharyngeal system . the analysis of engineered mouse mutants developing a phenotype resembling digeorge syndrome has revealed genes and signalling pathways crucial for this process . intriguingly , these mouse models reveal that interference with either of two distinct phases of pharyngeal apparatus development can contribute to the aetiology of digeorge syndrome . with regards to ad , furthermore , string 9.0 known and predicted protein - protein interactions for tbx1 identify sept5 which is also among 29 genes that are deregulated in pr5 tau transgenic mice ( jg and lim , manuscript submitted ) . the value of the enu37 strain lies in the absence of a perinatal phenotype which allows for studies which are not feasible with the complete knockout . 5730455p16rik ( riken cdna ) ; tcof1 ( treacher collins franceschetti syndrome 1 ) , responsible for the production of treacle , which plays a role in the formation of tissue in the face . the human disease is also autosomal dominant ; tbx1 ( t - box 1 gene ) t - box genes encode transcription factors involved in the regulation of developmental processes . chromosomal deletions in this region have been linked to neural - crest - related defects . a point mutation could potentially cause a mild neural phenotype ; senp7 ( sumo1/sentrin specific peptidase 7 gene ) involved in the posttranslational modification of proteins by the addition of ubiquitin - like sumo proteins ; ggt7 ( gamma - glutamyltransferase 7 gene ) : this gene is a member of a family that encodes enzymes involved in both the metabolism of glutathione and in the transpeptidation of amino acids ; changes in the activity of -glutamyltransferase may signal preneoplastic or toxic conditions in the liver or kidney in humans ; hkdc1 ( hexokinase domain containing 1 gene ) ; chrna4 ( cholinergic receptor , nicotinic , alpha polypeptide 4 gene ) : this gene encodes a nicotinic acetylcholine receptor . enu12/301 pedigree ( in short : enu12)enu12 mice , under mild stress , show sudden jerky movements when moving around the cage and appear to tremor constantly . some mice show what could be described as opisthotonus ( a type of spasm in which the head and heels arch backwards in extreme hyperextension and the body forms a reverse bow ) . the mice display varying degrees of severity ; even when severely affected , they are able to move freely and quickly around the cage . they occasionally fall to either side and circle around the cage in very fast movements when highly stressed , such as when the cage is changed . the enu12 phenotype can be recognised at weaning.deep sequencing identified an a to t substitution of bp 1408 in exon 10 of the kcnq1 gene that results in a lys to stop change of amino acid 435 . the kcnq1 gene encodes a voltage - gated potassium channel protein required for the repolarisation phase of the cardiac action potential . the gene product can form multimers with two other potassium channel proteins , kcne1 and kcne3 . inherited forms of a human condition known as long qt syndrome ( lqts ) , an abnormality in the cardiac ventricular repolarisation characterised by qt interval prolongation and abnormal t - waves on an electrocardiogram ( ecg ) , are most commonly associated with mutations in kcqn1 . some patients show only a cardiac defect ( romano - ward syndrome , rws , dominant ) ; others may suffer from severe deafness due to a lack of endolymph in the vestibular and auditory compartment ( jervell and lange - nielsen syndrome , jlns , recessive ) .the complete knockout is a model of jnls , but the mice show an additional circling ( shaker / waltzer ) and rapid head blobbing phenotype . introducing the familial a340e point mutation by a knock - in approach models rws ( cardiac phenotype , but no inner ear defect ) . this point mutation is in the amino - terminus of kcnq1 that contains the six transmembrane domains s1-s6 and the pore domain p . a comparative analysis of the strains allowed the authors to draw conclusions with regards to the etiology of the cardiac phenotype . the k434stop mutation in the enu12 mice is in the carboxy - terminus that contains four helical regions , of which two form coiled - coil assemblies . the enu12 strain has a phenotype similar to the complete knock - out demonstrating an essential role for the helical domain in kcnq1 function . enu12 mice , under mild stress , show sudden jerky movements when moving around the cage and appear to tremor constantly . some mice show what could be described as opisthotonus ( a type of spasm in which the head and heels arch backwards in extreme hyperextension and the body forms a reverse bow ) . the mice display varying degrees of severity ; even when severely affected , they are able to move freely and quickly around the cage . they occasionally fall to either side and circle around the cage in very fast movements when highly stressed , such as when the cage is changed . deep sequencing identified an a to t substitution of bp 1408 in exon 10 of the kcnq1 gene that results in a lys to stop change of amino acid 435 . the kcnq1 gene encodes a voltage - gated potassium channel protein required for the repolarisation phase of the cardiac action potential . the gene product can form multimers with two other potassium channel proteins , kcne1 and kcne3 . inherited forms of a human condition known as long qt syndrome ( lqts ) , an abnormality in the cardiac ventricular repolarisation characterised by qt interval prolongation and abnormal t - waves on an electrocardiogram ( ecg ) , are most commonly associated with mutations in kcqn1 . some patients show only a cardiac defect ( romano - ward syndrome , rws , dominant ) ; others may suffer from severe deafness due to a lack of endolymph in the vestibular and auditory compartment ( jervell and lange - nielsen syndrome , jlns , recessive ) . the complete knockout is a model of jnls , but the mice show an additional circling ( shaker / waltzer ) and rapid head blobbing phenotype . introducing the familial a340e point mutation by a knock - in approach models rws ( cardiac phenotype , but no inner ear defect ) . this point mutation is in the amino - terminus of kcnq1 that contains the six transmembrane domains s1-s6 and the pore domain p . a comparative analysis of the strains allowed the authors to draw conclusions with regards to the etiology of the cardiac phenotype . the k434stop mutation in the enu12 mice is in the carboxy - terminus that contains four helical regions , of which two form coiled - coil assemblies . the enu12 strain has a phenotype similar to the complete knock - out demonstrating an essential role for the helical domain in kcnq1 function . enu16/069 pedigree ( in short : enu16)with minimal stress , enu16 mice display a mild action tremor . with moderate stress the tremor worsens and the mice display an unusual , hypermetric gait in their hind limbs . upon restraining of the tail either one or both hind limbs show dramatic clasping , and when returned to the cage , it takes some time for the mice to regain control over the limbs with what looks to be a sporadic and short - term lack of control of the hind limbs . within seconds of being returned to the cage post restraint the gait appears to improve but can persist and be quite unsteady and waddle - like . the phenotype is noticeable from 4 to 5 weeks of age.deep sequencing identified an a to g substitution of bp 530 in exon 5 of the mpz ( myelin protein zero ) gene resulting in an asp to gly change of aa 121 . aliases of mpz are mpp , p - zero , and p0 , among others . schwann cells , the glia of the peripheral nervous system ( pns ) , play pivotal roles in the development and maintenance of the pns . in addition to forming the myelin sheath , schwann cells are involved in neuronal survival where they also provide support to axons during development and throughout adulthood . the mpz gene encodes a transmembrane glycoprotein that is the major structural protein of peripheral myelin . mutations in the mpz gene result in the autosomal dominant form of charcot - marie - tooth disease type 1 ( cmt1b ) . this domain is important for the homotypic interactions that convey the self - adhesive properties that are essential to maintain myelin compaction and integrity .the complete p0 knockout causes a severe demyelinating neuropathy , but the function of p0 is still not fully resolved . axons in their peripheral nerves are severely hypomyelinated , and a subset contain myelin - like figures and axons degenerate . a second strain is available from jackson laboratories : b6.cg-mpz/grsrj mice carry the spontaneous totterer mutation . homozygous mice are poor breeders , reproducing at most once or twice , and as females are poor mothers , use of the strain is limited . besides the totterer strain , for p0 there is only a complete knock - out available , and not a knock - in strain . the enu16 strain has an n121 g point mutation that is juxtaposed to the only known glycosylation site in the protein . we found however by immunohistochemistry and western blot analysis that the mutated protein ( p0 n121 g ) is expressed at a pattern and at levels similar to wild - type . this allows us to employ the enu16 strain in studies ( such as of adhesion ) that are not feasible with the complete knockout . with minimal stress , the tremor worsens and the mice display an unusual , hypermetric gait in their hind limbs . upon restraining of the tail either one or both hind limbs show dramatic clasping , and when returned to the cage , it takes some time for the mice to regain control over the limbs with what looks to be a sporadic and short - term lack of control of the hind limbs . within seconds of being returned to the cage post restraint the gait appears to improve but can persist and be quite unsteady and waddle - like . deep sequencing identified an a to g substitution of bp 530 in exon 5 of the mpz ( myelin protein zero ) gene resulting in an asp to gly change of aa 121 . aliases of mpz are mpp , p - zero , and p0 , among others . schwann cells , the glia of the peripheral nervous system ( pns ) , play pivotal roles in the development and maintenance of the pns . in addition to forming the myelin sheath , schwann cells are involved in neuronal survival where they also provide support to axons during development and throughout adulthood . the mpz gene encodes a transmembrane glycoprotein that is the major structural protein of peripheral myelin . mutations in the mpz gene result in the autosomal dominant form of charcot - marie - tooth disease type 1 ( cmt1b ) . this domain is important for the homotypic interactions that convey the self - adhesive properties that are essential to maintain myelin compaction and integrity . the complete p0 knockout causes a severe demyelinating neuropathy , but the function of p0 is still not fully resolved . axons in their peripheral nerves are severely hypomyelinated , and a subset contain myelin - like figures and axons degenerate . a second strain is available from jackson laboratories : b6.cg-mpz/grsrj mice carry the spontaneous totterer mutation . homozygous mice are poor breeders , reproducing at most once or twice , and as females are poor mothers , use of the strain is limited . besides the totterer strain , for p0 there is only a complete knock - out available , and not a knock - in strain . the enu16 strain has an n121 g point mutation that is juxtaposed to the only known glycosylation site in the protein . we found however by immunohistochemistry and western blot analysis that the mutated protein ( p0 n121 g ) is expressed at a pattern and at levels similar to wild - type . this allows us to employ the enu16 strain in studies ( such as of adhesion ) that are not feasible with the complete knockout . this overview of pedigrees with motor phenotypes reveals that mice are amenable to enu mutagenesis and that it is possible to establish interesting phenotypes . compared to the functional genomics approaches we have pursued in the past [ 99102 ] , enu mutagenesis is more directed as it not only identifies gene mutations that modulate an existing pathology but also has the added advantage of establishing mutant mouse strains at the same time . as outlined above , enu tends to introduce point mutations rather than causing deletions ; this results in the expression of mutant proteins at levels that are high enough to perform functional studies . a limitation in a more wide - spread application of enu mutagenesis though to mice is the high costs associated with establishing the mutants and identifying the underlying gene mutation and the fact that not all phenotypes are amenable to a robust and fast read - out . therefore , using the motor phenotype as a surrogate marker for memory functions ( as for the k3 transgene - dependent pedigrees ) is an avenue that can be pursued in the field of ad mouse models . what are the challenges of enu mutagenesis in transgenic models of neurodegeneration and what issues both practical and conceptual did we encounter while doing these studies ? in hindsight it appears to have been the preferred strategy to start with as many enu - mutagenised males for cross - breeding with k3 mice as possible in order to saturate the genome with mutations . also , we had initially planned to identify both dominant and recessive mutations , but considering the extensive breeding needed , we realised that such an undertaking is not affordable and therefore we concentrated solely on dominantly inherited traits . in principle the question that may be raised is which other transgenic models could be easily screened and used for enu mutagenesis . we believe that any strain with a phenotype that can be easily picked up is amenable to this strategy . in our case we have been using mice that are characterized by a robust tau - dependent motor and memory phenotype , so we assumed that any modification of the motor phenotype would also modify memory functions , a trait , which would be tested after the modification of the motor phenotype has been confirmed . another question is what the expectations are beyond finding possible new targets and how we see the field evolving in the years ahead . we have started to establish caenorhabditis elegans in the laboratory as a system complementary to mice and it seems that overall a mutagenesis screen in the roundworm system is easier . we still believe that there is a potential in applying enu mutagenesis to mouse models . these models might nicely complement ongoing concerted efforts such as those of the international knockout mouse consortium ( http://www.knockoutmouse.org/ ) . the main objectives of this consortium are to generate , archive , and distribute worldwide up to 12.000 conditional mutations across the mouse genome in mouse embryonic stem ( es ) cells . the main mutagenesis strategies pursued are : ( i ) conditional gene trapping a random approach for expressed genes ; ( ii ) conditional - targeted trapping a directed approach , used for expressed genes ; and ( iii ) conditional gene targeting a directed approach , used for non - expressed genes hopefully , these concerted efforts help in developing a cure for a disease for which only symptomatic treatment is available .

in patients with portal hypertension , bleeding from rectal varices is rare . however , it can be life - threatening . we report a case of massive bleeding from large rectal varices in a 59-year - old man with alcoholic cirrhosis . emergent transjugular intrahepatic portosystemic shunt ( tips ) placement was performed following failed local endoscopic therapy . despite normalization of the portosystemic pressure gradient , the patient had another episode of massive bleeding on the following day . embolization of the rectal varices via tips successfully stopped the bleeding . after the procedure , rapid decompensation of the cirrhosis led to severe encephalopathy , and death was observed . although tipss have been reported to be useful in controlling bleeding from rectal varices , our case illustrates the potential pitfalls in using this technique in the treatment of rectal variceal bleeding . tipss may not be always successful in controlling massive bleeding from large rectal varices , even after normalization of portal hypertension . tipss can also be associated with life - threatening complications that may lead to early mortality . modifier screening is a powerful genetic tool . while not widely used in the vertebrate system , we applied these tools to transgenic mouse strains that recapitulate key aspects of alzheimer 's disease ( ad ) , such as tau - expressing mice . these are characterized by a robust pathology including both motor and memory impairment . the phenotype can be modulated by enu mutagenesis , which results in novel mutant mouse strains and allows identifying the underlying gene / mutation . here we discuss this strategy in detail . we firstly obtained pedigrees that modify the tau - related motor phenotype , with mapping ongoing . we further obtained transgene - independent motor pedigrees : ( i ) hyperactive , circling enu 37 mice with a causal mutation in the tbx1 gene the complete knock - out of tbx1 models digeorge syndrome ; ( ii ) enu12/301 mice that show sudden jerky movements and tremor constantly ; they have a causal mutation in the kcnq1 gene , modelling aspects of the romano - ward and jervell and lange - nielsen syndromes ; and ( iii ) enu16/069 mice with tremor and hypermetric gait that have a causal mutation in the mpz ( myelin protein zero ) gene , modelling charcot - marie - tooth disease type 1 ( cmt1b ) . together , we provide evidence for a real potential of an enu mutagenesis to dissect motor functions in wild - type and tau mutant mice .

as the number of patients with unruptured cerebral aneurysms has increased , more attention has been given to the primary prevention of the aneurysm rupture4 ) . endovascular intervention , one of the major treatment modalities , has begun to catch up with and surpass the surgery in the number of cases treated , based on the favorable outcomes and reduced morbidity / mortality21220 ) . in addition , recently , patients have shown a preference for endovascular intervention over surgery because of the cosmetic and psychological aspects . scalpel surgeons are being asked to enhance their competitiveness and develop novel surgical devices and skills , such as minimally invasive keyhole surgery81519 ) , neuroendoscopic systems7 ) , indocyanine green ( icg ) fluorescence angiography518 ) , intraoperative neurophysiological monitoring16 ) , and intraoperative cerebral angiography1 ) . microscopic icg fluorescence angiography is very helpful for detecting and readjusting unexpected arterial occlusion and incomplete clipping of cerebral aneurysms . however , the usefulness of icg fluorescence angiography is limited when the microscopic view is poorly visualized or when icg can not absorb sufficient light to emit fluorescence . therefore , these limitations can be overcome using the endoscopic systems that provide sufficient light illumination and excellent visualization of the operative fields . thus , icg fluorescence angiography would be very useful to supplement the shortcomings of microscopic icg fluorescence . to date , there are 3 clinical reports regarding endoscopic icg fluorescence angiography from japan and europe51114 ) . we independently developed an endoscopic camera system for icg fluorescence angiography that can show the real - time simultaneous visible and fluorescent imaging . using this system , all procedures were approved by the local institutional animal care and use committee and were conducted according to international guidelines . a male crossbred swine ( landraceyorkshireduroc ) , aged 3 months and with a weight of 38.5 kg , was used in this study . anesthesia was induced intramuscularly with 5 mg / kg of zoletil and 2 mg / kg of xylazine . the swine was intubated and intravenous 0.9% normal saline was administered via the ear vein at a rate of 5 ml / kg / hr . anesthesia was maintained with 2% to 4% isoflurane at a rate of 23 l / min and oxygen / nitrogen at a rate of 3 l / min . vital parameters , such as arterial blood pressure , heart rate , and carbon dioxide levels , were continuously recorded . a 12 cm small craniotomy was performed at a distance of 2 cm from the midline and 3 cm anterior to the bregma . the icg dye was injected into an ear vein at a dose of 0.3 mg / kg ( 25 mg dissolved in 5 ml of sterile normal saline ) . the endoscopic icg system consists of an endoscope , a light source , a camera , a display , and a computer with software ( fig . 1 ) . although all types of endoscopes with the same size of optical adaptor are adjustable , an endoscope 4 mm in diameter and 30 in angle ( mgb endoskopische gerate gmbh berlin , berlin , germany ) was used in this study . the light source is composed of a white light source and a near infrared ( nir ) laser source ( fig . we developed a white light source that is installed with a hxp 120w/45c vis mercury lamp ( osram gmbh , munich , germany ) . we also developed a nir laser source that is installed with an 805 nm wavelength laser diode ( reallight , beijing , china ; fwhm < 2 nm ) . icg as a fluorophore binds to the hydrophobic core of human serum proteins , which shifts the absorption and emission spectra of icg . after intravenous injection of icg , the main peak of the absorption spectrum of icg moves to 805 nm , so we chose an 805 nm laser that has the same wavelength as the icg absorption peak6 ) . to deliver the combined light from the light source to the endoscope , we selected a 3 mm liquid light guide ( newport corporation , irvine , ca , usa ) . to simultaneously obtain the visible and icg fluorescence images , we used a fluorescence navigation surgery ( fns ) camera developed by korea electrotechnology research institute ( keri ) ( fig . this camera has two charge - coupled device ( ccd ) image sensors : one is for visible color imaging , and the other is for nir fluorescence imaging . a cube beam splitter was installed to divide the visible color image and the nir fluorescence image . the reflected visible color image passes through an infrared ( ir ) cut - off filter to the color - image sensor and the penetrated nir image goes to the mono - image sensor . the two images are processed on an image capture board and the data transfer board delivers the real time imaging to the computer by usb 3.0 interface . we used a 1634 mm zoom optical adaptor ( mgb endoskopische gerate gmbh berlin , berlin , germany ) to make the optical connection between the endoscope and the camera . the image monitoring program was developed by keri and can show and record single or simultaneous images of visible light and fluorescence and a visible / fluorescence overlay image . all procedures were approved by the local institutional animal care and use committee and were conducted according to international guidelines . a male crossbred swine ( landraceyorkshireduroc ) , aged 3 months and with a weight of 38.5 kg , was used in this study . anesthesia was induced intramuscularly with 5 mg / kg of zoletil and 2 mg / kg of xylazine . the swine was intubated and intravenous 0.9% normal saline was administered via the ear vein at a rate of 5 ml / kg / hr . anesthesia was maintained with 2% to 4% isoflurane at a rate of 23 l / min and oxygen / nitrogen at a rate of 3 l / min . vital parameters , such as arterial blood pressure , heart rate , and carbon dioxide levels , were continuously recorded . a 12 cm small craniotomy was performed at a distance of 2 cm from the midline and 3 cm anterior to the bregma . the icg dye was injected into an ear vein at a dose of 0.3 mg / kg ( 25 mg dissolved in 5 ml of sterile normal saline ) . the endoscopic icg system consists of an endoscope , a light source , a camera , a display , and a computer with software ( fig . although all types of endoscopes with the same size of optical adaptor are adjustable , an endoscope 4 mm in diameter and 30 in angle ( mgb endoskopische gerate gmbh berlin , berlin , germany ) was used in this study . the light source is composed of a white light source and a near infrared ( nir ) laser source ( fig . we developed a white light source that is installed with a hxp 120w/45c vis mercury lamp ( osram gmbh , munich , germany ) . we also developed a nir laser source that is installed with an 805 nm wavelength laser diode ( reallight , beijing , china ; fwhm < 2 nm ) . icg as a fluorophore binds to the hydrophobic core of human serum proteins , which shifts the absorption and emission spectra of icg . after intravenous injection of icg , the main peak of the absorption spectrum of icg moves to 805 nm , so we chose an 805 nm laser that has the same wavelength as the icg absorption peak6 ) . to deliver the combined light from the light source to the endoscope , we selected a 3 mm liquid light guide ( newport corporation , irvine , ca , usa ) . to simultaneously obtain the visible and icg fluorescence images , we used a fluorescence navigation surgery ( fns ) camera developed by korea electrotechnology research institute ( keri ) ( fig . this camera has two charge - coupled device ( ccd ) image sensors : one is for visible color imaging , and the other is for nir fluorescence imaging . a cube beam splitter was installed to divide the visible color image and the nir fluorescence image . the reflected visible color image passes through an infrared ( ir ) cut - off filter to the color - image sensor and the penetrated nir image goes to the mono - image sensor . the two images are processed on an image capture board and the data transfer board delivers the real time imaging to the computer by usb 3.0 interface . we used a 1634 mm zoom optical adaptor ( mgb endoskopische gerate gmbh berlin , berlin , germany ) to make the optical connection between the endoscope and the camera . the image monitoring program was developed by keri and can show and record single or simultaneous images of visible light and fluorescence and a visible / fluorescence overlay image . icg fluorescence began to appear on the cerebral cortex with endoscopy in approximately 25 seconds after icg dye injection , it became peak in 1 minute , then it became pale in approximately 5 minutes . visible color and icg fluorescent endoscopic images of cortical vessels were simultaneously observed on the display monitor with a high resolution . in addition , real - time merging images of the fluorescent cortical vessels could be made and marked in a purple color . the real - time merging of the visible color and fluorescent images corresponded well ( fig . since raabe et al.17 ) introduced microscopic icg fluorescence angiography for cerebrovascular surgery in 2003 , it has become a ubiquitous piece of equipment for aneurysm surgery as well as micro - anastomosis and surgery for arteriovenous fistulas and malformations . during aneurysm surgery , real - time identification of the blood flow in the vasculature within the operative field helps to improve surgical outcomes and reduce perioperative complications . this is because the repositioning and addition of clips can be promptly determined on the occlusion of parent arteries and perforators or incomplete clipping of the aneurysms . however , it is invasive and has a limitation to visualize the small perforators around the aneurysms in spite of the superiority in the acquisition of 3-dimensional imaging around the clipped aneurysms . in addition , supplementary facilities for intraoperative cerebral angiography are needed , and a time lag between the identification of compromise of the normal arteries and the repositioning of clips can result in an irreversible ischemic complication . on the other hand , microscopic icg angiography also has some shortcomings : vascular structures beyond the line of microscopic view are hard to visualize and icg fluorescence is weakly detected for the deeply located structures because of the limited spatial resolution and light illumination of microscopy . when keyhole surgery is performed , such limitations in microscopic icg angiography become more profound . to overcome the drawbacks of microscopic icg angiography in fact , endoscopic icg angiography has been applied in other surgical fields before it was introduced in the field of neurosurgery , based on the property of icg , which is circulated through the blood vessels , metabolized in the liver , and excreted via the bile duct into the intestines . examples of the surgical applications of this technique include the detection of the sentinel lymph nodes of patients with gastric cancer13 ) , visualization of bleeding points in the gastrointestinal tract9 ) , assessment of tissue perfusion and bile tree in hepatopancreatobiliary surgery22 ) , and evaluation of free flap perfusion3 ) . neuroendoscopy itself has been used mainly in skull base surgery via the transnasal approach and aneurysm surgery71021 ) . meanwhile , the use of endoscopic icg angiography in aneurysm surgery has recently been introduced5614 ) . the authors consider endoscopic icg angiography to be useful for confirming the occlusion of the aneurysms and the patency of blood flow in the parent arteries and perforators , which could not be visualized under the microscopy and its icg angiography . moreover , it is possible in the endoscopic icg angiography to better magnify the areas of interest5 ) , obtain longer icg fluorescence duration , and be more useful in the ruptured aneurysms11 ) , compared to the microscopic system . light illumination from microscopy alone the advantage of our endoscopic icg angiography system is that icg fluorescence and visible color imaging can be simultaneously visualized and merged in a real - time manner . previous microscopic or endoscopic icg angiography systems can not help but to demonstrate the visible color or nir fluorescence imaging one at a time such that surgeons may feel some difficulty in comparing the real structures with fluorescence images during the surgery . however , the size of our prototype camera is larger than the previous one , which is an improvement . first , an endoscope with smaller diameter and flexibility may be more useful because the operative space is very narrow without retracting brain tissue or drilling bony structures . as the diameter becomes smaller , imaging resolution and transmission of the light illumination and emitting fluorescence tend to be diminished . second , distinct from the endonasal approach , which is performed through the bony nasal cavity , endoscopy with icg angiography during aneurysm surgery reaches and stays around the targeted vascular structures intradurally adjacent to the brain under microscopic inspection . separate displays for microscopy and endoscopy may be detrimental and time - consuming because endoscopy can damage the adjacent cerebrovascular structures . a unified display system that can visualize both the microscopic and endoscopic imaging in one piece may be useful in terms of safety and convenience . different from the previous systems , it was possible to simultaneously display visible color and icg fluorescent images , and make a real - time merging images of them . technical supplementation and approval for the clinical implication are expected to support the application of aneurysm surgery as well as surgery for other cerebrovascular diseases .

fluoro refers to both fluorescent and fluorinated compounds . despite the shared prefix , there are very few fluorescent molecules that are soluble in perfluorinated solvents . this paucity is surprising , given that optical microscopy is a ubiquitous technique throughout the physical sciences and the orthogonality of fluorous materials is a commonly exploited strategy in synthetic chemistry , materials science , and chemical biology . we have addressed this shortage by synthesizing a panel of fluorofluorophores , fluorescent molecules containing high weight percent fluorine with optical properties spanning the visible spectrum . we demonstrate the utility of these fluorofluorophores by preparing fluorescent perfluorocarbon nanoemulsions . objectivemicroscopic indocyanine green ( icg ) angiography is useful for identifying the completeness of aneurysm clipping and the preservation of parent arteries and small perforators . neuroendoscopy is helpful for visualizing structures beyond the straight line of the microscopic view . we evaluated our prototype of endoscopic icg fluorescence angiography in swine , which we developed in order to combine the merits of microscopic icg angiography and endoscopy.methodsour endoscopic icg system consists of a camera , a light source , a display and software . this system can simultaneously display real - time visible and near infrared fluorescence imaging on the same monitor . a commercially available endoscope was used , which was 4 mm in diameter and had an angle of 30. a male crossbred swine was used.resultsunder general anesthesia , a small craniotomy was performed and the brain surface of the swine was exposed . icg was injected via the ear vein with a bolus dose of 0.3 mg / kg . visible and icg fluorescence images of cortical vessels were simultaneously observed on the display monitor at high resolution . the real - time merging of the visible and fluorescent images corresponded well.conclusionsimultaneous visible color and icg fluorescent imaging of the cortical vessels in the swine brain was satisfactory . technical improvement and clinical implication are expected .

red eye is a common eye sign / symptom presenting to the general physicians , other eye care workers , and the ophthalmologist . this symptom accounts for approximately 15% of consultations for ophthalmologists and 6% for general medical practitioners in eastern europe . it also accounted for 40% of all outpatients seen in bawku hospital ghana in 2004 and 10 district hospitals in pakistan . in nigeria , lawan reported 14.8% of patients attending their teaching hospital eye clinic presenting with red eye . studies done in port harcourt and ile - ife in nigeria showed that red eye accounted for 19.61% and 54.9% of pediatric ophthalmic eye diseases , respectively . a major cause of concern to health workers is the late presentation of patients with the condition at a standard health facility . oftentimes , in developing countries such as nigeria , individuals commence various forms of medications , both orthodox and traditional , which they consider as first aid measures . the causes of red eye are many including mild to serious conditions that may threaten vision . some even are managed as emergencies when it is being treated by an ophthalmologist . these include conjunctivitis , corneal ulcer , iritis , trauma , acute glaucoma , and use of traditional eye medicine ( tem ) . by far , the most common cause is conjunctivitis which could be infective or allergic . in a study done in ile - ife nigeria , among pediatric age group , ocular trauma was the most common cause of red eye , followed by allergic conjunctivitis and infections of the eye and its adnexa . however , in developing countries , individuals and communities often commence first aid before presenting themselves to the health practitioner . researchers in ghana studied the pattern of eye care services sought outside the orthodox health system . cataracts , eye injuries , and other ocular conditions were reportedly managed by herbalists , who often instilled the herbal mixtures directly into the eyes . the knowledge and attitude of a community regarding a disease often influence members practices including health - seeking behavior . studies carried out in developing countries of sub - saharan africa showed a sub - optimal level of knowledge , attitude , and perception of ocular conditions and eye health in general . in cape coast , ghana , a study of the eye care seeking behavior of the populace , showed 23.3% of respondents having practiced self - medication during the last episode of eye disorder whereas 5.5% visited a traditional healer . only 32.5% reported at a health facility for the management of the last episode of an eye disorder . the main reason for self - medication among respondents was a poor perception of the severity of the ocular condition . abubakar found 62.3% of secondary school students in kano , having poor knowledge and 58.2% with a poor attitude toward ocular disorders , especially blindness and its prevention . in limpopo province of south africa , the perception of ocular manifestation of hiv / aids was assessed among 2659 high school students with results showing a need for better health communication involving this population group . almost two - third ( 65.6% ) of respondents , felt an ophthalmologist or an optometrist should be consulted for ocular problems whereas 16.5% felt traditional healers could be considered . researchers in ile - ife , southwest nigeria , reported 49% of secondary school students participating in a screening exercise , to have allergic conjunctivitis ; 6% had infective conjunctivitis . more females than males had one form of ocular disorder or the other . among those with allergic conjunctivitis , only 12.5% had visited an eye specialist prior to conducting the research . in addition , health habits formed during this period is often carried on to adulthood and passed to the next generation , which can be largely influenced by their perception and attitude toward disease conditions when in school . early detection and quality treatment often positively influence the outcome of an episode of red eye . period of presentation at a health facility is dependent on several factors , including patients knowledge and perception of the symptom involved . no previous study has examined the perception and practice of senior secondary ( ss ) school students in sagamu , concerning the red eye . findings on perception and practice regarding health and related conditions can be tools to provide education and treatment plans , which ultimately improve patient care and quality of life . this study , therefore , planned to determine the attitude of ss school students in sagamu local government to red eye . the result will be useful to health planners in planning interventional programs to reduce blindness and visual impairment from red eye within the local government area ( lga ) , state , and the country at large and in the other developing countries . sagamu is a peri - urban area consisting of 15 wards with a diverse population in terms of ethnicity , occupation , age structure , and socioeconomic status . the town serves as a transit zone between the southwest and south - south regions of the country . ss school students attending public secondary schools in sagamu lga were recruited into the study . ss-3 students were exempted from participating in the study because their final examinations were on - going and were thus unavailable . a cross - sectional , descriptive study was carried out among 1082 ss ( ss ) school students in sagamu lga . using the formula for descriptive studies and assuming a prevalence of 50% ( since no previous study has been carried out on the subject matter in our environment ) as well as a nonresponse rate of 10% , a sample size of 420 was calculated . however , this was exceeded , and 1082 students were recruited into the study . multi - stage sampling technique was used for selection of study participants . the first stage involved the selection of three wards from the 15 existing wards in sagamu lga by simple random sampling . the second stage involved the selection of one ss school in each of the preselected wards also by simple random sampling . the third stage involved the selection of three arms from the five existing arms of ss-1 and ss-2 in each school . all consenting students in the selected arms were recruited into the study . a validated , semi - structured , self - administered questionnaire was used for data collection among the study participants . the questionnaire obtained information on sociodemographic data , perception , and attitude toward red eye from respondents . data were analyzed with ibm statistical package for the social sciences ( spss ) version 20 ( ibm corp . a score of 50% was graded as good whereas that < 50% was graded as poor . attitude scores were also calculated on a scale of 100% , with a good attitude being a score of 50% and above whereas a score of < 50% was graded as poor . chi - square test was used to test the association between categorical variables , with the level of significance ( p ) set at < 0.05 . approval was obtained from the zonal education office of the state ministry of education , science , and technology . this study followed the ethical standards issued by the nuremberg code and the declaration of helsinki on research in humans . participation was fully voluntary , and respondents were free to withdraw from the study if they so wished . sagamu is a peri - urban area consisting of 15 wards with a diverse population in terms of ethnicity , occupation , age structure , and socioeconomic status . the town serves as a transit zone between the southwest and south - south regions of the country . ss school students attending public secondary schools in sagamu lga were recruited into the study . ss-3 students were exempted from participating in the study because their final examinations were on - going and were thus unavailable . a cross - sectional , descriptive study was carried out among 1082 ss ( ss ) school students in sagamu lga . using the formula for descriptive studies and assuming a prevalence of 50% ( since no previous study has been carried out on the subject matter in our environment ) as well as a nonresponse rate of 10% , a sample size of 420 was calculated . the first stage involved the selection of three wards from the 15 existing wards in sagamu lga by simple random sampling . the second stage involved the selection of one ss school in each of the preselected wards also by simple random sampling . the third stage involved the selection of three arms from the five existing arms of ss-1 and ss-2 in each school . a validated , semi - structured , self - administered questionnaire was used for data collection among the study participants . the questionnaire obtained information on sociodemographic data , perception , and attitude toward red eye from respondents . data were analyzed with ibm statistical package for the social sciences ( spss ) version 20 ( ibm corp . , amonk , ny ) . a score of 50% was graded as good whereas that < 50% was graded as poor . attitude scores were also calculated on a scale of 100% , with a good attitude being a score of 50% and above whereas a score of < 50% was graded as poor . chi - square test was used to test the association between categorical variables , with the level of significance ( p ) set at < 0.05 . approval was obtained from the zonal education office of the state ministry of education , science , and technology . this study followed the ethical standards issued by the nuremberg code and the declaration of helsinki on research in humans . participation was fully voluntary , and respondents were free to withdraw from the study if they so wished . the female students were 593 ( 54.8% ) while males were 489 ( 45.2% ) in the study . many of the students were christians ( 66.5% ) and in ss-2 ( 51.1% ) [ table 1 ] . demographic characteristics of respondents majority ( 81% ) had heard of red eye and this was mainly from neighbors [ table 2 ] whereas 216 ( 19% ) had not . four hundred and fifteen ( 38.4% ) did not know the cause of red eye , while 99 ( 9.1% ) thought trauma was a cause , 147 ( 13.6% ) infection , 73 ( 6.7% ) allergy , 21 ( 1.9% ) sunlight whereas other causes such as drugs forms 327 ( 30.2% ) . two hundred and seventy - six ( 25.5% ) knew that they can be infected by someone with red eye , 632 ( 58.4% ) says they can not be infected by someone with red eye whereas 174 ( 16.1% ) did not know whether they could be so infected or not [ figure 1 ] . about 35% would instill onion when they have red eye [ figure 2 ] , followed by urine ( 21.9% ) and breast milk ( 12.9% ) . other things that they were likely to instill are salt water ( 4.6% ) , sugar water ( 4.6% ) , and battery water ( 1.5% ) whereas 19.4% will not use any of the substances . though majority says that a doctor is the one that takes care of red eye , 24 says self , 19 says their parent , 55 says chemist , 58 nurses whereas 12 says a herbalist can care for red eye . source of respondent 's information about red eye can someone with red eye infect you ? traditional medications used in red eye awareness of red eyes was associated with age ( p = 0.005 ) , but not with sex and religion [ table 3 ] . perception grade was good in 49 ( 4.5% ) and poor in 1033 ( 95.5% ) . five hundred and forty - three ( 50.2% ) felt that red eye could lead to blindness [ figure 3 ] . perception of red eye was associated with age ( p = 0.027 ) and class ( p 0.001 ) . the factors associated with attitude toward red eye are shown in table 3 ; this is statically significant for class ( p 0.001 ) . factors associated with awareness , perception , and attitude toward red eyes can red eye lead to blindness ? attitude refers to the feelings toward a subject , as well as any preconceived ideas ; one may have toward it . practice refers to the ways in which people demonstrate their knowledge and attitudes through their actions on the subject matter . it is not surprising that many of the respondents have heard of red eye because epidemic conjunctivitis called apollo is a very common cause of red eye in nigeria and , especially the west africa coast . in this study , majority heard about red eye from neighbors which show the impact of community on people 's health . that only 13.9% heard from health workers shows that there is a need for eye care workers to intensify health education , especially among students . only 13.6% were aware that red eye could be caused by infections which may also influence their attitude to red eye . this might explain why more than half of the students say they can not be infected by someone with red eye . it is comparable to findings from limpopo , south africa , where only 31.4% felt hiv infection and its sequelae could result in red eyes . most ( 96.2% ) respondents attitude toward red eye was poor , a finding quite different from that reported in kano , where 52.8% of students had a poor attitude toward severe ocular disorders . the observed difference might be due to the fact that this study focused solely on red eyes whereas the study carried out in kano assessed knowledge and attitude toward causes of blindness and its prevention . usually in the developing countries , people usually use traditional eye medication and unprescribed medications to treat their red eye . these medications that are being used for red eye as found in this study include onion , urine , breast milk , salt water , sugar water , and battery water . these can cause chemical conjunctivitis , infective conjunctivitis , especially gonococcal conjunctivitis when infected urine is used and if not detected and treated early , it can result in blindness . this finding also confirms the report of other researchers who have shown that people seek alternative eye care treatment instead of orthodox treatment with implications for ocular health . there was a report of a usage of herbal preparation on the eye with resultant blindness . health education done in schools during assembly especially by eye care workers will go a long way to help reduce this among the students and in the communities as these children will , in turn , tell their parents about what they are taught at school . furthermore , incorporation of school eye health program into the existing school health program will be of immense benefit . perception grade and attitude of this population were poor , this may mean that the perception and attitude to red eye , even though very common is low in the general population as the students are actually reflecting what they have learnt from their homes . intensive effort to enlighten the population will therefore help to reduce blindness / visual impairment in our community . these enlightenment programs can be taken to the religious houses , i.e. , mosques and churches for a wider coverage . we therefore conclude that secondary school students in sagamu have poor perception and attitude to red eye . appropriate eye health services and education should be included in school health services and early presentation to eye care centers for its treatment should be encouraged . auricular acupuncture is a diagnostic and treatment system based on normalizing the body 's dysfunction through stimulation of points on the ear . resulting amelioration of pain and illness is believed to be through the reticular formation and the sympathetic and parasympathetic nervous systems ( 1 ) . ear acupuncture , is an acupuncture technique similar to reflexology , and is speculated that the technique works because groups of pluripotent cells contain information from the whole organism and create regional organization centers representing different parts of the body , through recruitment of more cortex cells dedicated to specific areas of the body . thus stimulation of a reflex point in the ear can relieve symptoms of distant pathology with a reliable duration . rudimentary forms of acupuncture which probably arose during the stone age have survived in many parts of the world right down to present day . primitive sharp stones and bamboo were replaced by fish bones , bamboo clips and later various shapes of needles made of metal . when stones and arrows were the only tools of war , warriors wounded in war found that some diseases that affected them for many years were gone , as probably testify scars on the skin of the mummified body of similaun , italy . the bantus of south africa scratch certain areas of their skin to allay the symptoms of many illnesses , while in brazil there is a tribe whose method of treating illness is to shoot tiny arrows from a blowpipe to specific areas of the skin . the practice of cauterizing a part of the ear with a hot metal probe has also been reported among certain tribes in arabia . this is probably a vestige of the acupuncture practiced in ancient egypt and saudi arab . ( now in the british museum ) describes a system of channels and vessels in the body which approximates more closely to the chinese system of channels than to any known system of blood vessels , lymph vessels or nerves . the egyptologist alexandre varille ( 19091951 ) has documented that women in ancient egypt who did not want any more children , had their external ear pricked with a needle or cauterized with heat . gold earrings worn by mediterranean sailors were not just used as decorations , but were said to improve vision . hippocrates , the father of of greek medicine , reported that doctors made small openings in the veins situated behind the ear to facilitate ejaculation and reduce impotency problems . the greek physician galen introduced hippocratic medicine to the roman empire in the second century ce , and commented on the healing value of scarification at the outer ear . after the fall of roman empire , the medical records of egyptian , greek and roman medicine were best preserved in ancient persia and arabian world . included in these persian records were specific references to medical treatments for sciatic pains and sexual related disease produced by cauterization of the external ear . during renaissance sporadic clinical reports in europe the dutch east india company actively engaged in trade with china from 1600s to 1800s , and its merchants brought chinese acupuncture practices back to europe . doctors working with the company had become impressed by the effectiveness of needles and moxa , and cauterization of the external ear , or by cutting the veins behind the ears for relieving conditions such as sciatic pains and arthritis of the hip . in 1637 probably for the first time in europe was described by the portugese physician zacatus lusitanus the treatment of sciatic pain by cauterization of the ear after that bloodletting had failed . the italian anatomist and surgeon antonio maria valsalva ( 16661723 ) , who made the first modern anatomical description of the ear ; in 1717 published the aura humanus tractatus , where he describes the treatment of toothache by scarification of antitragus . in 1810 ignazio colla of parma , italy , reported the observation of a man stung by a bee in the antehelix which resulted in dramatic relief of pain in the legs , and in the same year dr cecconi , another italian physician , performed cauterization to help treatment of sciatic pain . in 1850 the french journal des connaissainces medico - chirurgicales reported 13 different cases of sciatic pain that had been treated by cauterization with a hot iron applied to the ear . but it was not until a century later that paul nogier rediscovered this type of treatment . in 1957 , dr paul nogier a physician resident in lyons , france , first presented his observations of the somatotopic correspondences of the ear . dr nogier ( 2 ) originated the concept of an inverted fetus map on the external ear ( fig . he developed this theory after noticing that some patients attending his clinic had a small scar from a burn on part of their ear . on inquiring into this , he was told that a very small area of their ear had been cauterized by a certain madame barrin for treatment of sciatic pain a treatment that they proved very rapid and effective . later his first great insight was the recognition of the homunculus , the man in the ear , the representation and anatomical correlation of the inverted fetus in the ear . points on the body , for example the knee , corresponded precisely with the fetal representation of the knee in the auricle . auriculotherapy following nogier 's theory uses the ear to help determine whether the right and left hemispheres of the brain are functioning as a dynamic whole , whether there are specific neurological , musculo - skeletal or organ systems that are in imbalance , and whether there are any blockages to treatment , such as scar tissue or emotional disorders and it should be a new diagnostic system too ( 2,3 ) . figure 1.this drawing illustrates the concept of an inverted fetus map on the external ear . then dr nogier noticed that there was a distinct change in the amplitude and dimension of the pulse when certain points on the auricle were stimulated . being able to detect the vas on the radial pulse of the patients left hand enables the practitioner to precisely determine the location of a point , whether there is a pathology in the region of the body that relates to specific points , and whether certain substances are indicated . accurate employment of the vas would be essential in diagnosis and treatment following the principles of nogier 's auricolomedicine . nogier collaborated with a group of medical colleagues who , in a spirit of cooperation and discovery , shared their experiences . one of those colleagues , dr jacques niboyet , convinced nogier to introduce his discoveries to the congress of the mediterranean society of acupuncture in february of 1956 . attending that congress was dr grard bachmann who published nogier 's research , translated into german , in a acupuncture journal in 1957 . this journal had an international circulation and it was not long before japanese acupuncturists became familiar with nogiers reflex system . the discovery of the system spread to china and led to intensive research by the chinese medical authorities at a time of renewed interest in traditional chinese medicine . after learning about the nogier ear charts in 1958 , a massive study was initiated by the nanjing army ear acupuncture research team . this chinese medical group verified the clinical effectiveness of the nogier approach and assessed the conditions of over 2000 clinical patients , recording which ear points corresponded to specific diseases . the outcome of that research was very positive and resulted in the utilization of this therapy by the was published an ear chart remarkably similar to that of dr nogier in 1958 ( 4 ) . nogier acknowledged that chinese traditional medicine had been using ear points for acupuncture prior to his discovery , but these had been considered empirical points for particular treatments and were not associated with a somatotopic representation of the homunculus in the ear . this oversight appears to have inhibited awareness of options laid open to recognize and treat other points in the ear following an anatomical relationship to the points already known at the time . later the american physician td oleson has published a very important paper that is a real milestone in ear acupuncture ( 5 ) . to experimentally evaluate the claims by french and chinese ear acupuncture that a somatotopic mapping of the body was represented in the external ear , 40 patients were examined to determine areas of their body where there was musculoskeletal pain . each patient was draped with a sheet and a physician conducting the auricular diagnosis had no prior knowledge of the patient 's medical condition , but simply examined the patient 's ear for areas of elevated skin conductivity or tenderness . the concordance between the established medical diagnosis and the auricular diagnoses was 75.2% ( 5 ) . these results thus supported the hypothesis that there is a somatotopoic organization of the body represented upon the human auricle , but represented following definite areas not meridian lines or other energetic concepts . in the last years modern clinical and basic research is confirming the efficacy of ear acupuncture mostly in the treatment of pain both acute and chronic ( 69 ) , and of anxiety related disorders ( 1012 ) . while the treatment of irritable bowel syndrome , obesity , smoke cessation , alcohol withdrawal and other types of substance abuse disease is still waiting definitive confirmation ( 1317 ) . basic research is trying to explain the effect of therapeutic reflexes induced by ear acupuncutre so behavioral analgesia produced by auricular acupuncture can be blocked by the opiate antagonist naloxone , indicating the role of endorphinergic systems in understanding the underlying mechanisms of auriculotherapy analgesia ( 18 ) ; and ear stimulation in healthy persons is associated with changed activity in the sympathetic and parasympathetic nervous system depending on the site of stimulation and period of observation ( 19 ) . auricolotherapy is a treatment diffusing in all over the world , and its patterns follow the principles of chinese acupuncture , revised and updated , with chinese maps of the ear ; the principles of paul nogier and also the principles of reflexology basing on somatotopic maps that do not recognize energetic - based stimulation , while just the evocation of a reflex stimulating precise areas of the ear ; moreover are used for stimulation of ear skin many different tools : finger acupressure , laser , electricity , different types of needles , magnetic balls , seeds . actually one of the many methodological problems with auricular acupuncture is that there are so many maps of the ear and little agreement exists regarding point location , lacking definitive anatomic study on ear skin and its somatotopic correspondences . another problem is that all correspondence or reflex systems do not correlate to the knowledge of anatomy and physiology based on the patterns of mainstream medicine ( 20 ) .

background : red eye is a very common presenting complaint in clinical practice among all age groups , including adolescents . health habits formed during adolescence is carried to adulthood and is often a consequence of their perception . this study , therefore , determined the perception of students toward the red eye.aim:to determine the perception of red eye and its associated factors among secondary school students in sagamu.methods:a cross - sectional descriptive study was carried out among 1082 senior secondary school students in sagamu local government area , using a semi - structured self - administered questionnaire . data were analyzed using spss version 20 . relevant descriptive and inferential statistics were calculated.results:the mean age of respondents was 15.27 1.48 years . there were more females ( 54.8% ) than males . majority ( 81% ) had heard of red eye , and this was mainly from neighbors ; 58.4% felt they could not contact red eye from an infected person . about 35% would instill onion if they had a red eye . about 50.2% felt red eye could lead to blindness . awareness of red eye was associated with age ( p = 0.005 ) , but not with sex and religion . among respondents , 95.5% and 96.2% had a poor perception as well as a poor attitude toward red eye , respectively.conclusion:the perception and attitude of senior secondary school students in sagamu to red eye is poor . appropriate eye health education and promotional services , including periodic eye examination of students , should be carried out in school health services . early presentation to eye care centers for its treatment should be encouraged . auricular acupuncture is a diagnostic and treatment system based on normalizing the body 's dysfunction through stimulation of definite points on the ear . rudimentary forms of acupuncture which probably arose during the stone age have survived in many parts of the world right down to present day . it was used in the ancient egypt , rome , greece and all the mediterranean area . it is a microacupuncture technique similar to reflexology , and was first described in france in 1950 by paul nogier who is considered the father of modern ear acupuncture . it was speculated that the technique works because groups of pluripotent cells contain information from the whole organism and create regional organization centers representing different parts of the body . nevertheless stimulation of a reflex point in the ear seems relieve symptoms of distant pathologies . modern research is confirming the efficacy of ear acupuncture for analgesia and anxiety related disease , while tobacco dependence and other substance abuse still need confirmation . actually main methodological problems with auricular acupuncture are that exist too many maps with little agreement regarding point location in the ear , and that the correspondence or reflex systems does not correlated with modern knowledge of anatomy and physiology .

in this dataview , we look at the sponsors of health care , providing statistics on business , household and government health spending . the companion article , national health expenditures , 1995 ( levit et al . , 1996 ) presents health care expenditure data in an accounting structure that describes the size , current growth and historical trends of health care by service , matched against the sources that pay the health care bill , such as private health insurance ( phi ) and government programs like medicaid and medicare . the national health expenditures ( nhe ) structure provides policymakers , researchers , and the public with valuable health care expenditure statistics . however , it does not provide information on the size and impact of rising health costs on the sponsors of health care . the accounting structure used in this dataview breaks apart the nhe to examine the effects of health care expenditures on the sponsoring sectors . business , households and government finance health care bill payers through taxes , premium payments ( for both private and public health insurance ) , and general revenues ; these sectors also make direct payments to providers . for 1990 through 1995 , we will present statistics on how much each sponsor spent on health care and the impact of these expenditures on their ability to pay . since 1993 , a combination of slower health care cost growth and an upswing in the economy has stabilized or eased the burden that business , households and government faced in financing health care . business , especially , is benefiting from the changing health care cost environment . during the same period , spending for health services and supplies ( hss ) ( a subset of nhe ) reached $ 957.8 billion in 1995 . business paid for 26 percent of hss , households paid for 34 percent , and the public sector paid for 38 percent . another component , non - patient revenues , made up the remaining 2 percent . over time , this relationship among the sponsors has changed . in 1965 , households were the primary sponsors of health care . since then households have been gradually paying for a smaller proportion of hss and business and the public sector has been paying for a larger proportion . however , by aggressively controlling their health care expenses , business decreased its share of hss during the 1990s . actions by the public sector were less dramatic with the result that the pubic sector share of health care costs increased significantly during this period after nearly 20 years of relative stability . by 1991 , business spent $ 249.4 billion on health care in 1995 , including $ 183.8 billion for employer - sponsored health insurance . the 1993 to 1995 average annual growth of 4.3 percent was the slowest since we began to measure business health spending in 1965 . the burden placed on business eased over the years 1993 through 1995 . during this time overall health care costs grew at a slow rate . also , enrollment in managed care plans grew . these plans generally charged lower premiums than traditional fee - for - service plans . in highly competitive markets , managed care plans also kept premiums low to increase enrollment and boost market share . this in turn forced traditional indemnity insurance companies competing with managed care plans to develop new low cost products or lose market share . these marketplace changes contributed to health care costs consuming less of the business 's compensation costs and profits . according the bureau of labor statistics ' employment cost index ( eci ) for civilian workers , the primary driver of the slowdown in benefits growth was the decline in the cost of health benefits . although a separate health insurance benefit index is not available , the eci program does publish employer compensation cost levels using current employment weights . based on that eci measure , average civilian employer health insurance costs fell 6.2 percent ( $ 1.29 to $ 1.21 per hour worked ) between march 1994 and 1995 . during the same period , wages and salary costs grew 0.5 percent , from $ 13.06 to $ 13.12 per hour worked . private health insurance is obtained primarily through employer - sponsored health plans and health plans purchased separately by individuals . in 1995 , private health insurance premiums continued to grow at a slow rate , increasing just 2.6 percent from the previous year . however , the employee share of employer - sponsored health insurance increased 8.1 percent from 1994more than twice the growth rate of employer contribution to premiums with the result that the share of health plan premium costs paid by employers inched further downward . in recent years , many employers shifted more of the premium burden to their employees by either requiring workers to contribute to their own health insurance premiums or by raising the required employee premium more rapidly than overall premium cost growth . one factor contributing to the premium cost shift from employers to employees was the migration of covered workers into lower cost managed care plans , such as health maintenance organizations , preferred provider organizations , and point of service plans . although employees generally incurred both reduced out - of - pocket costs and expanded services under a managed care plan , employers typically paid a smaller portion of the total premium for these plans than for traditional indemnity ( fee - for - service ) plans . therefore , it is likely that the rapid expansion of managed care enrollment in the 1990s had the effect of boosting the employee share of total premiums . the majority of spending ( $ 182.6 billion ) was for out - of - pocket health care expenses not covered by insurance and for copayments and deductibles . the remainder was spent on phi premiums ( $ 68.5 billion ) and premiums and contributions to the medicare trust funds ( $ 72.2 billion ) . the switch to managed care affected the level of household out - of - pocket payments . starting with the late 1980s , the growth in out - of - pocket expenses for health care has been the lowest since 1970 . this coincides with the increased enrollment in managed care plans that have lower deductibles and copayments than traditional fee - for - service plans . households have paid approximately 5.5 percent of their income after taxes for health care ( u.s . bureau of labor statistics , 1990 - 95 ) . according to consumer expenditure survey data , elderly households ( households with reference persons 65 years of age or over ) spend three times as much of their income after taxes on health care as non - elderly households . in 1995 , the public sector paid 38 percent , or $ 360.4 billion , of hss . the federal government paid $ 203.4 billion , while state and local government paid $ 157.0 billion . the portion of federal government revenues financing health care costs declined slightly in 1994 and 1995 after several years of significant growth . the decrease from 24.0 percent in 1993 to 22.8 percent in 1995 was primarily due to an increase in the federal revenues rather than a slowdown in federal health spending . medicare incurred expenditures of $ 187.0 billion in 1995 , up 11.6 percent from the previous year . the medicare program is funded through 3 sources : ( 1 ) payroll taxes paid by employers and households ; ( 2 ) premiums and income taxes on social security benefits paid by households ; and ( 3 ) income from the federal government . in this accounting scheme , federal government contributions to the medicare program include trust fund interest income , net changes in the trust fund balances and transfers from the general fund of the treasury ( board of trustees of the federal hospital insurance trust fund , 1996 and board of trustees of the federal supplemental insurance trust fund , 1996 ) . in 1995 , medicare hospital insurance ( hi ) benefit payments and administrative expenses exceeded income by $ 2.6 billion . this financing shortfall was met by redeeming $ 2.6 billion in treasury securities held by the hi trust fund . this necessity contributed to the increase in the government 's share of total expenditures in 1995 . the disparity between income and expenses is expected to grow rapidly , with the assets of the hi trust fund becoming exhausted about 2001 in the absence of corrective legislation .

for the period 1990 - 95 , we will present data on health care spending by business , households , and government . in addition , we will measure the relative impact of these expenditures on each sector 's ability to pay . in 1994 and 1995 , health care costs experienced the slowest growth in 3 decades . combined with healthy revenue growth , slow cost growth helped ease or stabilize the financing burden faced by business , households and government . we developed a sensitive and accurate analytical method for quantifying methyleugenol ( me ) in human serum . our method uses a simple solid - phase extraction followed by a highly specific analysis using isotope dilution gas chromatography - high resolution mass spectrometry . our method is very accurate ; its limit of detection is 3.1 pg / g and its average coefficient of variation is 14% over a 200-pg / g range . we applied this method to measure serum me concentrations in adults in the general u.s . population . me was detected in 98% of our samples , with a mean me concentration of 24 pg / g ( range < 3.1 - 390 pg / g ) . lipid adjustment of the data did not alter the distribution . bivariate and multivariate analyses using selected demographic variables showed only marginal relationships between race / ethnicity and sex / fasting status with serum me concentrations . although no demographic variable was a good predictor of me exposure or dose , our data indicate prevalent exposure of u.s . adults to me . detailed pharmacokinetic studies are required to determine the relationship between me intake and human serum me concentrations.imagesfigure 1figure 2figure 3figure 4figure 5figure 6figure 7

it can be developmental or acquired and rarely may be associated with temporomandibular joint ( tmj ) ankylosis . it has been only occasionally reported since then , probably due to its usually asymptomatic nature . in 1941 , hrdlicka reported the first cases of bmc in 21 specimens from an unspecified number of dried skulls , and in 1948 , sicher first reported this anomaly in a living person . honee and bloem described a case of bifid condyle in the cadaver of a 71-year - old patient . although this type of morphologic change is generally associated with trauma , conditions such as teratogenic drug use , genetic inheritance , infection and exposure to radiation can also cause the development of this anomaly . the first patient was a 14-year - old female with a history of extraction of lower decayed and painful left first molar done about 3 months earlier . the patient continued to have pain and recurrent swelling on and off after extraction and was managed by her treating dentist with medications . the patient presented to the department with dull pain and facial swelling in relation to lower left molar region extending to the angle of mandible . axial and coronal computed tomography ( ct ) images of bilateral tmj and mandible with multiplanar reformatting ( mpr ) were done for evaluating any pathologic fracture and tmj pathosis . these findings are best seen on the axial and sagittal images [ figure 1 ] . coronal and axial ct images demonstrate left bmc oriented anteroposteriorly with articular surface irregularity the second patient was a 12-year - old female patient , referred for ct examination for evaluation of mild facial asymmetry and suspected tmj ankylosis . she had a reduced degree of jaw opening since childhood , and subsequently developed midline deviation to the left along with difficulty in mastication . these images demonstrated sagittal splitting of the left mandibular condyle into medial and lateral condylar head . the first patient was a 14-year - old female with a history of extraction of lower decayed and painful left first molar done about 3 months earlier . the patient continued to have pain and recurrent swelling on and off after extraction and was managed by her treating dentist with medications . the patient presented to the department with dull pain and facial swelling in relation to lower left molar region extending to the angle of mandible . axial and coronal computed tomography ( ct ) images of bilateral tmj and mandible with multiplanar reformatting ( mpr ) were done for evaluating any pathologic fracture and tmj pathosis . these findings are best seen on the axial and sagittal images [ figure 1 ] . the second patient was a 12-year - old female patient , referred for ct examination for evaluation of mild facial asymmetry and suspected tmj ankylosis . she had a reduced degree of jaw opening since childhood , and subsequently developed midline deviation to the left along with difficulty in mastication . these images demonstrated sagittal splitting of the left mandibular condyle into medial and lateral condylar head . szentpetery et al , in their study of 1882 cadaveric skulls , found the incidence of bmc to be 0.48% . a report of four cases of bifid condyles presented by loh and yeo included one involving an edentulous cadaver . in a literature review of reported cases in living patients , subsequently , four cases by stefanou et al and two other cases have been reported . recently , artvinli and kansu ( 2003 ) and antoniades et al ( 2004 ) have reported the first two cases of trifid condyle in patients who also had bifid condyles on the other side . described the first case of bilateral bifid condyles in a living patient . since then 10 more cases have been reported . current literature review in living patients revealed a total of 45 cases of bifid condyle , out of which 11 cases are bilateral , giving a ratio of approximately 3:1 . however , if the survey of the dry skulls and cadaveric reports are included , then the total number is 84 cases , of which 15 are bilateral . they appear to be more common on the left side in unilateral cases by a ratio of 2:1 . most cases ( 67% ) are asymptomatic and are found on routine dental radiographic examination . however , some have been reported in patients presenting with tmj symptoms , swelling , trauma , or ankylosis . the embryologic theory suggests that it is due to the obstruction of the blood supply to the condyle or the persistence of the vascularized fibrous septa . another theory postulates trauma as the cause with disruption or dislocation of joint integrity due to birth trauma , condylar fractures or surgical condylectomy in separate studies conducted by loh and yeo and antoniades et al , it was found that most cases of bifid condyle were asymptomatic and not associated with any history of trauma .. the site of fracture of the mandibular condyle and its relation to the insertion of the lateral pterygoid muscles are factors determining the future development of bifid condyle . other causes that have been proposed include genetics , endocrine disturbances , infection , radiation , nutritional deficiencies , and exposure to teratogenic substances . support for the latter suggestion comes from the work of gundlach et al who experimentally induced bifid condyles in rats by injecting teratogenic substances such as n - methyl - n - nitrosourea and formhydroxamic acid in different concentrations , at various stages of pregnancy . the extent of bifid condyle may range from a shallow groove to discrete condylar heads and the orientation may be anteroposterior or mediolateral . it has been postulated that anteroposterior splitting usually occurs in patients with identifiable antecedent trauma , while mediolateral splitting is usually developmental in origin . but here in our patient we have seen that there was a history of trauma for the mediolaterally splitted condyle and the anteroposterior splitting was because of osteomyelitis and the patient denied any previous trauma in all her life . szentpetery et al . have suggested that when two condylar parts lie in the sagittal plane , trauma is indicated as the cause , and when the parts lie in the coronal plane , the persistence of the fibrous septa at the condylar cartilage is likely to be the cause . while this may be true for the majority of cases , some mediolateral bifid condyles have been reported following sagittal fracture through the condylar head . according to blackwood , two articulating surfaces of the bmc were divided by a groove and could be orientated mediolaterally or anteroposteriorly , characterizing a specific entity . in this case report , as postulated above , groove formation and presence of medial and lateral head of both condyles clearly demonstrated the formation of the bmc . majority of the cases are detected during routine radiographic examination . in most of the cases , however , bmc is reported to be associated with pain , swelling , restricted mouth opening and most commonly tmj clicking . in our case due to the lack of clinical symptoms , diagnosis is made by radiographic findings . ct scan is the best radiograph for detection of bmc because it allows for detailed evaluation of condylar morphology . however , bmc can also be seen on opg , but sometimes the overlapping of the anatomic structures can hide the bifidity . in summary , bmc , an anatomic variation of condyle is a rare anomaly whose etiology is unknown . in the present case , literature says that it is most commonly due to facial trauma in the early stages of development . patients with internal articular derangement should be treated with occlusal splints and arthroscopic surgery , while patients with associated articular ankylosis may need surgical condylectomy or arthroplasty . awareness of this abnormality will help to avoid mistaking it for a fracture or a tumor . our first case also illustrates the point that bmc may be associated with tmj ankylosis . such a case requires detailed clinical examination and evaluation by ct for further management and for prognosticating the outcome . this retrospective study included 343 glaucoma subjects who were examined at the glaucoma clinic of the asan medical center . patients who met our inclusion criteria of at least three years of follow - up and seven or more reliable vf measurements were consecutively enrolled by medical record review . eyes that underwent intraocular surgery ( including cataract and glaucoma filtering procedures ) during the follow - up period were excluded . at initial diagnosis , each patient received a comprehensive ophthalmologic examination that included a review of medical history for the presence of systemic diseases like systemic hypertension , diabetes , cerebrovascular accident , dyslipidemia , and migraine , measurement of best - corrected visual acuity to confirm that visual acuity was adequate for automated perimetry performance , slit - lamp biomicroscopy , goldmann applanation tonometry , gonioscopy , dilated fundoscopic examination using a 90- or 78-diopter lens , stereoscopic optic disc and retinal nerve fiber layer ( rnfl ) photography , vf examination by standard automated perimetry ( humphrey swedish interactive threshold algorithm standard strategy 24 - 2 ; carl zeiss meditec , dublin , ca , usa ) , and measurement of central corneal thickness ( cct ; dgh-550 instrument , dgh technology inc . , all included patients met the following criteria at the baseline exam : best - corrected visual acuity of 20 / 30 or better , with a spherical equivalent ( se ) within 5 diopters and a cylinder correction within + 3 diopters ; vf mean deviation ( md ) higher than -20 db ; and the presence of a normal anterior chamber and open - angle on slit - lamp and gonioscopic examinations . diagnosis of glaucoma was based on both the presence of typical glaucomatous changes in the optic disc and glaucomatous vf defect . thus , eyes with a glaucomatous optic disc and a normal vf were not defined as having glaucoma . glaucomatous optic disc change included increased cupping ( vertical cup - disc ratio more than 0.7 ) , a difference in the vertical cup - disc ratio ( more than 0.2 between the eyes ) , diffuse or focal neural rim thinning , disc hemorrhage , or rnfl defects . eyes defined as having glaucomatous vf defects met two of the following three criteria ( as confirmed by more than two reliable consecutive tests ) : 1 ) a cluster of three points with a probability of less than 5% on a pattern deviation map in at least one hemifield , including at least one point with a probability of less than 1% or a cluster of two points with a probability of less than 1% ; 2 ) a glaucoma hemifield test result outside the normal limit ; and 3 ) a pattern standard deviation result of less than 5% . reliable vf assessment was defined as a vf test with a false - positive error of less than 15% , a false - negative error of less than 15% , and a fixation loss of less than 20% . if at least one eye showed glaucomatous changes , that patient was included for analysis . iop - lowering medication was prescribed if the eyes showed glaucomatous optic disc changes or had both a glaucomatous optic disc and vf changes . all procedures conformed to the declaration of helsinki , and the study was approved by the institutional review board of the asan medical center . since glaucomatous damage involves both structural and functional changes that may not appear at the same time , glaucoma progression was determined by either structural or functional measures . structural progression was assessed using a stereoscopic optic disc and red - free rnfl photographs . two glaucoma experts ( krs and jhn ) independently assessed all of the photographs in order to estimate the glaucoma progression between the patients ' first and last visits . both graders were blind to each other 's progression assessments and to all clinical and vf information . photographs were presented in chronological order and with masking of patient identification , age , and test date . each grader viewed all photographs of each eye before making an assessment and was asked to determine the possible presence of a glaucomatous optic disc or of rnfl progression , as revealed by an increase in the extent of neuroretinal rim thinning , enhancement of disc excavation , any widening or deepening of a rnfl defect , and/or the appearance of new disc hemorrhage . if the opinions of the two observers differed , a third examiner ( dj ) made the final decision . commercial software ( guided progression analysis , carl zeiss meditec ) was used to determine progressive functional loss of the vf . vf defect progression was defined as a significant deterioration from the baseline pattern of deviation at three or more of the same test points and was evaluated on three consecutive examinations . if only one eye showed progression by either structural or functional assessment during the follow - up period , the patient was included in the upg group . in contrast , if both eyes showed progression , the patient was included in the bpg group . age , gender , and the prevalence of systemic diseases ( systemic hypertension , diabetes , cerebrovascular accident , migraine , and dyslipidema ) were compared between the upg and bpg groups . in the upg group , pre - treatment iop , mean post - treatment iop , cct , se , baseline vf md , vf pattern standard deviation , and progression rates were compared between the pe and the npe within the same patient . the progression rates ( md slope ) were determined by linear regression analysis of md values in serial vf analyses . in the bpg group , the same parameters were compared between the fpe and the spe within the same patient . normally distributed data were compared between the upg and bpg groups using unpaired t - tests . non - normally distributed data were compared using either the mann - whitney or wilcoxon signed rank test . this retrospective study included 343 glaucoma subjects who were examined at the glaucoma clinic of the asan medical center . patients who met our inclusion criteria of at least three years of follow - up and seven or more reliable vf measurements were consecutively enrolled by medical record review . eyes that underwent intraocular surgery ( including cataract and glaucoma filtering procedures ) during the follow - up period were excluded . at initial diagnosis , each patient received a comprehensive ophthalmologic examination that included a review of medical history for the presence of systemic diseases like systemic hypertension , diabetes , cerebrovascular accident , dyslipidemia , and migraine , measurement of best - corrected visual acuity to confirm that visual acuity was adequate for automated perimetry performance , slit - lamp biomicroscopy , goldmann applanation tonometry , gonioscopy , dilated fundoscopic examination using a 90- or 78-diopter lens , stereoscopic optic disc and retinal nerve fiber layer ( rnfl ) photography , vf examination by standard automated perimetry ( humphrey swedish interactive threshold algorithm standard strategy 24 - 2 ; carl zeiss meditec , dublin , ca , usa ) , and measurement of central corneal thickness ( cct ; dgh-550 instrument , dgh technology inc . , all included patients met the following criteria at the baseline exam : best - corrected visual acuity of 20 / 30 or better , with a spherical equivalent ( se ) within 5 diopters and a cylinder correction within + 3 diopters ; vf mean deviation ( md ) higher than -20 db ; and the presence of a normal anterior chamber and open - angle on slit - lamp and gonioscopic examinations . diagnosis of glaucoma was based on both the presence of typical glaucomatous changes in the optic disc and glaucomatous vf defect . thus , eyes with a glaucomatous optic disc and a normal vf were not defined as having glaucoma . glaucomatous optic disc change included increased cupping ( vertical cup - disc ratio more than 0.7 ) , a difference in the vertical cup - disc ratio ( more than 0.2 between the eyes ) , diffuse or focal neural rim thinning , disc hemorrhage , or rnfl defects . eyes defined as having glaucomatous vf defects met two of the following three criteria ( as confirmed by more than two reliable consecutive tests ) : 1 ) a cluster of three points with a probability of less than 5% on a pattern deviation map in at least one hemifield , including at least one point with a probability of less than 1% or a cluster of two points with a probability of less than 1% ; 2 ) a glaucoma hemifield test result outside the normal limit ; and 3 ) a pattern standard deviation result of less than 5% . reliable vf assessment was defined as a vf test with a false - positive error of less than 15% , a false - negative error of less than 15% , and a fixation loss of less than 20% . if at least one eye showed glaucomatous changes , that patient was included for analysis . iop - lowering medication was prescribed if the eyes showed glaucomatous optic disc changes or had both a glaucomatous optic disc and vf changes . all procedures conformed to the declaration of helsinki , and the study was approved by the institutional review board of the asan medical center . since glaucomatous damage involves both structural and functional changes that may not appear at the same time , glaucoma progression was determined by either structural or functional measures . structural progression was assessed using a stereoscopic optic disc and red - free rnfl photographs . two glaucoma experts ( krs and jhn ) independently assessed all of the photographs in order to estimate the glaucoma progression between the patients ' first and last visits . both graders were blind to each other 's progression assessments and to all clinical and vf information . photographs were presented in chronological order and with masking of patient identification , age , and test date . each grader viewed all photographs of each eye before making an assessment and was asked to determine the possible presence of a glaucomatous optic disc or of rnfl progression , as revealed by an increase in the extent of neuroretinal rim thinning , enhancement of disc excavation , any widening or deepening of a rnfl defect , and/or the appearance of new disc hemorrhage . if the opinions of the two observers differed , a third examiner ( dj ) made the final decision . commercial software ( guided progression analysis , carl zeiss meditec ) was used to determine progressive functional loss of the vf . vf defect progression was defined as a significant deterioration from the baseline pattern of deviation at three or more of the same test points and was evaluated on three consecutive examinations . if only one eye showed progression by either structural or functional assessment during the follow - up period , the patient was included in the upg group . in contrast , if both eyes showed progression , the patient was included in the bpg group . age , gender , and the prevalence of systemic diseases ( systemic hypertension , diabetes , cerebrovascular accident , migraine , and dyslipidema ) were compared between the upg and bpg groups . in the upg group , pre - treatment iop , mean post - treatment iop , cct , se , baseline vf md , vf pattern standard deviation , and progression rates were compared between the pe and the npe within the same patient . the progression rates ( md slope ) were determined by linear regression analysis of md values in serial vf analyses . in the bpg group , the same parameters were compared between the fpe and the spe within the same patient . normally distributed data were compared between the upg and bpg groups using unpaired t - tests . non - normally distributed data were compared using either the mann - whitney or wilcoxon signed rank test . a total of 343 korean patients were included in the final analysis , of which 182 were men and 161 were women . the mean follow - up period was 4.2 years . at baseline , 134 patients had unilateral glaucoma . among these 134 patients , 102 showed glaucomatous optic disc changes but a normal vf in the fellow eye , while the remaining 32 patients had a normal appearing optic disc and a normal vf in the fellow eye . among the 134 unilateral glaucoma patients , 25 patients showed unilateral progression during follow - up while 6 patients showed bilateral progression ; thus , 31 patients had either unilateral or bilateral progression while 103 patients were stable . among the 32 patients with unilateral glaucoma and a normal optic disc and vf in the fellow eye , none of the fellow eyes showed progression . at baseline , 209 patients had bilateral glaucoma , of which 43 showed progression with 25 showing bilateral progression . 1 . hence , the bpg group consisted of 31 patients ( 6 from the unilateral glaucoma at baseline group and 25 from the bilateral glaucoma at baseline group ) , while the upg group consisted of 43 patients ( 25 with unilateral glaucoma at baseline and 18 with bilateral glaucoma at baseline ) . the mean age was significantly higher in the upg group than in the bpg group ( 58.7 10.0 vs. 52.6 3.9 years , p = 0.035 ) . however , the prevalence of all analyzed systemic diseases was not different between the two groups ( table 1 ) . all pes in the upg group showed more severe clinical characteristics in terms of baseline vf parameters . however , baseline iop , mean follow - up iop , cct , and se were not significantly different between the pe and the npe ( table 2 ) . the progression rate determined by the md slope was compared between the fpe and the spe within the same patient in the bpg group . as expected , the progression rate was higher in the fpe , but other parameters did not differ significantly ( table 3 ) . when the pe in the upg group was compared to the fpe eye in the bpg group , the progression rate was significantly higher in the fpe in the bpg group , but no other parameters differed ( table 4 ) . according to the data from 343 patients analyzed in this study , 134 ( 39.1% ) presented with unilateral glaucoma at baseline . among these unilateral glaucoma patients , 76.1% showed glaucomatous optic disc changes in the fellow eye , while only 32 patients out of all glaucomatous patients ( 9.3% ) had completely unilateral glaucoma with both a normal optic disc and a normal vf in the fellow eye . among the 134 unilateral glaucoma patients , 23.1% showed progression during the follow - up period . the fellow eye of the 32 patients with unilateral glaucoma did not show progression during follow - up , although iop - lowering treatment was not performed in those eyes . among the 209 bilateral glaucoma patients , 20.6% showed progression . thus , the overall prevalence of progression was similar between patients with unilateral glaucoma and bilateral glaucoma . interestingly , 80.6% of patients with progressing disease in the unilateral glaucoma group demonstrated unilateral progression , while 58.1% of patients with progressing disease in the bilateral glaucoma group showed bilateral progression . chen and park showed that 6.2% of fellow eyes progressed over a period of 8.7 years . among 134 unilateral perimetric glaucoma patients , 6 ( 4.5% ) showed progression in the fellow eye in the present study . the results from the current study indicated no significant differences in the prevalence of any analyzed systemic disease between the upg and bpg groups . overall , the prevalence of systemic disease was very low in both groups , which may affect the statistical analysis and contribute to the lack of significance of the data . in contrast to the present data , a study by kim and kim showed that the prevalence of systemic disease was different between patients with unilateral and bilateral glaucoma . the discrepancy is likely due to the fact that systemic disease can affect the development of bilateral glaucoma , but the progression of disease is modulated by various factors thus reducing the effect of systemic disease on glaucoma progression . for instance , myopia is known to be a risk factor for the development of glaucoma but not for progression of the disease , although this remains under debate . when we compared the pe and the npe in the upg group , the pe showed a more advanced disease status at baseline . however , there was no difference in iop , cct , and se between the fpe and spe in the bpg group . in the comparison between the pe and npe in the upg group , iop similarly , iop did not differ between the fpe and the spe in the bpg group . as shown by mean baseline iop measurements , most of the patients in the current study had low iop and can be categorized as having normal - tension or low - tension glaucoma . whether a correlation exists between iop asymmetry and glaucoma severity in cases of normal - tension glaucoma has been under debate in previous studies . some studies have shown that higher iop is related to more advanced stages of glaucoma . however , the low - pressure glaucoma treatment study reported that iop asymmetry is unrelated to vf asymmetry . the present study revealed no differences in iop between the pe and the npe in the upg group or between the fpe and the spe in the bpg group . while iop reduction substantially decreased the progression rate in the collaborative normal - tension glaucoma study , some treated eyes also showed progression in that randomized clinical trial . thus , taken together , our results and those of previous studies suggest that factors other than iop , which may affect the progression of glaucoma with lower iop readings , should be investigated in future studies . furthermore , no differences were noted in baseline vf defect severity and iop values between the pe in the upg group and the fpe in the bpg group ; however , the progression rate was significantly higher in the fpe in the bpg group . the progression rate of the pe in the upg group was -0.70 db / year , which is approximately two times higher than the usual progression rates reported in previous randomized clinical trials ( -0.36 to -0.41 db / yr ) . the progression rate of the fpe in the bpg group was approximately five times higher ( -3.43 vs. -0.70 db / yr ) than that of the pe in the upg group . thus , we speculate that in simultaneously bilaterally progressing patients , both eyes progress faster than the pe in upg . in contrast , baseline vf defect severity was not different between the pe in the upg group and the fpe in the bpg group . first , the prevalence of systemic disease was determined by self - reported histories from the patients . second , the follow - up period ( mean 4.2 years ) was relatively short . considering the slow - progressing nature of glaucoma , this period may not be sufficient to observe the natural progression of the disease . some cases of unilateral glaucoma may eventually progress to bilateral glaucoma and some cases with a unilaterally progressing eye may show progression in the fellow eye over time . however , during the four years of observation in this study , unilateral glaucoma tended to progress only in the glaucomatous eye , while simultaneously bilaterally progressing patients showed much faster progression rates than those with a unilaterally progressing eye . no systemic factors analyzed in this study significantly differed in prevalence between the upg and bpg groups . therefore , glaucoma patients with simultaneous bilateral progression should be treated with caution in clinical practice because these cases can show relatively rapid progression .

bifid condyle is a rare anatomic variation of mandibular condyle . it can be symptomatic or diagnosed incidentally on routine radiographic examination . no definite etiologic factor has been identified . it is suggested that bifid condyle could be a developmental anomaly or secondary to trauma . we are reporting two cases of bifid mandibular condyle . both were diagnosed using computed tomography scan , which additionally revealed the associated pathosis in the angle of the mandible in first patient and the ankylosis of temporomandibular joint in the second patient . purposeto compare the clinical characteristics of unilaterally progressing glaucoma ( upg ) and simultaneously bilaterally progressing glaucoma ( bpg ) in medically treated cases.methodsprimary open angle glaucoma patients were classified as having upg or bpg according to an assessment of optic disc and retinal nerve fiber layer photographs and visual field analysis . risk factors including the presence of systemic diseases ( hypertension , diabetes , cerebrovascular accident , migraine , and dyslipidema ) were compared between the upg and bpg groups . baseline characteristics and pre- and post - treatment intraocular pressure ( iop ) were compared between the progressing eye ( pe ) and the non - progressing eye ( npe ) within the same patient in the upg group and between the faster progressing eye and the slower progressing eye in the bpg group.resultsamong 343 patients ( average follow - up period of 4.2 years ) , 43 were categorized into the upg group and 31 into the bpg group . the prevalence of all analyzed systemic diseases did not differ between the two groups . pes in the upg group had more severe pathology in terms of baseline visual field parameters than npes ( mean deviation -6.9 5.7 vs. -2.9 3.9 db , respectively ; p < 0.001 ) . however , baseline iop , mean follow - up iop , and other clinical characteristics were not significantly different between the pe and the npe in the upg group . the progression rate was significantly higher in the faster progressing eye in patients with bpg than in the pe for patients with upg ( -3.43 3.27 vs. -0.70 1.26 db / yr , respectively ; p = 0.014).conclusionsthere were no significant differences in the prevalence of systemic diseases between the upg and bpg groups . simultaneously bilaterally progressing patients showed much faster progression rates than those with a unilaterally progressing eye .

diabetes is the most common metabolic disease , with an increasing prevalence that reduces life expectancy to a third ( 1 , 2 ) . there are 200 million people with diabetes worldwide ( about 5% of adult population ) ( 3 ) . according to a nationwide study in 2001 , the prevalence of diabetes in iran was estimated at 4.67% among the population older than 20 years ( 4 ) . diabetes is usually associated with short - term complications such as hypoglycemia , and long - term complications like cardiovascular diseases , neuropathy , nephropathy , and retinopathy . amid patients with diabetes are twice exposed to the risk of psychiatric diseases compared to normal population , and according to some studies , one in every 5 patients with diabetes suffer from depression ( 5 , 6 ) . symptoms of depression were observed in 29% of men and 30.5% of women with diabetes in bangladesh villages ( 7 ) . in khamseh study , 71.8% of patients with diabetes had major depression ( 8) , and in sepehrmanesh study in kashan , this rate was 57.7% among women and 41% among men ( 9 ) . many factors can be affected by treatment of the disease , including physical function and daily activity , social relationships , quality of life , and personal mood . as the disease affects patient s self - confidence and increase his or her fear of worsening complications , the risk of psychiatric diseases increases ( 10 ) . depression is associated with increased risk of high blood glucose , complications of diabetes , cardiovascular diseases , and possibly patient s reduced compliance too ( 11 - 14 ) . furthermore , diabetic patients with depression have greater burden of treatment costs and referrals than those patients without depression ( 15 ) . various studies have shown that unlike normal people , patients with diabetes have lower quality of life , and this is much more pronounced in those suffering from complications of diabetes ; depression also has a significant effect on patients quality of life ( 16 - 18 ) . in a study in yazd , the mean score of quality of life in studied patients was 25.65 out of 60 , and in patients with complications like retinopathy or neuropathy , this score was lower compared to patients without these complications ( 19 ) . comorbidity of depression and diabetes can have a cumulative effect on people s functions ( 20 ) . diagnosis of depression in these patients is particularly important in order to reduce complications and improve quality of life . li et al . reported that depression was not diagnosed in 45% of diabetic patients ( 21 ) . depression leads to patient s noncompliance , blood glucose mismanagement , and other physical complications ( 11 , 19 , 22 ) . ciechanowski et al . reported that depressed patients are unable to comply with dietary regimen or manage blood glucose , and in this respect , they are clearly different from non - depressed patients ( 23 ) . with regard to the effect of diabetes with depression on the quality of life , in 2004 , filakovic et al . demonstrated that comorbidity of depression with diabetes further lowers patients quality of life by many folds ( 24 ) although several studies have been conducted on either quality of life or depression in diabetic patients , there are few studies on the relationship between quality of life and depression in diabetic patients . moreover , given that failure to diagnose depression in diabetic patients is commonplace , the need to identify and screen psychiatric disorders in patients with chronic diseases , especially diabetes , and the impact of depression on the quality of life , this article aimed to investigate the relationship between depression and quality of life in patients with diabetes using medical records at diabetics clinic in gorgan . the current survey was approved in the 46 ethics committee of golestan university of medical sciences ( no . 327123847 ) . in total , 330 eligible patients with diabetes were selected from patients admitted to the only specialized diabetic clinic in city of gorgan , north of iran , at 5th azar hospital , in the first 6 months of 2012 . inclusion criteria were confirmed diabetic record by the clinic specialist , age range of 25 - 75 years , having regular visits to the clinic , being under treatment with insulin and oral medication , not having type i diabetes , and not being treated for psychological health problems . patients were assured of the confidentiality of data and their informed consents were obtained . according to ethics committee guidelines , study ( 25 ) , the correlation between the depression and quality of life ( r = 0.39 ) , 95% confidence interval , and 90% power test , 66 diabetic patients with depression were needed . to achieve that and with respect to 20% estimation of depression in patients with diabetes ( 5 , 7 ) , 330 patients with type 2 diabetes clinic is the only specialized and state referral clinic in gorgan that is supported by 5th azar hospital and is also one of the 3 state hospitals belonged to the golestan university of medical sciences . since more than 80% of attended patients with diabetes to the health centers , had been visited by the aforementioned clinic ( with continuous access ) and according to the all patients with record ( n = 1150 ) , 330 subjects were finally selected using systematic sampling with an interval of 3 ( k = n / n = 3 ) , in the way that the first patient was randomly selected . then , a number was selected from 1 to 3 ( the second patient was selected ) . in other cases and with respect to the sampling interval as 3 , other samples were included ( such as number 5 , 8 to 989 ) . demographic questionnaire was completed in the presence of patients , and related data to the disease and its complications were obtained from their records . beck depression inventory ( bdi ) was used to assess depression , and the short version ( 26 items ) of the world health organization quality of life questionnaire ( whoqol - bref ) was used to assess quality of life . bdi contains 21 items , which evaluates signs of depression , including sadness , guilt , loss of interest , social isolation , and suicidal ideation . each item has rated on a 4-point likert scoring style from 0 for the lowest to 3 for the highest . each option is determined from mild to severe ( 0 for psychological health , and scores 1 , 2 , and 3 for mild , moderate , and severe disorders , respectively ) . overall depression score for each patient is found by summing up scores of all items , ranging from 0 to 63 , and higher scores indicate more severe depression , as follows : 0 to 15 means no depression , 16 to 31 mild depression , 32 to 47 moderate depression , and 48 to 63 severe depression . its reliability was determined through internal consistency using cronbach of 84% , and correlation was obtained 70% using split half method ( 26 ) . in this study , cronbach for the reliability of questionnaire for diabetic patients was found 0.77 , which indicates the questionnaire was suitable for this study . diez - quevedo et al . also studied it in diagnosing mental disorders in 1003 general hospital spanish inpatients in 2001 ( 27 ) . in the 26-item whoqol - bref , the first two items assess general health status and quality of life , and the next 24 items assess quality of life in 4 domains of physical health , psychological health , social relationships , and environment using 3 , 6 , 7 and 8 items , respectively . in this study the 5-point likert scale was applied for items 1 and 15 ( very bad , bad , not bad , good , and very good ) , items 2 , and 16 to 25 ( very unhappy , unhappy , not unhappy , happy , and very happy ) , items 3 to 14 ( not at all , a little , medium , high , and very high ) , and for item 26 ( never , rarely , occasionally , often , and always ) . scoring for items 3 , 4 , and 26 was in reverse . to compare domains and the first two items in depressed and non - depressed groups of patients , quality of life score for each patient ( xi ) was converted into scores between 0 and 100 using the following equation 2 ( 28 - 31 ) : validity and reliability of whoqol - bref have been reported favorable in many native and foreign studies ( 17 , 30 , 32 ) . in the present study , validity of this questionnaire was estimated according to internal consistency for the whole scale of 85% , physical health 78% , psychological health 80% , social relationships 69% , and environment 84% . the normality of qol scores and its dimensions were tested and confirmed by kolmogorov - smirnov test . where an item was missing , we substituted the mean of other items in the domain . also , when more than 2 items were missing from a domain , we did not calculate the domain score . the missing data in bdi questionnaire were replaced using median near points . to describe data , central and dispersion indicators such as mean and standard deviation were calculated . to compare qualitative characteristics of depressed and non - depressed patients , chi - square test was used . mean scores of qol and its sub - domains , age , and duration of disease in two groups were analyzed using independent t test . we conducted multiple linear regression analyses to assess prediction of qol scores and its relationship with other variables . in addition , we used scatter diagrams to visualize the relationship between dependent variable and each metric independent variable to be sure that they are linear . in addition , we also checked whether the relationship between dependent variable and categorical independent variables is homoscedastic . the current survey was approved in the 46 ethics committee of golestan university of medical sciences ( no . 327123847 ) . in total , 330 eligible patients with diabetes were selected from patients admitted to the only specialized diabetic clinic in city of gorgan , north of iran , at 5th azar hospital , in the first 6 months of 2012 . inclusion criteria were confirmed diabetic record by the clinic specialist , age range of 25 - 75 years , having regular visits to the clinic , being under treatment with insulin and oral medication , not having type i diabetes , and not being treated for psychological health problems . patients were assured of the confidentiality of data and their informed consents were obtained . according to ethics committee guidelines , study ( 25 ) , the correlation between the depression and quality of life ( r = 0.39 ) , 95% confidence interval , and 90% power test , 66 diabetic patients with depression were needed . to achieve that and with respect to 20% estimation of depression in patients with diabetes ( 5 , 7 ) , 330 patients with type 2 diabetes clinic is the only specialized and state referral clinic in gorgan that is supported by 5th azar hospital and is also one of the 3 state hospitals belonged to the golestan university of medical sciences . since more than 80% of attended patients with diabetes to the health centers , had been visited by the aforementioned clinic ( with continuous access ) and according to the all patients with record ( n = 1150 ) , 330 subjects were finally selected using systematic sampling with an interval of 3 ( k = n / n = 3 ) , in the way that the first patient was randomly selected . then , a number was selected from 1 to 3 ( the second patient was selected ) . in other cases and with respect to the sampling interval as 3 , other samples were included ( such as number 5 , 8 to 989 ) . demographic questionnaire was completed in the presence of patients , and related data to the disease and its complications were obtained from their records . beck depression inventory ( bdi ) was used to assess depression , and the short version ( 26 items ) of the world health organization quality of life questionnaire ( whoqol - bref ) was used to assess quality of life . bdi contains 21 items , which evaluates signs of depression , including sadness , guilt , loss of interest , social isolation , and suicidal ideation . each item has rated on a 4-point likert scoring style from 0 for the lowest to 3 for the highest . each option is determined from mild to severe ( 0 for psychological health , and scores 1 , 2 , and 3 for mild , moderate , and severe disorders , respectively ) . overall depression score for each patient is found by summing up scores of all items , ranging from 0 to 63 , and higher scores indicate more severe depression , as follows : 0 to 15 means no depression , 16 to 31 mild depression , 32 to 47 moderate depression , and 48 to 63 severe depression . its reliability was determined through internal consistency using cronbach of 84% , and correlation was obtained 70% using split half method ( 26 ) . in this study , cronbach for the reliability of questionnaire for diabetic patients was found 0.77 , which indicates the questionnaire was suitable for this study . diez - quevedo et al . also studied it in diagnosing mental disorders in 1003 general hospital spanish inpatients in 2001 ( 27 ) . in the 26-item whoqol - bref , the first two items assess general health status and quality of life , and the next 24 items assess quality of life in 4 domains of physical health , psychological health , social relationships , and environment using 3 , 6 , 7 and 8 items , respectively . in this study the 5-point likert scale was applied for items 1 and 15 ( very bad , bad , not bad , good , and very good ) , items 2 , and 16 to 25 ( very unhappy , unhappy , not unhappy , happy , and very happy ) , items 3 to 14 ( not at all , a little , medium , high , and very high ) , and for item 26 ( never , rarely , occasionally , often , and always ) . scoring for items 3 , 4 , and 26 was in reverse . to compare domains and the first two items in depressed and non - depressed groups of patients , quality of life score for each patient ( xi ) was converted into scores between 0 and 100 using the following equation 2 ( 28 - 31 ) : validity and reliability of whoqol - bref have been reported favorable in many native and foreign studies ( 17 , 30 , 32 ) . in the present study , validity of this questionnaire was estimated according to internal consistency for the whole scale of 85% , physical health 78% , psychological health 80% , social relationships 69% , and environment 84% . the normality of qol scores and its dimensions were tested and confirmed by kolmogorov - smirnov test . where an item was missing , we substituted the mean of other items in the domain . also , when more than 2 items were missing from a domain , we did not calculate the domain score . the missing data in bdi questionnaire were replaced using median near points . to describe data , central and dispersion indicators such as mean and standard deviation were calculated . to compare qualitative characteristics of depressed and non - depressed patients , chi - square test was used . mean scores of qol and its sub - domains , age , and duration of disease in two groups were analyzed using independent t test . we conducted multiple linear regression analyses to assess prediction of qol scores and its relationship with other variables . in addition , we used scatter diagrams to visualize the relationship between dependent variable and each metric independent variable to be sure that they are linear . in addition , we also checked whether the relationship between dependent variable and categorical independent variables is homoscedastic . a total of 330 diabetic patients ( 35.5% men , and 64.5% women ) were studied , with mean and standard deviation of age and history of disease of 50.6 9 and 5.4 4.5 years , respectively , and age range of 25 to 75 years . the prevalence of depression in all diabetic patients was 58.2% , of whom 124 patients ( 64.6% ) had mild depression , 56 ( 29.2% ) moderate depression , and 12 ( 6.2% ) severe depression . this was in 64.8% of men and 61.5% of women , with insignificant differences between them . depression was observed in 45.1% of the single diabetic patients and in 60.6% of married ones , with significant difference ( p = 0.039 ) . more than 50% of depressed diabetic patients and 62.5% of non - depressed diabetic patients were literate , and the difference between two groups was significant . however , there were insignificant differences between the two groups in terms of employment status ( table 1 ) . ( % ) . mean and standard deviation of depression score according to bdi was 29.4 10.1 in depressed patients , and 8.1 4.1 in non - depressed patients , with a significant difference between them ( p < 0.001 ) . comparison of the relationship between clinical characteristics and depression in patients with diabetes showed insignificant differences between the two groups with respect to dietary regimen , diabetes complications ( retinopathy , neuropathy , and nephropathy ) , and duration of the disease . however , hypertension was 13.9% more ( p < 0.001 ) , and physical activity 24.7% less in diabetic patients with depression , and the difference between groups was significant ( p = 0.01 ) ( table 2 ) . comparing scores of various domains of quality of life between depressed and non - depressed patients with diabetes using independent t test indicated significant lower mean scores in subscales of general health , quality of life in general , psychological health , environment , and overall score of quality of life in depressed diabetic patients at error level of 0.001 . however , in physical health domain , mean score was lower in depressed patients , with a significant difference ( p = 0.009 ) ( table 3 ) . linear regression model of prediction of quality of life score with independent variables of age , gender , duration of disease , hypertension , diabetes complications , education , marital status , occupation , physical activity , and depression score showed significant and inverse relationship between overall score of quality of life and variables of physical activity , diabetes complications or otherwise , and depression score ( p = 0.01 ) ( table 4 ) . the present study results are in line with the results in other studies , and suggest high prevalence of depression among patients with diabetes ( 7 - 9 , 19 ) . according to the present study , 58.2% of participants had moderate to severe depression and 15.5% had mild depression , i.e. , 73.7% of the patients are likely to have depression . in a study by nedjat et al . ( 28 ) , 28% of patients were depressed and 38% had mild depression , making a total of 66% . the rate of depression was 41.9% in larijani , and 53.3% in sepehrmanesh studies ( 9 , 19 , 21 ) . kahn et al . argue that not only depression could result from diabetes ; but also it is a risk factor for the onset of diabetes ( 22 ) . depression is associated with 60% increased risk of type 2 diabetes , while diabetes only causes an increased risk of depression by about 15% ( 23 ) . however , in the present study , there was an insignificant difference between sexes in the prevalence of depression , which is in line with results of pinquart et al . the mean overall quality of life score in depressed patients was 50.68 14.04 , which was significantly lower than non - depressed patients with 60.46 13.27 , and is close to the results found by nedjat et al . ( 28 ) . in psychiatric and habitat areas and overall score , the differences between groups was significant , and among depressed patients , mean score was lower , which is in line with other studies ( 6 , 25 , 28 ) . only in social relationships domain , the difference between the two groups was insignificant . perhaps , it could be attributed to rich relationships between patients and their family and relatives , which often become richer at times of disease . depression can affect physical outcomes such as myocardial infarction , life expectancy in cancer patients , and infections . also , it explains the effect of depression on immune system the most . other reasons for this effect can be due to the fact that depression in diabetic patients affects compliance , proper dietary regimen , smoking , and exercise ( 7 ) . in a study by lin et al . , it was confirmed that depressed patients perform more poorly than other patients in compliance with personal care , including proper nutrition , exercise , compliance with treatment and prevention ( 34 ) . in another study it was found that regarding incident disability and mortality , the effects of interaction between diabetes and depression seemed to be synergistic ( that is , greater than the additive main effects of diabetes and depression ) . compared depressed patients and 8 groups of patients with chronic diseases , and found that depressed patients showed greater reduction in the quality of life than the other 8 groups , and this effect remained , even after eliminating factors such as age , gender , and chronic diseases ( 35 ) . in the present study , high prevalence of comorbidity of depression with diabetes in patients indicates that effect of depression comorbidity on the quality of life can significantly affect outcomes of the disease , and early diagnosis of depression can improve quality of life ( 25 ) . study limitations included lack of measurement of hba1c , which could help assess better the patients blood glucose , and its effect on disease complications . it is , therefore , suggested that its measurement be taken into consideration in future similar studies . given the results of this and other studies , it seems that there is high prevalence of depression among patients with diabetes , and it has a significant effect on diabetes outcomes and quality of life . it is recommended that besides regular doctors visits in terms of physical problems , the psychiatric problems and quality of life of these patients should also be more considered . regular psychiatrist visits , or psychological assessments for screening mental disorders can help early diagnosis of these disorders . our survey weaknesses were as follow : 1 ) we used self - administered questionnaire , which could include patients bias , 2 ) participants may not have fully understood or have misinterpreted questions that might have biased the responses , and 3 ) we conducted a cross - sectional method that provided us with information for that particular issue in time . however , our study strong points can be mentioned as covering more than 80% of patients with diabetes in golestan province who were attended to the only specialized diabetes clinic supported by golestan university of medical sciences . we first tested the proposed strategy by designing a symmetric rna dna hybrid dx motif ( fig . , there are two parallel hetero - duplexes that are joined together at two points where strands cross over from one duplex to the other . the dx motif is composed of five nucleic acid strands : one central , long , two times repetitive dna strand ( l2 ) , two identical , short , peripheral dna strands ( s ) and two rna strands ( r ) . although strands l and s cross between duplexes , each r strand continuously extends through one duplex from one end to the other . a twofold rotational axis passes through the centre of the motif and is perpendicular to the tile plane . because of this symmetry , the two s strands are identical , as are the two r strands . each dx tile only needs three unique , component strands : l , r and s. strand r is an rna molecule , and strands l and s are both dna molecules . the dx tiles have single - stranded overhangs ( sticky ends ) at each end of the two duplexes . two neighbouring dx tiles can then associate with each other through hybridization of these sticky ends . the distance between the two crossover points in the tile is set as two turns ( 22 bp ) . to allow the dx tiles to tessellate a plane instead of forming one - dimensional arrays , it is essential to keep any two adjacent , interacting dx tiles coplanar , which requires the distance between the two adjacent crossover points of two interacting dx tiles to be n + 0.5 turns ( n is an integer ) . in the current design the self - assembly of rna dna hybrid nanostructures is a one - pot process . an aqueous - solution mixture of the component dna and rna strands at the designated ratio is slowly cooled from 70 c to 25 c over 24 h. in the process , the dna rna strands recognize and associate with each other , first into individual dx tiles and then into large assemblies . the assemblies were analysed by native polyacrylamide gel electrophoresis ( page ) and visualized by atomic force microscopy ( afm ) . in native page ( supplementary fig . s2 ) , all dna rna complexes appeared as sharp bands with the expected mobility , indicating that the hybrid complexes were formed and stable . large two - dimensional crystals and tubular structures were clearly visible ( fig . 1 ; supplementary fig . the apparent height of the single - layered two - dimensional crystals was 2.1 nm , a reasonable value for the diameter ( 2.5 nm ) of a - form duplexes as rna the two - dimensional crystals showed clear periodicities both along and perpendicular to their helical axes . the distance between two adjacent crossover points of two interacting dx tiles ( 2.5 turns or 29 bp , as indicated in fig . 1b , inset ) is important for ensuring that all dx tiles are coplanar in the final assemblies . if this distance deviates from this value , the helical nature of the duplexes would cause any two interacting tiles to be on different planes , and promote the formation of tubular structures . when this distance is decreased by 1 or 2 bp from 29 bp , the hybrid dx tiles are still able to assemble into large structures , which are predominantly tubular ( their heights are much higher than 2.5 nm , the diameter of an a - form duplex ) , and sometimes extended two - dimensional crystals . the tubular structures are occasionally ripped into monolayers , the regular patterns of which can be observed . when the distance increases by 1 bp or decreases by 3 bp , the hybrid dx tiles do not form any large , regular assemblies . overall , the formation of tubules is preferred over two - dimensional arrays in all cases , and rna dna hybrid dx tiles are more prone than pure dna dx tiles to form tubular structures . the strategy of using dna to program rna self - assembly is a general approach and can be used for the construction of a variety of structures . to demonstrate this capability , we engineered two rna dna hybrid star motifs . symmetric dna star motifs are a family of related nanostructures that have different numbers of branches ( 3 , 4 , 5 and 6 ) . each motif is assembled from three different strands : a long repetitive strand ( l ) , a medium strand ( m ) and a short peripheral strand ( s ) . all star motifs share the same strands m and s , but differ from one another in strand l. the sequence repeating time of the l strand determines the branch number of the motif . in the current work , the m strand is replaced by an rna strand ( r ) , resulting in rna dna hybrid star motifs . rna residues comprise half of the nanomotifs . when appropriate sticky ends are present at the peripheral ends of the duplexes , the hybrid motifs can further associate with each other into two - dimensional arrays ( figs 2 and 3 ) . the l strands contain unpaired , single - stranded loops ( shown in orange in figs 2 and 3 ; three or four bases long for the three- or four - pointed star , respectively ) at the centre . the short loops are long enough to prevent the branches from stacking with one another at the centre , and short enough to prevent the tiles from being too flexible . to prevent the intrinsic curvatures of the tiles from accumulating in one direction , a corrugation strategy was also applied12,33,34 . any two interacting tiles are separated by 4.5 turns , so the two adjacent tiles are related by a twofold rotational axis that is perpendicular to the molecular plane . this arrangement cancels the intrinsic curvatures of the tiles and promotes the tiles to form extended two - dimensional arrays . during slow cooling , the rna dna hybrid star tiles readily assemble into two - dimensional crystals , which can be visualized by means of afm imaging . the four - point star tiles assemble into periodic two - dimensional arrays ( fig . 2 ) with an apparent height of 2.2 nm . the cavities in the resulting two - dimensional crystals , to our surprise , are not squares . instead their dimensions are ( 27.2 1.6 nm ) ( 19 1.4 nm ) . this observation suggests that the overall geometry of the individual rna dna hybrid tile is a skewed four - point star or a four - leafed pinwheel ( fig . any two opposite leafs of the hybrid star are shifted away by 4 nm from one another in the direction perpendicular to the helical axes . the overall tile structure is quite different from that of its homo - dna counterpart . it is unlikely that this is due to a surface phenomenon of differential adsorption onto the mica substrate . the two faces of the two - dimensional arrays are identical to each other , as many twofold rotational symmetry axes ( for example , the twofold rotational axes indicated by the bold black arrows shown in figs 2b and 3b ) exist in the plane . a similar phenomenon , although less obvious , can be observed for the rna dna hybrid three - point star motif ( fig . its overall geometry is a skewed three - point star or three - leafed pinwheel with a threefold rotational symmetry . consequently , two - dimensional arrays of such tiles only have threefold , instead of sixfold , rotational symmetries . the strategy of dna - programmed rna self - assembly is not limited to two - dimensional self - assembly ; it can also be applied to three - dimensional self - assembly . to demonstrate the three - dimensional assembly each vertex has a connectivity of 3 and can be represented by a three - point star tile . the hybrid three - point star is similar to the one used for assembling trigonal two - dimensional arrays . however , the three - point star tiles in dodecahedra are significantly bent away from a planar structure . to provide enough flexibility for the tile to bend , the central single - stranded loops on the l strand are elongated from 3 to 11 bases . in addition , any two interacting tiles are designed to be separated by four turns ( an integral number of turns ) . at such a separation , any two interacting tiles will face the same side of the tile plane , and any intrinsic tile curvatures will accumulate in the same direction . in turn , this will promote the tiles to assemble into closed , discrete structures17 . similarly to two - dimensional self - assembly , the assembly process for three - dimensional structures is a simple one - pot process . dna hybrid dodecahedra have been characterized by dynamic light scattering ( dls ) and cryogenic electron microscopy ( cryoem ) imaging . the hydrodynamic radius of the hybrid complex is 20.7 1.3 nm as measured by dls . the value is consistent with the radius ( 20.5 nm ) of the circumscribed sphere of the hybrid dodecahedron model , assuming a pitch of 0.26 nm / bp and a diameter of 2.3 nm for the rna 4 ) . in the raw images , randomly distributed dodecahedral particles are observed , which are of the expected size . some small fragments and large aggregates also exist it is not clear whether those undesired structures are formed during the assembly process or result from damage accompanying sample preparation . the yield of the correctly formed particle is 40% , based on visual observation of the cryoem images ( this is significantly lower than the assembly yield of pure dna dodecahedrons ) . from the experimentally observed particles , a three - dimensional map of the rna dna hybrid dodecahedron is generated by single - particle three - dimensional reconstruction , a powerful technique routinely used by structural virologists35 . the map resolution was determined to be at 2.5 nm using the fourier shell correlation ( 0.5 threshold criterion ) of two three - dimensional maps independently built from half data sets . there is clear similarity between the computer - generated two - dimensional projections from the reconstructed structural model and the raw , individual particle images ( fig . 4e ) , and the class averages of raw particle images with similar views ( supplementary fig . the radius of the reconstructed dodecahedron model is 20.5 nm , consistent with both the design and the dls data . the skewness of the star motif is not observed from the reconstructed structural model , as it was in the two - dimensional arrays , presumably due to the low resolution of our cryoem data . when considering which strand of the overall assembly should be rna , our guideline was to make sure that every helical domain was composed of one dna strand and one rna strand . this ensures that all helical domains will be uniform , hybrid a - form duplexes . red strands in the tiles , but not for the green or black strands ( according to the colour code of the figures ) . theoretically , it is also possible to simultaneously use rna for the green and black strands and dna only for the red strand . it is also possible to use rna only for all three strands ( green , red and black ) . however , we have not experimentally explored this possibility , and it is not our intention , either . in general , rna strands are more difficult to prepare , much less stable , and much more expensive than dna strands with exactly the same sequence . thus , we decided to use the minimum number ( here only one ) of rna strands in the current study . for two - dimensional self - assembly of star motifs of either homo - dna or rna dna hybrid , the symmetry remains the same : three- or fourfold rotational symmetry ( figs 2 and 3 ) . in the design of the star motifs , each branch is an identical crossover structure and three- or fourfold rotational symmetries are expected . in the previous homo - dna structures , the star motif exhibits near d3 or d4 symmetry . those higher - degree symmetries are a surprising finding , and not the results expected from the design . dna structures is likely due to the subtle difference between a- and b - form duplexes at the central region of the tile ( supplementary fig . the base pairs stack directly onto one another and go through the centre of the dna helix . in contrast , base pairs in an a - form duplex are shifted from one another , and a cavity appears in the centre of the duplex when looking down the duplex . in addition , the negative charges on the phosphate groups are distributed differently along the different forms of the duplexes . electrostatic interactions would affect the homo- or hybrid duplexes differently , causing a structural adjustment to minimize the electrostatic repulsion . single - stranded loops ( 11 bases long ; the orange segments in strand l3 ) were used at the centre to provide enough flexibility for the motif to bend , but a dodecahedron formed instead of a tetrahedron . dna hybrid star motifs are more rigid than their corresponding homo - dna star motifs . the most important challenge for rna self - assembly is to rationally design and construct well - defined , stiff , branching motifs , the basic building blocks for nanoconstruction . to appreciate the level of challenge , a four - arm dna junction always adopts a right - handed , x - shaped structure . dna hybrid junction can be either right- or left - handed36,37 . compared with this challenge , it is simple and straightforward to adjust the repeating length for the different duplex forms . in the present study , we have also observed numerous different behaviours of the a- and b - form duplexes in nanoconstruction , including the strong tendency of hybrid dx to form tubular structures , the skewed appearance of the star motifs , and the surprising three - dimensional assembly results . star motifs ( with four - base - long sticky ends ) associate with one another and form low - mobility , high - molecular - weight species ( smears ) in the absence of divalent cations ( supplementary fig . under the same conditions , the corresponding homo - dna motifs will not associate with each other . dna hybrid structures in the current study were chemically less stable than the corresponding homo - dna structures . for example , homo - dna structures do not change noticeably under afm imaging after being stored for one week at 4 c , but the rna dna hybrid structures will degrade , and no well - defined nanoarray can be found under afm imaging after such storage . as well as the current approach for introducing rna into dna nanostructures , a more straightforward method is simply to create dna nanostructures with appended , single - stranded oligonucleotides with which rna strands can hybridize . furthermore , the rna moieties will be fully accessible to the surrounding solvent , harsh chemicals and degrading enzymes ( rnases ) , and are therefore prone to degradation . with the strategy reported here , it is possible to tightly fold rna deep inside the nanostructures to prevent such rnase accessibility ( this is in our study plan ) . in summary the presented strategy of using dna to program rna self - assembly comprises a versatile method for assembling rna molecules into a range of nanostructures . we believe that this strategy can be applied to all established dna nanomotifs and dna origami . some unexpected phenomena ( such as the fact that the star motifs are not straight ) have also emerged . to fully understand such phenomena and achieve tight structural controls , a systematic , theoretical modelling and experimental studies are necessary . we expect this strategy to provide a unified platform for assembling both rna and dna into large nanostructures with multifunctional rna modalities : sirnas , micrornas , antisense rnas , ribozymes and aptamers . dna nanoparticles for cellular deliveries of small rnas : aptamers for cellular targeting and other small rna rna molecules were transcribed with t7 rna polymerase from the corresponding dna templates , which were designed by the sequin38 computer program and purified with page . to form the designed nanostructures , rna and dna strands were combined according to the correct molecular ratios in a tris acetic edta mg ( tae / mg ) buffer and slowly cooled from 70 c to room temperature . after annealing , the samples were visualized by tapping - mode afm on a multimode afm with nanoscope iiia controller ( veeco ) in air at 22 c . for cryoem imaging , rna dna sample solutions were concentrated to 3 m , spread onto quantifoil grids , then plunge - frozen . data were recorded using a gatan 4k 4k charge - coupled device ( ccd ) camera in a philips cm200 transmission electron microscope . three - dimensional reconstructions of the dna dodecahedra used the single - particle image - processing software eman39 . an icosahedral symmetry for the polyhedra was established by processing the images assuming different symmetries and finding the symmetry that yielded a three - dimensional reconstruction consistent with the particle images . rna molecules were transcribed with t7 rna polymerase from the corresponding dna templates , which were designed by the sequin38 computer program and purified with page . to form the designed nanostructures , rna and dna strands were combined according to the correct molecular ratios in a tris acetic edta mg ( tae / mg ) buffer and slowly cooled from 70 c to room temperature . after annealing , the samples were visualized by tapping - mode afm on a multimode afm with nanoscope iiia controller ( veeco ) in air at 22 c . for cryoem imaging , rna dna sample solutions were concentrated to 3 m , spread onto quantifoil grids , then plunge - frozen . data were recorded using a gatan 4k 4k charge - coupled device ( ccd ) camera in a philips cm200 transmission electron microscope . three - dimensional reconstructions of the dna dodecahedra used the single - particle image - processing software eman39 . an icosahedral symmetry for the polyhedra was established by processing the images assuming different symmetries and finding the symmetry that yielded a three - dimensional reconstruction consistent with the particle images .

background : frequency of mood disorders in patients with chronic diseases , especially diabetes and its effects on life quality are dramatically increasing.objectives:this study aimed to investigate the relation between depression and quality of life in patients with diabetes.patients and methods : this is a cross sectional survey . subjects were selected from 330 eligible people referred to the only diabetes clinic in gorgan city during 6 months , using systematic random sampling . beak depression questionnaire and the brief questioner with 26 questions recommended by the world health organization ( whoqol - bref ) were used to measure depression and quality of life , respectively . data were analyzed through descriptive methods , chi - square , independent t test and linear regression model using spss16 ; moreover , p value < 0.05 was considered as significant.results:in total , 330 patients with diabetes ( 35.5 % male and 64.5% women ) were studied . the mean and standard deviation of their age and years involved with diabetes were 50.6 9.0 and 5.4 4.5 years , respectively . range of age was 25 - 75 years , as well . the prevalence of depression in all patients with diabetes was 58.2% ( 124 mild , 56 medium , and 12 with severe depression ) . hypertension was 13.9% more in diabetic patients with depression ( p value < 0.001 ) and physical activity in 24.7% of the cases was less with a meaningful difference ( p value = 0.01 ) . the mean and standard deviation of quality of life in diabetic patients with and without depression was 50.7 14 and 60.5 13.3 , respectively that was significant in two groups ( p < 0.0001).conclusions : the prevalence of depression is high in patients with diabetes and has a considerable impact on the consequences of diabetes and quality of life too . dna has recently been used as a programmable smart building block for the assembly of a wide range of nanostructures . it remains difficult , however , to construct dna assemblies that are also functional . incorporating rna is a promising strategy to circumvent this issue as rna is structurally related to dna but exhibits rich chemical , structural and functional diversities . however , only a few examples of rationally designed rna structures have been reported . herein , we describe a simple , general strategy for the de novo design of nanostructures in which the self - assembly of rna strands is programmed by dna strands . to demonstrate the versatility of this approach , we have designed and constructed three different rna dna hybrid branched nanomotifs ( tiles ) , which readily assemble into one - dimensional nanofibres , extended two - dimensional arrays and a discrete three - dimensional object . the current strategy could enable the integration of the precise programmability of dna with the rich functionality of rna .

inflammation frequently complicates intraocular surgery and can result in pain , elevated intraocular pressure , and cystoid macular edema ( cme).13 cme is the most common cause of vision loss after uncomplicated cataract surgery and although its pathogenesis remains incompletely understood , inflammation is a known cause.4 excessive or persistent postoperative inflammation may also lead to prolonged compromise of the blood topical corticosteroids are effective for reducing and treating inflammation in the setting of cataract surgery , and have a long track record of ophthalmic use , but are limited by side effects , including elevated intraocular pressure , delayed wound healing , and increase risk of infection . consequently , safer alternative anti - inflammatory agents to prevent and treat excessive postoperative inflammation and pain following cataract surgery would be advantageous . cyclo - oxygenase ( cox ) is an important enzyme in the inflammatory process and catalyzes the biosynthesis of prostaglandins and thromboxanes from arachidonic acid.1 two main isoforms , cox-1 and cox-2 , have been well characterized . cox-1 is constitutively expressed in most mammalian cells , including the kidney , gastrointestinal tract , platelets , and vascular endothelium , and plays a pivotal role in normal physiological function . cox-2 , on the other hand , is an inducible enzyme that is thought to be primarily responsible for inflammatory - mediated reactions . nonsteroidal anti - inflammatory drugs ( nsaids ) are potent inhibitors of cox enzymes and thereby the synthesis of prostaglandins . while the anti - inflammatory actions of corticosteroids are in part from the inhibition of phospholipase a2 preventing the release of arachidonic acid from membrane - bound phospholipids , nsaids act more downstream in the cascade and directly inhibit cox-1 and cox-2 enzymes . within the eye , it is firmly established that prostaglandins disrupt the blood ocular barrier , increase vasodilation , facilitate leukocyte migration , and promote pain.1 consequently , their inhibition should have favorable effects on both intraocular inflammation and pain . in support of this , several randomized , prospective , double - masked , and placebo - controlled studies have shown that topically applied indomethacin 1% , flurbiprofen 0.03% , ketorolac 0.4% and 0.5% , diclofenac 0.1% , nepafenac 0.1% , and bromfenac 0.09% reduce postoperative inflammation following cataract surgery.1 similarly , prospective randomized studies have demonstrated that diclofenac 0.1% , ketorolac 0.4% , nepafenac 0.1% , and bromfenac 0.09% reduce ocular discomfort after cataract surgery.1 of all the commercially available ophthalmic nsaids , ketorolac tromethamine ( ketorolac ) possesses the greatest number of studies supporting its efficacy in preventing and treating postoperative inflammation and pain after cataract surgery ( table 1).521 ketorolac 0.5% ( acular , allergan inc , irvine , ca ) is approved by the food and drug administration ( fda ) for seasonal allergic conjunctivitis , inflammation following cataract surgery , and ocular discomfort after refractive surgery.22 to reduce the incidence of burning and stinging , a 0.4% concentration of ketorolac ( acular ls , allergan inc ) was formulated , and appears to have a similar therapeutic effect.18,23 ketorolac 0.4% is approved by the fda for the reduction of ocular pain and burning following corneal refractive surgery . more recently , a preservative - free 0.45% preparation of ketorolac ( acuvail , allergan inc ) in carboxymethylcellulose was approved by the fda in 2009 for the treatment of pain and inflammation following cataract surgery and is dosed twice daily.24 the specific intent of this review is to assess the available evidence supporting the efficacy of acuvail in the treatment of pain and inflammation after cataract surgery . nsaids are a chemically heterogenous group of compounds that inhibit the formation of prostaglandins and lack a steroid nucleus biosynthetically derived from cholesterol . there are six major classes , but topical formulations are limited to the relatively water - soluble classes , ie , indole acetic , aryl acetic , and aryl propionic acids.25 ketorolac tromethamine is an aryl acetic acid derivative . the chemical name is ( )-5-benzoyl-2,3- dihydro-1h - pyrrolizine-1-carboxylic acid , compounded with 2-amino-2-(hydroxymethyl)-1,3-propanediol ( 1:1 ) . current preparations consist of a racemic mixture of r-(+ ) and s-( ) ketorolac tromethamine . when administered systemically , ketorolac has proven analgesic , anti - inflammatory , and antipyretic activity.1 its mechanism of action , as with all nsaids , is presumed to be due to inhibition of prostaglandin biosynthesis . however , systemic administration is not thought to achieve sufficient intraocular drug levels to inhibit prostaglandin synthesis completely in the ciliary body and iris.1 topical administration of ketorolac , on the other hand , reaches adequate levels to inhibit prostaglandin synthesis in these target tissues . in one study , after a single topical application of ketorolac 0.4% , a peak aqueous concentration of 57.5 ng / ml was achieved after 60 minutes.26 another study demonstrated mean aqueous concentration of 1079 ng / ml after a total of 12 doses of ketorolac 0.4% administered over 2 days.27 the original ophthalmic formulation of ketorolac is a 0.5% solution marketed as acular . a 0.4% formulation ( acular ls ) the 0.5% and 0.4% preparations are supplied as an isotonic aqueous mixture with a ph of 7.4 , and have an osmolality of approximately 290 mosmol / kg . however , both the 0.4% and 0.5% solutions contain benzalkonium chloride ( a preservative ) , the surfactant octoxynol-40 , and sodium edetate ( a metal - chelating agent ) , and are associated with a high incidence of burning and stinging on instillation ( as high as 40%).22,23,28 in an effort to increase ocular bioavailability , a new formulation of ketorolac was developed to preserve the efficacy of prior formulations , while enhancing tolerability with a less frequent dosing regimen.21 compared with the ketorolac 0.4% formulation , the concentration of acuvail is 12% greater ( 0.45% ) and carboxymethylcellulose is added to the emulsion to allow greater drug retention on the ocular surface and improve comfort and drug penetration . other key changes include lower ph and absence of surfactant , metal - chelating agents , and preservatives . as such , acuvail is supplied as a sterile isotonic preservative - free solution with a ph and osmolality of approximately 6.8 and 285 mosmol / kg , respectively , and is approved by the fda for treatment of pain and inflammation following cataract surgery . in a study by attar et al evaluating the pharmacokinetics of ketorolac 0.45% versus 0.4% , the addition of carboxymethylcellulose in combination with the 12% increase in concentration resulted in a 35% enhancement of bioavailability in the aqueous humor of the 0.45% preparation.29 furthermore , decreasing the ph from 7.4 to 6.8 in combination with addition of carboxymethylcellulose enhanced bioavailability in the aqueous humor by two - fold and in the iris - ciliary body by three - fold . at the time of writing , the pharmacokinetics of acuvail have not been assessed in humans , but both acular ls and acuvail formulations were directly compared after a single topical application of 35 l.29 peak concentration in the aqueous humor and iris - ciliary body was 389 ng / ml and 450 ng / g , respectively , for acuvail , and 211 ng / ml and 216 ng / g , respectively , for acular ls . this study concluded that acuvail delivered significantly higher concentrations of ketorolac to the aqueous humor and iris - ciliary body . waterbury et al directly compared the peak and trough intraocular levels of acuvail , bromfenac 0.9% ( xibrom , ista pharmaceuticals inc , irvine , ca ) , and placebo after three applications ( 35 l ) every 20 minutes in an animal model of lipopolysaccharide - induced inflammation.30 peak concentrations in the aqueous humor and iris - ciliary body were 738 ng / ml and 556 ng / g , respectively , for acuvail and 94 ng / ml and 46 ng / g , respectively , for bromfenac 0.9% . trough concentrations in the aqueous humor and iris - ciliary body were 127 ng / ml and 59 ng / g , respectively , for acuvail , and 17 ng / ml and 8 ng / g , respectively , for bromfenac 0.9% . the study concluded that acuvail achieved aqueous and iris - ciliary body concentrations that exceeded its inhibitory concentration of 50% ( ic50 ) values for cox-1 and cox-2 at both peak and trough ( table 2).1,26,31,32 in addition , while both nsaids inhibited lipopolysaccharide - induced aqueous prostaglandin e2 elevation , only acuvail significantly prevented vascular leakage , as measured by fluorescein isothiocyanate - dextran leakage at trough levels . although suggestive , the number of animals used in both studies was small , and therefore additional studies with larger sample sizes are needed to confirm these initial results . furthermore , both studies used new zealand white rabbits which lack pigment , blink infrequently , and have an unusually unstable blood aqueous barrier , so these results should be extrapolated to humans with caution . given the favorable pharmacokinetic data of acuvail , approval was granted for twice - daily dosing in contrast with four times daily dosing for acular ls . this reduced dosing regimen offers a distinct therapeutic advantage for acuvail because in addition to patient convenience , several studies have suggested increased patient compliance with less frequent dosing.33 the efficacy of acuvail was assessed in two identical multicenter , double - masked , randomized , placebo - controlled , parallel studies and specifically conducted to evaluate the effects of acuvail on relief of pain and inflammation after cataract surgery.21 the results of these parallel studies were analyzed together and collectively involved 511 patients . patients were randomized in a 2:1 ratio to receive either acuvail or vehicle in the operative eye for 16 days . patients were dosed twice daily the day before surgery . on the day of surgery , patients had one drop upon awakening , three drops each 20 minutes apart starting two hours before surgery , one drop before discharge , and one drop 12 hours after the first dose upon awakening , resulting in a total of six drops of the study medication . patients continued one drop twice daily of the study medication for 14 days after surgery . all patients underwent elective unilateral , uncomplicated , extracapsular cataract extraction with posterior chamber intraocular lens implantation . the primary efficacy endpoint was the percentage of patients with a summed ocular inflammation score ( sois ) of 0 for anterior chamber cell and flare on day 14 . the main secondary efficacy endpoint was the percentage of patients with no pain ( grade = 0 ) on postoperative day 1 . in this study , anterior chamber cell was graded on a six - point scale and anterior chamber flare was graded on a five - point scale . the sois was calculated as the sum of scores for anterior chamber cell and flare . to assess the degree of pain , patients called an interactive voice response system diary twice daily during the two weeks after the day of surgery and were instructed to rate their level of ocular pain on a five - point scale ( 0 = none , 1 = mild , 2 = moderate , 3 = severe , 4 = intolerable ) . the acuvail group had a significantly higher percentage of patients with a sois score of 0 compared with vehicle at days 7 and 14 . at day 7 , acuvail and vehicle had a sois score of 0 for 32% ( 102/318 ) and 17% ( 26/155 ) of patients , respectively ( p < 0.001 ) . at day 14 , acuvail and vehicle had a sois score of 0 for 53% ( 167/318 ) and 27% ( 41/155 ) of patients , respectively ( p < 0.001 ) . a pain score of 0 on day 1 was reported in 72% ( 233/322 ) of acuvail patients versus 40% ( 62/156 ) in vehicle patients ( p < 0.001 ) . the median time to postoperative ocular pain resolution was one day in patients treated with acuvail and two days in patients treated with vehicle ( p < 0.001 ) . although the combined results of these controlled studies demonstrate the efficacy of acuvail for the prevention and treatment of postoperative inflammation and pain after cataract surgery , these results should be interpreted with caution . the importance of achieving a median of one less day of pain with acuvail versus placebo needs to be assessed in the appropriate context of treatment cost , clinical impact , and in the absence of concomitant corticosteroid use . moreover , cell and flare were combined together in contrast with grading each outcome independently , and their summation may have amplified the treatment differences observed between the acuvail and vehicle group . in addition , because corticosteroids were not used concomitantly , no information can be discerned about the additive benefits of acuvail with a corticosteroid in regards to inflammation and pain following cataract surgery . in a study comparing the efficacy of ketorolac 0.5% with prednisolone acetate 1% , simone et al observed that prednisolone acetate was more effective at reducing intraocular inflammation by day 7 after cataract surgery than ketorolac , although this difference resolved by day 28.10 several studies have demonstrated an additive benefit of a topical nsaid with a corticosteroid and their combined use , therefore , is common in clinical practice.1 therefore , the results of this study can not be directly applied in the setting of concomitant corticosteroid use . nevertheless , previous studies have demonstrated an additive benefit of ketorolac 0.5% or 0.4% when used in conjunction with corticosteroids in reducing inflammation , pain , and cme following cataract surgery . thus , given the favorable pharmacokinetics of acuvail in comparison with these older formulations , similar therapeutic benefit may be likely.521 several in vitro studies indicate that ketorolac is the most potent inhibitor of cox-1 , while both amfenac ( active component in nevanac , alcon laboratories inc , fort worth , tx ) and bromfenac have been reported as being the most potent inhibitors of cox-2.1,26,31,32 bromfenac may be a 318 times more potent inhibitor of cox-2 than diclofenac , amfenac , and ketorolac.25,32 another study found that amfenac was a more potent inhibitor of cox-2 than bromfenac . therefore , the anti - inflammatory actions of nsaids are presumed to relate to their ability to inhibit this isoform . however , this paradigm has not been consistently demonstrated in clinical trials , and the possibility exists that cox-1 also plays an important role in inflammation and , in the presence of substrate , may readily convert arachidonic acids into prostaglandins . thus , the clinical importance of selective cox-1 versus cox-2 inhibition for ocular disease remains unproven . although ketorolac is approximately six times more potent as an inhibitor of cox-1 than cox-2 ( table 2 ) , it is nevertheless a potent inhibitor of cox-2 , with an ic50 in the range of 0.090.12 m ( 33.945.2 ng / ml).32 this allows ketorolac to inhibit cox-2 in the iris - ciliary body after topical application , an important fact that can be overlooked if the relative cox-1/cox-2 potencies of ocular nsaids are emphasized . on the other hand , ketorolac is an approximately 550 times more potent inhibitor of cox-1 than diclofenac , bromfenac , and amfenac . the ability to inhibit both isoforms of cox for short periods of time may be advantageous in allowing more rapid and complete inhibition of prostaglandin production , but long - term inhibition of cox-1 may not be desirable because it is involved in normal physiologic function . the combination use of topical nsaids and corticosteroids is sometimes referred to as synergistic in the literature . this clinical impression of synergy remains unproven and would seem unlikely , given the fact that both drug classes inhibit prostaglandin production ( figure 1 ) . synergy is defined , in general , as two or more agents working in combination to produce an effect that could not be obtained by each agent independently . a classic example of synergy involves penicillin and aminoglycoside antibiotics where use of both antibiotics in combination significantly lowers the ic50 of each antibiotic for a given organism . although a large , randomized , prospective study demonstrated that ketorolac 0.5% was more effective than dexamethasone sodium phosphate 0.1% solution in facilitating re - establishment of the blood aqueous barrier after surgery , differences in drug formulation and intraocular concentration preclude any conclusions about synergy.3436 furthermore , although many prospective studies have confirmed that the combination use of an nsaid and corticosteroid is superior to a corticosteroid alone for pain , inflammation , cme , and visual improvement after intraocular surgery , these findings can be explained by an additive effect of a second anti - inflammatory agent.1,17,37 the distinction between a synergistic effect and an additive effect has important implications because synergy implies that an nsaid used in combination with a corticosteroid provides a therapeutic effect that can not be replicated by simply increasing the dosing regimen of the corticosteroid . although topical administration of nsaids provides aqueous humor levels adequate to suppress prostaglandin synthesis in the iris and ciliary body , the ability to suppress prostaglandin synthesis in the retina / choroid is less certain . in an animal model , ketorolac 0.5% could not be detected in the vitreous after topical administration , but in a small prospective comparative study of patients undergoing vitrectomy , ketorolac 0.4% could be detected in the vitreous ( 2.8 ng / ml ) and reduced vitreous prostaglandin e2 levels.38,39 this observed reduction was presumably from inhibition of iris and ciliary body prostaglandin e2 production because the measured vitreous concentration of ketorolac was considerably less than the ic50 for both cox-1 and cox-2 inhibition , and therefore unlikely to have inhibited cox activity in retinal cells . accumulating evidence indicates that cox-2 has important implications for retinal disease.1,40 cox-2 is the predominant isoform in human retinal pigment epithelial cells and is significantly upregulated in response to proinflammatory cytokines.41 cox-2 is also present in choroidal neovascularization , as well as in other highly vascularized lesions , and its expression increases in diabetic retinopathy.1,42 in a variety of experimental systems , cox-2 inhibition suppresses angio - genesis.43 in this regard , both nepafenac 0.1% and bromfenac 0.09% could be detected in the rabbit retina after topical administration , and in one study nepafenac inhibited 55% of retinal prostaglandin synthesis.31,44 in another study , topical ketorolac 0.4% inhibited experimentally induced choroidal neovascularization and reduced both retinal prostaglandin e2 and vascular endothelial growth factor levels by > 30%.45 while interesting , these results were obtained in animal models and can not be directly extrapolated to humans . given the favorable pharmacokinetics of acuvail versus acular ls , it is worth speculating that clinically meaningful retinal cox inhibition may now be achievable . the overall incidence of treatment - related adverse events was significantly higher in the vehicle group ( 14% ) than in the acuvail group ( 6%).21 the most common adverse events reported were increased intraocular pressure , conjunctival hyperemia , and/or hemorrhage , corneal edema , ocular pain , headache , tearing , and blurred vision . the most common adverse events reported at a higher frequency than vehicle included increased intraocular pressure ( 5.8% versus 1.8% ) , conjunctival hemorrhage ( 1.2% versus 0.6% ) , and blurred vision ( 1.2% versus 0.6% ) , and were generally considered by the clinical investigators to be a consequence of the cataract procedure . adverse events of burning and stinging were low , with only 1.5% of patents in the acuvail group and 0.6% in the vehicle group reporting such an occurrence . when ketorolac 0.5% was topically applied in one eye three times daily , only five of 26 subjects had a detectable amount of ketorolac in their plasma ( range 10.722.5 ng / ml ) at day 10 . in contrast , when ketorolac 10 mg was administered systemically every six hours , peak plasma levels were around 960 ng / ml . given the possibility of systemic absorption after topical application , techniques such as lid closure and nasolacrimal occlusion may be used to decrease systemic exposure . as with other nsaids , acuvail may slow or delay healing , and in some susceptible patients , continued use of topical nsaids may result in epithelial breakdown , corneal thinning , corneal erosion , corneal ulceration , or corneal perforation . there exists the potential for cross - sensitivity to acetylsalicylic acid , phenylacetic acid derivatives , and other nsaids , and therefore caution should be used when treating individuals who have previously exhibited sensitivities to these drugs . therefore , it is recommended that topical nsaids be used with caution in patients with known bleeding tendencies or who are receiving anticoagulation . there are no adequate well controlled studies of topical nsaids in pregnant woman and , as such , these medications are classified as category c. because of the known effects of prostaglandin - inhibiting drugs on the fetal cardiovascular system ( closure of the ductus arteriosus ) , the use of topical nsaids should be avoided during late pregnancy . severe corneal toxicity has been reported with diclofenac 0.1% , ketorolac 0.5% , nepafenac 0.1% , and bromfenac 0.09%.1 although uncommon , these dramatic events are referred to as corneal melt . however , a definite link between nsaid use and corneal melt remains unproven , but it is still prudent to avoid nsaid use in patients with severe corneal surface disease . older formulations of ketorolac had an incidence of transient burning and stinging on instillation of approximately 20%40% for the 0.4% concentration and 40% for the 0.5% concentration.22,23 in contrast , acuvail was associated with substantially lower rates ( 1.5% ) of burning and stinging.21 most importantly , a significantly higher percentage of patients randomized to acuvail had a 3-line or more improvement in vision from baseline compared with those treated with vehicle.21 at least two previously published studies using ketorolac 0.4% demonstrated a similar beneficial effect upon visual acuity following cataract and vitreoretinal surgery.17,37 in patients undergoing routine vitreoretinal surgery , patients randomized to ketorolac 0.4% experienced an average 4.3-line improvement from baseline compared with a 2.5-line improvement with placebo.37 this greater improvement in vision in the ketorolac group was observed despite concomitant corticosteroid use in both groups . excessive postoperative inflammation and pain after cataract surgery can delay visual recovery and affect long - term outcomes . there is substantial evidence from well designed studies with sufficient numbers of patients that ketorolac formulations 0.4% and 0.5% effectively treat both inflammation and pain after cataract surgery . given the favorable pharmacokinetics , better tolerance , and reduced dosing requirement of acuvail , additional clinical studies comparing acuvail with other nsaids for the treatment of inflammation and pain after cataract surgery are indicated to confirm these promising results . malaria , which is caused by a protozoan parasite of the plasmodium genus , represents a serious global health concern given that nearly half of the world population is at risk of infection ( who , 2014 ) . although several anti - malarial drugs for treating the symptomatic stage ( or blood stage ) of malarial infection are commercially available ( antony and parija , 2016 ) , their efficacy has declined appreciably in the last few decades owing to widespread drug resistance developed by the parasite ( breman et al . , chloroquine was the first - line malaria treatment for many decades until drug - resistant p. falciparum strains became common . the drug causes a dose - dependent decrease in hemozoin formation ( chou and fitch , 1992 , slater and cerami , 1992 ) and an associated increase in toxic free heme in the food vacuole of the parasite ( combrinck et al . , 2013 , loria et al . , 1999 ) . over the past few decades , researchers have proposed many different mechanisms for chloroquine action , including 1 ) dna intercalation ( meshnick , 1990 ) , 2 ) alteration of digestive food vacuole ph ( yayon et al . , 1985 ) , 3 ) inhibition of heme polymerase ( yayon et al . , 1985 ) , and 4 ) formation of a toxic chloroquine - ferriprotoporphyrin ix complex ( sugioka et al . , yet , there is no consensus on the exact mechanisms by which chloroquine kills the parasite and under what circumstances they operate . shedding light on the possible actions of chloroquine is critical for developing more potent drugs or combination drug treatments against the parasite and for understanding how the parasite can develop resistance against them . here we used systems biology model and transcriptional profiles of p. falciparum to obtain information about metabolic and biological precursors that determine the physiological or pathophysiological state of the parasite . specifically , we used the p. falciparum transcriptomic data obtained by hu and colleagues during the intraerythrocytic development cycle ( idc ) at different concentrations of chloroquine ( hu et al . , 2010 ) . our primary goal was to use these data to investigate the effect of chloroquine on p. falciparum dna replication . we chose to study this effect because it has been proposed as a mechanism of chloroquine action in different plasmodium species ( gutteridge et al . , 1972 , meshnick , 1990 , polet and barr , 1968a , polet and barr , 1968b ) , in bacteria ( ciak and hahn , 1966 ) , and in bioassays examining the effect of chloroquine on rna and dna polymerases ( cohen and yielding , 1965 , o'brien et al . we first developed a method that integrates the information embedded in the transcriptome data with the whole - genome metabolic network model for p. falciparum ( fang et al . , 2014 ) to predict the metabolic phenotype corresponding to those transcription data . we validated the developed method by using a data set from an independent study that jointly capture transcriptomic and metabolomic data from p. falciparum , to correctly predict the metabolic phenotype tied to genetic perturbation of two krebs cycle enzymes ( ke et al . , 2015 ) . we then focused on the inhibition of dna replication in p. falciparum ( as a consequence of chloroquine treatment ) a critical metabolic phenotype ( ciak and hahn , 1966 , cohen and yielding , 1965 , polet and barr , 1968a ) , and used our method to simulate the effect of chloroquine on the metabolic fluxes of p. falciparum during the idc . we identified genes that were substantially altered in response to chloroquine treatment and linked to the inhibition of p. falciparum dna replication . the cohort of identified genes suggests that dna replication is inhibited by the downstream effect of heme accumulation . specifically , our analysis suggests that continuous accumulation of heme inhibits redox metabolism , carbon fixation , and pyrimidine metabolism , which leads to inhibition of dna replication and facilitates death of the parasite . our results provide a mechanistic explanation for why parasites with an efficient redox metabolism may have a lower sensitivity to chloroquine ( kasozi et al . , 2013 , lehane et al . , 2012 , metabolic flux profiles of p. falciparum strains 3d7 ( pf3d7 ) and dd2 ( pfdd2 ) during the idc were estimated by using transcriptome data previously reported by llinas and colleagues ( llinas et al . , 2006 ) , who extracted cdna from parasites cultured in erythrocytes and hybridized them to dna microarrays to yield hourly levels of gene expression for the pf3d7 ( or pfdd2 ) strain during the idc . the temporal resolution of these hourly gene expression profiles allowed us to make high - resolution time - dependent metabolic flux predictions during the idc . we used previously reported gene expression data ( hu et al . , 2010 ) to simulate the effect of chloroquine on the time - dependent metabolic fluxes of pf3d7 and pfdd2 during the idc . briefly , hu and colleagues ( hu et al . , 2010 ) administered chloroquine 18 h after erythrocyte invasion at concentrations of 41 , 72 , and 144 nm for pf3d7 and at 43 nm for pfdd2 , and obtained gene expression profiles every hour for 8 h during the idc following the chloroquine treatment . the model was identical to a previously published model ( fang et al . , 2014 ) , with one minor modification : we added reactions explicitly carried out by pdx2 ( pf11_0169 ) and pdx1 ( mal6p1.215 ) . this was done by 1 ) modifying an old reaction that incorrectly assumed that pyridoxal 5-phosphate ( plp ) was synthesized from ribulose 5-phosphate and 2 ) adding a new ( and separate ) reaction for pdx1 because experiments suggest that it can synthesize plp independent of the ammonia generated by pdx2 ( hanes et al . , 2008 ) . in the updated model , pdx2 hydrolyzes l - glutamine to yield glutamate and ammonia while pdx1 incorporates the ammonia generated by pdx2 to yield plp from the substrates ribose 5-phosphate and glyceraldehyde 3-phosphate ( gengenbacher et al . , 2006 ) , with the isomerization of ribose 5-phosphate to ribulose 5-phosphate as a side reaction . we used gene - to - reaction mappings available in the metabolic network model to obtain reaction expressions rexp , which depend on the expression of a gene ( or genes ) catalyzing a particular metabolic reaction . for a reaction catalyzed by a single enzyme , rexp was equal to the expression of the gene catalyzing that reaction however , if a reaction was associated with more than one gene , then their gene - to - reaction mappings were defined by using the boolean operators and and or . we implemented these operations by taking the minimal ( for the and operator ) and maximal ( for the or operator ) values of the associated gene expression data ( song et al . , 2014 ) . , 2014 ) that integrates hourly gene expression data with a whole - genome metabolic network model to estimate the hourly metabolic flux profiles of p. falciparum during the 48-h long idc period . to take advantage of additional studies that collect gene expression data as a function of genetic or chemical perturbations , we modified the method because such studies usually do not provide the same granularity and density of gene expression data to fully cover the entire idc . therefore , we aimed to develop a method that could utilize a reduced transcriptomic data set to predict alterations in metabolic flux profiles during the idc . briefly , we used the following steps to estimate metabolic fluxes under no - stress conditions : step 1 : estimate nutrient fluxes sufficient for the nominal growth rate ( 40% of the maximum growth rate).step 2 : estimate reaction fluxes vin for metabolite i corresponding to the nominal growth rate constrained by the nutrient fluxes obtained in step 1.step 3 : estimate reaction fluxes vit for reaction i at any time point t during the idc by incorporating time - dependent transcriptomic profiles under stoichiometric and nutrient uptake constraints . step 1 : estimate nutrient fluxes sufficient for the nominal growth rate ( 40% of the maximum growth rate ) . step 2 : estimate reaction fluxes vin for metabolite i corresponding to the nominal growth rate constrained by the nutrient fluxes obtained in step 1 . step 3 : estimate reaction fluxes vit for reaction i at any time point t during the idc by incorporating time - dependent transcriptomic profiles under stoichiometric and nutrient uptake constraints . the last step was achieved by globally minimizing for all reactions and time points the value of t that minimized the weighted absolute difference , ig|vitritvin| , where g represents the set of unidirectional reactions with known gene - to - reaction mapping . the factor rit = rexp , itmin(rexp , it)mean(rexp , it)min(rexp , it ) ; where , rexp , it represents reaction expression of ith reaction at time t obtained from the gene - to - reaction mapping of the p. falciparum metabolic network , according to the approach outlined in section 2.2 . this factor , rit , ensured that the mean of the ith reaction during the idc was equal to vin . , we estimated the organism - level impact of a drug on metabolism by using transcriptomic data obtained after drug treatment . the original formulation outlined above ( steps 13 ) can not be directly translated to stress conditions unless the complete transcriptomic profile for each stress condition during the entire idc is available ( fang et al . , 2014 ) . therefore , we introduced a parameter i to account for up- or down - regulation of individual reactions in response to stress to simulate the effect of exogenous stress on metabolic flux profiles during the idc . we modified the factor rit to iritmin(irit)mean(rit)min(irit ) , where i denotes the median relative change in the reaction expression of the ith reaction following chloroquine treatment , as observed in previous experiments ( hu et al . , 2010 ) . note that the mean of each individual modifying factor rit , i.e. , 1nj=1tnrij , obtained by summing the values across all time points in the idc , tracks with i ; that is , when the mean is less than one , greater than one , or equal to one , i<1 , i>1 , or i=1 , respectively . in other words , i increases or decreases the nominal flux of the ith reaction during the entire idc depending on the relative increase or decrease in the ith reaction expression . in principle , a drug may differentially affect gene transcription at different time points during the idc . here , we assumed that the median change in gene transcription after a stress treatment was representative of the transcriptional response throughout the idc . we used a previously published data set ( ke et al . , 2015 ) to validate our methodology to simulate the effect of external stress on metabolic fluxes during the idc . ke and colleagues ( ke et al . , 2015 ) constructed a p. falciparum mutant with two tricarboxylic acid ( tca ) cycle enzymes deleted ( idh/kdh ) , cultured wild - type and mutant parasites in erythrocytes , and obtained matched transcriptomic and metabolomic data . we used this data set because it describes changes in gene expression ( transcriptomics ) and phenotype ( metabolomics ) under the same experimental stress conditions . if the reaction fluxes estimated from previously reported transcriptome data ( ke et al . , 2015 ) adequately represent the reaction fluxes in vitro , then the predicted changes in reaction fluxes should be sufficient to predict the changes observed in the corresponding metabolomic data . we used a computer model of mitochondrial oxidative phosphorylation and the tca cycle ( wu et al . , 2007 ) to predict changes in tca cycle metabolites in response to changes in these estimated reaction fluxes ( ke et al . the kinetic parameters of this computer model were determined and validated by using extensive experimental data . we simulated wild - type reactions by running this model , with its default parameters , for 2000 s ( time required to reach steady - state condition ) and stored the concentrations of tca cycle metabolites . we performed simulations for the mutant parasite in the same way , albeit after scaling the model parameters that govern tca cycle enzyme activities by the estimated increase or decrease in reaction fluxes of those enzymes . we used a freely available software package called bisen ( vanlier et al . , 2009 ) to implement the tca cycle computer model ( wu et al . , 2007 ) . p. falciparum takes up nutrients to synthesize dna , rna , proteins , and phospholipids . these processes are not constitutively activated , because the parasite will synthesize them as needed throughout the idc . we used a validated in - house developed method ( fang et al . , 2014 ) to simulate time - dependent dna synthesis during the idc . for comprehensiveness , we introduce the constraint governing the rate of dna synthesis in the metabolic network model : cdatpdatp+cdctpdctp+cdgtpdgtp+cdttpdttpdna+(cdatp+cdctp+cdgtp+cdttp)diphosphate . here , datp , dctp , dgtp , and dttp refer to deoxyadenosine , deoxycytidine , deoxyguanosine , and deoxythymidine triphosphates , respectively . we refer the reader to the original article ( fang et al . , 2014 ) for further details on this method for simulating time - dependent p. falciparum macro - molecules during the idc . the criticality of a gene is determined by optimizing the following objective function : idnacimi , subjected to : smxnvnx1=0 and vlbvvub . here , dna is the gene set containing the metabolites that contribute to dna synthesis ( introduced above ) and ci is the contribution of the ith metabolite ( mi ) . in addition , s is the stoichiometry matrix , v is a vector containing all metabolic reactions of the p. falciparum metabolic network model , m is the number of metabolites in the network , n is the number of reactions in the network , and vlb and vub are the upper and lower bounds , respectively , of metabolic reactions . we deemed a reaction as critical if there was no dna synthesis after knocking out the reaction in silico . chloroquine administration inhibits dna replication in malaria parasites ( cohen and yielding , 1965 , polet and barr , 1968a ) . 2010 ) also show that metabolic genes critical for p. falciparum dna synthesis are suppressed by chloroquine treatment . we sought to identify the genes that are substantially altered in response to chloroquine and cause inhibition of p. falciparum dna synthesis . obvious candidates are genes that are down - regulated and critical for p. falciparum dna synthesis . however , not all such genes will have a pronounced effect on dna synthesis , which depends on the extent to which an individual gene is down - regulated and/or the individual contribution of that gene to the dna synthesis of the parasite . this suggests that many combinations of such genes may underlie the metabolic response associated with chloroquine treatment . , 1990 ) , we aimed at identifying the minimal number of genes ( hereinafter referred to as the core genes ) that are sufficient to completely account for the effect of chloroquine on dna synthesis by the parasite . we identified the core genes based on the bayesian information criteria ( bic ) score ( schwarz , 1978 ) , which is given by , nln(ssen)+kln(n ) , where sse=i=1n(victlvicqn)2max(vctl)min(vctl ) and n denotes the number of time points , k the number of genes being tested , vctl the estimated rate of dna synthesis under control conditions , and vcqn the estimated rate of dna synthesis after chloroquine treatment ( with =1 for the k genes being tested ) . we implemented an algorithm that starts with a set of size k=1 and increases the size of the test set as long as a given combination of genes for the new set ( size k+1 ) has a bic score lower than the minimal bic score of the previous size . we exhaustively repeated this process at each chloroquine dose ( 41 , 72 , and 144 nm for pf3d7 , and 43 nm for pfdd2 ) . we modified our previous approach ( fang et al . , 2014 ) to simulate the effect of external stress , as depicted in fig . 1 and described in the materials and methods section . we validated the methodology by demonstrating that reaction fluxes obtained in response to stress were sufficient to predict metabolic phenotypes corresponding to that stress . 2a shows the biochemical pathways incorporated into the computer model used to predict the functional consequences of altered fluxes of tca cycle enzymes in response to previously studied genetic perturbations ( ke et al . , 2015 ) . 2b shows the predicted alterations in tca cycle metabolites in response to genetic perturbations of tca cycle enzymes ( fig . 2a , red circles ) . we calculated the altered metabolite concentrations by scaling the control parameters in the tca cycle model ( wu et al . , 2007 ) by the median of the predicted changes in tca cycle enzyme activities ( fig . 2c ) . 2b , filled bars ) qualitatively captured the trends of relative change in experimentally measured tca cycle metabolites ( fig . 2b , open bars ) . 2b , filled bars ) was not within the margin of error of the experimental measurements ( fig . 2b , open bars ) . we examined all possible enzymes / transporters ( e.g. , citrate synthase , pyruvate dehydrogenase , aspartate aminotransferase , citrate - malate antiporter ) whose perturbation could yield mitochondrial citrate levels higher than those in the wild - type system . in each scenario , either the predicted citrate levels did not increase or the predicted oxaloacetate levels increased . therefore , based on our model analysis , the source of this increase in the experimentally reported citrate level appears to be non - mitochondrial in nature . in agreement with previous experimental observations ( cohen and yielding , 1965 , polet and barr , 1968a ) , our simulations suggested that chloroquine treatment results in dose - dependent inhibition of dna replication ( fig . a , blue diamonds ) and 43 nm ( pfdd2 , panel b , blue diamonds ) , the rate of dna synthesis during the ring stage ( the first 18 h after infection ) was less inhibited than it was during the late trophozoite and early schizont phases ( beyond 30 h after infection ) . these results are in agreement with previous observations ( yayon et al . , 1983 ) . because many studies suggest that pfdd2 is less sensitive to chloroquine than pf3d7 ( moneriz et al . , 2011 ) , dna synthesis might have been expected to be more inhibited in pf3d7 than in pfdd2 . in our simulations , however , a similar concentration of chloroquine resulted in greater dna synthesis inhibition in pfdd2 ( fig . 3b ; blue diamonds versus black circles , paired - sample t - test ; p < 0.0001 ) than in pf3d7 ( fig . 3a ; blue diamonds versus black circles , paired - sample t - test ; p = 0.15 ) . these differences likely stem from differences in the relative gene expression ratios of pfdd2 and pf3d7 . notably , the transcriptional profile for pfdd2 was more down - regulated than that for pf3d7 at a similar concentration of chloroquine . in other words , the percentage of down - regulated genes critical for p. falciparum dna synthesis was greater in pfdd2 than in pf3d7 ( table s1 ) . together with the highly similar ic50 values reported previously ( hu et al . , 2010 ) for pf3d7 ( 41 nm ) and pfdd2 ( 43 nm ) , these results suggest that the pfdd2 strain used here may have diverged from its true ( chloroquine - resistant ) phenotype . we found that 81 metabolic reactions in our network model are critical for p. falciparum dna synthesis . we denoted these reactions as critical because their elimination resulted in abolished dna synthesis ( see section 2.6 ) . of these 81 reactions , 64 had a known gene association whereas 17 did not . these genes belonged to pathways involved in glycolysis , pentose phosphate synthesis , pyruvate metabolism , purine metabolism , pyrimidine metabolism , redox metabolism , and mitochondrial metabolism . in the online supplement ( see spreadsheet ) , we provide detailed information , including the reaction name , chemical reaction , gene association , and pathway , of all reactions critical for p. falciparum dna synthesis . experimental observations ( cohen and yielding , 1965 , polet and barr , 1968a ) and the present simulations ( fig . 3 ) indicate that chloroquine treatment inhibits replication of p. falciparum dna , which in turn suggests that pathways essential for dna synthesis are inhibited . 4 shows the relative change in the amount of dna synthesized by the parasite during the idc period in response to chloroquine treatment . table s2 lists the actual amounts of synthesized dna for both strains under each simulated condition . we observed a dose - dependent decrease in the amount of dna synthesized by the parasite during the idc ( fig . 4 , red markers ) . we used bic scores ( schwarz , 1978 ) and employed the algorithm described in section 2.7 to identify core genes that caused this net decrease of synthesized dna . preventing only the core genes from being down - regulated by chloroquine was sufficient to restore the amount of dna synthesized to control values ( fig . 4 , green markers ) ; for these simulations , the reaction expressions of the core genes were set to be equal for control and chloroquine - treated conditions . a list of the core genes identified for each strain and chloroquine dose ( table s3 ) suggests that thioredoxin reductase and ribonucleotide reductase substantially affect the amount of synthesized dna . for pfdd2 , only two genes ( thioredoxin reductase and ribonucleotide reductase ) caused the nearly 50% reduction in the net amount of synthesized dna ( table s3 and fig . 4 ) . these genes were not mentioned in the original transcriptome analysis of hu and colleagues , who used a fundamentally different approach to interpret their transcriptome data ( hu et al . , 2010 ) . these authors focused on genes that were either co - regulated or showed strongly differential expression ( 8 log2-fold change ) in response to a drug . in contrast , our methodology of coupling transcriptomic changes with a metabolic network model can predict physiological outcomes as they relate to parasite metabolism , regardless of the magnitude of transcriptional change for an individual gene . transcriptional changes are not proportional to changes in metabolic reaction fluxes ( moxley et al . , 2009 ) . our analysis suggests that low - amplitude alterations in these identified genes ( table s3 ) were sufficient to cause a significant reduction in parasite dna synthesis . several existing methods integrate whole - genome metabolic networks and transcriptome data ( colijn et al . , 2009 , song et al . , 2014 , recently , we developed a method that predicts metabolic flux profiles of p. falciparum during the idc ( fang et al . the transcriptome data of p. falciparum obtained under different stress conditions ( e.g. , drug exposure , genetic mutation , etc . ) contain information about its functional state and can be used to predict qualitative alterations , if any , in metabolic flux profiles during the idc . however , such data are often collected at a limited number of time points ; for instance , hu and colleagues ( hu et al . , 2010 ) obtained p. falciparum transcriptome data at only 8 selected time points during the idc . currently , there is no approach to integrate limited data , such as those reported previously ( hu et al . , 2010 ) , with a whole - genome network to predict metabolic flux profiles during the idc . this is mainly because such an approach requires gene expression data throughout the idc to predict the corresponding metabolic flux profiles . therefore , we developed an approach that uses gene expression data obtained under a stress condition to predict alterations of metabolic fluxes that correspond to that condition . we validated the developed method by integrating transcriptome data obtained under a given stress condition and predicting metabolomic data corresponding to that stress ( ke et al . , , we simulated the effect of chloroquine on the metabolic flux profiles of p. falciparum during the idc . chloroquine inhibits the formation of hemozoin and leads to heme accumulation , which is toxic to the parasite ( fitch , 1998 ) . however , the specific mechanism underlying this toxicity is not known ( ginsburg et al . , 1998 , some experiments suggest inhibition of dna replication as a potential mechanism of chloroquine action ( cohen and yielding , 1965 , kwakye - berko and meshnick , 1989 , polet and barr , 1968a ) . we developed a method that successfully predicted a reduction in the ability of the parasite to synthesize dna in response to chloroquine treatment ( see fig . we identified twelve distinct genes that were substantially affected in pf3d7 and pfdd2 after chloroquine treatment ; importantly , preventing their down - regulation in response to chloroquine treatment was sufficient for restoring the p. falciparum dna synthesis inhibited by chloroquine treatment ( see table 1 ) . these genes belonged to pathways involved in glycolysis , redox metabolism , and pyrimidine metabolism . a closer look at these genes reveals a potential mechanism of chloroquine action that unifies previous theories and observations involving heme ( fitch , 1998 ) , dna replication ( polet and barr , 1968a ) , and oxidative stress ( lehane et al . , 2012 , radfar et al . , 5 , suggests that administration of chloroquine has two major metabolic effects : i.it interferes with the function of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , which reduces the synthesis of phosphoenolpyruvate , whose carbon fixation activity is essential for anaplerotic tca metabolism and redox balance ( storm et al . , 2014).ii.its interference of thioredoxin reductase ( trxr ) reduces conversion of oxidized thioredoxin trx ( s2 ) to reduced thioredoxin trx ( sh2 ) , which is essential for donating electrons to reactions catalyzed by thioredoxin peroxidase ( tpx ) and ribonucleotide reductase ( rnr ) . tpx is essential for converting h2o2 ( reactive ) to h2o ( harmless ) ( pannala and dash , 2015 ) , and rnr for converting ribonucleoside 5-diphosphates species ( adp , udp , gdp and cdp ) , to deoxy - species ( dadp , dudp , dgdp and dcdp ) which are used for dna synthesis ( munro and silva , 2012 ) . it interferes with the function of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , which reduces the synthesis of phosphoenolpyruvate , whose carbon fixation activity is essential for anaplerotic tca metabolism and redox balance ( storm et al . , 2014 ) . its interference of thioredoxin reductase ( trxr ) reduces conversion of oxidized thioredoxin trx ( s2 ) to reduced thioredoxin trx ( sh2 ) , which is essential for donating electrons to reactions catalyzed by thioredoxin peroxidase ( tpx ) and ribonucleotide reductase ( rnr ) . tpx is essential for converting h2o2 ( reactive ) to h2o ( harmless ) ( pannala and dash , 2015 ) , and rnr for converting ribonucleoside 5-diphosphates species ( adp , udp , gdp and cdp ) , to deoxy - species ( dadp , dudp , dgdp and dcdp ) which are used for dna synthesis ( munro and silva , 2012 ) . for example , our analysis points to trxr and gapdh as candidate molecules that determine the efficacy of chloroquine . here , we focused on candidates susceptible to a low dose of chloroquine because several pathways will be inhibited at higher concentrations ( see table s3 ; chloroquine = 144 nm ) . in addition , proteins that transport chloroquine out of food vacuoles should decrease the effective concentration of chloroquine . targeting these pathways while administering chloroquine treatment should enhance the efficacy of chloroquine at a given effective concentration . the hypothesis that chloroquine kills the malarial parasite by dna intercalation is decades old ( meshnick , 1990 ) . although it was the prime candidate mechanism of chloroquine from the 1950s to the 1970s , it has now largely been abandoned for the past two decades . our model analyses do not identify a drug target or targets ; however , they suggest that the effect of chloroquine administration leads to inhibition of dna synthesis . according to our model , the parasite responds to chloroquine by reducing the expression of genes critical for dna synthesis . although chloroquine could specifically intercalate with the identified core gene set in accord with the dna intercalation theory of chloroquine ( meshnick , 1990 , o'brien et al . , 1966a heme is a more plausible molecular inhibitor , given that it accumulates in large amounts within the parasite 's food vacuole and is capable of causing dna damage ( ishikawa et al . , 2010 , rahman et al . , 1997 ) , protein oxidation ( kitatsuji et al . , 2016 ) , nadph oxidation ( bodaness , 1983 ) , and apoptosis ( chiabrando et al . , 2014 ) . however , there is no direct evidence suggesting that heme causes any of these damages in p. falciparum . chloroquine , which was once a highly effective drug , is now abandoned in many malaria endemic regions because of widespread resistance . knowledge of this mechanism allowed us to identify metabolic pathways that could be exploited to increase chloroquine efficacy . for example , our model analyses suggest that administration of h2o2 would potentiate parasite killing by chloroquine , consistent with in vitro experiments ( malhotra et al . , similarly , our analyses suggest the hypothesis that targeting parasite - specific pathways that synthesize phosphoenolpyruvate and reduce thioredoxin would potentiate parasite killing . targeting these pathways , while administering amodiaquine ( a chloroquine analogue ) , should result in higher efficacy than administering chloroquine , especially because amodiaquine accumulates more than chloroquine in the parasite s food vacuole and has a mechanism similar to that of chloroquine ( hawley et al . , 1996 ) . our aim was to identify and investigate enzymes that contribute to decreased dna replication in response to chloroquine treatment . using a transcriptomics - based approach , we can identify cellular responses that are specific to chloroquine by expression data taken from chloroquine - treated parasites . although other antimalarial drugs may activate similar stress pathways , the details of the response will be specific to the particular conditions . the approach makes no assumptions on the underlying molecular actions that cause or affect the response . the effect of chloroquine on dna replication is likely to be indirect , being mediated by heme accumulation , which evokes multiple toxic effects in the cell . we simulated the effect of chloroquine over the entire idc based on the available transcriptome data taken from an eight - hour window during the trophozoite stage . ideally , for any specific inhibitor , it is desirable to have hourly transcriptional readouts to predict the effect of a systemic perturbation throughout the entire idc . in the case presented here , the largest effect of chloroquine occurs during the trophozoite stage and the protocol for collecting experimental transcriptomic data was designed to characterize transcriptional changes during this stage . our use of these data to identify changes in metabolite fluxes throughout the idc relative to untreated controls rests on the assumption that altering the expression of genes not associated with any biological function specific to a particular stage will have minimal physiological effects at other stages . this is an assumption that is specific to p. falciparum , because malaria relies on the execution of a highly regimented transcriptional program during the idc , which initiates specific stage - dependent cellular functions . these assumptions were ultimately corroborated because the observed metabolite phenotype changes are mainly associated with the trophozoite stage and metabolic alterations during previous time points were non - existent or immaterial to dna synthesis . herein , we investigated the potential mechanism by which chloroquine inhibits dna synthesis in p. falciparum and kills the parasite . we first developed an approach ( that requires limited transcriptome data ) to simulate the effect of exogenous stress on p. falciparum metabolic fluxes during the idc . we then developed an algorithm employing bayesian information theory to identify potential pathways that inhibit dna synthesis in response to chloroquine treatment . our analysis suggests that 1 ) chloroquine inhibits dna synthesis via inhibition of redox metabolism , carbon fixation , and anaplerotic tca cycle metabolism ; 2 ) p. falciparum may maintain dna synthesis after chloroquine treatment by preserving a reduced pool of thioredoxin and by invoking alternate mechanisms for synthesizing phosphoenolpyruvate , and 3 ) a drug or combination of drugs that target redox metabolism , carbon fixation , and heme accumulation could be an effective way to target the parasite . the method developed here used limited transcriptome data to identify pathways that underlie the inhibition of dna synthesis of p. falciparum in response to chloroquine treatment . this suggests that the developed method could also be suitable to simulate and capture the effect of other drugs ( or stress conditions ) on complex metabolic and physiological responses of p. falciparum , based solely on transcriptome data .

background : the purpose of this review was to provide a critical appraisal of the literature supporting the efficacy of ophthalmic ketorolac ( acuvail ) in the treatment of pain and inflammation after cataract surgery.methods:literature search and expert opinion of the authors.results:recent studies indicate greater intraocular drug levels in the anterior chamber and iris - ciliary body after topical application of acuvail in comparison with older formulations of ketorolac . a large randomized , multicenter , placebo - controlled study demonstrated significantly less inflammation and pain after cataract surgery using acuvail.conclusion:acuvail appears to be effective in reducing post - cataract surgery pain and inflammation . chloroquine , long the default first - line treatment against malaria , is now abandoned in large parts of the world because of widespread drug - resistance in plasmodium falciparum . in spite of its importance as a cost - effective and efficient drug , a coherent understanding of the cellular mechanisms affected by chloroquine and how they influence the fitness and survival of the parasite remains elusive . here , we used a systems biology approach to integrate genome - scale transcriptomics to map out the effects of chloroquine , identify targeted metabolic pathways , and translate these findings into mechanistic insights . specifically , we first developed a method that integrates transcriptomic and metabolomic data , which we independently validated against a recently published set of such data for krebs - cycle mutants of p. falciparum . we then used the method to calculate the effect of chloroquine treatment on the metabolic flux profiles of p. falciparum during the intraerythrocytic developmental cycle . the model predicted dose - dependent inhibition of dna replication , in agreement with earlier experimental results for both drug - sensitive and drug - resistant p. falciparum strains . our simulations also corroborated experimental findings that suggest differences in chloroquine sensitivity between ring- and schizont - stage p. falciparum . our analysis also suggests that metabolic fluxes that govern reduced thioredoxin and phosphoenolpyruvate synthesis are significantly decreased and are pivotal to chloroquine - based inhibition of p. falciparum dna replication . the consequences of impaired phosphoenolpyruvate synthesis and redox metabolism are reduced carbon fixation and increased oxidative stress , respectively , both of which eventually facilitate killing of the parasite . our analysis suggests that a combination of chloroquine ( or an analogue ) and another drug , which inhibits carbon fixation and/or increases oxidative stress , should increase the clearance of p. falciparum from the host system .

governments across the developed world are concerned with enabling older people to maintain their active contribution to society , and thereby their quality of life ( qol).1 qol has become a commonly used end point in the evaluation of multisector public policy , including health , social , community and environmental policy actions . for policy outcomes to be measured with any validity , measures of qol need to have social , as well as policy , relevance , to be meaningful to people 's lives , and to be carefully conceptualised and constructed . lawton26 developed a popularly cited quadripartite concept of qol , proposing that the good life ( qol ) may be represented by behavioural and social competence ( health , cognition , time use , social behaviour ) , perceptions of qol ( subjective evaluation of each domain of life ) , psychological well - being ( mental health , cognitive judgements of life satisfaction , positive - negative emotions ) and the external , objective , physical environment ( housing , economic indicators ) . however , there is no consensus about its conceptual definition or measurement,7 and most investigators have based their concepts on expert opinions rather than the perspectives of lay people . this has the consequence that there are few empirical data on the extent to which the items included in measurement scales have any relevance to people . thus , it is increasingly important to develop a multidimensional model and measure of quality of life , for use in descriptive and evaluative multisector policy research , which reflects the views of the population concerned , with cross - sectional and longitudinal applicability . elicitation of people 's own views of qol in this process is particularly important because qol is a subjective concept . survey and qualitative research with people aged 65 + , living at home in britain , reported that the central planks of qol emphasised by respondents were psychological well - being and positive outlook , having health and functioning , social relationships , leisure activities , neighbourhood resources , adequate financial circumstances and independence.710 this research led to the development of the older people 's quality of life questionnaire ( opqol ) , which is unique in being derived from the views of a representative sample of older people , cross - checked against theoretical models for assessment comprehensiveness . the aim here is to compare the psychometric properties of the opqol , with the casp-19 and whoqol - old among people 65 + participating in three national surveys of older people living at home in britain . two of these three surveys were cross - sectional , and the third was longitudinal ( see supplementary appendix 1 ) : ethnibus survey of people aged 65 + responding to two waves of the national ethni surveys ( http://www.ethnicfocus.com ) in 2008 . this is a rolling face - to - face interview survey with adults aged 16 + , living at home , based on focused enumeration , stratified random sampling of postcodes in britain , and statistically robust sampling of people in common ethnic minority groups in britain ; the response rate was 70% ( n=400 ) . ons survey of people aged 65 + responding to two waves of the office for national statistics ( ons ) national omnibus survey ( http://www.statistics.gov.uk ) in 2008 . this is a rolling face - to - face interview survey with adults aged 16 + , living at home , based on a stratified random sample of postcodes across britain ; the response rate was 61% ( n=589 ) . qol follow - up survey in 20072008 , of people living at home in britain , aged 65 + at baseline , who had responded to four ons national omnibus interview surveys . these were based on stratified random samples of postcodes across britain during 1999/2000 ; response was 77% ( n=999 ) at baseline and 58% among survivors ( n=287 ) at 20072008 follow - up . the qol follow - up survey is included here as the longitudinal design provided the opportunity to test the causal model of the opqol , as well as a willing sample for test - retest reliability assessment . the opqol was administered in all three surveys . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . the casp-1911 and the whoqol - old12 13 were administered in the two face - to - face interview surveys only ; it would have been too cognitively burdensome to have included all three scales in the postal , self - administration mode . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . this would not necessarily be applicable to populations in other countries , because it reflects close connections between new commonwealth countries and ethnic minority groups in the uk.15 descriptive analyses included frequencies , tests , and spearman 's r correlations . tests of scale reliability were applied in order to assess the extent to which scale items measure the same construct , with freedom from random error ( internal consistency ) . this is the strength of the association between each scale item and the full scale , item - item and item - total correlations . test - retest reliability of the stability of the newly developed opqol was assessed by mailing a second copy of the questionnaire to a random subsample of 50 follow - up qol survey respondents , 4 weeks after return of the first questionnaire ( response rate : 76%/38 ) . criterion ( concurrent ) validity is the independent corroboration that the scale is measuring what it intends to measure . this can only be measured by proxy with subjective measures , as there is no gold standard . proxy variables used here included independent self - ratings of qol overall and of qol domains ( health , social relationships , independence / control over life / freedom , home and neighbourhood , psychological / emotional well - being , financial circumstances , social and leisure activities ) . construct ( convergent and discriminant ) validity requires corroboration that scales measure the underlying construct they purport to measure . this was tested by assessing spearman 's r correlations between the qol scales and similar variables ( for convergent validity that the scale should correlate with similar or hypothesised variables ) and dissimilar variables ( for discriminant validity that there should be low correlations between scales and variables not expected to be associated ) . multiple regression was used to assess validity further by examining the ability of theoretically relevant variables to predict total qol scores . a hierarchical approach was used , with independent variables entered in their theoretical order of importance . statistical significance was set at p<0.05 . the variables entered did not correlate by more than 0.732 ; tests for multicollinearity were satisfied . the opqol was administered in all three surveys . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . the casp-1911 and the whoqol - old12 13 were administered in the two face - to - face interview surveys only ; it would have been too cognitively burdensome to have included all three scales in the postal , self - administration mode . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . this would not necessarily be applicable to populations in other countries , because it reflects close connections between new commonwealth countries and ethnic minority groups in the uk.15 tests of scale reliability were applied in order to assess the extent to which scale items measure the same construct , with freedom from random error ( internal consistency ) . this is the strength of the association between each scale item and the full scale , item - item and item - total correlations . test - retest reliability of the stability of the newly developed opqol was assessed by mailing a second copy of the questionnaire to a random subsample of 50 follow - up qol survey respondents , 4 weeks after return of the first questionnaire ( response rate : 76%/38 ) . criterion ( concurrent ) validity is the independent corroboration that the scale is measuring what it intends to measure . this can only be measured by proxy with subjective measures , as there is no gold standard . proxy variables used here included independent self - ratings of qol overall and of qol domains ( health , social relationships , independence / control over life / freedom , home and neighbourhood , psychological / emotional well - being , financial circumstances , social and leisure activities ) . construct ( convergent and discriminant ) validity requires corroboration that scales measure the underlying construct they purport to measure . this was tested by assessing spearman 's r correlations between the qol scales and similar variables ( for convergent validity that the scale should correlate with similar or hypothesised variables ) and dissimilar variables ( for discriminant validity that there should be low correlations between scales and variables not expected to be associated ) . multiple regression was used to assess validity further by examining the ability of theoretically relevant variables to predict total qol scores . a hierarchical approach was used , with independent variables entered in their theoretical order of importance . the variables entered did not correlate by more than 0.732 ; tests for multicollinearity were satisfied . just over half of each sample comprised women ( 52%/207 ethnibus , 55%/324 ons , 54%/154 qol follow - up ) . whereas most ethnibus respondents were aged 65<75 ( 91%/363 ) , just over half of ons omnibus ( 55%/326 ) , and less than a fifth of qol follow - up respondents ( 17%/47 ) , were aged 65<75 . thirty - eight per cent ( 152 ) of the ethnibus sample were indian , 29% ( 117 ) were pakistani , 22% ( 86 ) were black caribbean and 11% ( 45 ) were chinese . most , 94% ( 555 ) of the ons omnibus sample were white british ; all qol follow - up respondents were white british . in reflection of their younger age , more of the ethnibus than other respondents were married or cohabiting ( 58%/230 , 49%/285 , 49%/138 respectively ) . fewer ethnibus than other respondents were home - owners ( 532%/208 , 73%/429 , 85%/239 respectively ) and fewer lived alone ( 5%/19 , 48%/286 , 49%/137 respectively ) ( all differences were statistically significant at least at p<0.01 . ) for detailed characteristics of the samples , see supplementary table 1 . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . the reliability criterion for item - total correlations ( the correlation of the item with the scale total with that item omitted ) is that the item should correlate with the total scale by at least 0.20 . with three exceptions , the 35 full opqol items met this criterion for all three samples ( the exceptions were in the ethnibus sample with items 10 , 12 and 32 ; but as cronbach 's was not improved by their removal , and they performed well in validity tests , they were retained ) . six of the 19 casp items failed to meet this criterion ( ethnibus : items 1 , 2 , 5 , 17 , 18 ; ons : item 6 ) . fourteen of the 24 whoqol - old items failed this criterion in the ethnibus sample only . as expected , all items correlated more highly with similar , than dissimilar , items in the scales . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . the 4 week test - retest correlations , assessed among qol follow - up survey respondents , ranged from moderate to high ( r 0.4030.782 ) . lower correlations were explained by reported life changes in the intervening month , demonstrating the difficulties of test - retest exercises in older populations . respondents ' comments at follow - up about life changes in the last 4 weeks illustrate this:about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free;my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard;my daughter and her young son have now left our home and acquired her own house . we miss them a lot;my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free ; my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard ; my daughter and her young son have now left our home and acquired her own house . we miss them a lot ; my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. the criterion validity of all three qol scales was indicated by their moderate to strong , significant correlations with global self - rated qol : the spearman 's r correlations for the opqol by self - rated qol overall in each sample were ethnibus 0.347 , ons 0.602 and qol follow - up 0.659 . for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . all correlations were significant at least at p<0.01 , with the exception of whoqol - old in the ethnibus sample which was p<0.05 . the validity of the opqol was further supported by significant correlations between its subscales and the independent qol domain ratings , in theoretically expected , similar directions7 ( eg , opqol health and functioning subscale correlated with self - rated health : spearman 's r ethnibus 0.122 ( p<0.05 ) , ons omnibus 0.679 ( p<0.01 ) and qol follow - up 0.713 ( p<0.01 ) . there were no significant correlations with dissimilar pairs ( eg , health and religion ) , again as expected . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old sensory abilities subscale correlated significantly , again as expected , with self - rated health in the ons ( r 0.322 , p<0.01 ) , but not the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . for comparability , the same independent variables were entered into each model . on the basis of the literature,7 optimum scores on each measure were hypothesised to be associated with optimum qol : self - rated active ageing , independent self - ratings of qol domains , social activities and help from social network members , self - rated health status and physical functioning ( adl ) , age , sex , marital status and housing tenure . the qol follow - up sample also provided an opportunity to test the causal model underpinning the opqol . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . just over half of each sample comprised women ( 52%/207 ethnibus , 55%/324 ons , 54%/154 qol follow - up ) . whereas most ethnibus respondents were aged 65<75 ( 91%/363 ) , just over half of ons omnibus ( 55%/326 ) , and less than a fifth of qol follow - up respondents ( 17%/47 ) , were aged 65<75 . thirty - eight per cent ( 152 ) of the ethnibus sample were indian , 29% ( 117 ) were pakistani , 22% ( 86 ) were black caribbean and 11% ( 45 ) were chinese . most , 94% ( 555 ) of the ons omnibus sample were white british ; all qol follow - up respondents were white british . in reflection of their younger age , more of the ethnibus than other respondents were married or cohabiting ( 58%/230 , 49%/285 , 49%/138 respectively ) . fewer ethnibus than other respondents were home - owners ( 532%/208 , 73%/429 , 85%/239 respectively ) and fewer lived alone ( 5%/19 , 48%/286 , 49%/137 respectively ) ( all differences were statistically significant at least at p<0.01 . ) for detailed characteristics of the samples , see supplementary table 1 . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . the reliability criterion for item - total correlations ( the correlation of the item with the scale total with that item omitted ) is that the item should correlate with the total scale by at least 0.20 . with three exceptions , the 35 full opqol items met this criterion for all three samples ( the exceptions were in the ethnibus sample with items 10 , 12 and 32 ; but as cronbach 's was not improved by their removal , and they performed well in validity tests , they were retained ) . six of the 19 casp items failed to meet this criterion ( ethnibus : items 1 , 2 , 5 , 17 , 18 ; ons : item 6 ) . fourteen of the 24 whoqol - old items failed this criterion in the ethnibus sample only . as expected , all items correlated more highly with similar , than dissimilar , items in the scales . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . the 4 week test - retest correlations , assessed among qol follow - up survey respondents , ranged from moderate to high ( r 0.4030.782 ) . lower correlations were explained by reported life changes in the intervening month , demonstrating the difficulties of test - retest exercises in older populations . respondents ' comments at follow - up about life changes in the last 4 weeks illustrate this:about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free;my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard;my daughter and her young son have now left our home and acquired her own house . we miss them a lot;my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free ; my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard ; my daughter and her young son have now left our home and acquired her own house . we miss them a lot ; my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. the criterion validity of all three qol scales was indicated by their moderate to strong , significant correlations with global self - rated qol : the spearman 's r correlations for the opqol by self - rated qol overall in each sample were ethnibus 0.347 , ons 0.602 and qol follow - up 0.659 . for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . all correlations were significant at least at p<0.01 , with the exception of whoqol - old in the ethnibus sample which was p<0.05 . the validity of the opqol was further supported by significant correlations between its subscales and the independent qol domain ratings , in theoretically expected , similar directions7 ( eg , opqol health and functioning subscale correlated with self - rated health : spearman 's r ethnibus 0.122 ( p<0.05 ) , ons omnibus 0.679 ( p<0.01 ) and qol follow - up 0.713 ( p<0.01 ) . there were no significant correlations with dissimilar pairs ( eg , health and religion ) , again as expected . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old sensory abilities subscale correlated significantly , again as expected , with self - rated health in the ons ( r 0.322 , p<0.01 ) , but not the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . for comparability , the same independent variables were entered into each model . on the basis of the literature,7 optimum scores on each measure were hypothesised to be associated with optimum qol : self - rated active ageing , independent self - ratings of qol domains , social activities and help from social network members , self - rated health status and physical functioning ( adl ) , age , sex , marital status and housing tenure . the qol follow - up sample also provided an opportunity to test the causal model underpinning the opqol . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . this study describes the psychometric performance of a qol questionnaire , developed from the perspectives of older people themselves : the opqol . it was tested in two cross - sectional , and one longitudinal , surveys of older people across britain . the longitudinal survey enabled the opqol to be tested in a dynamic , ageing population and an assessment of its underlying model , although its self - administration mode necessitated the assessment of the opqol only ( and not the casp-19 or whoqol ) in this older sample . the surveys used statistically robust sampling methods , and the response rates were fairly to very good . the characteristics of respondents to the ons omnibus and ethnibus surveys ( and the qol survey at baseline ) were comparable with population estimates from the last census . the qol follow - up sample , by its longitudinal design , reflected the healthy survivors . also , although the sampling approach of the ethnibus survey was statistically robust , it used focused enumeration . there is no other practical methodology for attempting to obtain representative samples of people in ethnic minority groups in national samples . this study reported that ethnibus respondents obtained poorer ( worse ) qol scores than the other sample respondents , with the opqol , casp-19 and whoqol - old . this is not unexpected given that people in ethnic minority groups are often more economically disadvantaged than the wider population.15 further research is needed to examine whether differences in qol reflect real variations , methodology , and cultural variations in expectations or in reporting . ethnic minority groups in britain live in a wide range of different communities , and their diversity may also have affected responses in some way . it should also be noted that the standard question for ethnic status used , largely reflected britain 's new commonwealth groups , and may not be appropriate for use in other countries . multiple regression models supported its validity and underlying constructs . despite the ethnibus sample 's consistently worse qol scores , compared with the other samples , the casp-19 and whoqol - old did not meet all criteria for internal consistency ( reliability ) in the ethnically diverse ethnibus sample . the casp-19 and whoqol - old also had relatively large numbers of items that failed to meet the reliability criterion for item - total scale correlations ; they frequently failed correlation tests for validity in the ethnibus sample . this may have been due to this sample 's ethnic diversity , or because the casp-19 and whoqol - old were not sufficiently sensitive . the opqol is currently being tested with older people living in italy ; initial results for cultural equivalence and understanding are positive ( personal communication , dr claudio bilotta , university of milan).what is already known on this subjectincreasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age.qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions.what this study addsthis study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness.the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . increasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age . qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions . this study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness . the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . myiasis , the infestation of organs and tissues of human and animals with fly larvae , is a common phenomenon especially in people of tropical and subtropical areas . in spite of occasional reports of obligatory myiasis , the majority of reported cases are facultative myiasis and more than a dozen families of flies can cause this form ( gullan and durden 2009 ) . phoridae , commonly known as humpback or scuttle flies , is the most important family of dipteran that causes myiasis and various problems for human . in this family genus megaselia , perhaps the largest genera of living organisms , has a wide variety of life styles and polyphagus diet ( disney 2008 ) . among identified species of this genus , it has been unwittingly carried around the world by human ( disney 2008 ) and previously has been reported from alborz province in iran ( zamani 2009 ) . adults of scuttle flies which are very small ( 24 mm in length ) , can explore a large variety of environmental and ecological habitats . females of these flies are highly attracted to strong of foul odors and lay their eggs on different decomposing materials such as fruits , stole , meat , excrement and carrions ( disney 2008 ) . the scuttle flies which are important agent of human facultative myiasis , exhibit a greater diversity of larval habitat than other insects . these flies can explore a large variety of environmental and ecological niches ( disney 2008 ) . in spite of predation and parasitation of some arthropods by this species ( costa et al . 2007 ) , previous studies showed them as a cause of human myiasis ( francesconi lupi 2012 ) . moreover , they have already been a polyphagus species and may be harmful to health as they become vector of pathogens ( disney 2008 ) . 1978 , carpenter and chastain 1992 , hira et al . 2004 ) and intestinal ( kaneko et al . 1975 , sing et al . however , the urogenital form is rarely seen and all of reported cases are restricted to females . so the study of men clinical manifestation as well as morphology of third instars larvae of these flies seems to be important . the aim of this paper is to report a case of man urogenital myiasis involving and to discourse the importance of its diagnosis and management in clinical science . an 18 year - old man living in zanjan city , northwest of iran , presented to ayatollah mosavi hospital at zanjan university of medical sciences with history of difficulty in urination and maggot discharging . , he was medicated by venlafazine , alpirazolam and nortriptrin . physical examination and sonography of cortex of bladder and kidneys he received mebendazole ( 2 mg tablet ) orally and washed his urogenital area with solution of xylocain ( 2% ) and iodine ( 1% ) . the urine was yellow in color and laboratory examination showed microhematuria , proteinuria and leukocyturia . the larvae were forwarded to the department of medical entomology and vector control for further studies . sonography of cortex of bladder with , small echogenous floating particles microscopic examination showed that all larvae were alive , whitish in color and pale intestinal content in the third terminal part ( fig . one larva was preserved in 70 % ethylic alcohol and the other cultured in nutrient rich medium to complete its life cycle but it died after 1 day . the length and width of larvae ranged from 6.8 to 6.9 and 0.68 to 0.69 mm respectively ( fig . precision microscopic examination of larvae revealed the presence of short spinous ( peglike ) processes on the integument . the larval head was noncapsulate , being reduced to mouth hooks and supporting cephalopharyngeal skeleton which was developed and supported large areas of the head ( fig . the anterior spiracles were located on each latero - posterior edge of the prothorax each had about 810 rays . a pair of posterior spiracles protruded dorsally on the 12 segment each appears with large and slender plate . in addition to their anterior and posterior union the dorsal and lateral trunks were joined by transverse connectives ( fig . 4 ) . third instars larva of megaselia scalaris anterior section of third instars larva of megaselia scalaris posterior section of third instars larva of megaselia scalaris based on characters and previous descriptions of researchers ( sukantason et al . 2004 , disney 2008 ) the larvae were definitely identified as megaselia scalaris , facultative myiasis parasite belonging to the phoridae family . the voucher specimens were retained in the department of entomological systematics at university of tarbiat modarres . urogenital myiasis , the infestation of genitourinary organs by maggots , based on the anatomical location could be classified as external or internal myiasis . external urogenital myiasis is clinically , epidemiologically , and entomologically similar to wound myiasis but it affects women more commonly . internal urogenital myiasis is a rare event and occurs when the maggot reaches an internal genitourinary organ . all described cases are considered as accidental cases and uncommon myiasis agents are associated with m. scalaris , psychoda albipenis and muscoid flies ( francesconi and lupi 2012 ) . several previous researchers reported cases of wound ( disney 2008 ) , intestinal ( mazyard and rifaat 2005 ) and nasocominal myiasis ( hira et al . 2004 ) , due to m. scalaris . however , the published cases of genital myiasis are very few in number and these reports refer to patients of various ages . the first report of human urogenital myiasis due to this species was reported in 1978 by disney and kurahashi and then other cases were reported ( ramalingam et al . 1997 , delir et al . 1999 , perez - eid c , mouffok 1999 , rodriguez and , rashid 2001 , passos et al . 2002 ) . there is a paucity of such information from this country , probably because specimens tend to be discarded without study , due to lack of experience in identifying fly larvae and in the absence of reference center . we hope that our report will stimulate others to present similar clinical findings and to contribute to the pool of knowledge on the subject in iran . to the best of our knowledge , so we hope that in the coming future special aspects of this species will be studied in iran . the identification of larvae of megaselia sp posed difficulties here because none could be reared to adult for conformation based on adult morphology . the passing of live m. scalaris larvae in the urine indicated infestation of the urogenital system . the reported infestations ( priyanond et al . 1973 , biery et al . 1989 ) were normally , a secondary consequence of some obstructions to the normal flow of the urine , such as stone formation in the bladder . our microscopic examination showed that the larvae were probably mature ; at least second to third instars transition , and may have been seeking a place to pupate . it is worth mentioning that identification of the immature larvae of parasite causing myiasis posed difficulties particularly the parasite with little available knowledge . however , the available morphological characters of the larvae especially the cephalopharyngeal skeleton and the structure of spiracles can be used in differentiation of specimens . however the global warming and the globalization of commerce have established conditions suitable for expand their wider distribution . the flies infesting the patients probably originated from a natural population near home in the residential area . it s likely that female of megaselia deposited her eggs on the underwear clothing or directly on the urogenital area after being attracted by urine odor . the rational treatment of genitourinary myiasis is to remove the offending larvae . in many cases when the diagnosis is done several substances such as antiseptic and anesthetic solutions have been used in the form of lavage ( francesconi and lupi 2012 ) . it seems , in our case , oral administration of 2 mg mebendazole and washing the urogenital area with solution of xylocain ( 2% ) and iodine ( 1% ) , cured the myiasis . since the urogenitoury tract has inaccessible area , removed of maggots is difficult by conventional instruments , to overcome this problem we used urogenital lavage with xylocaine and iodine solution in our patient . xylocaine causes spastic paralysis in parasites straight muscles and this way the paralyzed larva was easily removed . medical personnel should take care of susceptible patients especially those with nocturnal enuresis , need to bear in mind the possibility of infestation with scuttle flies larvae , be able to make a prompt diagnosis of myiasis and implement relevant intervention to prevent tissue infestation . prevention of this condition is important and involves use of insect repellents and insecticides for control of fly population , adequate protective clothing , good skin and wound hygiene to keep flies from reaching the skin , covering open wounds , change dressing daily , hang clothes to dry in bright sunlight and/or iron them and improve hygiene and sanitation . because the size of gravid flies is very small besides , so much attention is paid to large muscoid flies that may cause myiasis , it is likely that the small phorids are unnoticed or ignored , which may lead to an increase in the occurrence of phorid myiasis . we need to emphasize that this myiasis rarely occurs in healthy individuals and neglected personal hygiene is the single most important factor in human infestation . nowadays the global warming and commerce globalization change the geographical map of scuttle flies and observation of the scarce case of urogenital myiasis in iran , represents their wide distribution . since m. scalaris can possess considerable larval habitat , its creation of various clinical forms of myiasis is imminent . therefore , we hope that our reported case will stimulate researches to present similar findings and we expect several aspects of this species will be studied in future . in addition medical personell should take special attention to susceptible cases and be able to diagnose myiasis as well as implicate relevant interventions to prevent new infections .

backgroundmost measures of quality of life ( qol ) are based on expert opinions . this study describes a new measure of qol in older age , the older people 's qol questionnaire ( opqol ) , which is unique in being derived from the views of lay people , cross - checked against theoretical models for assessment of comprehensiveness . its performance was assessed cross - sectionally and longitudinally . it was compared with two existing qol measures in the cross - sectional studies in order to identify the optimal measure for use with older populations.methodsdata were taken from three surveys of older people living at home in britain in 20072008 : one population survey of people aged 65 + , one focused enumeration survey of ethnically diverse older people aged 65 + , one follow - up of a population survey of people aged 65 + at baseline in 1999/2000 . measures were qol ( using opqol , control , autonomy , satisfaction , pleasure - 19 items ( casp-19 ) , world health organization quality of life questionnaire - version for older people ( whoqol - old ) ) , health , social and socioeconomic circumstances . the casp-19 and whoqol - old were not administered to the longitudinal sample in order to reduce respondent burden.resultspsychometric tests were applied to each qol measure . the opqol , casp-19 and whoqol - old performed well with the cross - sectional samples ; however , only the opqol met criteria for internal consistency in the ethnibus samples.conclusionthe opqol is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . further research is needed to examine whether differences by ethnicity reflect real differences in qol , methodological issues , variations in expectations or cultural differences in reporting . myiasis is the infestation of organs and tissues of human and animals with fly larvae . this article reports an 18 year - old man with urogenital myiasis , the passing of live megaselia scalaris larvae in the urine , from zanjan city , northwest of iran . we discourse the importance of diagnosis and management of urogenital myiasis in medicine .

although malaria is preventable and curable , it still causes high morbidity and mortality ( 1 ) . according to the recent 2013 who report , globally an estimated of 3.4 billon people are at risk of malaria . in this report , who estimated 207 million malaria cases and 627,000 deaths occurred globally in 2012 ( 2 ) the majority of the global burden of human malaria is caused by plasmodium falciparum and p. vivax ( 3 ) . p. falciparum is the most deadly plasmodium species responsible for about 90% of malaria deaths , mainly in africa ( 4 ) and p. vivax is the most cause of malaria infection in the world ( 1 ) . p. vivax is accountable for 2540% of the annual bouts of malaria worldwide ( 4 ) . in iran , 2,714,648 individuals ( 4% of the total population ) mainly living in southern provinces namely sistan and baluchistan , kerman and hormozgan are at risk of malaria ( 5 ) . p. vivax is the most prevalent species reported among the malaria patients in iran annually ( 6 ) . however , a considerable decrease of malaria cases has been reported within the past few years in iran . since malaria elimination program has commence from a few years ago in the country ( 7 ) , for steady continuation of the program rapid and accurate diagnosis of malaria parasites play an important role in opportune case finding and treatment which result in on time control and elimination of the infection . conventional microscopic examination of giemsa stained thick and thin blood smears has been accepted as golden standard method for malaria diagnosis up to now . although malaria microscopy contains some advantages including cost , availability and relative sensitivity ( 810 ) , it bears some disadvantages such as time consuming and labor intensity ( 9 ) . the who has recently reiterated the urgent need for simple and cost - effective diagnostic tests for malaria to overcome the deficiencies of light microscopy and clinical diagnosis ( 10 , 11 ) . based on who advise rapid diagnostic tests can be replaced with microscopic method in remote and isolated areas particularly when trained and skilled personnel is not available ( 1214 ) . utilizing parasite lactate dehydrogenase ( pldh ) in rdts has shown better sensitivity for diagnosing low level of parasitemia in comparison with other malaria proteins . moreover , the amount of pldh indicates to metabolically presence of p. vivax due to short stability of pldh in the body ( 13 ) . pldh plays role of a coenzyme due to involving the oxidation of lactate to pyruvate with nicotinamide adenine dinucleotide ( nad ) ( 15 ) . inhibition of the malarial ldh enzyme prevents the production of atp and results to death of the plasmodium parasites ( 13 ) ; it becomes an attractive drug target candidate ( 16 ) . the genetic diversity of pldh might influence its drug target candidacy and the sensitivity of rdt kits . as far as we know , until now the genetic variation of pldh gene in p. vivax and p. falciparum infections were not reported in iran . this study aimed to detect the polymorphism of pldh gene from iranian strains of p. vivax and p. falciparum . obviously , understanding such polymorphism is important for designing or improving rdt kits . it can also give information about the molecular details of p. falciparum ldh ( pfldh ) and p. vivax ldh ( pvldh ) genes for designing a new drug . totally 43 whole blood samples were collected from p. vivax and p. falciparum infected patients in sistan and baluchestan province located in southeast of iran from 2012 to 2013 . sistan and baluchestan province is bordered with afghanistan and pakistan to the east and oman sea in south . thirty - three samples for p. vivax and 10 samples for p. falciparum were confirmed positive by light microscopic examination of giemsa stained thick and thin blood smears . one ml of blood was collected into tubes containing edta anticoagulant , placed immediately at 20 c for further analysis . dna was extracted from 200 l of whole blood samples of 33 p. vivax and 10 p. falciparum malaria infected patients using , accu - prep kit , genomic dna extraction kit ( bioneer , seoul , korea ) based on the manufacturer instructions . nucleotide sequences corresponding to pvldh and pfldh genes were amplified using the following sets of primers using conventional pcr . pvldh gene amplification was conducted using : forward : 5-atgacgccgaaacccaaaat-3 and reverse : 5-acctttaaatgagcgccttcat-3 , on the other hand pfldh gene was also amplified by f : 5-agatggcaccaaaagcaaaaat-3 and r : 5-acctttaagctaatgccttcat-3. pvldh primers were designed based on p. vivax sal-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) strains from genbank whereas pfldh primers designed based on the reference sequence p. falciparum 3d7 ( xm_001349953.1 ) strain in genbank . dna was extracted from whole blood of a healthy person living in non - endemic area as a negative control for using in amplification process . pcr reaction was performed in 25l reaction volumes containing 1lof each forward and reverse primers ( 10 pmol ) , 10 l of ready to use master mix ( ampliqon , denmark ) contains ( tris - hcl ph 8.5 , 1.5 mm mgcl2 , dntps and taqdna polymerase ) , 3 l of genomic dna samples and 10 l distilled water . pcr cycle parameters for pvldh gene amplification were as follows : 5minutes initial denaturation at 95 c followed by 30 cycles with 30 s at 95 c , 30 at 56 c , 1 at 72 c and final extension at 72 c for 5 min . all the pcr parameters were the same for pfldh gene amplification except the annealing temperature was 58 c . the fragment sizes of pcr products were determined using 1 kb dna ladder marker ( solis biodyne , estonia ) . twenty - two sequences including 15 p. vivax and 7 p. falciparum were analyzed to investigate polymorphism in pvldh and pfldh genes respectively . these genes were sequenced by applied biosystems 3730/3730xl dna analyzers , ( bioneer , seoul , korea ) using sanger method . nucleotide sequences of pvldh and pfldh were aligned and compared using clustal w2 software ( embl - ebi , http://www.ebi.ac.uk/tools/msa/clustalw2/ ) . pvldh gene sequences were compared with genbank sequences of p. vivax belem ( dq060151.1 ) and p. vivax sai-1 ( xm_001615570.1 ) . on the other hand , pfldh gene sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) . moreover , amino acid sequences related to each samples of p. vivax and p. faliparum were derived using expasy translate tool ( http://web.expasy.org/tran-slate/ ) . pvldh amino acid sequences were compared with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) whereas pfldh amino acid sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains registered in genbank . finally phylogenic tree was prepared to illustrate the distance among sequences of isolates using average distance ( ad ) method in clustal w2 jalview software ( http://www.eb-i.ac.uk/ ) . thelactate dehydrogenase gene from iranian plasmodium strains were submitted with the accession numbers of ( km226649-km226654 and km226656-km226664 ) for p. vivax , and ( km226665-km226671 ) for p. falciparum in genbank ( blast ) . dna was extracted from 200 l of whole blood samples of 33 p. vivax and 10 p. falciparum malaria infected patients using , accu - prep kit , genomic dna extraction kit ( bioneer , seoul , korea ) based on the manufacturer instructions . nucleotide sequences corresponding to pvldh and pfldh genes were amplified using the following sets of primers using conventional pcr . pvldh gene amplification was conducted using : forward : 5-atgacgccgaaacccaaaat-3 and reverse : 5-acctttaaatgagcgccttcat-3 , on the other hand pfldh gene was also amplified by f : 5-agatggcaccaaaagcaaaaat-3 and r : 5-acctttaagctaatgccttcat-3. pvldh primers were designed based on p. vivax sal-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) strains from genbank whereas pfldh primers designed based on the reference sequence p. falciparum 3d7 ( xm_001349953.1 ) strain in genbank . dna was extracted from whole blood of a healthy person living in non - endemic area as a negative control for using in amplification process . pcr reaction was performed in 25l reaction volumes containing 1lof each forward and reverse primers ( 10 pmol ) , 10 l of ready to use master mix ( ampliqon , denmark ) contains ( tris - hcl ph 8.5 , 1.5 mm mgcl2 , dntps and taqdna polymerase ) , 3 l of genomic dna samples and 10 l distilled water . pcr cycle parameters for pvldh gene amplification were as follows : 5minutes initial denaturation at 95 c followed by 30 cycles with 30 s at 95 c , 30 at 56 c , 1 at 72 c and final extension at 72 c for 5 min . all the pcr parameters were the same for pfldh gene amplification except the annealing temperature was 58 c . the fragment sizes of pcr products were determined using 1 kb dna ladder marker ( solis biodyne , estonia ) . twenty - two sequences including 15 p. vivax and 7 p. falciparum were analyzed to investigate polymorphism in pvldh and pfldh genes respectively . these genes were sequenced by applied biosystems 3730/3730xl dna analyzers , ( bioneer , seoul , korea ) using sanger method . nucleotide sequences of pvldh and pfldh were aligned and compared using clustal w2 software ( embl - ebi , http://www.ebi.ac.uk/tools/msa/clustalw2/ ) . pvldh gene sequences were compared with genbank sequences of p. vivax belem ( dq060151.1 ) and p. vivax sai-1 ( xm_001615570.1 ) . on the other hand , pfldh gene sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) . moreover , amino acid sequences related to each samples of p. vivax and p. faliparum were derived using expasy translate tool ( http://web.expasy.org/tran-slate/ ) . pvldh amino acid sequences were compared with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) whereas pfldh amino acid sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains registered in genbank . finally phylogenic tree was prepared to illustrate the distance among sequences of isolates using average distance ( ad ) method in clustal w2 jalview software ( http://www.eb-i.ac.uk/ ) . thelactate dehydrogenase gene from iranian plasmodium strains were submitted with the accession numbers of ( km226649-km226654 and km226656-km226664 ) for p. vivax , and ( km226665-km226671 ) for p. falciparum in genbank ( blast ) . a 955 bp band was observed in gel electrophoresis of pcr products of pfldh and pvldh amplified genes ( fig . 1 and fig . the amplified pvldh gene was yielded approximately 955 base pairs , coding for 316 amino acids . fifteen of the amplified genes were sequenced to analyze the genetic variation of pvldh gene using clustal w2 software . after comparing the sequences with the chromatogram with p. vivax sal-1 reference sequence , two single nucleotide substitution were detected at 666 , 899 positions from g to c and c to t respectively ( fig . thirteen of the 15 isolates displayed 100% nucleotide sequence homology with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) ( table 1 & fig . was brought an amino acid change from ( t , neutral polar amino acid to me , non - polar amino acid ) whereas the nucleotide substitution at 666 positions from g to c did not result any change in amino acid ( fig . dna was extracted from 10 p. falciparum confirmed whole blood samples and pfldh gene was amplified using specific primers . dna sequences of pfldh gene displayed three nucleotide substitutions at 36 , 814 and 891positions from a to g , g to a and g to a respectively ( fig . five of the 7 isolates had 100% nucleotide homology with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains submitted in genbank ( table 3 ) . : accession number , if : iran - baluchistan falciparum only one of the nucleotide changes at 814 positions from g to a was brought an amino acid change from aspartic acid ( d , acidic polar amino acid to n , neutral polar amino acid ) ( fig . 7 ) . the rest six isolates showed 100% amino acid homology withpfmzr-1 and pf3d7 strains from genbank ( table 4 ) . : accession number , if : iran - baluchistan falciparum the nucleotide homology between pvldh and pfldh in iranian isolates of p. vivax and p. falciparum was 75.876% . all p. vivax ldh nucleotide sequences had 75.79% homology with six of p. falciparum isolates . the amino acids sequence homology between pvldh and pfldh iranian isolates were 90.4% exception of one isolate which had 90.76% homology . generally , the amino acids sequence homology between pvldh and pfldh iranian isolates were more than 90% . the amplified pvldh gene was yielded approximately 955 base pairs , coding for 316 amino acids . fifteen of the amplified genes were sequenced to analyze the genetic variation of pvldh gene using clustal w2 software . after comparing the sequences with the chromatogram with p. vivax sal-1 reference sequence , two single nucleotide substitution were detected at 666 , 899 positions from g to c and c to t respectively ( fig . thirteen of the 15 isolates displayed 100% nucleotide sequence homology with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) ( table 1 & fig . was brought an amino acid change from ( t , neutral polar amino acid to me , non - polar amino acid ) whereas the nucleotide substitution at 666 positions from g to c did not result any change in amino acid ( fig . 5 ) . dna was extracted from 10 p. falciparum confirmed whole blood samples and pfldh gene was amplified using specific primers . dna sequences of pfldh gene displayed three nucleotide substitutions at 36 , 814 and 891positions from a to g , g to a and g to a respectively ( fig . five of the 7 isolates had 100% nucleotide homology with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains submitted in genbank ( table 3 ) . : accession number , if : iran - baluchistan falciparum only one of the nucleotide changes at 814 positions from g to a was brought an amino acid change from aspartic acid ( d , acidic polar amino acid to n , neutral polar amino acid ) ( fig . 7 ) . the rest six isolates showed 100% amino acid homology withpfmzr-1 and pf3d7 strains from genbank ( table 4 ) . the nucleotide homology between pvldh and pfldh in iranian isolates of p. vivax and p. falciparum was 75.876% . all p. vivax ldh nucleotide sequences had 75.79% homology with six of p. falciparum isolates . the amino acids sequence homology between pvldh and pfldh iranian isolates were 90.4% exception of one isolate which had 90.76% homology . generally , the amino acids sequence homology between pvldh and pfldh iranian isolates were more than 90% . pldh antigen is assumed to be a specific marker for the presence of viable plasmodium in blood , and is used for screening in malaria - endemic countries ( 17 ) . inhibition of the malarial ldh enzyme prevents the producing atp and causes death of the plasmodium parasites,(13 ) so it becomes an attractive drug target candidate ( 16 ) . therefore , the protein obtained from this gene can be used in any diagnostic test ( 18 ) . diversity in the pldh gene may influence specificity and sensitivity of rdts in any malaria endemic area . investigation of polymorphism in p. vivax and p. falciparum lactate dehydrogenase gene can lead to produce more specific and sensitive rdts kit . the nucleotide homology among 15 pvldh sequences of p. vivax was 100% with the exception of two isolates displayed 99.9% homology ( table 1 , 2 & fig . 100% pvldh nucleotide sequence homology was reported among chinese p. vivax , sal-1 and belem ( 19 ) . another study done in china reported 99.89% nucleotide identity of chinese isolates with belem strain ( 20 ) . this point out iranian pvldh nucleotide sequences had more homology with belem strain than chinese isolates . reported pvldh genes from chinese p. vivax anhui isolates had more than 99% sequence homology compared with strains in gene bank ( 21 ) . this outcome strongly agreed with findings from our study , which also showed more than 99% homology with all compared p. vivax strains registered in genbank . in the present study , pvldh gene sequences showed two nucleotide substitutions with one resulted an amino acid change from t , neutral polar amino acid to i , non - polar amino acids . antigen variability is unlikely to explain variability in implementation of rdts detecting pldh in p. falciparum , p. vivax cases ( 22 ) . in contrast to our finding , shin et al . in korea reported one snp which did not bring any change in amino acid ( 23 ) jianget al . in china also reported a single nucleotide difference at the position 666 between pvldh gene and p. vivax belem ( dq060151)(24 ) . pvldh genes from iranian isolates of p. vivax were displayed more nucleotide changes than korean and chinese isolates . in earlier chinese studies from jianghuai region and anhui isolates of p. vivax , there were no nucleotide changes among isolates ( 21 , 25 ) . compared to these reports the nucleotide changes among pvldh from iranian isolates of p. vivax was higher than both jianghuai region and anhui isolates of p. vivax . talman et al . reported four different type of dna sequence of p. vivax from 10 isolates ; the mutations were synonymous ( 22 ) . in our study , less number of nucleotide changes was seen and the mutations were not synonymous . fourteen isolates had the same amino acid sequences with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) . this finding was agreed with a study conducted in china , which reported 100% pvldh gene homology among chinese isolates , p. vivax sal - i and belem ( 19 ) . studies done in korea and china from korean and hainan isolates respectively , also reported 100% amino acid homology with p. vivax belem ( dq060151.1 ) ( 20 , 23 ) . this indicated rdts produced from korean and chinese isolates can be used in iran . on the other hand , pfldh homology among iranian strains of p. falciparum was 100% with the exception of two isolates . in contrast to our finding , talman et al . reported no variability among all sequences p. falciparum ( n = 49 ) in worldwide isolates of plasmodium spp ( 22 ) . five of the seven isolates had 100% nucleotide identity with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains registered in genbank . iranian pfldh genes and reference sequence ( pf3d7 ) had high homology about 99.9%100% ( table 1 , 2 & fig . 8) . this indicated pfldh gene is relatively conserved and can be a good target for antimalarial drug and producing rdt . in our study , these amino acid sequences also had 100% homology with p. falciparum mzr-1 and p. falciparum 3d7 strains from gen - bank . our finding was supported by the study conducted in indonesia , which reported 100% amino acid sequences between indonesian pfldh and pf 3d7 reference sequence ( 26 ) . in this study however , only the substitution at 891 positions from g to a was brought an amino acid change from aspartic acid to asparagine ( d , acidic polar amino acid to n , neutral polar amino acid ) . in madagascar , two snps at 73 and 814 positions among the 137 dna sequences of p. falciparum isolates were displayed . the nucleotide change in 10 isolates at 814 position resulted in an amino acid change ( d , acidic polar amino acid to n , neutral polar amino acid ) . in addition , another amino acid change ( at codon 25 : q , neutral polar amino acid to a , basic polar amino acid ) was seen due to the snp at 73 position ( 18 ) . the position of nucleotide change ( 814bp ) and the resulted amino acid change ( aspartic acid ( d ) to asparagine ( n ) in one of the isolates in our study was the same with the madagascar study . the nucleotide change at 814 positions in our study might be a single nucleotide polymorphism given the madagascar study snp report at the same position . iranian pfldh demonstrated less number of amino acid changes in comparison with the report that released from madagascar study . the nucleotide sequences homology between iranian isolates of pvldh and pfldh were 75.7976% . in china , jiang et al . akbulut et al . also reported 74.8% homology between pvldh and pfldh ( 27 ) . compared to jiang et al . and akubulut et al . report , the homology of iranian pvldh and pfldh was high . in our study , the amino acid sequences homology among iranian isolates of pvldh and pfldh was 90.4% with the exception of one isolate . reported 89.5% and 90.2% amino acid sequence homology between pvldh and pfldh respectively ( 23 , 28 ) . this indicated the amino acid homology between pvldh and pfldh genes from iranian isolates of p. vivax and p. falciparum were higher than previously reports . generally , in our study the amino acid homology between pvldh and pfldh was more than 90% . pldh gene from iranian p. falciparum and p.vivax isolates displayed 98.8100% homology with 13 nucleotide substitutions . this relatively stability indicated pvldh and pfldh genes can be a good antimalaria target and used for producing rdt kits . this indicated some techniques like drug discovery , vaccine development and other activities , which were applied on p. falciparum , could also be tried for p. vivax . the homology among pldh of p. vivax and p. falciparum should be further investigated with large enough sample size . in general , before using pldh for producing rdt kits the genetic variation of this gene should be investigated since its polymorphism varies with geographical locations . the fundamental problems with the personality disorders ( pd ) diagnostic system in the previous version of dsm , such as all - or - nothing diagnostic categories , considerable heterogeneity within categories , extensive overlap or comorbidity among categories , indistinct boundaries with normal personality , and incomplete coverage of personality psychopathology , led to the revision of the dsm approach ( 1 , 2 ) . since 2000 , after the revision of dsm - iv , pd researchers largely agree that personality pathology should be represented dimensionally rather than categorically ( 3 ) . thus , many alternative dimensional models of personality have been considered ( 4 - 7 ) , and ongoing research has been performed to delineate the conceptual and empirical structure of personality traits in the pathological range ( 6 , 8) . finally , a multidimensional system was proposed for representing and diagnosis of personality disorder features in dsm-5 . the new approach for the assessment of personality pathology identifies core impairments in levels of personality functioning , pathological personality traits , and prominent personality pathology types ( 9 ) . however , there is wide consensus that severity assessment is essential for any dimensional system of personality psychopathology ( 10 ) . moreover , the dsm-5 personality disorders work group has proposed impairments in personality functioning as the central element of pd ( 11 ) . thus , in the dimensional model of personality disorder any pd ( schizotypal , antisocial , borderline , narcissistic , obsessive - compulsive and avoidant ) is associated with fundamental disturbances of self and interpersonal relations , problems with identity , self - direction , empathy and intimacy . the review of literature showed that levels of personality functioning or self - other approach for diagnosis are informative in determining both the type and the severity of personality pathology ( 12 - 14 ) . the severity of impairment in personality functioning has also been shown to be important in planning treatment and predicting its outcome . despite the good advantages and empirical background of levels of personality functioning , practical studies in clinical settings with specific pds and cross - cultural studies in this field also , researchers need to determine which levels of personality functioning better predict and explain severity than others . the purpose of the current study was firstly , to examine the relationship between dsm-5 levels of personality functioning on an iranian sample with antisocial and borderline personality disorders , and second , to explore which levels of personality functioning in patients with antisocial and borderline personality disorders can better predict severity . of the 300 participants , 252 individuals with antisocial ( n = 122 ) and borderline personality disorders ( n = 130 ) were selected from september to november 2013 . with regard to = 0.05 and based on partial r = 0.095 , the sample size for antisocial personality disorder ( aspd ) was calculated to be 119 . for borderline personality disorder ( bpd ) , and were 0.05 and 80% , respectively , and four variables were tested by multiple regression analysis . based on partial r = 0.1 , the adequate sample size for bpd was 113 . participants were selected from prisoners ( 48.8% ) , outpatients ( 16.5% ) and inpatients ( 7.5% ) . they were recruited from tehran prisons , and clinical psychology and psychiatry centers of razi psychiatric hospital and taleghani general hospital , tehran , iran . inclusion criteria were diagnosis of antisocial and borderline personality disorders ( for personality disorders ) , being at least 18 years old , having at least secondary education , and the exclusion criteria were presence of a psychotic disorder , presence of severe mood disorder , presence of mental retardation , and presence of physical condition that impairs the person 's mental state . participants were 90.5% males and 9.5% females , aged 18 to 60 years ( sd = 1.37 ) , with guidance school degree education level or higher . disorders of axis i ; 106 patients ( 42.06% ) had no impairment , 78 patients ( 30.97% ) had a history of substance - related disorders , 35 patients ( 13.88% ) had mood disorder , 15 patients ( 5.96% ) had anxiety disorders , 8 patients ( 3.17% ) had had a history of psychotic spectrum disorder and 10 patients ( 3.96% ) had other disorders . data gathering measurements included psychological reports , scid - ii - pq , scid - ii , and dsm-5 levels of personality functioning checklist . the scid and its versions are considered to be the most comprehensive of the structured diagnostic interviews , which are currently available . in fact , in 1987 they were new and wide range of instruments , by spitzer , gibbon , williams and built in compliance with the criteria of the dsm - iv ( 16 ) . this instrument has been established as the gold standard for reliable assessment of psychiatric disorders . inter - rater reliability for scid - i was above 70 for mood , anxiety , schizophrenic disorders and alcohol abuse ; it was somewhat lower for a few other disorders ( 17 ) , while for scid - ii it was reported between 0.48 and 0.98 for the categorical diagnoses ( cohen s ) and 0.90 to 0.98 for the dimensional judgments ( intra - class correlation coefficient ) ( 18 ) . cronbach s was found between 0.71 and 0.94 for the scid - ii personality disorder scales ( 18 ) . due to high accuracy of the diagnostic criteria and extraordinary compliance with dsm - iv criteria , translated to and adapted with different languages . in iran scid - ii and scid - ii - pq the duration of the scid - i is 30 to 90 minutes , the duration of the scid - ii is 30 to 60 minutes . severity was determined in this study by the scores obtained from scid - ii . levels of personality functioning refer to the core capacities of personality - related self and interpersonal functioning and determining the severity of any impairment in these areas ( 12 ) . the dsm-5 levels of personality functioning include identity , self - direction , empathy and intimacy . the levels of personality functioning scale use each of these elements to differentiate five levels of impairment on a continuum of severity ranging from no impairment , i.e. healthy functioning ( level = 0 ) , to extreme impairment ( level = 4 ) ( 11 ) . the more impairment in these areas , the more likely that person has pd . in preliminary analysis of a sample of 424 psychiatric patients , morey et al . ( 14 ) found that a score of greater than three ( out of five ) on a short five - item scale , had a sensitivity of 79% and a specificity of 54% for a semi - structured interview diagnosis of pd . ( 20 ) have translated dsm-5 levels of personality functioning scale to farsi , and developed a semi - structured interview . inter - rater reliability for semi - structured interview of dsm-5 levels of personality functioning items was above 0.80 . the dsm-5 levels of personality functioning scale domains correlation with dsm - iv was between 0.30 and 0.69 . the duration of the semi - structured interview for dsm-5 levels of personality functioning is 15 to 25 minutes . in the implementation process , the researchers included three post - graduates of clinical psychology . to avoid probable bias outcome from these people , they were not informed of the exact goal of the research in detail and they were told that the research goal was to study personality disorders . they were entirely uninformed of the concerned disorder types . to control the probable bias , the research associates began to collect data periodically per step while they were blinded to the outcome of the previous or next step . the colleagues were trained to use these instruments . after training under the supervision of the researcher , some people were actually interviewed , and the interviewers bug was fixed . as mentioned above , there were two groups of patients ( patients with personality disorders and normal subjects ) . prior to the research onset , the participants were fully informed and provided a written consent . to avoid fatigue and to increase motivation in subjects , study for each subject was conducted during two days . in the days following the completion of the demographic questionnaire furthermore , cases , which had both symptoms of antisocial and borderline personality disorders , were examined by the structured clinical interview for personality disorders ( scid - ii ) . after the definitive diagnosis of antisocial personality disorder and borderline , they were invited to attend the next stage of the interview process based on dsm-5 levels of personality functioning . ethics approval was obtained from the university of social welfare and rehabilitation sciences research ethics committee , and the registered ethical code was 92/801/3110/2/a . we computed bivariate correlations between the dsm-5 levels of personality functioning and aspd , and bpd symptoms . to examine which levels of personality functioning in patients with antisocial and borderline personality disorders can better predicted severity , stepwise regression analysis was used , in which scid - ii results ( aspd and bpd severity ) were the dependent variables and levels of personality functioning were the predictors . of the 300 participants , 252 individuals with antisocial ( n = 122 ) and borderline personality disorders ( n = 130 ) were selected from september to november 2013 . with regard to = 0.05 and based on partial r = 0.095 , the sample size for antisocial personality disorder ( aspd ) was calculated to be 119 . for borderline personality disorder ( bpd ) , and were 0.05 and 80% , respectively , and four variables were tested by multiple regression analysis . based on partial r = 0.1 , the adequate sample size for bpd was 113 . participants were selected from prisoners ( 48.8% ) , outpatients ( 16.5% ) and inpatients ( 7.5% ) . they were recruited from tehran prisons , and clinical psychology and psychiatry centers of razi psychiatric hospital and taleghani general hospital , tehran , iran . inclusion criteria were diagnosis of antisocial and borderline personality disorders ( for personality disorders ) , being at least 18 years old , having at least secondary education , and the exclusion criteria were presence of a psychotic disorder , presence of severe mood disorder , presence of mental retardation , and presence of physical condition that impairs the person 's mental state . participants were 90.5% males and 9.5% females , aged 18 to 60 years ( sd = 1.37 ) , with guidance school degree education level or higher . disorders of axis i ; 106 patients ( 42.06% ) had no impairment , 78 patients ( 30.97% ) had a history of substance - related disorders , 35 patients ( 13.88% ) had mood disorder , 15 patients ( 5.96% ) had anxiety disorders , 8 patients ( 3.17% ) had had a history of psychotic spectrum disorder and 10 patients ( 3.96% ) had other disorders . data gathering measurements included psychological reports , scid - ii - pq , scid - ii , and dsm-5 levels of personality functioning checklist . the scid and its versions are considered to be the most comprehensive of the structured diagnostic interviews , which are currently available . in fact , in 1987 they were new and wide range of instruments , by spitzer , gibbon , williams and built in compliance with the criteria of the dsm - iv ( 16 ) . this instrument has been established as the gold standard for reliable assessment of psychiatric disorders . inter - rater reliability for scid - i was above 70 for mood , anxiety , schizophrenic disorders and alcohol abuse ; it was somewhat lower for a few other disorders ( 17 ) , while for scid - ii it was reported between 0.48 and 0.98 for the categorical diagnoses ( cohen s ) and 0.90 to 0.98 for the dimensional judgments ( intra - class correlation coefficient ) ( 18 ) . cronbach s was found between 0.71 and 0.94 for the scid - ii personality disorder scales ( 18 ) . due to high accuracy of the diagnostic criteria and extraordinary compliance with dsm - iv criteria , translated to and adapted with different languages . in iran scid - ii and scid - ii - pq the duration of the scid - i is 30 to 90 minutes , the duration of the scid - ii is 30 to 60 minutes . severity was determined in this study by the scores obtained from scid - ii . levels of personality functioning refer to the core capacities of personality - related self and interpersonal functioning and determining the severity of any impairment in these areas ( 12 ) . the dsm-5 levels of personality functioning include identity , self - direction , empathy and intimacy . the levels of personality functioning scale use each of these elements to differentiate five levels of impairment on a continuum of severity ranging from no impairment , i.e. healthy functioning ( level = 0 ) , to extreme impairment ( level = 4 ) ( 11 ) . the more impairment in these areas , the more likely that person has pd . in preliminary analysis of a sample of 424 psychiatric patients , morey et al . ( 14 ) found that a score of greater than three ( out of five ) on a short five - item scale , had a sensitivity of 79% and a specificity of 54% for a semi - structured interview diagnosis of pd . ( 20 ) have translated dsm-5 levels of personality functioning scale to farsi , and developed a semi - structured interview . inter - rater reliability for semi - structured interview of dsm-5 levels of personality functioning items was above 0.80 . the dsm-5 levels of personality functioning scale domains correlation with dsm - iv was between 0.30 and 0.69 . the duration of the semi - structured interview for dsm-5 levels of personality functioning is 15 to 25 minutes . the scid and its versions are considered to be the most comprehensive of the structured diagnostic interviews , which are currently available . in fact , in 1987 they were new and wide range of instruments , by spitzer , gibbon , williams and built in compliance with the criteria of the dsm - iv ( 16 ) . this instrument has been established as the gold standard for reliable assessment of psychiatric disorders . inter - rater reliability for scid - i was above 70 for mood , anxiety , schizophrenic disorders and alcohol abuse ; it was somewhat lower for a few other disorders ( 17 ) , while for scid - ii it was reported between 0.48 and 0.98 for the categorical diagnoses ( cohen s ) and 0.90 to 0.98 for the dimensional judgments ( intra - class correlation coefficient ) ( 18 ) . cronbach s was found between 0.71 and 0.94 for the scid - ii personality disorder scales ( 18 ) . due to high accuracy of the diagnostic criteria and extraordinary compliance with dsm - iv criteria , translated to and adapted with different languages . in iran scid - ii and scid - ii - pq the duration of the scid - i is 30 to 90 minutes , the duration of the scid - ii is 30 to 60 minutes . levels of personality functioning refer to the core capacities of personality - related self and interpersonal functioning and determining the severity of any impairment in these areas ( 12 ) . the dsm-5 levels of personality functioning include identity , self - direction , empathy and intimacy . the levels of personality functioning scale use each of these elements to differentiate five levels of impairment on a continuum of severity ranging from no impairment , i.e. healthy functioning ( level = 0 ) , to extreme impairment ( level = 4 ) ( 11 ) . the more impairment in these areas , the more likely that person has pd . in preliminary analysis of a sample of 424 psychiatric patients , morey et al . ( 14 ) found that a score of greater than three ( out of five ) on a short five - item scale , had a sensitivity of 79% and a specificity of 54% for a semi - structured interview diagnosis of pd . ( 20 ) have translated dsm-5 levels of personality functioning scale to farsi , and developed a semi - structured interview . inter - rater reliability for semi - structured interview of dsm-5 levels of personality functioning items was above 0.80 . the dsm-5 levels of personality functioning scale domains correlation with dsm - iv was between 0.30 and 0.69 . the duration of the semi - structured interview for dsm-5 levels of personality functioning is 15 to 25 minutes . in the implementation process , the researchers included three post - graduates of clinical psychology . to avoid probable bias outcome from these people , they were not informed of the exact goal of the research in detail and they were told that the research goal was to study personality disorders . they were entirely uninformed of the concerned disorder types . to control the probable bias , the research associates began to collect data periodically per step while they were blinded to the outcome of the previous or next step . the colleagues were trained to use these instruments . after training under the supervision of the researcher , some people were actually interviewed , and the interviewers bug was fixed . as mentioned above , there were two groups of patients ( patients with personality disorders and normal subjects ) . prior to the research onset , the participants were fully informed and provided a written consent . to avoid fatigue and to increase motivation in subjects , study for each subject was conducted during two days . in the days following the completion of the demographic questionnaire furthermore , cases , which had both symptoms of antisocial and borderline personality disorders , were examined by the structured clinical interview for personality disorders ( scid - ii ) . after the definitive diagnosis of antisocial personality disorder and borderline , they were invited to attend the next stage of the interview process based on dsm-5 levels of personality functioning . ethics approval was obtained from the university of social welfare and rehabilitation sciences research ethics committee , and the registered ethical code was 92/801/3110/2/a . we computed bivariate correlations between the dsm-5 levels of personality functioning and aspd , and bpd symptoms . to examine which levels of personality functioning in patients with antisocial and borderline personality disorders can better predicted severity , stepwise regression analysis was used , in which scid - ii results ( aspd and bpd severity ) were the dependent variables and levels of personality functioning were the predictors . data are presented as mean standard deviation ( sd ) or no ( % ) . the mean and standard deviation scores of the levels of personality functioning in antisocial and borderline personality disorder groups are showed in table 2 . bivariate correlation between dsm-5 levels of personality functioning ( identity , self - directedness , intimacy , empathy ) and antisocial and borderline personality symptoms are presented in table 3 . as seen in table 3 , positive and significant correlations were found between antisocial personality symptoms and dsm-5 levels of personality functioning . the total levels are highly correlated with aspd symptoms ( p value < 0.001 ) . in borderline personality disorder , except identity and empathy , other variables had significant correlations with bpd symptoms ( p value < 0.001 and < 0.05 ) . to examine the relationship between dsm-5 levels of personality functioning and aspd and bpd severity , a stepwise regression analysis was conducted , where aspd and bpd severity were the dependent variables and levels of personality functioning ( identity , self - directedness , intimacy , empathy ) were the independent variables . the results of regression analysis for levels of personality functioning and aspd severity are presented in table 4 . these results indicate that approximately 21.7% , 31.4% , and 33% of the variance of the aspd severity could be accounted by identity , intimacy and self - directedness , respectively . the data showed that identity , intimacy , and self - directedness significantly predicted antisocial personality disorder severity . the results of regression analysis for levels of personality functioning and bpd severity are displayed in table 5 . the results showed that intimacy and empathy were good predictors of borderline personality disorder severity . this means that approximately 16.5% and 25.1% of the variance of the bpd severity could be accounted for by these variables . the first aim of this study was to examine the dsm-5 levels of personality functioning on an iranian sample with antisocial and borderline personality disorders . the study findings revealed that all levels of personality functioning had significant positive correlations with aspd . this means that dsm-5 levels of personality functioning had a good relationship with antisocial personality disorder symptoms of an iranian sample . also , the findings indicate that except identity and intimacy , other variables ( self - directedness and empathy ) had a significant correlation with borderline personality symptoms . it seems that the study population could be one of the reasons for these findings . previous studies indicated that severity is the most important single predictor of concurrent and prospective dysfunction in assessing personality psychopathology ( 11 , 21 , 22 ) . therefore , the second aim of this study was to explore which levels of personality functioning in patients with antisocial and borderline personality disorders can better predict severity . the stepwise regression analysis results showed that identity , intimacy , and self - directedness are positively and significantly correlated with antisocial personality disorder severity , indicating that those with higher scores on these variables tend to have higher symptoms of aspd . the analysis showed that intimacy and empathy had a significant association with borderline personality disorder severity . it means that interpersonal level of personality functioning ( intimacy and empathy ) could explain the severity on patients with bpd in this study . in addition , the results showed that the dsm-5 levels of personality functioning are an essential part of antisocial and borderline personality disorders . also , the results revealed that levels of personality functioning were the same in other cultures and the iranian sample . the present study is one of the first in which researchers examined the dsm-5 levels of personality functioning relationship with severity , and is the first study on iranian patients with personality disorders based on the dsm-5 approach . overall , our findings showed that the levels of personality functioning are a significant predictor of antisocial and borderline personality disorders severity . nevertheless , there are a number of important limitations in this work and future research is required to overcome such limitations . firstly , the results are based on a relatively small number of cases and so caution should be taken in interpreting the data . secondly , data was gathered by a semi - structured interview designed to assess a dimensional model of personality disorders , and future work should focus on other relevant instruments . the third limitation of the current study was the nature of the sample , which was drawn from antisocial and borderline personality disorders . thus , future research should examine the role of axis i disorders in personality disorders severity . fifth , most participants in this study were males . therefore , other research is needed to investigate dsm-5 levels of personality functioning and severity in females . seventh and finally , our work focused on the assessment of dsm-5 levels of personality functioning and severity in adults .

background : parasite lactate dehydrogenase ( pldh ) is extensively employed as malaria rapid diagnostic tests ( rdts ) . moreover , it is a well - known drug target candidate . however , the genetic diversity of this gene might influence performance of rdt kits and its drug target candidacy . this study aimed to determine polymorphism of pldh gene from iranian isolates of p. vivax and p. falciparum.methods:genomic dna was extracted from whole blood of microscopically confirmed p. vivax and p. falciparum infected patients . pldh gene of p. falciparum and p. vivax was amplified using conventional pcr from 43 symptomatic malaria patients from sistan and baluchistan province , southeast iran from 2012 to 2013.results:sequence analysis of 15 p. vivax ldh showed fourteen had 100% identity with p. vivax sal-1 and belem strains . two nucleotide substitutions were detected with only one resulted in amino acid change . analysis of p. falciparum ldh sequences showed six of the seven sequences had 100% homology with p. falciparum 3d7 and mzr-1 . moreover , pfldh displayed three nucleotide changes that resulted in changing only one amino acid . pvldh and pfldh showed 75%76% nucleotide and 90.4%90.76% amino acid homology.conclusion:pldh gene from iranian p. falciparum and p. vivax isolates displayed 98.8100% homology with 13 nucleotide substitutions . this indicated this gene was relatively conserved . additional studies can be done weather this genetic variation can influence the performance of pldh based rdts or not . background : fundamental problems with personality disorders ( pd ) diagnostic system in the previous version of dsm , led to the revision of dsm . therefore , a multidimensional system has been proposed for diagnosis of personality disorder features in dsm-5 . in the dimensional approach of dsm-5 , personality disorders diagnosis is based on levels of personality functioning ( criteria a ) and personality trait domains ( criteria b).objectives : the purpose of this study was firstly , to examine the dsm-5 levels of personality functioning in antisocial and borderline personality disorders , and second , to explore which levels of personality functioning in patients with antisocial and borderline personality disorders can better predicted severity than others.patients and methods : this study had a cross sectional design . the participants consisted of 252 individuals with antisocial ( n = 122 ) and borderline personality disorders ( n = 130 ) . they were recruited from tehran prisoners , and clinical psychology and psychiatry centers of razi and taleghani hospitals , tehran , iran . the sample was selected based on judgmental sampling . the scid - ii - pq , scid - ii and dsm-5 levels of personality functioning were used to diagnose and assess personality disorders . the data were analyzed by correlation and multiple regression analysis . all statistical analyses were performed using the spss 16 software.results:firstly , it was found that dsm-5 levels of personality functioning have a strong correlation with antisocial and borderline personality symptoms , specially intimacy and self - directedness ( p < 0.001 ) . secondly , the findings showed that identity , intimacy and self - directedness significantly predicted antisocial personality disorder severity ( p < 0.0001 ) . the results showed that intimacy and empathy were good predictors of borderline personality disorder severity , as well ( p < 0.0001).conclusions : overall , our findings showed that levels of personality functioning are a significant predictor of personality disorders severity . the results partially confirm existing studies .

homeostasis of the brain is maintained owing to its rigidly controlled communication with the peripheral tissues . entry of metabolites from the periphery to the brain is controlled by the blood brain barrier ( bbb ) . the major structural constituents of the bbb are the cerebral microvascular endothelial cells , and their barrier function relies on so- called tight - junctions ( tjs ) , consisting of transmembrane components : junctional adhesion molecule ( jam)-1 , occludin , and the claudins and intracellular proteins : zo-1 , zo-2 , and zo-3 , which link transmembrane proteins to the actin filaments of cytoskeleton and in this way improve stability and functioning of the tj . adherent junctions which are located in the basal region below the tjs , also contribute to the barrier function . cadherins stabilize adhesion between neighboring endothelial cells , while intracellularly , catenins link cadherins to the cytoskeleton ( fig . 1 ) . physically , the tjs limit free paracellular diffusion of low molecular weight compounds and make the transcellular transport of larger molecules dependent on specific transport systems , which can be grouped accordingly to the class of molecules transported ( hawkins and davis 2005 ; carvey et al . these transport systems are located in endothelial cells , and are modulated both intrinsically and by other cells of the neurovascular unit : astrocytes and pericytes ( simard and nedergaard 2004 ) . fine - tuning of the transport involves its polarization by differential location of the transport systems in the luminal versus abluminal membranes , which holds in particular for the different amino acid transport systems ( hawkins et al . two ultimate and complementary goals are reached : ( i ) control of the inflow and outflow of metabolic precursors and products , ( ii ) prevention of entry to the brain of undesired compounds.fig . 1composition of the tight junction and adherence junction which collectively restrict the paracellular passage of solutes across the bbb composition of the tight junction and adherence junction which collectively restrict the paracellular passage of solutes across the bbb the sections below describes the evolution of views on the role of bbb changes in the pathogenesis of diseases associated with increased exposure of the brain to blood - derived ammonia . studies on bbb penetration by different compounds in he models : a historical account section gives a historical perspective on the experimental studies on ammonia- and he - induced changes in bbb penetration of different compounds , without emphasis on the underlying mechanisms . transcellular passage of different molecules across the endothelium : roles of active transport section of the review will elaborate on the relatively well explored subject of modulation of transcellular passage , which represents active transport of medium- to large - molecules , and channel- or transporter - mediated ion fluxes across the capillary endothelial cell membranes . bbb leakage induced by ammonia and inflammatory molecules : new vistas on the underlying mechanisms section is devoted to the new findings regarding the mechanisms underlying alterations in the paracellular transport which is defined as bbb leakage , the role of which in ammonia neurotoxicity has so far been underestimated . pioneering studies pertinent to the effect of ammonia on bbb permeability were performed on animals with portacaval anastomosis ( pca)a model which mimics the condition of portal - systemic shunting in patients with liver cirrhosis . ( 1975 ) showed that bbb in pca rats is leaky to horseradish peroxidase ( hrp ) . this observation has been confirmed by sumner ( 1982 ) in a similar experimental setting , and by others using different bbb permeability markers and/or he models : by zaki ( 1983 ) also in pca rats who measured amino acid influx using the oldendorf perfusion technique ( oldendorf 1971 ) , and by horowitz et al . ( 1983 ) in galactosamine - induced animal model of acute liver failure ( alf ) , where permeability changes to aminoisobutyric acid were measured . however , other contemporary animal studies often performed in similar he models and using similar markers , revealed no brain vascular permeability changes . examples include the absence of changes of sucrose and methyl - aminoisobutyric acid permeation in galactosamine induced he ( lo et al . 1987 ) , and to mannitol or ions in the pca model ( sarna et al . 1977 ; alexander et al . transcellular passage of different molecules across the endothelium : roles of active transport and bbb leakage induced by ammonia and inflammatory molecules : new vistas on the underlying mechanisms sections , controversies about the bbb status as assessed with different compounds have lasted until the present time , with bbb changes being either confirmed ( wang et al . incoherent results were also obtained with regard to the passage of ammonia through the bbb , as monitored with n - labeled ammonia ( pet technique ) . lockwood et al . ( 1991 ) showed that ammonia enters the brain more easily in advanced he patients than in healthy controls . ( 2010 ) did not see any differences in bbb permeability for ammonia between patients with and without liver failure . ( 2007 ) observed increased ammonia accumulation in cirrhotic patients , but in their hands the increase was solely attributable to increased blood ammonia content . understanding of the effects of hyperammonemia on ammonia passage will require separate analysis of the two different forms of ammonia . at physiological ph overwhelming proportion of ammonia occurs as a cation ( warren 1962 ) , so it enters the brain mainly by a transcellular route , using an array of potassium channels and transporters or by substituting other cations with similar hydrated radius ( ott and larsen 2004 ) . one article indicated the presence of a specific nh4 carrier the rhesus associated glycoprotein rhcg in the brain capillaries ( huang and liu 2001 ) but its location ( luminal vs. abluminal side ) and functionality remains to be confirmed . however , recently the pericellular penetration by gaseous ammonia is being taken under consideration as a significant alternative ( ott and larsen 2004 ) . it is not known which of the two routes would be affected under excessive ammonia load . on the top of these controversies , increased vesicular transport across endothelial cells and swelling of astrocytic end - feet has been observed in different he models ( pilbeam et al . 1983 ) , with tjs remaining intact ( kato et al . it would thus appear that altered transcellular passage maybe a frequent phenomenon , albeit bbb changes in he are often too subtle to be detected with markers of gross bbb leakage . pca in rats is associated with amino acid imbalance in csf and brain due to enhanced blood to brain transport of tryptophan and other members of large neutral amino acid group ( lnaa ) ( james et al . in addition , increased concentration of aromatic amino acids ( aaa ) was found in brains of rats with pca , while the level of branched - chain amino acids was decreased ( smith et al . the above observations prompted a hypothesis that these alterations may contribute to impaired neurotransmission in he by producing ( i ) excessive amounts of neurotransmitters from which they derive , and/or ( ii ) false instead of authentic neurotransmitters , which are similar in structure but are either not active at the postsynaptic membrane , or their activity differs from their true counterparts ( curzon et al . 1975 ) . hypothesis appears attractive , because aaa are also precursors of these false modulators : tyrosine for octopamine ; phenylalanine for phenylethanolamine . ( 1982 ) showed elevated brain octopamine and phenylethanolamine levels in the brains of pca rats , and hilgier et al . however , the contribution of these false neurotransmitters to the neurotransmission imbalance associated with he has insofar not been examined in more detail . a plausible explanation for the ammonia - induced increase of blood brain aaa transport activity was proposed by james and colleagues ( 1979 ) who hypothesized that during hyperammonemia , increased brain glutamine ( gln ) production is followed by increased gln efflux from the brain , resulting in increased inward transport of these amino acids . this inference has been proven directly in studies in which increased tryptophan ( try ) uptake in exchange with gln via the l - transport system was recorded in cerebral capillary microvessels isolated from pca rats ( cangiano et al . vice versa , release of newly loaded gln from the capillaries was promoted by the try and leucine ( leu ) , and the effect was more pronounced when the capillaries were isolated from taa rats or following their incubation with ammonia than in control preparations ( hilgier et al . ( 1985 ) showed that treatment of pca rats with an inhibitor of gln synthesis , methionine sulfoximine ( mso ) , reduced the increased accumulation of the aaa in the brain in a manner correlated with increased ammonia accumulation . ( 1993 ) showed that administration of mso to pca rats normalized amino acid imbalance ascribed to excessive gln production . hyperammonemia was shown to be directly responsible for pca - induced alterations in the metabolism and transport of amino acids ( jessy et al . 1990 ) , including elevated brain try content and rise in the brain level of a serotonin metabolite , 5-hydroxyindoleacetic acid . these effects appeared to be due to ammonia - induced functional impairment of lnaa transport at the bbb . in rats in which ha was executed by urease administration , the impairment was found closely correlated with the rise in brain gln content ( bachmann and colombo 1983 ) , while in the cortical capillaries the increased try - gln exchange could be related to the raised -glutamyl - transpeptidase ( ggt ) activity ( stastn et al . 1988 ) . because ggt participates in lnaa transport and its activity was found to be increased in brain capillaries from hyperammonemic rats , a hypothesis has been put forward that ggt is involved in triggering the outward transport of the excess of gln from brain ( gorgievski - hrisoho et al . 1986 ) . in this way , enhanced activation of ggt could contribute to raised try and other lnaa levels as observed in rats with taa - induced alf ( hilgier et al . the above speculations were confirmed in a follow up study from the author s laboratory showing that ggt affects the l system - mediated amino acid exchange ( hilgier et al . the bbb transport of the cationic amino acids arginine ( arg ) and ornithine ( orn ) was investigated in different he models , and contradictory results have been obtained . zaki et al . ( 1984 ) showed a 30% increase in the brain uptake of arg in the galactosamine model of hepatic failure ; however , the effect was not specific to this amino acid and possibly secondary to bbb leakage also revealed by high molecular weight markers . by contrast , arg uptake from blood to brain was found decreased in chronic he ( pca ) rats ( zanchin et al . 1979 ) and in rats with thioacetamide ( taa)-induced he ( albrecht et al . 1996 ) . with regard to orn , increased brain uptake index of this amino acid coincident with its increased content in the blood was found in the taa model of he ( albrecht and hilgier 1986 ; albrecht et al . increased bbb transport of orn in the taa model has been considered as auto - protective response and in the same line has been speculated to facilitate intracerebral therapeutic action of the ammonia - trapping drug , l - ornithine - l - aspartate ( lola ) ( albrecht et al . . however , the benefits of orn may not apply to he in a chronic setting , where blood to brain transport of orn appears to remain unchanged ( zanchin et al . the mechanism underlying alterations of bbb transport of arg and orn has been hypothesized to involve changes in the basic amino acid transporter y activity and competition between these two amino acids for the transport site ( albrecht et al . 1996 ) , but experimental evidence in support of this hypothesis has not been provided as yet . the effects of he on arg transport are also likely to be mediated by gln , which accumulates intracerebrally in consequence of increased ammonia influx ( cooper and plum 1987 ) , overloading different cellular and subcellular compartments of the cns ( albrecht 2010 ) . it has been shown that gln added exogenously reduces no generation in the brain by inhibiting arg transport via the arg / gln exchanger , ylat2 , and that this effect is potentiated when ammonia is infused directly to the brain ( hilgier et al . if the above mechanism operates not only in the cns cells but also in the cerebral capillary endothelial cells forming the bbb , enhanced gln accumulation would modulate arg transport in these cells . the final outcome of this interaction would depend on whether gln accumulates intra- or extra - cellularly . a hypothesis that such an interaction may occur is supported by the observation that , gln infusion in the absence of hyperammonemia impairs cerebrovascular co2 reactivity , most likely by reducing arg availability and no synthesis , because co - infusion of arg counteracts the effect caused by glutamine ( okada et al . consistent with the role of arg / gln exchange at the bbb , our preliminary data indicate that ammonia increases the expression of the ylat2 transporter in a cerebral capillary endothelial cell line ( manuscript in preparation ) , as it does in the brain in the course of ha in situ ( zieliska et al . further studies on the mechanisms and pathophysiological implications of the changes in arg or orn influx to the brain are warranted in view of the proven or suspected contributions of the amino acids to the pathogenesis of he . arg is a precursor of no , a compound whose increased accumulation is engaged in the inflammatory response of the brain to ammonia ( jalan et al . 2011 ) , and in ammonia - induced brain swelling ( hussinger and grg 2010 ) , while decreased no synthesis has been implicated in impairment of cognition associated with prolonged hyperammonemia ( felipo 2006 ) . moreover , ha increases arg uptake to the different cell types within the cns ( rao et al . 1998 ) , and he in the taa model stimulates arg conversion to the neurotransmitter amino acids glu and gaba as measured in the whole brain ( albrecht and hilgier 1986 ) and in synaptosomes derived from these rats , which is likely to alter the balance between the inhibitory and excitatory neurotransmission ( albrecht et al . evaluation of the contribution of changes in arg transport across the bbb to the availability of this amino acid in the brain can not be accomplished without accounting for the variability in blood arg content in the different hyperammonemic models . the plasma arg level was shown to be decreased in pca rats ( zanchin et al . 1979 ) , but was elevated in rats subjected to prolonged hyperammonemia ( ishihara et al . 1998 ) , and fluctuated from increase to decrease during the development of taa - induced he ( albrecht and hilgier 1986 ) . orn plays a role in ammonia detoxification and gives rise to polyamines which exert hepato- and neuroprotection ( sikorska et al . treatment with lola , where orn contributes to urea formation , reduces blood ammonia level and in consequence improves the general condition of he patients ( kircheis et al . , increased brain uptake of orn as found in the taa model of he would further promote protection ( albrecht et al . orn also contributes in some degree to the biosynthesis of the neurotransmitter amino acids glu and gaba ( shank and campbell 1983 ) . similar to arg , conversion of its product orn to glu / gaba is stimulated during he ( albrecht and hilgier 1986 ; albrecht et al . however , implications for this increased conversion for neurotransmission imbalance associated with he are not known . taurine ( tau ) is a sulfur amino acid largely implicated in osmoregulatory and neuroprotective responses of the brain in various diseases , including hyperammonemia and he ( bosman et al . volume regulatory properties of tau are thought to be of particular importance in the case of brain edema , a major consequence of hyperammonemia , which results from impaired water homeostasis followed by swelling of astrocytes ( blei 2005 ) . he but not ha was associated with elevated blood content and increased brain uptake from blood to brain of tau , which collectively contributed to the increase of tau level in cerebral cortex ( hilgier et al . similar observation that liver failure induces elevation of tau in the blood were also made by other authors ( hamberger and nystrm 1984 ; zimmermann et al . because increased passage of tau was not due to massive breakdown of bbb ( as manifested by the absence of penetration of l - aspartate , which is not transported by intact capillary endothelial cells ) , it was believed to reflect activation of a tau transport system ( hilgier et al . of note in this context , treatment of an endothelial cell line with ammonia led to up - regulation and increased function of tau transporter ( blanger et al . hyperammonemia by affecting bbb transport of different substances and molecules can also lead to disturbances in cerebral energy homeostasis . hepatic encephalopathy evoked by pca was demonstrated to be associated with decreased brain glucose use and energy metabolism ( dejoseph and hawkins 1991 ) , and a similar effect was noted in rats with taa - induced he ( hilgier et al . brain uptake index of glucose was reduced after pca in rats ( sarna et al . 1979 ; crinquette et al . 1982 ) and this decrease was almost entirely due to the decrease in plasma glucose concentrations ( mans et al . glut-1 , the principal glucose transporter at the bbb responsible for supplying cns cells with blood - borne glucose was demonstrated to be induced by alf ( blanger et al . 2006 ) . since inhibition of glucose oxidative metabolism and subsequent activation of cerebral glycolysis are a hallmark of brain energy metabolism in he animals ( zwingmann et al . 2003 ; rao and norenberg 2001 ) , increased expression of glut-1 maybe considered as a compensatory response aimed at supporting higher glycolysis and maintaining brain atp levels . creatine ( cr ) a key substrate of the creatine / phosphocreatine / creatine kinase pathway is involved in regeneration of atp and in this way it also contributes to brain energy metabolism . moreover , cr was shown to affect gaba - ergic neurotransmission by acting as partial agonist on post - synaptic gaba(a ) receptors ( cupello et al . 2008 ) and to be crucial in dendritic and axonal elongation ( braissant et al . 2002 ) . exposure to ammonia was shown to generate a deficiency in cr in cns cells and to lead to neuronal cell loss , while co - treatment with cr was neuroprotective under ammonia exposure , but only in the presence of astrocytes ( braissant 2002 ) . ammonia treatment was demonstrated to increase cr uptake in cultured microcapillary brain endothelial cells ( blanger et al . pca in rats is associated with amino acid imbalance in csf and brain due to enhanced blood to brain transport of tryptophan and other members of large neutral amino acid group ( lnaa ) ( james et al . in addition , increased concentration of aromatic amino acids ( aaa ) was found in brains of rats with pca , while the level of branched - chain amino acids was decreased ( smith et al . the above observations prompted a hypothesis that these alterations may contribute to impaired neurotransmission in he by producing ( i ) excessive amounts of neurotransmitters from which they derive , and/or ( ii ) false instead of authentic neurotransmitters , which are similar in structure but are either not active at the postsynaptic membrane , or their activity differs from their true counterparts ( curzon et al . 1975 ) . hypothesis appears attractive , because aaa are also precursors of these false modulators : tyrosine for octopamine ; phenylalanine for phenylethanolamine . ( 1982 ) showed elevated brain octopamine and phenylethanolamine levels in the brains of pca rats , and hilgier et al . however , the contribution of these false neurotransmitters to the neurotransmission imbalance associated with he has insofar not been examined in more detail . a plausible explanation for the ammonia - induced increase of blood brain aaa transport activity was proposed by james and colleagues ( 1979 ) who hypothesized that during hyperammonemia , increased brain glutamine ( gln ) production is followed by increased gln efflux from the brain , resulting in increased inward transport of these amino acids . this inference has been proven directly in studies in which increased tryptophan ( try ) uptake in exchange with gln via the l - transport system was recorded in cerebral capillary microvessels isolated from pca rats ( cangiano et al . vice versa , release of newly loaded gln from the capillaries was promoted by the try and leucine ( leu ) , and the effect was more pronounced when the capillaries were isolated from taa rats or following their incubation with ammonia than in control preparations ( hilgier et al . ( 1985 ) showed that treatment of pca rats with an inhibitor of gln synthesis , methionine sulfoximine ( mso ) , reduced the increased accumulation of the aaa in the brain in a manner correlated with increased ammonia accumulation . ( 1993 ) showed that administration of mso to pca rats normalized amino acid imbalance ascribed to excessive gln production . hyperammonemia was shown to be directly responsible for pca - induced alterations in the metabolism and transport of amino acids ( jessy et al . 1990 ) , including elevated brain try content and rise in the brain level of a serotonin metabolite , 5-hydroxyindoleacetic acid . these effects appeared to be due to ammonia - induced functional impairment of lnaa transport at the bbb . in rats in which ha was executed by urease administration , the impairment was found closely correlated with the rise in brain gln content ( bachmann and colombo 1983 ) , while in the cortical capillaries the increased try - gln exchange could be related to the raised -glutamyl - transpeptidase ( ggt ) activity ( stastn et al . 1988 ) . because ggt participates in lnaa transport and its activity was found to be increased in brain capillaries from hyperammonemic rats , a hypothesis has been put forward that ggt is involved in triggering the outward transport of the excess of gln from brain ( gorgievski - hrisoho et al . 1986 ) . in this way , enhanced activation of ggt could contribute to raised try and other lnaa levels as observed in rats with taa - induced alf ( hilgier et al . the above speculations were confirmed in a follow up study from the author s laboratory showing that ggt affects the l system - mediated amino acid exchange ( hilgier et al . the bbb transport of the cationic amino acids arginine ( arg ) and ornithine ( orn ) was investigated in different he models , and contradictory results have been obtained . zaki et al . ( 1984 ) showed a 30% increase in the brain uptake of arg in the galactosamine model of hepatic failure ; however , the effect was not specific to this amino acid and possibly secondary to bbb leakage also revealed by high molecular weight markers . by contrast , arg uptake from blood to brain was found decreased in chronic he ( pca ) rats ( zanchin et al . 1979 ) and in rats with thioacetamide ( taa)-induced he ( albrecht et al . 1996 ) . with regard to orn , increased brain uptake index of this amino acid coincident with its increased content in the blood was found in the taa model of he ( albrecht and hilgier 1986 ; albrecht et al . increased bbb transport of orn in the taa model has been considered as auto - protective response and in the same line has been speculated to facilitate intracerebral therapeutic action of the ammonia - trapping drug , l - ornithine - l - aspartate ( lola ) ( albrecht et al . . however , the benefits of orn may not apply to he in a chronic setting , where blood to brain transport of orn appears to remain unchanged ( zanchin et al . the mechanism underlying alterations of bbb transport of arg and orn has been hypothesized to involve changes in the basic amino acid transporter y activity and competition between these two amino acids for the transport site ( albrecht et al . 1996 ) , but experimental evidence in support of this hypothesis has not been provided as yet . the effects of he on arg transport are also likely to be mediated by gln , which accumulates intracerebrally in consequence of increased ammonia influx ( cooper and plum 1987 ) , overloading different cellular and subcellular compartments of the cns ( albrecht 2010 ) . it has been shown that gln added exogenously reduces no generation in the brain by inhibiting arg transport via the arg / gln exchanger , ylat2 , and that this effect is potentiated when ammonia is infused directly to the brain ( hilgier et al . if the above mechanism operates not only in the cns cells but also in the cerebral capillary endothelial cells forming the bbb , enhanced gln accumulation would modulate arg transport in these cells . the final outcome of this interaction would depend on whether gln accumulates intra- or extra - cellularly . a hypothesis that such an interaction may occur is supported by the observation that , gln infusion in the absence of hyperammonemia impairs cerebrovascular co2 reactivity , most likely by reducing arg availability and no synthesis , because co - infusion of arg counteracts the effect caused by glutamine ( okada et al . consistent with the role of arg / gln exchange at the bbb , our preliminary data indicate that ammonia increases the expression of the ylat2 transporter in a cerebral capillary endothelial cell line ( manuscript in preparation ) , as it does in the brain in the course of ha in situ ( zieliska et al . further studies on the mechanisms and pathophysiological implications of the changes in arg or orn influx to the brain are warranted in view of the proven or suspected contributions of the amino acids to the pathogenesis of he . arg is a precursor of no , a compound whose increased accumulation is engaged in the inflammatory response of the brain to ammonia ( jalan et al . 2011 ) , and in ammonia - induced brain swelling ( hussinger and grg 2010 ) , while decreased no synthesis has been implicated in impairment of cognition associated with prolonged hyperammonemia ( felipo 2006 ) . moreover , ha increases arg uptake to the different cell types within the cns ( rao et al . 1998 ) , and he in the taa model stimulates arg conversion to the neurotransmitter amino acids glu and gaba as measured in the whole brain ( albrecht and hilgier 1986 ) and in synaptosomes derived from these rats , which is likely to alter the balance between the inhibitory and excitatory neurotransmission ( albrecht et al . evaluation of the contribution of changes in arg transport across the bbb to the availability of this amino acid in the brain can not be accomplished without accounting for the variability in blood arg content in the different hyperammonemic models . the plasma arg level was shown to be decreased in pca rats ( zanchin et al . 1979 ) , but was elevated in rats subjected to prolonged hyperammonemia ( ishihara et al . 1998 ) , and fluctuated from increase to decrease during the development of taa - induced he ( albrecht and hilgier 1986 ) . orn plays a role in ammonia detoxification and gives rise to polyamines which exert hepato- and neuroprotection ( sikorska et al . treatment with lola , where orn contributes to urea formation , reduces blood ammonia level and in consequence improves the general condition of he patients ( kircheis et al . , increased brain uptake of orn as found in the taa model of he would further promote protection ( albrecht et al . orn also contributes in some degree to the biosynthesis of the neurotransmitter amino acids glu and gaba ( shank and campbell 1983 ) . similar to arg , conversion of its product orn to glu / gaba is stimulated during he ( albrecht and hilgier 1986 ; albrecht et al . however , implications for this increased conversion for neurotransmission imbalance associated with he are not known . taurine ( tau ) is a sulfur amino acid largely implicated in osmoregulatory and neuroprotective responses of the brain in various diseases , including hyperammonemia and he ( bosman et al . volume regulatory properties of tau are thought to be of particular importance in the case of brain edema , a major consequence of hyperammonemia , which results from impaired water homeostasis followed by swelling of astrocytes ( blei 2005 ) . he but not ha was associated with elevated blood content and increased brain uptake from blood to brain of tau , which collectively contributed to the increase of tau level in cerebral cortex ( hilgier et al . similar observation that liver failure induces elevation of tau in the blood were also made by other authors ( hamberger and nystrm 1984 ; zimmermann et al . because increased passage of tau was not due to massive breakdown of bbb ( as manifested by the absence of penetration of l - aspartate , which is not transported by intact capillary endothelial cells ) , it was believed to reflect activation of a tau transport system ( hilgier et al . of note in this context , treatment of an endothelial cell line with ammonia led to up - regulation and increased function of tau transporter ( blanger et al . hyperammonemia by affecting bbb transport of different substances and molecules can also lead to disturbances in cerebral energy homeostasis . hepatic encephalopathy evoked by pca was demonstrated to be associated with decreased brain glucose use and energy metabolism ( dejoseph and hawkins 1991 ) , and a similar effect was noted in rats with taa - induced he ( hilgier et al . 1991 ) . brain uptake index of glucose was reduced after pca in rats ( sarna et al . 1979 ; crinquette et al . 1982 ) and this decrease was almost entirely due to the decrease in plasma glucose concentrations ( mans et al . glut-1 , the principal glucose transporter at the bbb responsible for supplying cns cells with blood - borne glucose was demonstrated to be induced by alf ( blanger et al . 2003 ; rao and norenberg 2001 ) , increased expression of glut-1 maybe considered as a compensatory response aimed at supporting higher glycolysis and maintaining brain atp levels . creatine ( cr ) a key substrate of the creatine / phosphocreatine / creatine kinase pathway is involved in regeneration of atp and in this way it also contributes to brain energy metabolism . moreover , cr was shown to affect gaba - ergic neurotransmission by acting as partial agonist on post - synaptic gaba(a ) receptors ( cupello et al . 2008 ) and to be crucial in dendritic and axonal elongation ( braissant et al . exposure to ammonia was shown to generate a deficiency in cr in cns cells and to lead to neuronal cell loss , while co - treatment with cr was neuroprotective under ammonia exposure , but only in the presence of astrocytes ( braissant 2002 ) . ammonia treatment was demonstrated to increase cr uptake in cultured microcapillary brain endothelial cells ( blanger et al . recent studies confirmed the view that hyperammonemia produces subtle changes in bbb integrity and partly unraveled the underlying mechanism . brain extravasation and edema in azoxymethane - induced alf were found to be secondary to tight junction ( tj ) protein degradation mediated by activation of matrix metalloproteinase-9 ( mmp-9 ) ( nguyen et al . specifically , it has been shown that tj proteins occludin and claudin-5 are significantly degraded in the brains of mice with galactosamine - induced alf , and this effect was reversed by treatment with inhibitor of mmp-9 , gm6001 ( chen et al . 2009 ) . a recent study delineated the most likely sequence of events linking activation of mmp-9 to occludin degradation in alf mice ; the intermediate steps include transactivation of epidermal growth factor receptor ( egfr ) and p38 mapk / nfb ( mitogen - activated protein kinase / nuclear factor - kappa b ) ( chen et al . 2011 ) . ( 2011 ) observed that progression of intracranial pressure in the course of alf is strictly correlated with the increase in bbb permeability and mmp-9 content . basing on this study the authors proposed a sequence of events of alf - induced brain damage , in which increase in bbb permeability is an initial step leading to vasogenic edema followed by ammonia excitotoxicity and cytotoxic edema . inflammatory molecules , including cytokines ( il-1 and/or il-6 ) and tumor necrosis factor - alpha ( tnf- ) are increased in plasma during acute and chronic liver failure in patients ( tilg et al . 2007 ) , and in animals with experimentally - induced he ( jiang et al . 2009 ) . circulating levels of tnf- correlate positively with the severity of he ( odeh et al . 2005 ) , moreover , its involvement in the development of intracranial pressure in patients with alf was demonstrated ( jalan et al . plasma il-6 level was also found well correlated with the severity of he and morbidity of the patients ( sheron et al . because massive breakdown of bbb is not observed during he , it is believed that the effects of inflammatory cytokines are transduced to the cns by vaso - active agents such as nitric oxide or prostanoids , which are synthesized by bbb - forming endothelium ( licinio and wong 1997 ) . brain barrier permeability in alf animals in the galactosamine ( lv 2010 ) and apap model ( wang et al . 2011 ) and in human alf patients ( lv 2010 ) , by disrupting tjs and inducing loss of the tj - associated protein occludin ( lv 2010 ) . data presented in this review provide considerable evidence that ammonia alters the passage of different molecules across the bbb , both by the transcellular route representing active or facilitated transport , and paracellularly , which occurs due to changes in the integrity of bbb constituents and thus reflects bbb leakage . as discussed above , increased bbb permeability adds a vasogenic component to the cytotoxic brain edema associated with he ( cauli et al . the effects of ammonia on the carrier - mediated transport of different molecules by the cerebral endothelial cells have been studied in considerable detail and the outlines of the changes in amino acid or energy metabolite transport are relatively well described . by contrast , the transcellular transport has long been given little consideration , mainly because in most he models , the ammonia - or he - induced changes have been too subtle and spatially restricted to be visualized by standard light- and electron microscopic techniques . the advent of more sensitive techniques has made it possible to identify the changes in tj proteins and their environment in a microscale , and provided tools to bridge the observations to the molecular mechanisms underlying the bbb leakage . further studies in this direction should allow to distinguish between the bbb changes in he which are induced directly by ammonia and those related to inflammatory toxins , mostly cytokines . one aspect deserving consideration in the future studies is the potential role of free radicals of oxygen and nitrogen , which have been found to be generated in excess by ammonia in different models and cell types of the cns and are responsible for the oxidative / nitrosative stress ( ons ) ( bemeur et al . 2010 ; hussinger 2010 ; skowroska et al . preliminary results from our laboratory disclosed that ons markers accumulate in an ammonia - treated brain microvascular endothelial cell line and increase permeability of these cells to a high molecular weight marker ( skowroska et al . this line of investigation appears attractive in view of the fact that ons causes bbb dysfunction in brain pathologies of varying etiology and severity ( lehner et al . many of the intracellular derangements known to be induced by ammonia in the cells within the cns or in peripheral tissues are likely to hold for the bbb - forming cerebral vascular endothelial cells , and may converge with events triggered in the different cells by ons . of note activation of the p38 mapk / nfb pathway which underlies mmp-9-induced tj protein damage ( chen et al . 2011 ) , is also involved in ammonia - induced oxidative damage of astrocytes ( jayakumar et al . other targets may include , for instance , altered nrf2-mediated synthesis of heme oxygenase i , an effect common to the response to various blood brain barrier damaging conditions ( lehner et al . 2011 ) and to the ammonia - induced ons in astrocytes ( warskulat et al . clearly , the above described mechanisms do not exhaust the list of possibilities that are worth further investigation . data presented in this review provide considerable evidence that ammonia alters the passage of different molecules across the bbb , both by the transcellular route representing active or facilitated transport , and paracellularly , which occurs due to changes in the integrity of bbb constituents and thus reflects bbb leakage . as discussed above , increased bbb permeability adds a vasogenic component to the cytotoxic brain edema associated with he ( cauli et al . 2011 ) . the effects of ammonia on the carrier - mediated transport of different molecules by the cerebral endothelial cells have been studied in considerable detail and the outlines of the changes in amino acid or energy metabolite transport are relatively well described . by contrast , the transcellular transport has long been given little consideration , mainly because in most he models , the ammonia - or he - induced changes have been too subtle and spatially restricted to be visualized by standard light- and electron microscopic techniques . the advent of more sensitive techniques has made it possible to identify the changes in tj proteins and their environment in a microscale , and provided tools to bridge the observations to the molecular mechanisms underlying the bbb leakage . further studies in this direction should allow to distinguish between the bbb changes in he which are induced directly by ammonia and those related to inflammatory toxins , mostly cytokines . one aspect deserving consideration in the future studies is the potential role of free radicals of oxygen and nitrogen , which have been found to be generated in excess by ammonia in different models and cell types of the cns and are responsible for the oxidative / nitrosative stress ( ons ) ( bemeur et al . preliminary results from our laboratory disclosed that ons markers accumulate in an ammonia - treated brain microvascular endothelial cell line and increase permeability of these cells to a high molecular weight marker ( skowroska et al . this line of investigation appears attractive in view of the fact that ons causes bbb dysfunction in brain pathologies of varying etiology and severity ( lehner et al . many of the intracellular derangements known to be induced by ammonia in the cells within the cns or in peripheral tissues are likely to hold for the bbb - forming cerebral vascular endothelial cells , and may converge with events triggered in the different cells by ons . of note activation of the p38 mapk / nfb pathway which underlies mmp-9-induced tj protein damage ( chen et al . 2011 ) , is also involved in ammonia - induced oxidative damage of astrocytes ( jayakumar et al . other targets may include , for instance , altered nrf2-mediated synthesis of heme oxygenase i , an effect common to the response to various blood brain barrier damaging conditions ( lehner et al . 2011 ) and to the ammonia - induced ons in astrocytes ( warskulat et al . clearly , the above described mechanisms do not exhaust the list of possibilities that are worth further investigation . focal and diffuse mesangial proliferative glomerulonephritis reveal the most common histologic lesions observed on renal biopsies from patients with iga nephropathy ( igan ) . however , histologic variability is also observed in this disease , which is the same as with lupus nephritis . in some cases , endocapillary hypercellularity is accompanied by segmental duplication of the glomerular basement membrane , and there is rarely diffuse endocapillary proliferation with lobular accentuation and multiple double contours , resembling type i membranoproliferative glomerulonephritis ( mpgn ) . tanaka and waga have reported a biopsied patient with acute igan following the formation of keloid scars due to a burn injury . however , further reports regarding the correlation between igan and burn injury are rare . a 46-year - old male patient with scrotal and pedal edema was admitted to our department in august 2012 . laboratory examination showed proteinuria ( 4 + ) and more than 775 red blood cells in the urine without casts . this patient had been treated for his 2nd- to 3rd - degree burn injuries in a local burn center . he had no inhalation injury or sepsis during the whole treatment process . at the admission time , he had a body temperature of 36.6c , a pulse rate of 75 beats / min , a respiratory rate of 16/min , and a blood pressure of 118/80 mm hg . physical examination revealed cutaneous convalescence after full - thickness burns of both hands , the coronal plane of the body and both legs . the skin showed fuscous , slight exudation , bleeding cracks , mild scaling and itch during the treatment process ( fig . the laboratory test results of the urine and blood for this patient are listed in table 1 . he was diagnosed as having nephrotic syndrome on the basis of a diminished serum albumin level of 23 g / l and an increased proteinuria excretion of 10.0 g/24 h. during the treatment course , microbiological examination of wound secretion was performed twice a week and no infection was observed . a percutaneous renal biopsy was performed on september 11 , 2012 ( day 57 after burn injury ) to explore the cause of proteinuria . the examination under light microscopy revealed a diffuse and moderate proliferation of mesangial cells and endothelial cells without observable crescents in 20 glomeruli . arterioles presented as normal status . neither tubular atrophy nor interstitial fibrosis was found , as shown in figure 2a c . immunofluorescence revealed iga ( 3 + ) , trace igm , c3 ( 3 + ) , igg ( ) , c4 ( ) and c1q ( ) in a coarse granular pattern with mesangial and capillary distribution ( fig . 2d , e ) , and inconspicuous staining for igm along the glomerular capillary walls in a granular pattern . in addition , this patient showed negative staining of hbsag and hbcag . the mesangial hypercellularity and increased mesangial matrix were present between the glomerular basement membrane and glomerular endothelium . the thickened capillary wall was composed of basement membrane - like materials , interposed mesangial cells , and electron - dense immune - type deposits ( fig . based on the proliferative properties of glomerular lesions , the patient was started on therapy with oral prednisone at a dose of 1 mg / kg / day . after treatment , the excretion of urinary protein revealed a significant decrease from 10.0 to 5.20 g/24 h in 2 weeks . the patient had been followed up to date and showed normal renal function and mild proteinuria . the glomerular filtration rate is increased after burn injuries , which is accompanied by vascular dysfunction and increased cardiac output . renal disease and acute kidney injury remains prevalent and is associated with the increased mortality of patients with severe burn injuries . this case shows the pattern of diffuse proliferative type i mpgn - like symptoms with mesangial interposition and double contours of the glomerular basement membrane present on periodic acid - schiff and silver stains ( fig . the following reasons are considered as a close correlation between burn injury and the onset of igan . first , this patient did not have any urinary abnormalities or chronic kidney diseases before and at the early stage of burn injury . second , nephrotic syndrome was observed at the 2nd week after the injury , and the patient did not have a history of infection such as inflammation of the respiratory tract although concurrent nephrotic syndrome in convalescence was observed . in addition , the patient suffered from skin problems with slight exudation , bleeding cracks , mild scaling and itch during the treatment process ( fig . although a causal relationship between igan and burn injury has not been established , an association of cutaneous lesions after burn injury with igan has been reported in a few patients [ 2 , 4 ] . tanaka and waga mentioned a biopsied patient with acute igan following the formation of keloid scars due to burn injury . the patient revealed a rapid recovery after complete removal of the scars without any medication . wang et al . have reported that acute igan is closely related to high - voltage electrical burn injury . in this study , all those case reports revealed a close relationship between igan and burn injury . because of trauma , infection , tissue necrosis , allogeneic plasma transfusion , increased concentrations of circulating immune complexes were observed . this situation is similar to the immune dysfunction of igan . both have inherent genetic predisposition and external infections , which may induce igan . if the pathogenesis of igan is not correlated with the concurrent burn injury , what is the cause of the nephrotic sydrome ? we hypothesize that an autoimmune dysregulation due to a sequestered antigen induced by skin infection and pruritus after burn injury may have an important role in the pathogenesis of igan . the persistent skin lesions with dry , itch and crusting properties can trigger iga deposition and promote the depositions in the mesangium and subendothelium of the patient . after serious burns , the immune system is disordered , the immune complex is increased in the circulation , and macrophages are excessively activated . numerous studies have provided evidence that burns upregulate inflammatory cytokines such as tnf- and il-1 . those cytokines can interfere with the hemodynamics of intraglomerular microcirculation , the coagulation - fibrinolysis system and the infiltration of inflammatory cells after burns . nephritis is a rare complication of burn injury , and once this happens , it will affect the recovery and prognosis of the patient . regular urine analysis will contribute to the early detection and diagnosis of glomerular diseases while treating burn injury . it is difficult to measure the blood pressure of burned patients , but blood pressure should be monitored as early as possible . because of wound exudation , infection and other reasons , the patients with large burned areas often have some degree of hypoalbuminemia . once persistent hypoalbuminemia exist , the burned patients need further examination to diagnose whether they have chronic glomerulonephritis or nephrotic syndrome . if the burned patient has nephrotic syndrome , enough glucocorticoid should be administered in order to alleviate proteinuria effectively and for the recovery of burn wounds . the casual relationship between the onset of igan and burn injury remains speculative and needs to be explored further .

ammonia is a neurotoxin involved in the pathogenesis of neurological conditions associated with hyperammonemia , including hepatic encephalopathy , a condition associated with acute(alf ) or chronic liver failure . this article reviews evidence that apart from directly affecting the metabolism and function of the central nervous system cells , ammonia influences the passage of different molecules across the blood brain barrier ( bbb ) . a brief description is provided of the tight junctions , which couple adjacent cerebral capillary endothelial cells to each other to form the barrier . ammonia modulates the transcellular passage of low - to medium - size molecules , by affecting their carriers located at the bbb . ammonia induces interrelated aberrations of the transport of the large neutral amino acids and aromatic amino acids ( aaa ) , whose influx is augmented by exchange with glutamine produced in the course of ammonia detoxification , and maybe also modulated by the extracellularly acting gamma - glutamyl moiety transferring enzyme , gamma - glutamyl - transpeptidase . impaired aaa transport affects neurotransmission by altering intracerebral synthesis of catecholamines ( serotonin and dopamine ) , and producing false neurotransmitters ( octopamine and phenylethylamine ) . ammonia also modulates bbb transport of the cationic amino acids : the nitric oxide precursor , arginine , and ornithine , which is an ammonia trap , and affects the transport of energy metabolites glucose and creatine . moreover , ammonia acting either directly or in synergy with liver injury - derived inflammatory cytokines also evokes subtle increases of the transcellular passage of molecules of different size ( bbb leakage ) , which appears to be responsible for the vasogenic component of cerebral edema associated with alf . iga nephropathy ( igan ) is the most common primary glomerulonephritis worldwide , accounting for approximately 3040% of patients undergoing renal biopsy in asia . the characteristic and diagnostic lesion of igan is the deposition of glomerular iga . the morphological lesions observed by light microscopy are extremely variable . a causal relationship between igan and burn injury has not been established , and the correlation between them is not clear if they appear at the same time . we have explored the cause of severe proteinuria of a chinese patient with burns of 2nd or 3rd degree after a gas leakage accident 2 weeks ago . the diffuse proliferative glomerulonephritis of this patient revealed type i membranoproliferative glomerulonephritis - like symptoms . moreover , this patient showed a sensitive response to prednisone . this case report demonstrates the intrinsic relationship between kidney disease and burn injury , which will facilitate a feasible treatment strategy for proteinuria after burn injury .

chronic total occlusion of coronary arteries ( cto ) is observed in 35%50% of patients with significant coronary artery disease ( cad ) undergoing diagnostic angiography . percutaneous coronary intervention ( pci ) for cto has become a widely accepted treatment strategy and accounts for up to 20% of all pcis [ 36 ] . successful pci of cto has been associated with improved left ventricular ( lv ) systolic function , reduced anginal symptoms , increased exercise capacity , decreased need for bypass surgery , and , most importantly , an increase in survival . qt dispersion ( qtd ) , first introduced by campbell et al . , is the maximum inter - lead variation between the longest and shortest qt intervals recorded in the standard 12-lead electrocardiogram ( ecg ) . ventricular contraction and recovery are recorded as qt interval in ecg ; hence , qtd represents the regional heterogeneity of ventricular repolarization duration in the 3-dimensional structure of ventricular myocardium [ 1012 ] . it has been previously demonstrated by many studies that the defect in myocardial microvascular perfusion was presented as an increase in qtd [ 1315 ] and the qtd was decreased following successful revascularization . heart rate variability ( hrv ) has been shown to be a reliable non - invasive technique for the quantitative analysis of the activity of the components of the autonomic nervous system . analysis of hrv consists of a series of measurements of successive rr interval variations of sinus origin , which provide information about autonomic tone . increased sympathetic tone may increase the incidence and severity of arrhythmias in the setting of ischemia . the significance of autonomic factors for arrhythmogenesis following early reperfusion of ctos has not yet been well established . the myocardial defect and/or ongoing myocardial ischemia in cto patients may lead to impairment in ventricular repolarization . the change in autonomic tone in these patients may also be related to ventricular arrhythmias . the aim of this study was to assess short - term changes in qtd and hrv parameters after successful pci of cto patients . in this prospective , observational study , conducted from april 2011 to february 2013 , we enrolled 139 successfully revascularized patients with cto . all patients underwent physical examination , chest x - ray , ecg , and transthoracic echocardiographic evaluation . cto was defined as the lumen compromise resulting in either thrombolysis in myocardial infarction ( timi ) flow grade 0 or 1 , with a likely duration of > 3 months . all patients included had a native vessel occlusion estimated to be of at least 3-month duration on the basis of a history of sudden chest pain , a previous myocardial infarction ( mi ) in the same target vessel territory , or the time between diagnosis made on coronary angiography and pci . we excluded patients with mi within the previous 6 months , second or third - degree atrioventricular conduction disturbances , atrial fibrillation or flutter , frequent ( > 10/min ) ventricular extrasystoles , sinus node disease , lv hypertrophy , permanent st changes on ecg , permanent cardiac pacemaker , abnormal serum electrolyte levels , congenital long - qt syndrome , an ecg with more than 6 missing leads , and patients taking drugs that modify the qt interval . after pci , all patients were prescribed lifelong aspirin ; in addition , clopidogrel was prescribed for at least 12 months in all participating sites . procedural success was defined as successful recanalization and dilation of at least 1 cto per patient with or without stent implantation , residual stenosis of < 50% , and timi flow > 2 . the study protocol was approved by the institutional ethics committee , and all patients provided written informed consent . the qt intervals and qtd were measured manually from the standard ecgs available before and after pci . standard 12-lead ecg was recorded at a 25 mm / s paper speed and a gain of 10 mm / mv ( montara instrument eu 250 electrocardiograph , milwaukee , wi , usa ) . all ecgs were scanned at a 600 dpi resolution and computer - based analysis was performed by 2 independent cardiologists who were blind to the timing of the ecgs and patients data . to measure qtd , qt interval was defined as the interval between beginnings of qrs complex to the point the t wave returned to isoelectric line and for each lead the mean of qt interval in 4 consecutive beats was measured . when the u wave was present , the end of the t wave was defined as the nadir of the curve between the t and the u waves . dispersion of the qt interval was measured as the difference between the maximum and minimum mean qt intervals recorded in any of the 12 leads of the standard ecg . for each patient , qtd was measured twice ; once 612 hours before , and once 6 hours after pci . both the qt interval and qtd were rate - corrected with a modification of bazett s formula ( corrected qt interval qt / square root of the rr interval ) ( qtcd ) . after completing the computer - based measurements , qtd was tested to identify intra - observer variability in 25 randomly selected patients using the bland - altman method . the 95% limit of agreement for qtd was acceptable ( 7.4 and 7.1 ms , respectively ) ( figure 1a ) . the 95% limit of agreement for qtd was 9.2 and 5.8 ms , respectively ( figure 1b ) . hrv was analyzed using data from holter recordings ( synetec version 1.10 , ela medical , montrouge , france ) , which were started on hospital admission . r data from holter recordings were assessed using power spectral analysis before and within 24 hours after pci . we calculated time - domain hrv indices of standard deviations of the normal - to - normal qrs intervals ( sdnn ) , square roots of the mean squared differences of successive n n intervals ( rmssd ) , and percentage of consecutive rr differences > 50 ms ( pnn50 ) . for frequency domain hrv indices , we used the fourier transform method for the spectral measurements , and the heart rate spectrum between 0.003 and 0.40 hz was defined as total energy ( ms ) . this energy was divided into 2 components : low frequency ( lf : 0.040.15 hz ) and high frequency ( hf : 0.160.40 hz ) . continuous variables are reported as mean standard deviations ( sd ) and categorical variables are expressed as percentages . comparison of categorical and continuous variables between the 2 groups was performed using the test and unpaired t - test , respectively . the qt intervals and qtd were measured manually from the standard ecgs available before and after pci . standard 12-lead ecg was recorded at a 25 mm / s paper speed and a gain of 10 mm / mv ( montara instrument eu 250 electrocardiograph , milwaukee , wi , usa ) . all ecgs were scanned at a 600 dpi resolution and computer - based analysis was performed by 2 independent cardiologists who were blind to the timing of the ecgs and patients data . to measure qtd , qt interval was defined as the interval between beginnings of qrs complex to the point the t wave returned to isoelectric line and for each lead the mean of qt interval in 4 consecutive beats was measured . when the u wave was present , the end of the t wave was defined as the nadir of the curve between the t and the u waves . dispersion of the qt interval was measured as the difference between the maximum and minimum mean qt intervals recorded in any of the 12 leads of the standard ecg . for each patient , qtd was measured twice ; once 612 hours before , and once 6 hours after pci . both the qt interval and qtd were rate - corrected with a modification of bazett s formula ( corrected qt interval qt / square root of the rr interval ) ( qtcd ) . after completing the computer - based measurements , qtd was tested to identify intra - observer variability in 25 randomly selected patients using the bland - altman method . the 95% limit of agreement for qtd was acceptable ( 7.4 and 7.1 ms , respectively ) ( figure 1a ) . the 95% limit of agreement for qtd was 9.2 and 5.8 ms , respectively ( figure 1b ) . hrv was analyzed using data from holter recordings ( synetec version 1.10 , ela medical , montrouge , france ) , which were started on hospital admission . r data from holter recordings were assessed using power spectral analysis before and within 24 hours after pci . we calculated time - domain hrv indices of standard deviations of the normal - to - normal qrs intervals ( sdnn ) , square roots of the mean squared differences of successive n n intervals ( rmssd ) , and percentage of consecutive rr differences > 50 ms ( pnn50 ) . for frequency domain hrv indices , we used the fourier transform method for the spectral measurements , and the heart rate spectrum between 0.003 and 0.40 hz was defined as total energy ( ms ) . this energy was divided into 2 components : low frequency ( lf : 0.040.15 hz ) and high frequency ( hf : 0.160.40 hz ) . continuous variables are reported as mean standard deviations ( sd ) and categorical variables are expressed as percentages . comparison of categorical and continuous variables between the 2 groups was performed using the test and unpaired t - test , respectively . the mean patient age was 58.39.6 years , 118 ( % 84 ) were male and 59 ( % 42 ) had diabetes mellitus . the cohort included 47 patients ( 34% ) with prior mi and 14 patients ( 10% ) with a history of congestive heart failure . at the baseline , most of the patients were on aspirin ( n=139 , 100% ) , angiotensin - converting enzyme ( ace ) inhibitors ( n=98 , 70% ) , lipid - lowering medications ( n=113 , 81% ) , and -blockers ( n=124 , 89% ) however , use of nitrate ( n=29 , 20% ) , calcium channel blockers ( n=23 , 16% ) , and angiotensin receptor blockers ( n=34 , 24% ) was limited . both qtd and qtcd showed significant improvement following successful revascularization of cto ( 55.8314.79 vs. 38.8711.69 ; p<0.001 and 61.0216.28 vs. 42.9213.41 ; p<0.001 , respectively ) . regarding hrv parameters , the revascularization of the left coronary artery ( lad ) ( n=38 ) resulted in a decrease in hrv indices , including sddn , rmssd , and pnn50 , but none of them reached statistical significance . similar findings were observed after revascularization of the circumflex branch of the left coronary artery ( cx ) ( n=28 ) and right coronary artery ( rca ) ( n=73 ) lesions ( table 3 ) . we investigated changes in qtcd and hrv parameters in cto patients undergoing successful percutaneous revascularization . the major findings of this study are : ( 1 ) successful revascularization may improve qtcd in patients with cto , ( 2 ) the revascularization in cto lesions does not seem to have a significant impact on hrv , and ( 3 ) the impact on hrv does not change with the intervention to the lad , cx , or rca . myocardial necrosis and reversible myocardial ischemia both impact qtd . a direct relationship between the prolongation of the qt interval and myocardial ischemia has been reported by roukema et al . , who observed increased qtd in patients with exercise - induced myocardial ischemia . in experimental animal studies and in human studies it has been shown that the qt interval shortened in acutely hypoperfused areas , whereas in infarcted myocardium there was a prolonged repolarization time associated with qt prolongation on the ecg . the heterogeneity of the ventricular excitability was presumed to increase the propensity for arrhythmic manifestations and arrhythmic death , especially in patients with previous mi or history of cad . reported that increased qtd is related to susceptibility to reentry ventricular tachyarrhythmias , independent of degree of lv dysfunction or clinical characteristics of the patient , suggesting that the simple , noninvasive measurement of this interval from a standard 12-lead ecg significantly contributes to identifying patients at risk for life - threatening arrhythmias after a previous mi . the strong heart study of assessment of qt interval and qtd for prediction of all - cause cardiovascular ( cv ) mortality showed that qtcd was a strong predictor of all - cause mortality and a weaker predictor of cv mortality , and that qtd is a significant predictor of cv mortality . in the present study we showed that successful revascularization of ctos resulted in significant decrease in qtd and qtcd . our results are consistent with data reported by yunus et al . , who assessed qtd in patients with ischemia due to 1-vessel cad without prior mi and who underwent successful pci . however , our patient population included patients with only ctos and demonstrated the improvement in qtd in this challenging group . we suggest that the electrophysiological mechanism of action is based on a decrease in ischemia - induced prolongation of conduction and in dispersion of conduction times . the improvement in qtd with pci in may suggest a role for revascularization in achieving more homogenous repolarization and , perhaps , greater clinical stability in cto patients . hrv analysis is a safe and convenient method for the evaluation of the function of the autonomic nervous system activity in accordance with guidelines for standardization . significant decrease in autonomic tone has been shown to be an independent predictor of mortality in patients with cad . it has been shown that hrv indices decreased following coronary revascularizations . in our study , although there was a trend in decrease in some hrv parameters following pci of cto , none of the parameters reached statistical significance . similar results were also reported by szwoch et al . , but they repeated the hrv analysis after 3 months and observed improvement in most of the parameters . we speculate that the decrease in hrv parameters in early periods following recanalization of ctos could be transient and might be associated with acute endothelial injury during the procedure or microembolization to the vascular bed that was previously protected from ischemia by collateral support . consistent with the results of a previous study , localization of totally occluded coronary arteries in this study did not have significant impact on hrv results after the revascularization procedure . because the sinus node blood supply primarily comes from this artery , a significant change in hrv following successful pci to rca might be expected . the reason may not only be the incomplete disclosed mechanisms of hrv , but also a common impact of each artery on the sinus node artery or other mechanisms that might be responsible from hrv changes in addition to changes in blood supply . the main limitations of our study were lack of long - term follow - up and heterogeneous patient population with concomitant diseases . nearly half of our patients ( 42% ) had diabetes . diabetes itself may be associated with depressed hrv due to diabetic autonomic neuropathy and may result in reduced hrv , inducing hemodynamic changes that involve adrenergic activation and vagal tone reduction . however , our patient group may not be representative of the overall patient population with cad . we analyzed hrv parameters before and after pci , but we did not measure respiratory rate . in addition , because hrv was affected by various factors such as age of the patient and the use of ace inhibitors and -blockers , these factors might have affected our results [ 3436 ] . in conclusion , successful pci of cto patients may improve ventricular repolarization abnormalities , and thus may reduce the incidence of ventricular arrhythmias . early assessment of hrv parameters does not seem to be changed following recanalization of cto lesions . ocular cicatricial pemphigoid ( ocp ) is an uncommon , chronic autoimmune disease that affects mucous membranes , particularly the conjunctiva.1 the disease typically results in chronic conjunctivitis and causes conjunctival and corneal scarring , which can result in limbal stem cell deficiency and blindness.2 adequate control of ocular inflammation usually requires systemic as well as topical immunosuppressants . commonly used systemic medications include prednisone , methotrexate , mycophenolate mofetil ( mmf ) , and cyclophosphamide.3 the porphyrias are metabolic disorders caused by defective enzymes within the heme synthetic pathway.4 these defective enzymes cause an accumulation of intermediates from the heme synthetic pathway that results in various clinical manifestations.4,5 porphyria cutanea tarda is the most common of the porphyrias and results from a deficiency in uroporphyrin decarboxylase , which is the fifth enzyme in the heme synthetic pathway.6 this deficiency results in the accumulation of porphyrins in the liver and plasma . on exposure to light with a wavelength near 400 nm , the porphyrins enter an excited state that can lead to the damage of proteins , lipids , and basement membranes.6 this process results in blisters , fibrosis , and scarring of the skin in areas of the body exposed to sunlight.6 park et al7 have reported a case of a 31-year - old with cicatricial conjunctivitis who was biopsy negative for ocp and was later diagnosed with porphyria cutanea tarda ; in this case the patient s clinical symptoms significantly improved after initiating phlebotomy treatments . the present authors report a similar case , in which pathology and direct immunofluorescence confirmed a diagnosis of ocp and where the patient s clinical condition also improved significantly upon the diagnosis and treatment of porphyria cutanea tarda . a 64-year - old caucasian male complaining of redness and tearing for 3 years in both eyes was referred for evaluation of cicatricial conjunctivitis . he had been treated with tobramycin and dexamethasone ophthalmic ointment in both eyes as needed and doxycycline 100 mg by mouth daily with no improvement in his symptoms . on slit lamp examination the patient had subconjunctival fibrosis , symblepharon , forniceal foreshortening , and trichiasis in both eyes ( figure 1 ) . examination of the corneas revealed multiple punctate epithelial erosions . a schirmer s test was performed without anesthesia , showing 22 mm of wetting in the right eye and 13 mm in the left after 5 minutes . direct immunofluorescence studies of the conjunctival biopsy specimen revealed immunoglobulin g4 deposits in the basement membrane zone of the junctional area ( figure 2 ) , consistent with ocp . given the findings of subconjunctival fibrosis and symblepharon formation in both eyes , the patient was diagnosed with bilateral stage iii pemphigoid . the patient was started on methotrexate 15 mg by mouth weekly and prednisone 20 mg by mouth daily . the patient demonstrated gradual improvement in the conjunctival inflammation after starting the methotrexate and prednisone . the patient was subsequently tapered off the prednisone , while the methotrexate 15 mg by mouth weekly was continued . after 4 months of treatment with the methotrexate , the patient s conjunctival inflammation began to worsen and his regimen was subsequently changed from methotrexate to mmf 1000 mg by mouth twice daily . approximately 3 months after initiating mmf treatment , the patient was diagnosed with porphyria cutanea tarda . the patient s conjunctival inflammation appeared stable following initiation of the phlebotomy treatments and the mmf was subsequently discontinued . approximately 6 weeks following discontinuation of the mmf , the patient returned with mildly increased conjunctival injection and trichiasis in both eyes . epilation was performed and the patient was started on 1% prednisolone acetate ( one drop in both eyes twice daily ) . the patient s conjunctival inflammation stabilized and he was tapered down to one drop of 1% prednisolone acetate in both eyes once daily . since initiation of the phlebotomy treatments , the patient s conjunctival inflammation and subconjunctival fibrosis has remained quiescent for 4 months without requiring mmf ( figure 3 ) . ocp is believed to be an autoimmune disease of genetic predisposition , and it is likely that a second - hit environmental trigger is required to initiate onset of the disease.1 it has been thought this could include chemical exposure or microbial environmental triggers . the present case suggests that the patient s porphyria could be a causal factor associated with the ocp and could even have been the environmental trigger that stimulated the disease to occur . the porphyrins are present in plasma and therefore they would be present in the ocular surface vasculature.5 the ocular surface is constantly exposed to light . exposure to ultraviolet light would lead the porphyrins to enter into an excited state , resulting in inflammation and damage to the ocular surface . this process could be the trigger to either initiate or exacerbate ocp . in the present case , the patient s conjunctival inflammation was observed to significantly improve following initiation of treatment for his porphyria . a similar response was observed in the aforementioned case reported by park et al,7 although that particular patient was biopsy negative for ocp and was positive for hepatitis c virus infection ; in addition , the patient remained on methotrexate . the patient in the present case was unique in that he was biopsy positive for ocp and his clinical improvement was significant enough after starting phlebotomy treatments that his mmf was discontinued . the authors consider that this case , as well as the case reported by park et al,7 sheds new light on the search for the etiology of ocp and the subsequent treatment options for patients with this disease .

backgroundqt dispersion ( qtd ) , which is a measure of inhomogeneity of myocardial repolarization , increases following impaired myocardial perfusion . its prolongation may provide a suitable substrate for life - threatening ventricular arrhythmias . we investigated the changes in qtd and heart rate variability ( hrv ) parameters after successful coronary artery revascularization in a patient with chronic total occlusions ( cto).material / methodsthis study included 139 successfully revascularized cto patients ( 118 men , 21 women , mean age 58.39.6 years ) . qtd was measured from a 12-lead electrocardiogram and was defined as the difference between maximum and minimum qt interval . hrv analyses of all subjects were obtained . frequency domain ( lf : hf ) and time domain ( sdnn , pnn50 , and rmssd ) parameters were analyzed . qt intervals were also corrected for heart rate using bazett s formula , and the corrected qt interval dispersion ( qtcd ) was then calculated . all measurements were made before and after percutaneous coronary intervention ( pci).resultsboth qtd and qtcd showed significant improvement following successful revascularization of cto ( 55.8314.79 to 38.8711.69 ; p<0.001 and 61.0216.28 to 42.9213.41 ; p<0.001 ) . the revascularization of lad ( n=38 ) , cx ( n=28 ) and rca ( n=73 ) resulted in decrease in hrv indices , including sddn , rmssd , and pnn50 , but none of the variables reached statistical significance.conclusionssuccessful revascularization of cto may result in improvement in regional heterogeneity of myocardial repolarization , evidenced as decreased qtcd after the pci . the revascularization in cto lesions does not seem to have a significant impact on hrv . a 64-year - old caucasian male complaining of redness and tearing for 3 years in both eyes was referred for evaluation of cicatricial conjunctivitis . ocular cicatricial pemphigoid was suspected and this diagnosis was confirmed through biopsy . the patient s condition showed moderate improvement following treatment with methotrexate and mycophenolate mofetil . the patient was later diagnosed with porphyria cutanea tarda and phlebotomy treatments were subsequently initiated . the patient s ocular symptoms improved further after he began receiving these phlebotomy treatments , and conventional treatment was discontinued . the authors hypothesize that circulating porphyrins activated by ultraviolet light could be the cause of the ocular cicatricial pemphigoid in this patient .

the incidence of endometrial cancer has remained stable , but the number of deaths annually from this disease has doubled since 1987 . despite the fact that many gynecologists believe endometrial cancer is harmless , when compared stage by stage , 5-year survival is identical to that of cervical cancer , which is considered virulent . thus , we are obligated to reassess the screening , diagnostic , staging , and therapeutic aspects and most importantly the debate on lymphadenectomy during hysterectomy . currently , an extremely low prevalence is inferred for lymph node involvement in stages ia , ib , and grade 1 neoplasm , which comprises 40% to 60% of newly diagnosed patients . furthermore , it is postulated that in early disease stages and when regional nodes are clinically unaffected , nodes should only be sampled for prognostic significance and not removed radically in the vain hope of curing the patient . indeed , evidence from laboratory studies has shown that many lymphatic and lymphatico - venous shunts that bypass regional lymph nodes exist and allow an early stage lymphatic and hematogenous dissemination of malignant cells . on the other hand , removal of lymph nodes if they are grossly positive , lessening the tumor burden , should decrease the amount of suppressive tumor antigens present in the host and reduce the amount of adjunctive therapy required to treat residual disease . indeed , ineffective nodes are no longer useful to the host but now contribute to increased tumor - induced immunologic suppression . for tumors infiltrating the inner third of the myometrium , the actual risk of node metastases is substantial for grade 3 histologic type tumors , and for the middle third invasion , the risk of node metastases is substantial for grade 2 and 3 lesions . for tumors infiltrating the outer third , thus , the risk is ignorable for superficial invasion and substantial for deep myometrial invasion for all grades ( 20% to 45% ) . the negligible risk of node metastases ( 0% to 4% ) is also valid for inner one - third myometrial involvement for grades 1 and 2 , for middle one - third involvement with grade 1 tumors , and for all aforementioned conditions with no vascular space involvement and no spread to the cervix or adnexa , or both , with histologic type adenocarcinoma and adenoacanthoma . this group comprises 75% of patients . nevertheless , the common belief among gynecologists of just to take out the uterus as it is small and well differentiated is not valid as 10% to 15% of the grade 1 lesions have substantial myometrial invasion exposing the patient to substantial risk for node metastases and thereby necessitating intraoperative assessment of myometrial invasion in those instances as well . thus , the need for lymph node assessment during surgery is the most challenging issue among gynecologists . several methods of lymph node dissection have been proposed for different situations although these differ from one surgeon to the next . the adequacy of clinical assessment of lymph nodes and sampling lymphadenectomy has been a subject of controversy for years . meticulous inspection and palpation bidigitally are refined exercises for the gynecologic oncologist , but slightly more than one half of the suspicious pelvic and paraaortic nodes reveal metastases while only 5% of the unsuspicious nodes bear malignant cells . thus , one should bear in mind the possibility of undetected metastases of regional nodes by traditional methods during surgery . in addition , some tumor cells can only be detected by immunohistochemical analysis and thereby pass unrecorded . thus , the real incidence of tumor involvement might have been underestimated due to the technique used for pathologic assessment and limited node dissection . do we need to do complete dissection for positive detection , because negative staging does not necessarily identify a subset that does not need further therapy is another debate . on the other hand , the concern about what would be the false negativity rate to be accepted by the surgeon and the patient ? is another important issue . hence , being the most prevalent gynecological cancer , endometrial cancer does not require a radical hysterectomy but lymph node evaluation in its early stage . as gynecologic oncology surgeons have gained more experience and skill in different techniques and procedures , and more interest in minimally invasive surgery , the laparoscopic approach has found applications in the treatment of endometrial cancer . with the application of video and improvement in the armamentarium of video , laparoscopic surgery changed from being performed in an operating room by one man frustrated because of the lack of the ability of other operating room staff to follow and participate in this surgical procedure , to being performed in an amphitheater , allowing not only the assistant and other operating room staff to see the actual procedure and follow the sequences but these advances resulted in accelerated improvement in operative laparoscopy and proved to be an important aid to education because of the advantage of using monitors during the operation and taping the surgery for repetitive education . laparoscopic - assisted surgical staging ( lass ) of early stage endometrial cancer , although remaining a controversial subject , is an attractive alternative to the traditional laparotomic approach . nevertheless , one should bear in mind that the first purpose of the staging is to provide a common language and uniformity for comparison instead of aiding therapy and management issues . staging must be the first aim despite there still existing some conflicting ideas regarding the system of staging . laparoscopy has a similar success rate when compared with laparotomic staging , and it can be accepted as less invasive . studies have shown that the lymph node yield from laparoscopy is equivalent to that of laparotomy . feasibility of laparoscopic staging in incompletely staged endometrial cancer patients is another subject of interest . although laparoscopic pelvic and paraaortic lymphadenectomy is accepted as feasible , randomized studies are still awaited to standardize its indications and applications . the patient population consisted of 52 early stage endometrial cancer patients who presented to akdeniz university obstetrics and gynecology department between 1998 and 2002 . all underwent staging surgery comprising total hysterectomy , bilateral salpingo - oophorectomy , and pelvic lymph node sampling by the same 2 surgeons ( cgz , ts ) , using the same technique and instruments . the patients were randomly assigned to the surgical approach and informed before surgery about the type of surgery they were to undergo . one group comprised 26 patients managed by traditional laparotomic surgery , while the other comprised 26 patients who underwent laparoscopic surgery . the patients who were clinically thought to have advanced disease were not included in the study . the main outcomes studied were operative time , blood transfusion , intraoperative and postoperative complications , duration of hospital stay , number of lymph nodes obtained and lymph node positivity . statistical analysis was performed by the mann whitney u test , and p values < 0.05 were considered statistically significant . the laparotomy group had an average age of 54.9 ( range , 36 to77 ) and an average gravidity of 3.8 ( range , 0 to 9 ) . the laparoscopy group had an average age of 56.6 ( range , 40 to 72 ) and an average gravidity of 3.6 ( range , 0 to 8) . body mass indexes were not significantly different between laparotomy and laparoscopy groups , being 26.2 vs 24.4 respectively . the majority of the patients had stage i disease ( 67.3% ) , and endometrioid type of endometrial cancer was more common ( tables 1 , 2 , and 3 ) . the number of lymph nodes was not significantly different between the groups ( p>0.05 ) . the laparoscopic group had an average number of 18.2 ( range , 9 to 31 ) , and the laparotomic group 21.1 ( range , 9 to 38 ) . two ( 7.7% ) patients in the laparoscopy group and 4 ( 15.4% ) in the laparotomy group had pelvic lymph node metastasis . eleven patients ( 42.3% ) in the laparoscopy group and 10 ( 38.5% ) in the laparotomy group were later scheduled for adjuvant radiation therapy . study groups compared according to stage histologic grades of the study groups histologic types of endometrial cancer in the study groups the laparoscopic group had significantly shorter hospitalization than did the laparotomy group ( 4.1 vs 8.2 days , z 1.96 , p<0.05 ) . operative time of laparoscopy being close to that of laparotomy was encouraging ( 155 vs 144 minutes , p>0.05 ) . in terms of postoperative and intraoperative complications , the laparoscopic approach had none , while wound complications occurred in 5 patients in the laparotomy group , of which one was evisceration and needed reoperation for closure . eight units of red blood cell suspension were transfused to the laparotomy group patients and 6 units to the laparoscopy group patients . nowadays , the laparoscope allows us to carry out almost any procedure that can be done by laparotomy in gynecologic practice . laparoscopically assisted vaginal hysterectomies and laparoscopic total hysterectomies for benign conditions are being applied widely even in patients with large uteri . however , in the management of gynecologic cancers , the laparoscope had a lesser role , until pelvic and paraaortic lymph node dissections became feasible in the 1990s . following this achievement , the laparoscope has gained importance among gynecologic oncologists especially in patients with early endometrial and cervical cancers . every surgeon was doubtful about the outcome of this type of surgery . because of such doubt , many institutions established pilot studies and some prospective small - scale trials . at any rate , some were successful with speeded up publication encouraging surgeons to switch to this type of surgery . laparoscopically assisted vaginal hysterectomy with lymph node evaluation is now an alternative treatment for endometrial cancer in properly selected patients . the goal is to minimize the morbidity of treatment instead of satisfying surgeons ' new enthusiasm to use the technique . laparoscopically assisted surgical staging for endometrial cancer in experienced hands can be performed with equal success and safety with minimal morbidity . it has the advantages of less pain , early resumption of normal activities , and overall improved quality of life . many authors are in agreement that it results in significantly less blood loss and shorter hospitalization . scribner et al compared laparoscopy and laparotomy in a similar number of patients as in our study . they concluded that although the early hospital discharge is an advantage , longer surgical time and higher anesthetic costs of the laparoscopic approach offset this gain , and total costs appear not to differ statistically . in contrast to scribner 's study , gemignani et al , found the overall charges in the laparoscopy group significantly lower than that of laparotomy group , considering fewer postoperative complications seen with the laparoscopic approach , a factor that lowers overall hospital charges . in these 2 studies , the laparoscopy group had a significantly longer operating room time ( 237 vs 157 minutes in scribner 's study and 214 vs 144 minutes in gemignani 's study ) ; however , in our study , the duration of the operation for the 2 groups was similar ( 155 vs 144 minutes ) . another randomized study comparing the laparoscopicvaginal approach with the conventional abdominal approach for treatment of patients with endometrial cancer was performed including 70 patients with endometrial cancer figo stage i - iii . thirty - seven patients were treated in the laparoscopic group versus 33 patients in the laparotomy group . lymph node dissection was performed in 25 patients by laparoscopy and in 24 patients by laparotomy . blood loss and yield of pelvic and paraaortic lymph nodes , duration of surgery , and incidence of postoperative complications were similar for both groups . while comparing laparotomy and laparoscopy , the number of residual nodes following lymphadenectomy has also been studied because lymph nodes left in situ might have had microscopic metastases . in a review by lecuru and taurelle , laparoscopic pelvic lymphadenectomy was declared able to retrieve 90% to 95% of the nodes , which was similar to that of laparotomy . laparoscopic paraaortic lymphadenectomy can be substituted for its open counterpart regarding lymph node yield and accuracy in recovery of positive nodes as well . port - site metastases are one of the most often addressed issues in patients undergoing a laparoscopic procedure for any kind of gynecologic cancer . dragging cancerous tissue through a small incision and exfoliation from the surface and implantation into the healing wounds are the main reasons for an increased likelihood of metastases or recurrences at that sites . however , in gynecologic cancers port - site recurrences are always associated with either disseminated intraperitoneal disease or cyst rupture . the question of contamination and increasing tumor growth because of co2 laparoscopy and the effect of pneumoperitoneum on survival remains obscure . however , surgeons should take some preventive measures , such as avoiding cyst rupture , gentle handling of the cancerous tissue , avoiding the rupture of the lymph node capsule , and including the port sites in the radiation field in patients undergoing postoperative irradiation . in this study morbidities related only to laparoscopic surgery are of some concern , and injuries are generally related to trocar installation . these morbidities are mostly bowel , vascular , and bladder injuries . increasing numbers of advanced laparoscopic applications like expanded lymph node dissections and more radicalness in treating gynecologic cancers have caused surgeons to face other complications as well . injury to vein tributaries during laparoscopic lymphadenectomy may cause major hemorrhage from the adjacent veins . it might not be easy sometimes to overcome such bleeding even during laparotomy because of the depth of the location of bleeding , preventing easy access to the area . a laparoscope with 5-to 7-fold magnification could sometimes be superior in identifying such deep bleeding locations ; however , difficulty in suturing or unavailability of vascular clamps may prevent the appropriate approach . in our study group , almost 90% of the patients in the laparoscopy group had stage i or ii disease following surgical staging , whereas only 70% of the patients in the laparotomy group had early stage disease . however , the breakout of stage iii subsets showed us that such patients are not identifiable before surgery , and therefore this could be incidental because we never felt that we 're glad that we opened this patient ; it would be dreadful if we put the scope for the first time during surgery . we conclude that the laparoscope seems to be very useful in a select group as it reduces postoperative morbidity offering quick recovery with the same success and efficacy . however , experience is of the utmost importance , and there is no way to start scoping without proper training . feeling ready does not mean that one will be good in the operating room , and thus being supervised should not be overlooked before integrating such a procedure into clinical practice . shorter hospital stay and less postoperative morbidity are advantages of laparoscopic surgery . duration of laparoscopic surgery does not seem to be different from that of laparotomy .

objective : the aim of this study was to evaluate the feasibility of laparoscopy in the management of early stage endometrial cancer.methods:fifty-two patients with endometrial cancer who underwent surgical staging consisting of total hysterectomy , bilateral salpingo - oophorectomy with pelvic lymph node dissection , and cytology between 1998 to 2002 were included in the study . laparotomy and laparoscopy were randomly offered to patients upon admittance.results:of 52 patients , 26 underwent laparotomy and the remaining 26 underwent laparoscopic staging surgery . no significant difference existed between the demographic characteristics of the 2 groups . the mean number of harvested lymph nodes was 18.2 in the laparoscopic group and 21.1 in the laparotomic group ( p>0.05 ) . pelvic lymph node metastases were detected in 7.7% of the patients in the laparoscopy group and 15.4% in the laparotomy group , and the difference was not significant . adjuvant radiotherapy was applied later to 42.3% of the laparoscopy group and 38.5% of the laparotomy group . operative morbidity was higher in the laparotomy group mainly because of postoperative wound infection , and the patients in the laparotomy group had a longer hospital stay.conclusion:laparoscopic surgery is a method that can be applied as well as laparotomy in the management of endometrial cancer . lymph node number and detection of lymph node metastasis did not differ significantly in laparotomic and laparoscopic approaches . wound infections were more frequent in laparotomies . a mild catalytic asymmetric direct fluoro - arylation of styrenes has been developed . the palladium - catalyzed three - component coupling of selectfluor , a styrene and a boronic acid , provides chiral monofluorinated compounds in good yield and in high enantiomeric excess . a mechanism proceeding through a pd(iv)-fluoride intermediate is proposed for the transformation and synthesis of an sp3 c f bond .

genetic analysis of phenotypes and diseases has traditionally followed two approaches : family - based linkage analysis and population - based association studies . while in linkage analysis it is the co - segregation of alleles in families that is measured , population - based studies use non - random associations between phenotypes and alleles in populations to identify causative genes . linkage analysis has proven to be immensely successful as a means of identifying genes for a number of single gene diseases with simple mendelian inheritance ( eg see omim database ) . complex diseases are multifactorial , polygenic and often characterised by late age of onset , incomplete penetrance , locus heterogeneity and environmental exposures and , despite significant efforts , have not been amenable to family - based mapping . linkage disequilibrium ( ld ) is an important aspect of genetic association studies and is generated in a population through mutation , selection , drift , non - random mating and admixture . allelic associations due to ld are significant and are correlated with physical distance within small genomic regions but decay over time due to recombination [ 2 - 4 ] . ld - based association studies have been successful in both fine scale mapping and initial disease gene mapping in homogeneous populations that have undergone recent bottlenecks ( eg hirschsprung disease in mennonites , bardet- beidle syndrome in bedouins ) . allelic associations can result either from direct functional effects of the alleles tested or indirectly through non - random associations between the allele measured and nearby functional alleles . since functional alleles in most genes are still unknown and are indeed an object of the research , ld is an important feature of how genes can be screened for alleles that alter disease risk . thus , there has been substantial focus on the extent of ld across the genome and the definition of statistical methods for disease gene mapping using ld [ 9 - 11 ] . in large cosmopolitan populations , however , ld may be difficult to detect when the mutation is old , since the amount of remaining ld may be small . additionally , false - positive associations due to population stratification are important confounders in ld - based association studies . the process of admixture itself creates ld between all loci , linked and unlinked , that have different allele frequencies in the parental populations . the magnitude of admixture linkage disequilibrium ( ald ) in an admixed population depends on the allele frequency differential between the parental populations , the level of admixture , the admixture dynamics , the time since admixture and the recombination rate between the loci . while ald between unlinked markers decays rapidly ( within two to four generations ) the exponential decrease in ald with genetic distance facilitates the differentiation of ald that is high between markers that are close together and genetically linked , from ald generated at unlinked loci . thus , if the parental populations differ in a trait or disease due to different frequencies of risk alleles , it should be possible to identify the loci containing these alleles using admixture mapping ( am ) [ 12 - 14 ] . the european colonial period that started in the late 1400s brought together in the new world populations that had been geographically isolated , namely , europeans , west africans and native americans . given the recent and common origin of all human populations , this admixture had only a small average effect on the gene pools of these new populations . in other words , for most genomic regions , the pre - colonial ( or parental ) populations had similar allele frequencies and , at these , admixture was of little consequence . at some other loci , however , there had been some change in allele frequency in the time since the separation of parental populations and it is at these loci where admixture has had an important effect . since populations like african americans , african caribbeans and mexican americans were formed in the recent past , allelic associations in these populations that were created by admixture extend over large distances . admixed populations represent a useful resource for mapping complex - disease genes by using this long - range ald , which requires fewer markers to screen the genome than other populations or approaches . understanding the genetic consequences of admixture is important because it can be both a confounding factor and a source of statistical power in gene identification studies . two models of admixture dynamics have been described to represent the extremes of the process by which an admixed population is formed : the continuous gene flow ( cgf ) model and the hybrid isolation ( hi ) model . in the hi model , admixture occurs immediately in a single generation without further contribution from either parental population , hence , ald is generated in a single generation and gradually decays in successive generations through independent assortment and recombination between loci . few false - positive results are thus expected in an association study under the hi model . alternatively , the cgf model represents a situation where admixture occurs at a steady rate in each generation , with contributions from one ( or all ) of the parental populations into the admixed population . ald under the cgf model increases in each generation , since new admixture is constantly occurring . a point will be reached , however ( when the admixture proportion = 0.5 ) , where continued admixture will actually decrease the ald , since added gene flow will result in the conversion of the admixed population into the introgressing parental population . figure 1 shows the amount of ald expected under these two models for linked and unlinked loci . for both models , association between markers simulation studies have shown that populations that have a demographic history more consistent with the cgf model of admixture retain ald over larger chromosomal regions and show significant associations between unlinked marker loci . while associations between unlinked markers could potentially lead to false - positives , conditioning upon parental admixture allows the distinction between associations arising due to true linkage and those due to cgf stratification to be made , thereby providing greater power for detecting ald over larger chromosomal distances . the amount of admixture linkage disequilibrium ( ald ) expected under the continuous gene flow ( cgf ) and hybrid isolation ( hi ) models of admixture for unlinked loci and loci linked at 5 cm . the results shown are for two loci with = 0.54 and 0.49 , and with 50 per cent admixture in the first generation for the hi model and 1.9 per cent admixture for 36 generations under the cgf model ( equivalent to 50 per cent total ) . ald under the hi model decreases for both linked and unlinked loci , whereas ald under the cgf model for both linked and unlinked loci increases initially and then decreases ( adapted from pfaff et al . , 2001 ) there are several ways in which admixture can be an important resource in the elucidation of genetic factors that contribute to the risk of common disease . common diseases often have environmental components to their risk , and the clinical phenotype results from currently unknown interactions between environmental factors and underlying genotypes . decomposing the sources of variation it is possible to distinguish between the genetic and environmental explanations for ethnic differences in disease risk ( and investigating the mode of inheritance ) , by studying the relationship of disease risk to individual admixture [ 14,17 - 19 ] . for example , recent studies have demonstrated a strong relationship between proportional west african ancestry and the risk of systemic lupus erythematosus in admixed populations in trinidad . several common diseases ( eg hypertension , diabetes , obesity , prostate cancer and osteoporosis ) have differences in risk among population groups ( see table 1 ) . in situations where these differences have a genetic basis , genes underlying these differences can be identified by testing for locus ancestry by conditioning on parental admixture . as detailed by shriver et al . , this approach has a greater statistical power than family linkage studies for mapping polygenic traits . estimates of biogeographical ancestry ( bga ) , the proportional ancestry levels of an individual , can be used in conjunction with measured environmental effects for investigating the roles of environmental and inherited risks underlying complex traits [ 18 - 20 ] . it is important to recognise that associations between individual admixture and disease risk might reflect correlations between bga and socio - cultural variables and exposures . for example , hypothetically , if bga and years of education were to be correlated , hypertension might be correlated with bga , even though the causal risk factor was years of education or vice versa . diseases with possible genetic components based on ethnic differences in disease rates and hence amenable to admixture mapping admixture - based methods rely on using suitable markers and estimates of allele frequencies from appropriately identified parental populations . since ald is fairly new and extends over larger distances , fewer markers are required for am studies . markers informative for ancestry have been used in several contexts and have been referred to as ' ideal , ' ' private ' and ' unique ' . informativeness of such markers can be measured as the allele frequency differential ( ) , which is the absolute value of the difference of a particular allele between populations . microsatellites and insertion / deletion polymorphisms with > 0.3 were recently called ' ethnic - difference markers ' ( edms ) suitable for mapping by admixture linkage disequilibrium ( mald ) . additionally , markers with high and very high log likelihood allelic ratio ( llar ) between populations have been designated ' population specific alleles ' ( psas ) . this report followed from earlier work where markers with large allele frequency difference were identified to be appropriate for admixture studies , and most ( > 95 per cent ) of the arbitrarily identified biallelic markers had < 50 per cent . thus , the authors proposed that ideal psas should have > 50 per cent and also indicated that for multiallelic loci , a composite could be estimated as one half the summation of the absolute value of allelic frequency differences for all alleles at that locus . it has also been shown that markers with lower values , of approximately 30 per cent , can provide up to 80 per cent power for detecting associations at distances of 5 cm with a large enough sample size ( n = 1,000 ) . pfaff et al . , suggested referring to markers suitable for admixture studies as ' ancestry informative markers ' ( aims ) , given that the central feature of these markers is the ancestry information content ( f ) . the present authors agree that the term aim more accurately describes these markers and does so using language that is less likely to be misunderstood and misinterpreted . marker information content ' f ' denotes the locus - specific fst and is a value representative of the differentiation between two populations at a single locus . simulation studies for estimating the information content of markers with varying levels of f have shown that for 1,000 markers with average information content for ancestry at 40 per cent between two ancestral subpopulations , approximately 80 per cent of the information about ancestry can be extracted from an initial genome screen . after initial identification of regions showing admixture , more markers can be typed in these regions to increase extraction of information to nearly 100 per cent . it is well established , however , that only 5 - 15 per cent of the total genetic variation results from differences among human populations [ 30 - 32 ] . moreover , most alleles are shared between populations , and alleles common in one population are also common in other populations . thus , most genetic markers are unaffected by admixture and it is imperative to choose markers that show high levels of d ( and f ) between the parental populations . recent studies by several groups have focused on identifying panels of markers suitable for admixture studies . one notable study screened 744 microsatellite markers for composite d values and llar in four different populations and identified a genome spanning set of 315 markers ( average spacing 10 cm , 0.3 ) for mapping in african americans and 214 markers ( average spacing of 16 cm , 0.25 ) for mapping in hispanics . a dna pooling method was used to identify 151 aims ( microsatellites and short insertion / deletion polymorphisms ) , with > 0.3 for mapping in mexican american populations to distinguish between european - american and native - american contributions . ninety - seven aims were identified for mapping in african - american populations that show limited variation within africa . additional resources are available for obtaining marker frequency , and genotype and haplotype information , from the snp consortium ( tsc ; http://snp.cshl.org ) , the national center for biotechnology information 's ' dbsnp ' website ( http://www.ncbi.nlm.nih.gov/snp ) , the marshfield database ( http://research.marshfieldclinic.org/genetics/default.htm ) and the ongoing hapmap project . since the amount of ald created is proportional to the level of admixture in a population , it is important briefly to review studies on admixture levels across populations . those populations that are likely to be useful for admixture studies include african americans , mexican americans , cubans and puerto ricans in the usa , african caribbeans , various latin american populations , various groups in central and south america and the caribbean islands , anglo indians in india and ' coloured ' populations of south africa . various statistical approaches have been used to estimate admixture proportions in these populations and have been reviewed in detail elsewhere . these include a least squares method , a weighted least squares method and likelihood methods . a recent review of admixture studies and admixture proportions of various latin american populations is provided by sans . african americans are a well - studied group with substantial european and west african contributions and a smaller native american contribution . a survey of current literature indicates that european admixture ranges from 3.5 per cent in the gullah sea islanders of south carolina , to 28 per cent in new orleans . admixture estimates in african - american populations can be highly variable across the usa , which is likely to reflect local variation in the demographic histories and social norms . us hispanics form a complex socio - political conglomerate including puerto ricans , cubans , spanish americans , mexican americans . the proportional contributions from parental europeans are estimated to be the largest , followed by a substantial native american ancestry and varying amounts of west african ancestry . in a sample of mexican americans from arizona , the admixture estimates obtained using a weighted least squares method showed 29 4 per cent native american , 68 5 per cent european and 3 2 per cent west african contribution . a recent study reports the following estimates for a hispanic population from the san luis valley , colorado : 62.7 2.1 per cent european , 34.1 1.9 per cent native american , 3.2 1.5 per cent west african . in puerto ricans from new york city , the estimates obtained were 53.3 2.8 per cent european , 29.1 2.3 per cent west african , 17.6 2.4 per cent native american . in a separate mexican - american population sample from california , european ancestry was estimated to be 60 per cent and native american contribution was estimated at 40 per cent . from these results , it is evident that , when studying any new admixed population sample , it is important to accurately determine the proportional contributions and not to rely on previously obtained estimates from a similar population . additionally , it is instructive to have information on the levels of stratification related to admixture that are present in the population under consideration . traits and diseases more prevalent in one population than in others are amenable to admixture analysis and some examples are listed in table 1 . most of the diseases shown in this table have a complex aetiology affected by multiple genes and environmental factors . earlier studies focused on admixed populations as units of analysis in exploring relationships between ancestry and phenotypes . these authors showed that non - insulin - dependent ( type 2 ) diabetes mellitus prevalence is correlated with admixture proportions among a selection of populations with varying levels of native american ancestry . data like these provide compelling evidence for frequency differences in risk modifying alleles , but such data have not been collected for many diseases . another related approach is to test for individual admixture - phenotype correlations within an admixed population . correlations between ancestry and phenotypes have been detected and reported by various authors [ 14,17 - 19,44,45,47 ] . a prerequisite for testing ancestry / phenotype correlations is the presence of stratification related to admixture , which will be evident in variation in individual ancestry levels . figure 2 shows the distribution of bga estimates from three examples of hispanic population samples , puerto ricans from new york , mexicans from tlapa , mexico and hispanics from the san luis valley , colorado . substantial variation is observed in all three samples . with the san luis valley group , more variability is observed on the european - native american axis , while the new york group is more variable on the european - west african axis . following the argument of chakraborty and weiss , admixture proportions should be correlated with diseases / traits that differ in populations due to underlying genetic differences . in each of these population samples , strong positive correlation was observed between individual ancestry and skin pigmentation measured as melanin index ' m ' or lightness index ' l ' ( figures 3a , 3b and 3c ) . a significant negative correlation was also observed between the proportion of west african ancestry and bone mineral density ( bmd ) in the puerto rican sample . proportion west african ancestry and skin pigmentation ( measured as melanin index ) in individuals is also correlated in african americans from washington dc and african caribbeans from the uk , but not in european americans from state college , pennsylvania ( figure 4 ) . recently , correlations have been observed between proportion west african ancestry and lower insulin sensitivity , higher fasting insulin and acute insulin response to glucose in a combined sample of african - american and european - american children . in a separate sample of african - american females , west african admixture is associated with body mass index , fat mass , fat - free mass and bmd . it is important to keep in mind that ancestry - pheno - type correlations are dependent on both the existence of functional alleles at different frequencies in parental populations , and significant stratification related to admixture . although most admixed populations tested to date are structured , there is variation in the amount of stratification present , and this structure should be tested for explicitly when investigating a new population . each vertex represents a parental population , which for this plot are europeans , west africans and native americans . the three populations shown are hispanics from the san luis valley ( blank circles ) , puerto ricans from new york city ( grey diamonds ) and mexicans from tlapa , mexico ( grey triangles ) ( adapted from bonilla , 2003 ) the relationship between proportional ancestry and skin pigmentation in three hispanic populations . for all populations , proportional ancestry was estimated using the maximum likelihood ( ml ) method ( adapted from bonilla , 2003 ) . ( a ) percent native american ancestry versus lightness index ( l ) in hispanics from the san luis valley , colorado ( ancestry estimated using 22 aims ) . ( b ) percent native american ancestry versus melanin index in mexicans from tlapa , mexico ( ancestry estimates using 29 aims ) . ( c ) percent african ancestry versus melanin index ( m ) in puerto ricans from new york city ( ancestry estimated using 35 aims ) the relationship between percent african ancestry and skin pigmentation in three populations . percent african ancestry ( obtained using 34 aims and calculated by the maximum likelihood ( ml ) method ) and the melanin index ( m ) are shown for three populations , european americans from state college , pennsylvania ( diamonds ) , african americans from washington , dc and state college , pennsylvania ( squares ) and african caribbeans from britain ( triangles ) . 2003 ) theoretical and experimental studies have explored the parameters that characterise and affect admixture studies . the acronym mald was proposed to designate the mapping method proposed originally by chakraborty and weiss , which exploited the long range allelic associations created through ald . parameters critical for mald include the genetic distance between markers and disease locus ( ) ; number of generations since admixture ( t ) ; proportion of admixture ( m ) from one parental population ; the allele frequency differential ( ) between parental populations ; and sample size ( n ) . simulation studies suggest that sample sizes of 200 - 300 patients , typed for 200 - 300 evenly spaced markers , each having allele frequency differentials > 0.3 , have a > 95 per cent chance of locating the causative gene , when there has been no new admixture from the parental population in the last four generations and no other sources of population structure or sample heterogeneity . other approaches proposed for using admixture include a method based on the transmission disequilibrium test ( tdt ) that assesses excess transmission of alleles derived from high - risk ancestors to affected offspring of parents who are heterozygous at the marker locus , containing one allele from each of two ancestral populations . a second tdt - based likelihood approach was developed that compared the transmission of haplotypes with non - transmission in affected offspring in an admixed population following a multipoint method . it obtained a likelihood statistic to determine the significance of various models under different scenarios . one fundamental limitation of mald as initially described and in its early extensions , is the effects of stratification on causing false - positive association . marker data at all loci are combined to estimate ancestry of alleles at each locus . when allelic ancestry at marker loci is known , this approach is analogous to a linkage analysis , hence the term am is more appropriate than mald for describing this method and to distinguish it from ld approaches . the underlying variation in ancestry of chromosomes of mixed descent is modelled to extract all of the information about linkage that is generated by admixture . for example , where a locus is assumed to account for variation in skin pigmentation between two parental groups , eg west africans and europeans , individuals can be classified according to whether they have 0 , 1 or 2 alleles of west african descent at this locus . by comparing these three groups for mean pigmentation level , holding all other factors constant , variation in pigmentation can be observed depending upon the number of alleles of west african ancestry in an individual . controlling for parental admixture eliminates association of the trait with ancestry at unlinked loci . by removing the background effects of ancestry , it is possible to observe the locus - specific effects on a trait / disease . allelic ancestry at a locus is inferred from the marker by using the conditional probability of each allelic state given the ancestry - specific allele frequencies . a complex hierarchical model with many nuisance parameters this is implemented using the admixmap program ( at http://www.lshtm.ac.uk/eph.eu/geneticepidemiologygroup/htm ) , which follows a bayesian approach with markov chain simulation , and incorporates the admixture of each individual 's parents and the random variation of ancestry on chromosomes inherited from each of the parents in the model . variation in individual admixture introduces population stratification , which in turn can inflate the number of significant associations that are observed and is a potential confounder in association studies [ 29,57 - 59 ] . various statistical approaches have been developed to detect and control for stratification within a population sample [ 14,15,17,42,60 - 62 ] . for example , the dt / d0 test examines the relationship between the observed ld and the predicted ald between unlinked marker pairs for detecting structure within the sample . using individual ancestry as a conditioning variable in analysis of variance tests the bayesian approaches implemented by mckeigue et al . and pritchard et al . offer an advantage over classical maximum likelihood based methods by allowing for missing genotype and ancestry data and modelling admixture hierarchically . methods have been developed to control for parental admixture and to account for uncertain bga estimation . admixture - based methods rely on using suitable markers and estimates of allele frequencies from appropriately identified parental populations . since ald is fairly new and extends over larger distances , fewer markers are required for am studies . markers informative for ancestry have been used in several contexts and have been referred to as ' ideal , ' ' private ' and ' unique ' . informativeness of such markers can be measured as the allele frequency differential ( ) , which is the absolute value of the difference of a particular allele between populations . microsatellites and insertion / deletion polymorphisms with > 0.3 were recently called ' ethnic - difference markers ' ( edms ) suitable for mapping by admixture linkage disequilibrium ( mald ) . additionally , markers with high and very high log likelihood allelic ratio ( llar ) between populations have been designated ' population specific alleles ' ( psas ) . this report followed from earlier work where markers with large allele frequency difference were identified to be appropriate for admixture studies , and most ( > 95 per cent ) of the arbitrarily identified biallelic markers had < 50 per cent . thus , the authors proposed that ideal psas should have > 50 per cent and also indicated that for multiallelic loci , a composite could be estimated as one half the summation of the absolute value of allelic frequency differences for all alleles at that locus . it has also been shown that markers with lower values , of approximately 30 per cent , can provide up to 80 per cent power for detecting associations at distances of 5 cm with a large enough sample size ( n = 1,000 ) . pfaff et al . , suggested referring to markers suitable for admixture studies as ' ancestry informative markers ' ( aims ) , given that the central feature of these markers is the ancestry information content ( f ) . the present authors agree that the term aim more accurately describes these markers and does so using language that is less likely to be misunderstood and misinterpreted . marker information content ' f ' denotes the locus - specific fst and is a value representative of the differentiation between two populations at a single locus . simulation studies for estimating the information content of markers with varying levels of f have shown that for 1,000 markers with average information content for ancestry at 40 per cent between two ancestral subpopulations , approximately 80 per cent of the information about ancestry can be extracted from an initial genome screen . after initial identification of regions showing admixture , more markers can be typed in these regions to increase extraction of information to nearly 100 per cent . it is well established , however , that only 5 - 15 per cent of the total genetic variation results from differences among human populations [ 30 - 32 ] . moreover , most alleles are shared between populations , and alleles common in one population are also common in other populations . thus , most genetic markers are unaffected by admixture and it is imperative to choose markers that show high levels of d ( and f ) between the parental populations . recent studies by several groups have focused on identifying panels of markers suitable for admixture studies . one notable study screened 744 microsatellite markers for composite d values and llar in four different populations and identified a genome spanning set of 315 markers ( average spacing 10 cm , 0.3 ) for mapping in african americans and 214 markers ( average spacing of 16 cm , 0.25 ) for mapping in hispanics . a dna pooling method was used to identify 151 aims ( microsatellites and short insertion / deletion polymorphisms ) , with > 0.3 for mapping in mexican american populations to distinguish between european - american and native - american contributions . ninety - seven aims were identified for mapping in african - american populations that show limited variation within africa . additional resources are available for obtaining marker frequency , and genotype and haplotype information , from the snp consortium ( tsc ; http://snp.cshl.org ) , the national center for biotechnology information 's ' dbsnp ' website ( http://www.ncbi.nlm.nih.gov/snp ) , the marshfield database ( http://research.marshfieldclinic.org/genetics/default.htm ) and the ongoing hapmap project . since the amount of ald created is proportional to the level of admixture in a population , it is important briefly to review studies on admixture levels across populations . those populations that are likely to be useful for admixture studies include african americans , mexican americans , cubans and puerto ricans in the usa , african caribbeans , various latin american populations , various groups in central and south america and the caribbean islands , anglo indians in india and ' coloured ' populations of south africa . various statistical approaches have been used to estimate admixture proportions in these populations and have been reviewed in detail elsewhere . these include a least squares method , a weighted least squares method and likelihood methods . a recent review of admixture studies and admixture proportions of various latin american populations is provided by sans . african americans are a well - studied group with substantial european and west african contributions and a smaller native american contribution . a survey of current literature indicates that european admixture ranges from 3.5 per cent in the gullah sea islanders of south carolina , to 28 per cent in new orleans . admixture estimates in african - american populations can be highly variable across the usa , which is likely to reflect local variation in the demographic histories and social norms . us hispanics form a complex socio - political conglomerate including puerto ricans , cubans , spanish americans , mexican americans . the proportional contributions from parental europeans are estimated to be the largest , followed by a substantial native american ancestry and varying amounts of west african ancestry . in a sample of mexican americans from arizona , the admixture estimates obtained using a weighted least squares method showed 29 4 per cent native american , 68 5 per cent european and 3 2 per cent west african contribution . a recent study reports the following estimates for a hispanic population from the san luis valley , colorado : 62.7 2.1 per cent european , 34.1 1.9 per cent native american , 3.2 1.5 per cent west african . in puerto ricans from new york city , the estimates obtained were 53.3 2.8 per cent european , 29.1 2.3 per cent west african , 17.6 2.4 per cent native american . in a separate mexican - american population sample from california , european ancestry was estimated to be 60 per cent and native american contribution was estimated at 40 per cent . as with african - american populations , there is substantial variation across populations . from these results , it is evident that , when studying any new admixed population sample , it is important to accurately determine the proportional contributions and not to rely on previously obtained estimates from a similar population . additionally , it is instructive to have information on the levels of stratification related to admixture that are present in the population under consideration . traits and diseases more prevalent in one population than in others are amenable to admixture analysis and some examples are listed in table 1 . most of the diseases shown in this table have a complex aetiology affected by multiple genes and environmental factors . earlier studies focused on admixed populations as units of analysis in exploring relationships between ancestry and phenotypes . these authors showed that non - insulin - dependent ( type 2 ) diabetes mellitus prevalence is correlated with admixture proportions among a selection of populations with varying levels of native american ancestry . data like these provide compelling evidence for frequency differences in risk modifying alleles , but such data have not been collected for many diseases . another related approach is to test for individual admixture - phenotype correlations within an admixed population . correlations between ancestry and phenotypes have been detected and reported by various authors [ 14,17 - 19,44,45,47 ] . a prerequisite for testing ancestry / phenotype correlations is the presence of stratification related to admixture , which will be evident in variation in individual ancestry levels . figure 2 shows the distribution of bga estimates from three examples of hispanic population samples , puerto ricans from new york , mexicans from tlapa , mexico and hispanics from the san luis valley , colorado . substantial variation is observed in all three samples . with the san luis valley group , more variability is observed on the european - native american axis , while the new york group is more variable on the european - west african axis . following the argument of chakraborty and weiss , admixture proportions should be correlated with diseases / traits that differ in populations due to underlying genetic differences . in each of these population samples , strong positive correlation was observed between individual ancestry and skin pigmentation measured as melanin index ' m ' or lightness index ' l ' ( figures 3a , 3b and 3c ) . a significant negative correlation was also observed between the proportion of west african ancestry and bone mineral density ( bmd ) in the puerto rican sample . proportion west african ancestry and skin pigmentation ( measured as melanin index ) in individuals is also correlated in african americans from washington dc and african caribbeans from the uk , but not in european americans from state college , pennsylvania ( figure 4 ) . recently , correlations have been observed between proportion west african ancestry and lower insulin sensitivity , higher fasting insulin and acute insulin response to glucose in a combined sample of african - american and european - american children . in a separate sample of african - american females , west african admixture is associated with body mass index , fat mass , fat - free mass and bmd . it is important to keep in mind that ancestry - pheno - type correlations are dependent on both the existence of functional alleles at different frequencies in parental populations , and significant stratification related to admixture . although most admixed populations tested to date are structured , there is variation in the amount of stratification present , and this structure should be tested for explicitly when investigating a new population . each vertex represents a parental population , which for this plot are europeans , west africans and native americans . the three populations shown are hispanics from the san luis valley ( blank circles ) , puerto ricans from new york city ( grey diamonds ) and mexicans from tlapa , mexico ( grey triangles ) ( adapted from bonilla , 2003 ) the relationship between proportional ancestry and skin pigmentation in three hispanic populations . for all populations , proportional ancestry was estimated using the maximum likelihood ( ml ) method ( adapted from bonilla , 2003 ) . ( a ) percent native american ancestry versus lightness index ( l ) in hispanics from the san luis valley , colorado ( ancestry estimated using 22 aims ) . ( b ) percent native american ancestry versus melanin index in mexicans from tlapa , mexico ( ancestry estimates using 29 aims ) . ( c ) percent african ancestry versus melanin index ( m ) in puerto ricans from new york city ( ancestry estimated using 35 aims ) the relationship between percent african ancestry and skin pigmentation in three populations . percent african ancestry ( obtained using 34 aims and calculated by the maximum likelihood ( ml ) method ) and the melanin index ( m ) are shown for three populations , european americans from state college , pennsylvania ( diamonds ) , african americans from washington , dc and state college , pennsylvania ( squares ) and african caribbeans from britain ( triangles ) . theoretical and experimental studies have explored the parameters that characterise and affect admixture studies . the acronym mald was proposed to designate the mapping method proposed originally by chakraborty and weiss , which exploited the long range allelic associations created through ald . parameters critical for mald include the genetic distance between markers and disease locus ( ) ; number of generations since admixture ( t ) ; proportion of admixture ( m ) from one parental population ; the allele frequency differential ( ) between parental populations ; and sample size ( n ) . simulation studies suggest that sample sizes of 200 - 300 patients , typed for 200 - 300 evenly spaced markers , each having allele frequency differentials > 0.3 , have a > 95 per cent chance of locating the causative gene , when there has been no new admixture from the parental population in the last four generations and no other sources of population structure or sample heterogeneity . other approaches proposed for using admixture include a method based on the transmission disequilibrium test ( tdt ) that assesses excess transmission of alleles derived from high - risk ancestors to affected offspring of parents who are heterozygous at the marker locus , containing one allele from each of two ancestral populations . a second tdt - based likelihood approach was developed that compared the transmission of haplotypes with non - transmission in affected offspring in an admixed population following a multipoint method . it obtained a likelihood statistic to determine the significance of various models under different scenarios . one fundamental limitation of mald as initially described and in its early extensions , is the effects of stratification on causing false - positive association . marker data at all loci are combined to estimate ancestry of alleles at each locus . when allelic ancestry at marker loci is known , this approach is analogous to a linkage analysis , hence the term am is more appropriate than mald for describing this method and to distinguish it from ld approaches . the underlying variation in ancestry of chromosomes of mixed descent is modelled to extract all of the information about linkage that is generated by admixture . for example , where a locus is assumed to account for variation in skin pigmentation between two parental groups , eg west africans and europeans , individuals can be classified according to whether they have 0 , 1 or 2 alleles of west african descent at this locus . by comparing these three groups for mean pigmentation level , holding all other factors constant , variation in pigmentation can be observed depending upon the number of alleles of west african ancestry in an individual . controlling for parental admixture eliminates association of the trait with ancestry at unlinked loci . by removing the background effects of ancestry , it is possible to observe the locus - specific effects on a trait / disease . allelic ancestry at a locus is inferred from the marker by using the conditional probability of each allelic state given the ancestry - specific allele frequencies . a complex hierarchical model with many nuisance parameters this is implemented using the admixmap program ( at http://www.lshtm.ac.uk/eph.eu/geneticepidemiologygroup/htm ) , which follows a bayesian approach with markov chain simulation , and incorporates the admixture of each individual 's parents and the random variation of ancestry on chromosomes inherited from each of the parents in the model . variation in individual admixture introduces population stratification , which in turn can inflate the number of significant associations that are observed and is a potential confounder in association studies [ 29,57 - 59 ] . various statistical approaches have been developed to detect and control for stratification within a population sample [ 14,15,17,42,60 - 62 ] . for example , the dt / d0 test examines the relationship between the observed ld and the predicted ald between unlinked marker pairs for detecting structure within the sample . using individual ancestry as a conditioning variable in analysis of variance tests , it is possible to eliminate association of the trait with unlinked alleles . offer an advantage over classical maximum likelihood based methods by allowing for missing genotype and ancestry data and modelling admixture hierarchically . methods have been developed to control for parental admixture and to account for uncertain bga estimation . several theoretical and practical studies indicate that am approaches promise to be suitable for identifying genes causing complex diseases . methodological advancements have been made to offset the potential problems arising from association between unlinked loci by conditioning on parental admixture , and to detect and correct for population stratification . use of bayesian am can take into consideration various uncertainties , including missing data values for estimating admixture proportions , and can overcome problems arising out of mis - specification of parental allele frequencies and promises to be an effective tool for admixture studies . this method , which is different from the classical disequilibrium - based approach that is more commonly used , is perhaps more suitable for disease gene mapping in admixed populations and has already been successfully used for mapping . table 2 summarises recent studies showing associations between ancestry and phenotypes / diseases and instances where am was used to identify genes . currently , the primary impediment to exhaustive am genome scans is the lack of verified aim panels . sufficient numbers of markers are available as candidate aims , but effort and resources are required to confirm these markers and to generate accurate parental allele frequencies . it seems inevitable that more such studies will be carried out in the near future to utilise the immense potential of this approach . diseases showing ancestry - phenotype correlation a = r ; b = risk ratio ; c = rank - order correlation . we thank dr paul mckeigue and dr esteban parra for helpful discussions on the subject . we also acknowledge helpful comments from an unknown reviewer . this work was supported in part by grants from nih acyl - coa thioesterases ( acots ) represent a group of enzymes that metabolise acyl - coa esters to their corresponding non - esterified fatty acid and coenzyme a ( coash ) . in addition to ' acyl - coa thioesterases ' , these enzymes also have been described as ' acyl - coa hydrolases ' , ' acyl - coa thioester hydrolases ' and ' deacylases ' . the reaction catalysed by acot enzymes is very important during fatty acid metabolism . as such acyl - coa thioesterase expression is widespread and activity is detectable within many tissues and cell types . acot activity helps to regulate cellular pools , as well as proper ratios , of activated fatty acyl - coas ( acyl - coa esters ) , free fatty acids ( non - esterified fatty acids ) and coash . these enzymes are found within a number of subcellular locations , including peroxisomes , mitochondria and the cytosol . peroxisomal and mitochondrial acots are thought to play a major role in fatty acid degradation via -oxidation , which provides energy through the citric acid cycle , as well as in ketone body formation . in order for a fatty acid to be transported into the peroxisome for -oxidation , it must first be activated to its coenzyme a ester by acyl - coa synthetases . some acyl - coa esters undergo -oxidation much more slowly than others -- which can effectively sequester large amounts of coash and limit the activity of acyl - coa synthetases and the rate of subsequent fatty acid -oxidation . under these conditions , acots may act to increase the levels of free coash , thereby restoring fatty acid -oxidation . mammalian acots are capable of metabolising a wide variety of compounds , including short- , medium- and long - chain acyl - coas . they can also metabolise polyunsaturated fatty acyl - coas , branched - chain acyl - coas and aromatic - acyl - coas . these compounds are not only utilised during -oxidation and fatty acid degradation , but also have roles in inflammation , ion channel opening , signal transduction , lipid biosynthesis and gene regulation . ten human acot genes have been identified to date , compared with 13 in mice and 12 in rats . these genes can be divided into two groups , based upon differences in sequence and structure . these enzymes are members of the /-hydrolase fold enzyme superfamily , which includes other esterase - activity - exhibiting enzymes such as carboxylesterases and lipases . type ii acots make up the second group ; these enzymes are members of the ' hot dog ' fold enzyme superfamily , which includes a wide variety of functionally diverse proteins . despite catalysing the same reaction , type i and type ii enzymes share no similarity in terms of structure or sequence this observation is reminiscent of the cytochrome p450 ( cyp ) and nitric oxide synthase ( nos ) gene families . studies with purified nos proteins revealed that they possess p450-like activity ; however , detailed alignment analyses showed < 15 per cent amino acid identity , thereby confirming that these two gene families were not evolutionarily related . it was thus concluded that this was an example of convergent evolution . in other words , during the evolution of enzyme activities needed at a particular subcellular location ( probably more than 1 billion years ago ) mother nature needed a ' p450-like activity ' and assigned that task to the nearest protein , which happened to be an nos . the revised nomenclature for human , mouse and rat acyl - coa thioesterase genes was established by hunt et al . and has been accepted by the hugo gene nomenclature committee . in recent years , a number of human acots have been recombinantly expressed and enzymatically characterised , although the exact physiological function and importance of many of these enzymes remain unknown . to date , a total of ten genes have been identified within the human genome , all of which exhibit acyl - coa thioesterase activity ( table 1 ) . these ten genes can be sub - divided into two distinct groups , type i and type ii , based on sequence and structural homology . type i acots comprise the smaller group , which contains only four genes : acot1 , acot2 , acot4 and acot6 . the larger group includes six genes , and these members are referred to as type ii . there is no sequence homology between the two groups , and the translated proteins have very different domain architecture , indicating two distinct , evolutionarily unrelated , origins ( figure 1 ) and , most likely , an example of convergent evolution . list of all human ( acot ) with full gene name , aliases , chromosomal location , isoforms , ncbi refseq mrna accession number , ncbi refseq protein accession number and total amino acid number ( aa # ) domain organisation of human type i and type ii acyl - coa thioesterases ( acots ) . abbreviations : acth , acyl - coa thioester hydrolase domain ; baat , bile acid coa : amino acid n - acyltransferase ; el , esterase - lipase domain ; hd , ' hot dog ' fold domain ; start , steroidogenic acute regulatory protein ( star)-related lipid transfer ( start ) domain ; them , thioesterase superfamily member . the type i proteins , acot1 , acot2 , acot4 and acot6 , share a high degree of sequence homology ( table 2 ) . acot1 and acot2 are the most closely related , having 98 per cent amino acid sequence identity . acot6 is the least homologous , but still shares 54 - 57 per cent sequence identity with the other members . the four type i genes form an 80 kilobase gene cluster on chromosome 14q24.3 , indicating that they most likely have arisen as the result of gene duplication . amino acid sequence identity between type i and type ii human acyl - coa thioesterases ( acots ) . homology was considered not significant and left blank if the observed identity was less than 15 per cent abbreviations : baat , bile acid coa : amino acid n - acyltransferase ; them , thioesterase superfamily member a similar situation is observed within the mouse and rat orthologues acot1 , acot2 , acot3 , acot4 , acot5 and acot6 , which are clustered on chromosomes 12 d3 and 6q31 , respectively . a cladogram depicting the evolutionary relationships observed between human , mouse and rat type i proteins is shown in figure 2a . this cladogram clearly shows that , prior to the mammalian radiation approximately 75 million years ago ( mya ) , there was a single ancestral acot1 ( or -2 ) gene ; after the mammalian radiation but before the mouse - rat split at approximately 15 mya , this single ancestor divided twice to become four rodent acot functional genes ( now named acot1 , acot3 , acot4 and acot5 ) . by contrast , after the mammalian radiation , the human single ancestor duplicated into a second functioning gene , leading to acot1 and acot2 . on the other hand , acot4 and acot6 existed before the mammalian radiation and cladograms depicting the evolutionary relationships between human , mouse ( m ) and rat ( r ) orthologous protein sequences for type i ( a ) and type ii ( b ) acots . human carboxylesterase 1 ( ces1 ) is an unrelated esterase that was added simply to ' ground ' the tree . in the mouse , the acot3 , acot4 and acot5 enzymes each exhibit specific expression patterns and enzyme substrate specificity . the lack of a human orthologue to mouse acot3 and acot5 was suggested to be the result of convergent evolution ; it was hypothesised that convergent , functional evolution led to a reduced need for multiple enzymes in humans . the protein basic local alignment search tool ( blastp ) analysis of all non - redundant protein sequences , however , failed to identify orthologues for acot3 or acot5 in non - rodent species . this finding suggests that acot4 was an ancestral gene encoding an acot4 enzyme with broad substrate recognition , and that gene duplication events led to acot1 ( or -2 ) , and that further duplication events led to the mouse acot3 and acot5 genes , encoding the acot3 and acot5 enzymes , which have similar substrate specificity to that of mouse acot4 and human acot4 . analysis of the genomic acot6 sequence indicates transcription of a complete mrna containing three exons ; however , a full - length mrna is not detectable . subsequent translation begins at a methionine located within the second exon , producing a truncated protein that lacks the n - terminal acyl - coa thioester hydrolase domain found within other type i proteins . the truncated protein contains all residues required for catalysis and could therefore still produce an active acyl - coa thioesterase . the blast results indicate that bile acid coa : amino acid n - acyltransferase ( baat ) is the only enzyme to share significant sequence homology with type i acots . baat is known to participate in the transfer of bile acids from the acyl - coa thioester to either glycine or taurine , which are used for bile salt secretion . baat contains the same domain architecture and acot1 , acot2 and acot4 ; however , the catalytic triad found within the esterase - lipase domain contains a cysteine , instead of the highly conserved serine residue . this substitution may explain the difference in activity ( ie transferase versus hydrolase ) observed between the two enzymes . despite sharing catalytic domains , other /-hydrolase fold enzyme superfamily esterases , such as carboxylesterase 1 ( ces1 ) and acetylcholinesterase ( ache ) , share very little sequence homology with their acyl - coa thioesterase relatives . ces1 has thus been added as an evolutionarily unrelated gene to figure 2a simply to ' ground ' the dendrogram . none of the type ii proteins shares more than 25 per cent identity and many do not share any significant sequence similarity ( table 2 ) . unlike the type i genes , type ii acots are widely distributed throughout the genome . with the exception of acot13 , all type ii proteins contain double ' hot dog ' domains , which may have evolved as a gene duplication event that allowed for the accommodation of bulky substrates . acot7 shares 19 per cent identity with acot9 , 22 per cent with acot11 and 24 per cent identity with acot12 . acot11 and acot12 share 51 per cent sequence identity and both enzymes contain a c - terminal steroidogenic acute regulatory protein ( star)-related lipid transfer ( start ) domain , which seems to be specific to mammalian evolution . the n - terminal region of both acots is unrelated to any of the other start - containing enzymes , suggesting that these proteins may be the result of a gene fusion event between an acyl - thioesterase and start domain - containing sequences . like acot8 , blast results did identify 22 per cent sequence identity with thioesterase superfamily member 4 ( them4 is the official name ) -- also known as akt c - terminal modulator protein ( ctmp ) . this small degree of homology suggests that the two proteins may share common evolutionary ties . another protein , thioesterase superfamily member 5 ( them5 ) shares more than 37 per cent identity with them4 but lacks significant sequence homology with acot13 . similar to acot13 , both them4 and them5 contain a single ' hot dog ' domain . furthermore , type ii proteins , them4 and them5 do not share sequence homology with other ' hot dog ' domain superfamily members . the ' hot dog ' fold - containing proteins possess highly conserved structural elements that are created by highly divergent sequences . this high degree of divergence makes evolutionary comparisons difficult without three - dimensional structures , because conservation of structural interactions is not directly associated with residue conservation . a cladogram depicting the evolutionary relationships observed between human , mouse and rat type ii protein is shown in figure 2b . the mouse genome contains an additional type ii acot , acot10 , which shares approximately 95 per cent mrna nucleotide identity with acot9 . unlike acot3 and acot5 , however , which are found in both mouse and rat , acot10 has only been identified in mice , indicating that the gene duplication event occurred less than 17 mya -- that is , after the mouse - rat split . in mouse , acot9 is located at chromosomal location x f3 and acot10 is found on 15 a2 . the amplification and movement of genes to a new chromosomal location is often facilitated by transposable elements , as opposed to duplications occurring within close proximity on a chromosome , which are attributed to errors during meiosis . the remaining seven type ii acot genes are highly conserved among human , mouse and rat , confirming that these genes were all present in the ancestor prior to the mammalian radiation . type i and type ii acyl - coa thioesterases represent two structurally distinct enzyme groups . an unrooted dendrogram -- created using protein sequences from both type i and type ii enzymes , closest - related homologues ( baat , them4 and them5 ) , distant /-hydrolase fold homologues ( ces1 , ache ) and -actin ( actb ) as an unrelated sequence -- exemplifies how dissimilar the two groups are from one another ( figure 3 ) . it also illustrates how the type ii proteins must have originated from a common , evolutionarily distant , predecessor ( as reflected in individual branch proximity to the origin of the tree ) . bile acid acetyl co - a : amino acid n - acyltransferase ( baat ) , thioesterase superfamily members 4 and 5 ( them4 and them5 ) are the closest - related homologues to type i ( baat ) and type ii sequences ( them4 and them5 ) . type i acot branches are highlighted in blue , whereas type ii are in red . acot1 , acot2 and acot4 contain an n - terminal acyl - coa thioester hydrolase domain ( pfam 04775 ) . this domain does not participate directly in catalysis , but studies indicate that the n - terminus plays an important role in regulating enzyme activity . all type i proteins also contain a c - terminus esterase - lipase superfamily domain . all residues required for catalysis are located within this domain , which is also known as the /-hydrolase fold . proteins that share this common structural element make up the functionally diverse /-hydrolase fold superfamily of enzymes . within this family are a number of other distantly related proteins that exhibit esterase activity , including carboxylesterases , ache and lipases . the superfamily has diverged greatly and many members share very little sequence homology but are considered evolutionarily linked , based on structural homology . the enzyme activity is dependent on a catalytic triad composed of highly conserved serine , histidine and aspartate residues found within the active site . the crystalline structure for acot2 indicates that the enzyme is most likely biologically active as a monomer . the structure also reveals that the n - terminal acyl - coa thioester hydrolase domain is connected to the active site by a long loop ; it is postulated that this contributes substrate specificity , although this has yet to be verified experimentally . genomic analysis suggests that acot6 should encode a similar 3 exon transcript ; however , the only detectable transcript is truncated and utilises a start codon located within the 3 ' end of exon two , resulting in the synthesis of a truncated protein . in addition to the four protein - coding genes , there is one intronless pseudogene , referred to as acot4p , located on chromosome 19q13.12 . acotp4 is most likely the result of retroviral action that has inserted this dna segment elsewhere in the genome . acot1 is cytosolic and primarily responsible for the metabolism of long - chain acyl - coas . acot2 and acot1 can metabolise a similar range of substrates ; however , acot2 contains an n - terminal mitochondrial targeting signal which directs its transport to mitochondria . acot4 contains a c - terminal peroxisomal targeting signal , directing transport to peroxisomes , where it exhibits high activity against succinyl - coa , as well as a wide variety of short - chain dicarboxylic - coas . as previously mentioned , acot6 lacks the n - terminal acyl - coa thioester hydrolase domain . although studies have yet to confirm the enzymatic activity of acot6 , the fact that it contains a complete c - terminal esterase - lipase domain supports its role as a functional esterase . acot6 is predicted to be cytosolic , due to a missense mutation which alters the peroxisomal targeting signal that is found in the acot6 mouse ortholog . the subcellular localisation and substrate specificity for each enzyme suggest that each plays an important , non - redundant function within the cell . the type ii enzymes are more distantly related than type ii acots , as indicated by the relatively low degree of sequence conservation observed between proteins . despite the low degree of sequence homology , the enzymes are structurally related and capable of metabolising similar substrates . acot7 , formerly called brain acyl - coa hydrolase ( bach ) , has been extensively characterised , owing to recent studies that indicate arachidonoyl - coa as a preferred substrate . the metabolism of arachidonoyl - coa by acot7 gives rise to arachidonic acid and coenzyme a. arachidonic acid serves as a precursor for a number of compounds associated with inflammation , including prostaglandins , leukotrienes and thromboxanes . acot8 is found in peroxisomes , where it metabolises a diverse range of acyl - coa compounds . acot8 also has high activity towards branched- and methyl - branched chain acyl - coas . acot9 contains a mitochondrial - targeting signal and metabolises medium- to long - chain acyl - coas . acot9 exhibits the highest activity towards myristoyl - coa , which can then be used by n - myristoyltransferase to modify proteins posttranslationally . expression of mouse acot11 , formerly referred to as brown fat - inducible thioesterase ( bfit ) , increases after cold shock . this protein was found to play a role in regulating thermogenesis , which helps to maintain body temperature . the start domain in animals is commonly found within homeodomain transcription factors , where lipid - binding regulates transcription . the binding of lipids to the start domain in acot11 or acot12 acts as a mechanism for regulating activity and for allowing for rapid enzyme activation . acot13 , previously called thioesterase superfamily member 2 ( them2 ) , differs from the other type ii acots , in that it contains a single ' hot dog ' domain . were recently determined using recombinant protein ; results indicate that acot13 prefers medium- to long - chain - acyl - coa thioesters . this finding suggests that the start domains , identified in acot11 and acot12 , could have a similar effect on enzyme activity . recent in vitro kinetic activity studies revealed that them4 is a broad - range , high - activity acyl - coa thioesterase . similar to acot13 , them4 preferentially metabolises medium- to long - chain acyl - coa thioesters . as such , we recommend that the enzyme be renamed acot14 according to the accepted nomenclature established by hunt et al . . acyl - coa thioesterase activity has not been verified for them5 ; however , activity is probably based on the high degree of sequence identity with them4 , but the exact role that this protein might play in fatty acid metabolism has yet to be determined . the human acyl - coa gene family represents a diverse group of enzymes that catalyse the hydrolysis of acyl - coa thioesters to their corresponding free fatty acids and coenzyme a. these enzymes are involved in a number of cellular processes but are primarily thought to play an important role in lipid metabolism . despite catalysing the same enzymatic reaction , the acyl - coa thioesterase family members can be divided into two distinct groups , type i and type ii . the two groups do not share common structural elements or sequence homology and are therefore considered analogous , rather than homologous , enzymes ; this is probably another example of convergent evolution . type i proteins belong to the /-hydrolase fold superfamily , whereas type ii proteins fall into the ' hot dog ' fold superfamily . to date , four type i and six type ii genes have been identified within the human genome . there are two additional genes , them4 and them5 , which gene products share both structural and sequence homology with other type ii proteins . them4 was identified as having acyl - coa thioesterase activity and should be considered as a new family member . in the future , perhaps them5 should also be considered as a type ii acot family member . recent studies have greatly expanded the current understanding of acyl - coa thioesterases ; however , many precise physiological functions are not completely understood and warrant further investigation . we would like to thank our colleagues for valuable discussion and critically reviewing this manuscript . this work was supported , in part , by the following nih grants ; r01ey17963 and r21aa017754 ( v.v . ) and p30es06096 ( d.w.n . ) .

admixture is an important evolutionary force that can and should be used in efforts to apply genomic data and technology to the study of complex disease genetics . admixture linkage disequilibrium ( ald ) is created by the process of admixture and , in recently admixed populations , extends for substantial distances ( of the order of 10 to 20 cm ) . the amount of ald generated depends on the level of admixture , ancestry information content of markers and the admixture dynamics of the population , and thus influences admixture mapping ( am ) . the authors discuss different models of admixture and how these can have an impact on the success of am studies . selection of markers is important , since markers informative for parental population ancestry are required and these are uncommon . rarely does the process of admixture result in a population that is uniform for individual admixture levels , but instead there is substantial population stratification . this stratification can be understood as variation in individual admixtures and can be both a source of statistical power for ancestry - phenotype correlation studies as well as a confounder in causing false - positives in gene association studies . methods to detect and control for stratification in case / control and am studies are reviewed , along with recent studies showing individual ancestry - phenotype correlations . using skin pigmentation as a model phenotype , implications of am in complex disease gene mapping studies are discussed . finally , the article discusses some limitations of this approach that should be considered when designing an effective am study . the acyl - coa thioesterase gene ( acot ) family encodes enzymes that catalyse the hydrolysis of acyl - coa thioester compounds , also known as activated fatty acids , to their corresponding non - esterified ( free ) fatty acid and coenzyme a ( coash ) . these enzymes play a very important role in lipid metabolism by maintaining cellular levels and proper ratios of free and activated fatty acids , as well as coash . within the acyl - coa family there are two distinct subgroups , type i and type ii . despite catalysing the same reaction , the two groups are not structurally similar and do not share sequence homology , strongly suggesting convergent evolution . this suggestion is further supported if one compares the human with the mouse and rat acot gene families . to date , four human type i acots have been identified which belong to the /-hydrolase fold enzyme superfamily . type ii acots fall into the ' hot dog ' fold superfamily . there are currently six human type ii genes ; however , two homologous proteins , thioesterase superfamily members 4 ( them4 ) and 5 ( them5 ) share common type ii structural features and , in the case of them4 , acyl - coa thioesterase activity -- suggesting that the family may be larger than previously realised . although recent studies have greatly expanded the current understanding of these proteins and their physiological importance , there are a number of members whose functions are relatively unexplored and which warrant further investigation .

a colony was established from soybean and cowpea field collections of n. viridula in stoneville , ms , during the spring and the summer of 2010 . this colony has been maintained in culture for 5 years at the usda - ars national biological control laboratory . after approximately 40 generations in culture , three orange morph adult males were detected in the colony . the orange morph males were paired with green females at a two females per male sex ratio to represent a parent population ( f0 ) , and allowed to reproduce using the rearing methods described by rojas and morales - ramos ( 2014 ) . cages constructed from clear plastic boxes ( l 320 w 260 h 100 mm , part no . 048-c , pioneer packing , dixon , ky ) were modified with ten windows ( 27 mm dia . ) on the sides and four windows ( 65 mm dia . ) on the top all covered with nylon screen ( mesh 500 m ) . all adults in this study were reared in an environmentally controlled room at 26 1 c , 50 5% rh , and 14 h photophase and provisioned with peanuts , broccoli , a diet supplement , and fresh bean pods and a diet supplement that was replaced at 3 days intervals . eggs were collected daily and hatching nymphs ( f1 ) were allowed to develop under same conditions . the number of males and females of each coloration type was recorded for each generation in this study . resulting f1 adults were paired and reared at the same conditions to obtain the f2 generation . resulting f2 adults consisted of green females and green and orange males . only f2 orange males ( app . 25% of the total numbers ) were retained to mate with f2 females ( at two females per male sex ratio ) to produce the f3 . eggs were collected daily and enclosing nymphs were reared to the adult stage under the same conditions described above . because the f3 colony had become considerably larger , only resulting f3 adults from the first three emerging dates were counted and sexed . phenotype and sex frequencies observed in each generation were compared with frequencies expected , assuming that the orange color phenotype was determined by a single sex - linked recessive allele as reported by follett et al . the test was used to compare observed versus expected frequencies within each generation cross . resulting f3 adults were divided to form two different colonies : orange type and green type . the pure orange - type colony was established using solely f3 orange - type adults . the f4 pure orange - type colony was used to generate a pure orange - type f5 . the reproductive potential of the f5 pure orange colony was compared with that of the pure green stock ( parental ) colony . newly emerging f5 orange - type adults were collected and grouped in cohorts of the same emerging date . a total of three cohorts were obtained from the green - type stock colony ( 209 adults total ) and four cohorts from the f5 orange type ( 168 adults total ) . adults were reared as described above and were monitored for oviposition and mortality , which were recorded daily . egg masses collected each day were recorded , labeled , and allowed to develop for 3 days before counting the eggs . embryos in developing eggs at 3 days old showed noticeable red eye coloration , which made them visible and allowed discrimination between fertilized and unfertilized eggs . the fertility ratio was calculated as fertile eggs / total eggs and fertility ratio was compared between color types using the z test . the mean number of eggs per mass was compared between treatments using anova and the student s t test . fertility tables were produced for each type using the methods reported by portilla et al . the f1 generation resulted in only green morphs ( 590 and 684 ) as expected from a recessive orange allele of single gene character . however , the f2 generation produced 672 green females , 351 green males , 298 orange males , and no orange females . these ratios were not significantly different to those expected for a recessive allele of a sex - linked gene ( table 1 ) and were consistent with follett et al . the first three cohorts of the f3 generation consisted of 345 green females , 346 green males , 100 orange females , and 85 orange males . the difference between observed and expected ratios were significant ( =8.067 , df = 3 , p = 0.046 ) ( table 1 ) , but this difference could be explained by significant deviations from the expected sex ratio of the f3 orange types ( |z| = 2.38 , p = 0.0087 ) resulting in a slight bias of 1.17 females per male . a similar female bias sex ratio was observed by follett et al . ( 2007 ) and this bias was explained as higher mortality in males occurring during their development . table 1.expected progeny ratios and observed frequencies in resulting f1 , f2 , and f3 generations after cross of orange males and green females in parent generation ( f0).expected phenotype ratios in progenyobserved phenotype frequencies in progenygeneration : parent and ( progeny)assumed genotypesg g o o g g o o f0 ( f1)xx x xy0.50.500590684006.936f1 ( f2)xx x xy0.50.2500.2567235102984.653f2 ( f3)0.5 xx x xy + 0.5 xx x xy0.3750.3750.1250.125345346100858.067*degrees of freedom ( df ) = 3 ; values with * show significant deviation from expected ratios.f2 green males were removed leaving only orange males to cross with green females . expected progeny ratios and observed frequencies in resulting f1 , f2 , and f3 generations after cross of orange males and green females in parent generation ( f0 ) . degrees of freedom ( df ) = 3 ; values with * show significant deviation from expected ratios . mean oviposition rate ( sem ) measured as eggs per female per day was significantly higher in green - type stock colony females ( 2.76 0.29 ) as compared with f5 orange - type females ( 1.17 0.27 ) ( |t| = 2.456 ; df = 222 ; p = 0.0148 ) . there was no significant difference in the number of eggs per egg mass between the parental stock green ( 27.14 1.26 ) and f5 orange ( 26.39 1.62 ) types . also , the proportion of fertile eggs was not significantly different between green ( 0.718 0.0094 ) and orange ( 0.726 0.0058 ) types . however , green - type females produced significantly higher number of egg masses per day ( 2.09 0.16 ) than orange type females ( 0.71 0.15 ) ( |t| = 6.14 ; df = 222 ; p < 0.0001 ) . the reproductive output of green - type females was higher as measured by the ro value ( 20.67 ) compared with orange - type females ( 13.71 ) ( fig . in addition to the reduced expected phenotypic ratio in natural populations ( consisting mostly of males due to the sex linked and recessive nature of the orange allele ) , the lower reproductive output by orange types may be an explanation for their rarity in natural populations . 1.cumulative lxmx ( reproductive output ) from fertility tables of green and pure orange types of nezara viridula . cumulative lxmx ( reproductive output ) from fertility tables of green and pure orange types of nezara viridula . peritoneal dialysis ( pd ) therapy has increased in popularity since the end of the 1970s . the method was developed as an alternative to hemodialysis ( hd ) presenting a patient survival rate equivalent to hd and better preservation of residual renal function . currently , the two principal causes of technique failure in order of importance are ( a ) peritonitis , this important medical problem can also represent nearly 16% of the causes of death ; ( b ) ultrafiltration failure , a multifactorial complication that can affect up to 40% of patients after 3 years of therapy . these proinflammatory stimuli can induce lymphokine secretion by macrophages , which in turn , activate fibroblasts . fibroblast activation has been associated with structural alterations in the peritoneal membrane of varying intensity . these alterations can be seen in figure 1 which was extracted from a submitted study of our group . in this prospective controlled study in 20 nonuremic wistar rats , peritoneal fibrosis occurs after exposure to glucose - based pd solutions and regardless the use of simvastatin . eps is a clinical syndrome that leads to persistent or recurrent intestinal obstruction , with or without inflammatory parameters of peritoneal thickening , sclerosis , calcification , and encapsulation , and can be inferred by clinical symptoms and radiology , but confirmed only by direct visualization with laparotomy [ 2 , 3 ] . incidence of eps is heterogenous and has been reported to vary from 6 to 20% in eight years depending on the region . causes of inflammation in peritoneal dialysis range from traditional factors to those related to chronic kidney disease per se as well as from the peritoneal dialysis treatment itself . uremia is a factor present in all pd patients and generates an inflammatory state causing stress on the peritoneum due to the formation of carbonyl products . it accelerates the formation of advanced glycation end products ( ages ) that induces an upregulation of the receptors of advanced glycation end products ( rage ) . the peritoneal dialysis catheter is the first proinflammatory factor associated to pd with which the patient comes into contact . after implantation in the peritoneum , the catheter can induce an inflammatory reaction as was demonstrated by flessner et al . . in addition , the catheter can occasionally be the site of bacterial biofilm formation . several pd solutions are available on the market today , and all are , to varying degrees , associated with peritoneal inflammation . such inflammation is generated by several characteristics of these solutions , varying from low ph , presence of lactate , hyperosmolality , increased glucose concentration , presence of glucose degradation products ( gdp ) and advanced glycation end products ( ages ) , and icodextrin metabolites , among others [ 6 , 7 ] . currently available glucose - based pd solutions the glucose load offered daily by a traditional pd prescription usually ranges from 120 g to 400 g. the majority of pd solutions prescribed today markedly acidify ph to nearly 5.7 in approximately 2 to 3 minutes . this ph decreases viability of neutrophils and mesothelial cells , thus decreasing cytokine production and phagocytosis capacity . its bioincompatibility with the peritoneal membrane is well known as well as its capacity to stimulate the production of fibroblast growth factors contributing to peritoneal fibrosis . some studies have associated the osmotic agent with eps development , while others have shown it to be distinct , confirming its safety even with long - term utilization . even experimental studies with rats addressing this question are compromised by the increased -amylase activity in these animals . the presence of this enzyme in plasma and in the peritoneal cavity provokes a rapid drop in peritoneal icodextrin concentration . chronic exposure to high glucose load in traditional pd solution induces significant inflammation of the peritoneal membrane . these solutions induce several proinflammatory factors such as pga , vascular endothelial growth factors ( vegfs ) , fibroblast growth factor ( tgf-1 ) , ages , and upregulation of rages . glucose degradation products ( gdps ) , such as methylglyoxal , glyoxal , and 3-deoxyglucosone generated during the heat sterilization process , increase inflammation by inducing oxidative stress , which thus causes damage to mesothelial cells and leads to apoptosis and mesothelial denudation . substituting traditional solutions for more biocompatible solutions it has been suggested for some years that the pathway of transforming growth factor 1/smad plays a part in the development of peritoneal fibrosis . the latter is recognized as playing a role in angiogenesis , a histological characteristic that allows for differentiation from simple peritoneal fibrosis to eps . the endothelial system is another known factor with potent profibrotic characteristics and plays a role in the development of peritoneal fibrosis . this system can be activated by two receptors , endothelial receptors a and b. however , endothelial receptor b apparently does not play a role in peritoneal membrane thickening in experimental studies inducing deficiency of endothelial receptor b. finally , and of extreme importance , infectious peritonitis is an obvious cause of peritoneal inflammation and is associated with eps development . gram - positive organisms remain as the more prevalent peritonitis agents over the past decades representing up to 60% of cases followed by gram - negative organisms . however , the prevalence of peritonitis due gram - negative organisms is growing fast with the development of efficient strategies to control gram - positive infections . despite all efforts made over the past decades , all the above - mentioned factors contribute to the release of proinflammatory cytokines such as interleukin 1 ( il 1 ) , tumor necrosis factor ( tnf- ) , il-6 , and il-18 . thickening and cubic transformation of mesothelial cells occurs and is more accentuated in the parietal peritoneum . human peritoneal mesothelial cells ( hpmcs ) also suffer structural alterations and prominent transdifferentiation of hpmc to myofibroblasts occurs . histological alterations of the peritoneal membrane observed in eps cases are nonspecific and are masked by the alterations commonly observed in patients with ultrafiltration failure and infectious peritonitis over the long term . the most common findings are fibrin deposition , fibrous capsule formation , perivascular bleeding , interstitial fibrosis , and the presence of tissue granulation with vascular proliferation . submesothelial tissue thickening also occurs with an increase in deposition of mesothelial conjunctive tissue [ 19 , 20 ] . fibrosis is characterized by the accumulation of extracellular matrix ( ecm ) , resulting in disequilibrium between synthesis and degradation . expression of collagen types 1 and 3 is significantly increased as well as collagen type 4 . one of these clinical manifestations is ultrafiltration ( uf ) failure and can occur in up to 30% of patients on pd after five years of treatment . one of the presentations of uf failure occurs due to the increase in pores in the peritoneal membrane , which in turn accelerates small - solute transport dissipating the osmotic gradient necessary to maintain adequate fluid balance . this increase in vascular surface is observed in conjunction with an increase in density of interstitial fibers . these findings help justify the increase in transport of small molecules , while the alterations in the uf coefficient are only moderate . in addition to uf failure , clinical manifestations such as severe malnutrition , subocclusion or intestinal occlusion , and ascites suggest the presence of eps even after discontinuation of pd . prescribing more hypertonic glucose solutions is a common strategy to counter this drop in uf , primarily where there is no available icodextrin . this intensifies and perpetuates inflammatory disturbances , with a direct impact on dialysis adequacy and fluid balance . the final consequence is the inevitable transfer to hd . despite all damage to the peritoneal membrane with therapies performed today , large observational studies have shown an important evolution in pd patient survival when compared to hd over the past years . pd initiation increases inflammatory stimuli for the chronic kidney patient such as the presence of the peritoneal catheter , use of bioincompatible solutions , and possible infectious peritonitis . these manifestations are frequently observed and can range from difficulties in obtaining an adequate fluid balance until the dreaded encapsulant peritoneal sclerosis . an understanding of the mechanisms involved in peritoneal inflammation is fundamental for the development of new strategies . this knowledge can provide not only a better technique survival , but also improvements in patient survival and a better quality of life .

nezara viridula adult coloration can vary , including a rare orange - colored type ( i.e. , n. viridula f. aurantiaca ) . in november 2015 , three nezara viridula males displaying orange coloration were found in an established colony in stoneville , ms . the objectives of this study were to determine if alleles of these orange types conformed to the allele characteristics previously reported for n. viridula f. aurantiaca and to determine if there were any differences in reproductive output compared with the green - colored type . the three orange - type males were crossed with green - type females to produce a hybrid f1 . the f1 progeny was allowed us to cross to produce an f2 . the f2 progeny consisted of 672 green females , 351 green males , 298 orange males , and 0 orange females . these ratios did not differ significantly from the expected 50:25:25:0 ratios for a single recessive sex linked allele for color phenotype . the f2 cross of green females and orange males produced an f3 consisting of 345 green females , 346 green males , 100 orange females , and 85 orange males . these ratios also conformed to the expected ratios ( 0.375:0.375:0.125:0.125 ) with the exception of orange males , which numbers were slightly lower than expected . the pure orange type n. viridula produced significantly less egg masses ( 0.71 0.15 ) per day than green types ( 2.09 0.16 ) and their reproductive output , measured as net reproductive rate ( ro ) , was lower in orange ( 13.71 ) compared with green ( 20.67 ) types . peritoneal dialysis therapy has increased in popularity since the end of the 1970s . this method provides a patient survival rate equivalent to hemodialysis and better preservation of residual renal function . however , technique failure by peritonitis , and ultrafiltration failure , which is a multifactorial complication that can affect up to 40% of patients after 3 years of therapy . encapsulant peritoneal sclerosis is an extreme and potentially fatal manifestation . causes of inflammation in peritoneal dialysis range from traditional factors to those related to chronic kidney disease per se , as well as from the peritoneal dialysis treatment , including the peritoneal dialysis catheter , dialysis solution , and infectious peritonitis . peritoneal inflammation generated causes significant structural alterations including : thickening and cubic transformation of mesothelial cells , fibrin deposition , fibrous capsule formation , perivascular bleeding , and interstitial fibrosis . structural alterations of the peritoneal membrane described above result in clinical and functional changes . one of these clinical manifestations is ultrafiltration failure and can occur in up to 30% of patients on pd after five years of treatment . an understanding of the mechanisms involved in peritoneal inflammation is fundamental to improve patient survival and provide a better quality of life .

the incidence of this cancer varies throughout the world with the highest rates in developed communities ( 1 ) . the gold standard for detection and surveillance of bladder cancers are cystoscopy and urine cytology ( 2 ) . despite its benefit , cytology has low sensitivity , particularly for detection of low grade cancers and cystoscopy is a costly , invasive procedure . hence , there is a need to develop other simple , non - invasive diagnostic methods that can be used to prolong the intervals between cystoscopies and for disease monitoring ( 3 ) . an ideal bladder cancer screening and monitoring test should be non - invasive , easy to perform , and have high sensitivity and specificity . in recent years various markers these include bladder tumor antigen ( bta ) , nuclear matrix protein 22 ( nmp22 ) , survivin , telomerase , and cytokeratins ( cks ) ( 2 , 4 ) . cks are a family of more than 20 intermediate filament proteins expressed in cells of epithelial origin as well as endothelial cells . cks1 - 8 are the type ii group that comprise neutral to basic proteins whereas cks 9 - 20 are a type i group that include acidic proteins ( 5 ) . epithelial tumors largely maintain the features of specific ck expression of their normal epithelial origin ; therefore , these molecules have been extensively used as immunohistochemical markers in diagnostic tumor pathology ( 6 ) . these three markers are expressed by most types of carcinomas , including breast , prostate , lung , colon and ovarian ( 6 , 7 ) . their main use as immunohistochemical markers is to monitor treatment , evaluate response to therapy , and provide early prognostic information on tumor progression and metastasis formation ( 8).circulating cks result from release of intact proteins from rapidly proliferating tumor or dead cells ( 9 ) . several monoclonal anti - cks antibodies are available that recognize ck8 , ck18 and ck19 ( 10 ) . the presence of ck18 fragments in cancerous epithelial cells has been reported ( 5 , 11 ) . termed m30 , a specific monoclonal antibody is available , as well as another one , m65 that recognizes total soluble ck18 fragments . by using these monoclonal antibodies it is possible to determine different circulating forms of ck18 in plasma or serum ( 12 , 13).the m30 antibody detects ck18 fragments that contain a neo - epitope at positions 387 - 396 generated by the action of caspases 3 , 7 and 9 which are activated during the early stages of apoptosis . the m65 antibody also detects cleaved fragments , but can not distinguish between the full - length protein and its fragments ( 14 ) . thus , the m65 enzyme - linked immunosorbent assay ( elisa ) theoretically measures both caspase cleavage ( apoptosis ) and cellular release of intact ck18 ( necrosis ) . the potential diagnostic and prognostic significance of circulating m30 and m65 has been investigated in patients with non - small cell lung cancer ( nsclc ) and in colon cancer ( 15 , 16 ) . their levels correlated with disease stage and recurrence . in other malignancies such as lung cancer ( 17 ) and pancreatic cancer ( 18 ) , high pretreatment ck18 levels indicated a larger tumor burden and less favorable prognosis . a number of clinical trials on breast ( 19 , 20 ) , prostate ( 21 ) , small cell lung ( 22 ) and testicular ( 23 ) cancers used both m30 and m65 as biomarkers of cell death from a variety of different chemotherapeutic agents . it has been claimed that the m30 assay has both predictive value of drug response ( 24 ) and prognostic value for survival ( 17 , 25 ) . m30 and m65 have been used as markers of host tissue toxicity in a number of different clinical conditions such as acute myocardial infarction ( 26 ) , chronic liver disease ( 27 ) and hepatitis c ( 28 ) . however , there is scant information on the prognostic application of these markers in bladder cancer . the current study aims to characterize baseline levels and post - surgical changes of circulating serum m30 and m65 levels in patients with tcc , and to examine their correlation with various clinical features . this study enrolled 60 tcc patients ( 49 male and 11 female ) aged 35 - 85 years who referred to nemazi hospital , shiraz , iran . tumor staging was determined according to the tumor - node - metastases ( tnm ) classification and grading was confirmed by a pathologist . all protocols were approved by the ethics committee at shiraz university of medical sciences and informed consent was obtained from the patients . blood samples were collected before and 7 - 10 days after operation and the obtained sera were stored at -70c until analyzed . the level of ccck18 and total ck18 in sera were determined using the m30- apoptosense elisa and m65-elisa assay kits ( peviva , sweden ) , respectively , according to the protocols described by the manufacturer . briefly , 25 l of standard solutions , low and high controls , and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m5 as catcher antibody . then , 75 l of the diluted horseradish peroxidase ( hrp)-conjugated monoclonal antibody m30 as detector was added . the samples were then incubated for 4 hours at room temperature with constant shaking , after which excess unbound conjugate was removed by five washing steps . color development was then achieved by the addition of 200 l of tmb ( 3 , 3 , 5 , 5-tetramethyl benzidine ) solution , and incubation for 20 minutes in the dark . the reaction was stopped by the addition of 50 l of stop solution and the absorbance measured in elisa reader ( anthos 2020 , australia ) at 450 nm . total level of ccck18 in the samples was measured through plotting a standard curve of known concentrations of the antigen against absorbance . with respect to m65elisa , monoclonal m5 and m6 antibodies directed against two different epitopes of ck18 in both its intact and caspase - cleaved forms was used . twenty five l of standard solution , controls and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m6 , followed by addition of diluted hrp - conjugated monoclonal antibody m5 as detector . the procedure was continued as described for m30 elisa method and then ck18 quantity was calculated through plotting the standard curve . correlation between ccck18 ( m30 ) and ck18 ( m65 ) levels and clinical characteristics of the patients were evaluated . statistical analysis was performed using graphpad prism version5.00 for windows ( graphpad software , san diego , ca , usa ) . relationship between m30 or m65 levels in pre- and post - operative sera were analyzed by paired t - test two - tailed with wilcoxon signed rank test . spearman correlation test was used when determining an association between either m30 or m65 and age , and mann - whitney test was used for the relation between m30 or m65 level or m30:m65 ratio with gender or when comparing the markers between two groups . when there were more than two groups , the nonparametric kruskal - wallis was performed to test for significant differences among the groups . to compare m30 and m65 pre - operative and post - operative levels , unpaired t - test with welch s correction was performed . this study enrolled 60 tcc patients ( 49 male and 11 female ) aged 35 - 85 years who referred to nemazi hospital , shiraz , iran . tumor staging was determined according to the tumor - node - metastases ( tnm ) classification and grading was confirmed by a pathologist . all protocols were approved by the ethics committee at shiraz university of medical sciences and informed consent was obtained from the patients . blood samples were collected before and 7 - 10 days after operation and the obtained sera were stored at -70c until analyzed . the level of ccck18 and total ck18 in sera were determined using the m30- apoptosense elisa and m65-elisa assay kits ( peviva , sweden ) , respectively , according to the protocols described by the manufacturer . briefly , 25 l of standard solutions , low and high controls , and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m5 as catcher antibody . then , 75 l of the diluted horseradish peroxidase ( hrp)-conjugated monoclonal antibody m30 as detector was added . the samples were then incubated for 4 hours at room temperature with constant shaking , after which excess unbound conjugate was removed by five washing steps . color development was then achieved by the addition of 200 l of tmb ( 3 , 3 , 5 , 5-tetramethyl benzidine ) solution , and incubation for 20 minutes in the dark . the reaction was stopped by the addition of 50 l of stop solution and the absorbance measured in elisa reader ( anthos 2020 , australia ) at 450 nm . total level of ccck18 in the samples was measured through plotting a standard curve of known concentrations of the antigen against absorbance . with respect to m65elisa , monoclonal m5 and m6 antibodies directed against two different epitopes of ck18 in both its intact and caspase - cleaved forms was used . twenty five l of standard solution , controls and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m6 , followed by addition of diluted hrp - conjugated monoclonal antibody m5 as detector . the procedure was continued as described for m30 elisa method and then ck18 quantity was calculated through plotting the standard curve . correlation between ccck18 ( m30 ) and ck18 ( m65 ) levels and clinical characteristics of the patients were evaluated . statistical analysis was performed using graphpad prism version5.00 for windows ( graphpad software , san diego , ca , usa ) . relationship between m30 or m65 levels in pre- and post - operative sera were analyzed by paired t - test two - tailed with wilcoxon signed rank test . spearman correlation test was used when determining an association between either m30 or m65 and age , and mann - whitney test was used for the relation between m30 or m65 level or m30:m65 ratio with gender or when comparing the markers between two groups . when there were more than two groups , the nonparametric kruskal - wallis was performed to test for significant differences among the groups . to compare m30 and m65 pre - operative and post - operative levels , unpaired t - test with welch s correction was performed . sera from 60 patients with tcc of the bladder were assessed for m30 and m65 serum levels . abbreviation : nd , not determined . a wide range of m30 values ( 25 - 724 u / l ) was observed in total patients , with a mean of 148 16 u / l , median : 118 u / l ) and females ( 152 50 u / l , median : 122 u / l ) . patients were categorized into three groups according to age : 35 - 49 , 50 - 69 and 70 years . the levels of the soluble m30 and m65 molecules was determined in patients sera pre- ( n = 60 ) and post - operation ( n = 26 ) by elisa method . differences between pre- and post - operative m30 and pre- and post - operative m65 were not significant ( ns ) . m65 levels also showed a wide range of values ( 80 - 1375 u / l ) with a mean of 318 34 the m65 level in males was 305 35 u / l compared to females ( 376 108 u / l ; p = 0.53 ) . the level of this molecule significantly correlated with age ( p = 0.03 , spearman r = 0.27 ) and appeared to increase with age . as shown in table 1 , the majority of patients had stages t1 and t2 ( 61.7 % ) and grades iii and iv ( 55% ) tumors . figure 2 shows the results of m30 and m65 analyses in patients according to tumor stage . as shown , there were no significant differences between m30 levels in patients with different tumor stages . similarly , the level of m30 showed no significant difference in patients with various tumor grades ( figure 3 ) . in contrast to m30 , m65 levels showed a significant relation to tumor stage and grade . as shown in figure 2 , the quantity of this molecule in patients with t3/t4 stages ( 350 42 u / l ) was higher than patients with t1/t2 stages ( 293 45 u / l ; p < 0.038 ) . patients with tumor grades iii / iv had higher levels of m65 ( 383 58 u / l ) compared to those with grades i / ii tumors ( 231 26 u / l ; p = 0.04 ; figure 3 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor stages ( p= 0.038 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor grades ( p= 0.04 ) . as shown in figure 4 , this ratio for all patients was 0.54 0.04 . there was no significant difference in ratio between males ( 0.55 0.05 ) and females ( 0.46 0.09 ) . this ratio was 0.84 0.20 for patients aged 35 - 49 years , 0.53 0.04 for 50 - 69 year - old patients and 0.41 0.04 for patients 70 years of age ( p < 0.02 ) . this ratio was 0.61 0.04 in patients with stage t1 , which decreased to 0.260.008 in patients with stage t4 disease ( p < 0.002 ) . as shown in figure 5 , the m30:m65 ratio was lower in patients with t3/t4 stages ( 0.38 0.04 ) compared to those with t1/t2 stages ( 0.59 0.06 ; p < 0.002 ) . similarly , we observed a lower m30:m65 ratio in patients with grades iii / iv ( 0.48 0.07 ) compared to grades i /ii tumors ( 0.64 0.04 ; p < 0.01 ; figure 5 ) . data presented the mean m30:m65 ratio standard error in all patients according to sex and age . a significant higher m30:m65 ratio in younger group of patients ( 30 - 49 years ) vs. older ones ( 70 years ) was observed ( p = 0.02 ) . data presented the mean m30:m65 ratio standard error in patients with different stages and grades . a significant lower m30:m65 ratio in patients with higher stages ( p = 0.002 ) and grades of tcc ( p = 0.01 ) was observed . as shown in figure 1 , the m30 level in patients after surgery was 133 19 u / l which showed no significant difference with its pre - operative value ( 148 30u / l ) in this group of patients . the corresponding value for post - operative m65 levels was 240 21 u / l compared to its pre - operative level ( 319 63 there was a correlation between pre - operative m30 and m65 ( p < 0.0001 , spearman r = 0.51 figure 6a ) and post - operative m30 and m65 values ( p < 0.02 , spearman r = 0.45 ; figure 6b ) . however , we found no significant difference in m30 or m65 levels before and after surgery . the m30:m65 ratios before and after surgery showed a significant correlation in older patients ( 70 years ; p = 0.009 ) . a significant correlation between pre - operative m30 and m65 levels ( p < 0.0001 , spearman r = 0.51 ) and post - operative values ( p = 0.023 , spearman r = 0.45 ) was observed . a wide range of m30 values ( 25 - 724 u / l ) was observed in total patients , with a mean of 148 16 u / l , median : 118 u / l ) and females ( 152 50 u / l , median : 122 u / l ) . patients were categorized into three groups according to age : 35 - 49 , 50 - 69 and 70 years . the levels of the soluble m30 and m65 molecules was determined in patients sera pre- ( n = 60 ) and post - operation ( n = 26 ) by elisa method . differences between pre- and post - operative m30 and pre- and post - operative m65 were not significant ( ns ) . m65 levels also showed a wide range of values ( 80 - 1375 u / l ) with a mean of 318 34 u / l compared to females ( 376 108 u / l ; p = 0.53 ) . the level of this molecule significantly correlated with age ( p = 0.03 , spearman r = 0.27 ) and appeared to increase with age . as shown in table 1 , the majority of patients had stages t1 and t2 ( 61.7 % ) and grades iii and iv ( 55% ) tumors . figure 2 shows the results of m30 and m65 analyses in patients according to tumor stage . as shown , there were no significant differences between m30 levels in patients with different tumor stages . similarly , the level of m30 showed no significant difference in patients with various tumor grades ( figure 3 ) . in contrast to m30 , m65 levels showed a significant relation to tumor stage and grade . as shown in figure 2 , the quantity of this molecule in patients with t3/t4 stages ( 350 42 u / l ) was higher than patients with t1/t2 stages ( 293 45 u / l ; p < 0.038 ) . patients with tumor grades iii / iv had higher levels of m65 ( 383 58 u / l ) compared to those with grades i / ii tumors ( 231 26 u / l ; p = 0.04 ; figure 3 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor stages ( p= 0.038 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor grades ( p= 0.04 ) . we determined the ratio of m30 to m65 for each patient . as shown in figure 4 , this ratio for all patients was 0.54 0.04 . there was no significant difference in ratio between males ( 0.55 0.05 ) and females ( 0.46 0.09 ) . this ratio was 0.84 0.20 for patients aged 35 - 49 years , 0.53 0.04 for 50 - 69 year - old patients and 0.41 0.04 for patients 70 years of age ( p < 0.02 ) . this ratio was 0.61 0.04 in patients with stage t1 , which decreased to 0.260.008 in patients with stage t4 disease ( p < 0.002 ) . as shown in figure 5 , the m30:m65 ratio was lower in patients with t3/t4 stages ( 0.38 0.04 ) compared to those with t1/t2 stages ( 0.59 0.06 ; p < 0.002 ) . similarly , we observed a lower m30:m65 ratio in patients with grades iii / iv ( 0.48 0.07 ) compared to grades i /ii tumors ( 0.64 0.04 ; p < 0.01 ; figure 5 ) . data presented the mean m30:m65 ratio standard error in all patients according to sex and age . a significant higher m30:m65 ratio in younger group of patients ( 30 - 49 years ) vs. older ones ( 70 years ) was observed ( p = 0.02 ) . data presented the mean m30:m65 ratio standard error in patients with different stages and grades . a significant lower m30:m65 ratio in patients with higher stages ( p = 0.002 ) and grades of tcc ( p = 0.01 ) was observed . as shown in figure 1 , the m30 level in patients after surgery was 133 19 u / l which showed no significant difference with its pre - operative value ( 148 30u / l ) in this group of patients . the corresponding value for post - operative m65 levels was 240 21 u / l compared to its pre - operative level ( 319 63 there was a correlation between pre - operative m30 and m65 ( p < 0.0001 , spearman r = 0.51 figure 6a ) and post - operative m30 and m65 values ( p < 0.02 , spearman r = 0.45 ; figure 6b ) . however , we found no significant difference in m30 or m65 levels before and after surgery . the m30:m65 ratios before and after surgery showed a significant correlation in older patients ( 70 years ; p = 0.009 ) . a significant correlation between pre - operative m30 and m65 levels ( p < 0.0001 , spearman r = 0.51 ) and post - operative values ( p = 0.023 , spearman r = 0.45 ) was observed . in the present study we have examined sera from tcc patients for levels of circulating m30 and m65.during apoptosis ck18 is cleaved by caspases and subsequently released into the extracellular environment and blood ( 9 , 29 ) . these fragments can be detected by elisa using the m30 monoclonal antibody which recognizes the ck18asp396 neo - epitope . the m65 assay is based on two antibodies , m5 and m6 , directed against two different epitopes of ck18 . all ck18 fragments that contain epitopes in the 300 to 390 amino acid region of the protein are recognized . in addition to apoptosis , m65 is thought to measure intact ck18 released during cell necrosis ( 30 - 32 ) . therefore , ck18 levels can be considered as a surrogate marker of cell death activity in tumors and non - tumor conditions . determining its level in patients can be useful for diagnosis of tumor recurrence , prognosis and monitoring . additionally , in some experiments circulating ck18 has been used to assess the efficiency of different anticancer drugs during chemotherapy ( 14 ) . olofsson et al . suggested that the ck18 marker could be useful for early prediction of the response to chemotherapy in breast cancer and a useful biomarker for clinical trials ( 33 ) . circulating ck18 was considered as a biomarker of chemotherapy - induced cell death in testicular cancer ( 23 ) . post - surgical plasma ck18 levels showed a correlation with tumor recurrence and presence of residual disease in colorectal cancer ( 34 ) . the first report of using ck18 as a diagnostic value in tcc patients was in 2002 by ramazan sekeroglu et al . these researchers used a solid - phase two - site chemiluminescence assay to measure ck18 levels . the results suggested that serum ck18 could not be a diagnostic or screening tool in early stages of bladder cancer , but was helpful in diagnosis of higher tumor stages . they determined that a significant relationship between urinary nmp 22 , a tumor marker of bladder cancer , and ck18 levels existed which suggested that nmp22 and ck18 were useful markers for diagnosis and monitoring of tcc . levels of urinary ck18 significantly differed according to pathological grade and stage of patients tumors ( 36 ) . in the phase i study of intravesical adenoviral transduction of human bladder cells with human interferon- ( ad - ifn- ) treatment in patients with bladder cancer ( 37 ) , significant apoptosis and necrosis in the patients tumors this study was the first to suggest that analysis of urinary m30 and m65 levels might be used as a potential surrogate biomarker for tumor cell death and prognosis after treatment of non - muscle invasive bladder cancer with any therapeutic agent . however , to the best of our knowledge no studies examined both m30 and m65 levels in serum of tcc patients . in the current study , we evaluated m30 and m65 serum levels in a group of iranian patients with tcc . we sought to have an insight regarding the relationship of these markers to patient characteristics and prognostic factors such as tumor stage and grade . moreover , we measured the changes in the quantity of these markers in a number of our patients after surgery to determine their value for disease monitoring . the results of the study showed a significant correlation between m30 and m65 levels in patients prior to surgery . the levels of m65 , but not m30 , were significantly related to stage and grade of patients tumors which emphasized the importance of cell necrosis in tcc biology . higher levels of m65 in patients with greater stages and grades might suggest the relation of this biomarker with tumor progression . these results were consistent with results obtained by ramazan sekeroglu et al . who reported that serum ck18 could be helpful in the diagnosis of higher stage tumors ( 35 ) . in previous studies , serum levels of m30 and/or m65 significantly correlated with tumor stage in breast ( 24 ) and colorectal cancers ( 38 ) . unlike these studies , ozturk et al . observed no difference in serum m30 and m65 levels between patients with stages iii and iv of locally advanced head and neck tumors ( 39 ) . . showed that in colorectal cancer patients , despite the tendency for m65 to decrease with increasing tumor grade , differences between the groups did not reach statistical significance as withm30 ( 34 ) . we measured post - operative m30 and m65 levels to determine a possible relation of these marker levels with tumor burden . ( 38 ) on colorectal cancer patients that showed good correlation with m30 and m65 levels in the plasma of patients before and after surgical resection . in addition , we measured m30 and m65 levels only once after surgery ; possibly , by measuring these markers at different time intervals and in a higher number of patients , different results would be obtained . various studies conducted on different tumors compared the extent of apoptosis to total cell death by calculating the m30:m65 ratio . this ratio might be an important factor to select an appropriate treatment strategy for patients . the m30:m65 ratio decreased in endometrial cancer stages iii and iv when compared with stage ii , which indicated less apoptosis and/or more necrosis during tumor progression ( 14 ) . in colorectal cancer ( 38 ) our results showed a relationship between this ratio and age , tumor stage and grade . this finding was in line with the positive correlation obtained in this study between m65 levels and age which might suggest a predominance of apoptosis in younger patients versus necrosis in older patients . the pre - operative m30:m65 ratio has shown a tendency to decrease with increase in tumor stage and tumor grade . in this regard , because m30 is considered an indicator of apoptosis , it can be assumed that in tcc patients with more aggressive tumors the rate of apoptosis may be lower than those with less aggressive tumors and cell death is mostly due to necrosis in these types of tumors . we did not find a significant difference between the pre- and post - operative m30:m65 ratio in total patients ( p = 0.08 ) . however , a correlation between the pre - and post - operative ratio in patients 70 years was observed . in conclusion , the serum levels of m65 , but not m30 , showed a significant correlation with stage and grade of patients tumors . the pre - operative m30:m65 ratio has shown a tendency to decrease with increase in tumor stage and tumor grade . the significantly decreased ratio after surgery in the older group of patients may imply the importance of this ratio for tumor monitoring in this group of patients . further studies on a larger number of patients along with follow - up of the patients for tumor recurrence and presence of residual disease will determine the exact value of these markers for tcc monitoring . spinal dural arteriovenous fistula ( sdavf ) is a rare neurological disease with an estimated frequency of 510 cases per million per year . it is caused by a subdural shunt between a radiculomeningeal artery and a radicular vein , leading to impaired venous drainage from the spinal cord and the arterialization of the vein due to increased venous pressure . the time to diagnosis is often delayed due to unspecific neurological complaints to as long as 1015 years after the onset of symptoms . therefore , 40% of patients are severely disabled at the time of diagnosis . the most common location of an sdavf is in the lower thoracic and lumbar region and it is accompanied by complaints in the lower extremities . sdavf at cervical level ( c - sdavf ) is extremely rare , accounting for approximately 2% of the sdavf patients and can lead to quadriplegia and respiratory insufficiency . early clinical features are nonspecific and include gait claudication , worsened by exercise . by the time of the diagnosis , the triad consisting of ( 1 ) motor deficits ( leg / arm weakness , muscle cramps ) , ( 2 ) sensory disturbances ( paresthesia , numbness , pins and needles sensation , back pain ) , and ( 3 ) cauda syndrome ( micturition problems , erectile dysfunction , anal sphincter disturbances ) are present in 70% of the patients . characteristic mri features include ( 1 ) engorged perimedullary veins , ( 2 ) central medullary edema , and ( 3 ) swelling of the caudal segments of the spinal cord . in the absence of the findings , diagnosis is difficult to make solely based on clinical symptoms and is usually erroneous . differential diagnoses may include medullary pathology ( myelitis , medullary tumors , disc herniation ) or peripheral pathology ( sensory polyneuropathy , chronic inflammatory demyelinating polyneuropathy ) ( table 1 ) . evidence of bladder or bowel dysfunction which share no evident relation with the site of medullary pathology could be very useful in suggesting the diagnosis . the association of cauda symptoms should be considered a red flag in sdavf and can guide the physician toward the correct diagnosis . the treatment involves either superselective catheterization with endovascular embolization of the fistula or neurosurgical occlusion of the arterialized vein . if successful , the treatment usually stops the progression of symptoms or leads to an improvement of neurological deficits . therefore , it is of utmost importance that the diagnosis is established as soon as possible in order to avoid permanent handicap . a 56-year - old man complained of sensory symptoms ( pins and needles sensation ) and pain in both legs and the right arm , which progressively worsened in about 4 months . the patient had difficulty performing his daily work as a bus driver and noticed impaired foot sensation and arm weakness during changing gears and pressing the pedals . the patient 's past medical history was unremarkable except for hypertension and hypercholesterolemia , both treated with statins and oral antihypertensive drugs . neurological examination showed generalized weakness in both lower limbs and the right arm ( medical research council grade 4 ) with hyperreflexia and bilateral plantar reflexes . sensory exam showed generalized sensation loss ( involving both dorsal columns and the spinothalamic tract ) and subjective pins and needles sensations in both feet and the right arm . given the association of both lower and upper limb involvement and hyperreflexia , a cervical lesion was suspected . the patient underwent diagnostic brain and spinal cord mri , which showed an elongated intramedullary hyperintensity from the foramen magnum to c4 on t2-weighted images ( fig 1 ) with a small contrast enhancement at the junction between the brainstem and the cervical spinal cord . transverse myelitis was suspected and the patient was treated with a high intravenous corticosteroid dose [ 7 , 8 , 9 , 10 ] . the patient developed quadriplegia with severe generalized weakness and sensation loss in both legs and both arms , inspiratory muscle weakness , dysphonia , and high urinary retention . an allergic reaction was excluded and the patient was transferred to an intensive care unit for assisted breathing . a cervical mri was performed showing varicose dilatations , irregular cervical veins , and central medullary edema ( fig 2 ) characteristic of a cervical sdavf [ 4 , 5 , 6 ] . superselective angiography confirmed the presence of an sdavf and the patient successfully underwent open spine surgery with ligation and coagulation of the arterialized communicating vein . ten days after the surgical treatment , he almost completely regained neurological functions and was further referred to an intensive rehabilitation program in a specialized clinic . by the time of discharge from the neurology ward 2 weeks after surgery , he could walk more than 30 m with rollator support and only had mild numbness in the feet and some difficulty voiding . sdavf may present with a wide range of clinical features such as motor and sensory deficits often associated with a cauda equina syndrome . neurological deficits are due to perimedullary venous congestion leading to intramedullary edema or ischemia in severe cases or direct compression of the nerve roots at the level of the fistula . abnormal communication between a radiculomeningeal artery and a radicular vein leads to increased venous pressure in the dural sleeve , impairing normal venous return from the intramedullary capillaries from and below the level of the fistula , causing medullary edema . furthermore , decreased pressure gradient at capillary level accounts for decreased gas exchange and medullary hypoxia . an acute increase in venous hypertension due to sudden volume or pressure overload may exacerbate neurological deficits by hampering the already decreased venous drainage at the level of the sdavf causing transitory medullary ischemia . previous publications report clinical worsening related to an acute increase in intrathoracic and venous pressure such as during singing , valsalva maneuvers , or abdominal muscle compression during exercise . in addition to the current paper , several publications report rapid worsening of symptoms in patients receiving corticosteroids intravenously for other presumed diagnoses ( most commonly myelitis ) [ 12 , 13 , 14 , 15 , 16 ] . although the exact mechanism which leads to worsening of spinal cord dysfunction is not yet understood , it is possible that both corticosteroid and intravenous saline in which these are administered could play a role . in addition to anti - inflammatory effects , corticosteroids can also play a role in regulating water and mineral metabolism through their mineralocorticoid effects , causing fluid retention . this can compromise the already fragile venous return into the dural sac , leading to medullary hypoxia and further worsening the sdavf symptoms . furthermore , steroids administered via an intravenous line placed in the upper extremity eventually drain via the cephalic veins into the brachiocephalic veins . the azygos and semiazygos veins , which collect venous blood from the spinal cord , are also tributaries of the brachiocephalic veins . therefore , a rapid rise in venous return from the upper extremities into the brachiocephalic vein could also directly diminish the venous return from the medullary level and exacerbate symptoms . this theory is supported by the mri findings before and after corticoid treatment in our patient . the cervical mri before intravenous treatment shows no evident perimedullary venous abnormalities , whereas after treatment , engorged veins can clearly be identified in the subdural space , as evidence of acute venous hypertension in the subdural sac ( fig 3 ) . we would like to conclude this study by stressing out the importance of early diagnosis and treatment in patients with sdavf . therefore , the presence of red flags can be extremely useful in guiding the physician toward the correct diagnosis . acute neurological worsening after intravenous steroid administration for any presumed diagnosis should be considered such a red flag and raise the suspicion of an sdavf .

backgroundvarious markers are suggested for diagnosis and monitoring of transitional cell carcinoma of the bladder ( tcc ) , including cytokeratins ( cks).objectivesin the present study , the circulating ck18 ( m65 ) and its caspase - cleaved form , ccck18 ( m30 ) , have been investigated in a group of patients with tcc.patients and methodsserum samples were obtained from 60 patients before surgical resection , among which the samples of 26 patients after resection were also included . we measured the levels of soluble m30 and m65 molecules by enzyme - linked immunosorbent assay . the relation between these markers and patients clinical characteristics was evaluated.resultsm30 and m65 in total patient sera were 148 16 u / l and 318 34 u / l , respectively . a correlation existed between pre - operative m30 and m65 levels ( p < 0.0001 , spearman r = 0.51 ) . m65 , but not m30 , showed a significant relation to tumor stage and grade . the m65 quantity in patients with t3/t4 tumor stages ( 350 42 u / l ) was higher than that of patients with t1/t2 stages ( 293 45u / l ; p < 0.038 ) . patients with tumor grades iii / iv also showed higher levels of m65 compared to patients with tumor grades i / ii ( p < 0.04 ) . the m30:m65 ratio in all patients was 0.54 0.04 . there was a lower m30:m65 ratio in patients with t3/t4 stage tumors and those with tumor grades iii / iv ( p < 0.02 ) . the m30 ( 133 19 u / l ) and m65 levels ( 240 21 u / l ) after surgery did not significantly differ compared to their pre - operative values . however , a correlation between the pre- and post - operative m30:m65 ratio in patients 70 years was seen ( p = 0.009).conclusionsthese data suggested a relationship of both m65 and the m30:m65 ratio to tumor progression which might imply their importance in tcc monitoring . subdural arteriovenous fistula ( sdavf ) is a rare condition characterized by clinical manifestations ranging from mild bilateral sensory deficits to quadriplegia . the diagnosis is often delayed due to unspecific neurological symptoms , initially diagnosed as polyneuropathy or myelopathy . the diagnosis can be delayed for as long as 115 years . the following report describes a cervical sdavf case initially misdiagnosed as myelitis transversa and treated with intravenous steroids . a 56-year - old male presented with sensory deficits and mild leg and right arm weakness . cervical mri showed a central medullary hyperintense lesion with contrast enhancement . after metabolic , infectious , and malignant causes were excluded , myelitis transversa was presumed and the patient was treated intravenously with methylprednisolone . shortly after that , he developed quadriplegia . cervical mri imaging showed engorged cervical perimedullary vessels , which were not visible on the initial mri . the diagnosis was revised and a sdavf identified . prompt surgical treatment led to a complete recovery . the effect of intravenous steroids in sdavf is controversial . acute clinical worsening after steroid administration is previously reported in several publications ; however , due to the paucity of clinical studies on sdavf , this effect remains mostly overlooked or unknown . the findings in this patient support the causative relation between sdavf clinical worsening and steroid administration . we propose that acute clinical worsening under steroids in patients initially diagnosed with myelitis should raise suspicion of an sdavf .

the world health organization ( who ) has estimated that about 17 million people die annually from cardiovascular disease worldwide , which accounts for approximately 33% of all deaths . in addition , 9.4 million deaths are attributed to complications of hypertension . according to who statistics in 2008 , hypertension was diagnosed in almost two - fifths of people aged 25 years and older worldwide . if current trends continue , the number of patients with hypertension will increse by 500 million and reach a total of 1.56 billion in 2025 . there are about 639 million hypertensive people , which equals almost 75% of the population in developing countries with limited resources . these hypertensive persons also have a very low awareness of their condition , as well as poor blood pressure ( bp ) control . non - optimal bp control accounts for 66.6% of all strokes and 50% of coronary artery disease cases . furthermore , hypertension is known to be one of the main risk factor of deaths in south asia . according to a study conducted between december 2011 and march 2012 in kabul , afghanistan the prevalence of hypertension was 46.2% for the age group of 40years and older . several epidemiological studies have reported an association between proteinuria and a poor prognosis in hypertensive patients . the burden of proteinuria and its association with hypertension have not been studied in the adult hypertensive population of afghanistan . therefore , this study was aimed at determining the prevalence of proteinuria , and the association of different variables with proteinuria , among adult hypertensive patients attending an outpatient clinic in afghanistan . five hundred fifty - five patients with a high bp recorded at an outpatient clinic in andkhoy , afghanistan from december 2014 to may 2015 . the case subjects included patients aged 18 years and older , who were receiving treatment for newly diagnosed arterial hypertension , defined as seated systolic / diastolic hypertension 140/90 mm hg at rest on the day of the study visit . patients who had engaged in strenuous physical activity in the preceding 24 h , as well as female participants who were pregnant or menstruating , were ineligible because of the likely presence of false - positive results . the subjects were evaluated during their clinic visit , and the study protocol included assessment of data obtained from each patient , including demographic characteristics , body mass index ( bmi ; weight in kilograms divided by height in meters squared ) , bp pattern , cardiovascular history , presence of cardiovascular risk factors , comorbidity and current drug - therapy . the study was approved by the scientific review committee of balkh regional hospital . for this study , diabetes was defined as a fasting blood sugar level of 126 mg / dl , at the visit or self - reported use of hypoglycaemia medications . the results were obtained by using the reagent tests strips of the accu - trend plus kit for cholesterol / glucose ( roche diagnostic usa ) . the definitions of obesity varies across studies , even among from the same country . for this study , overweight was defined as a bmi of 25 to < 30 kg/ m and obesity as bmi 30 . dipstick screening for proteinuria and glycosuria were performed with the reagent strips ( roche diagnostic usa ) . the urinary protein levels of dipstick urinary analyses were recorded as follows ; negative ( ) , trace ( + / ) or proteinuria ( 1 + , 2 + , 3 + or 4 + ) . cigarette smoking was assessed through a face to face interview . patients were grouped into three categories : ( i ) patients who had smoked 100 cigarettes in his or her lifetime and currently smokes cigarettes ( current smoker ) ; ( ii ) patients who had smoked at least 100 cigarettes in his or her lifetime but had stopped smoking at the time of the interview ( past smokers ) ; ( iii ) patients who have never smoked , or who had smoked < 100 cigarettes in his or her lifetime ( non - smoker ) . other factors that were considered were regular aerobic physical activity a minimum of 30 min for 5 days / week or vigorous physical activity for a minimum of 20 min for 3 days / week . the results from this measurement were entered onto each patient s case report form . univariate and multivariate logistic regression analyses were performed to observe the association between proteinuria and the risk factors as well as target organ lesions . odds ratios ( ors ) with 95% confidence intervals ( cis ) were estimated by using a logistic regression model . all the prognostic valuables with p < 0.05 in a univariate logistic regression analysis were entered into a multivariate logistic regression analysis in order to determine independent predictors . a p value of < 0.05 was considered statistically significant . all analyses were performed with the spss 20.0 software package ( spss , chicago , il , usa ) . five hundred fifty - five patients with a high bp recorded at an outpatient clinic in andkhoy , afghanistan from december 2014 to may 2015 . the case subjects included patients aged 18 years and older , who were receiving treatment for newly diagnosed arterial hypertension , defined as seated systolic / diastolic hypertension 140/90 mm hg at rest on the day of the study visit . patients who had engaged in strenuous physical activity in the preceding 24 h , as well as female participants who were pregnant or menstruating , were ineligible because of the likely presence of false - positive results . the subjects were evaluated during their clinic visit , and the study protocol included assessment of data obtained from each patient , including demographic characteristics , body mass index ( bmi ; weight in kilograms divided by height in meters squared ) , bp pattern , cardiovascular history , presence of cardiovascular risk factors , comorbidity and current drug - therapy . the study was approved by the scientific review committee of balkh regional hospital . for this study , diabetes was defined as a fasting blood sugar level of 126 mg / dl , at the visit or self - reported use of hypoglycaemia medications . the results were obtained by using the reagent tests strips of the accu - trend plus kit for cholesterol / glucose ( roche diagnostic usa ) . the definitions of obesity varies across studies , even among from the same country . for this study , overweight was defined as a bmi of 25 to < 30 kg/ m and obesity as bmi 30 . dipstick screening for proteinuria and glycosuria were performed with the reagent strips ( roche diagnostic usa ) . the urinary protein levels of dipstick urinary analyses were recorded as follows ; negative ( ) , trace ( + / ) or proteinuria ( 1 + , 2 + , 3 + or 4 + ) . cigarette smoking was assessed through a face to face interview . patients were grouped into three categories : ( i ) patients who had smoked 100 cigarettes in his or her lifetime and currently smokes cigarettes ( current smoker ) ; ( ii ) patients who had smoked at least 100 cigarettes in his or her lifetime but had stopped smoking at the time of the interview ( past smokers ) ; ( iii ) patients who have never smoked , or who had smoked < 100 cigarettes in his or her lifetime ( non - smoker ) . other factors that were considered were regular aerobic physical activity a minimum of 30 min for 5 days / week or vigorous physical activity for a minimum of 20 min for 3 days / week . the results from this measurement were entered onto each patient s case report form . univariate and multivariate logistic regression analyses were performed to observe the association between proteinuria and the risk factors as well as target organ lesions . odds ratios ( ors ) with 95% confidence intervals ( cis ) were estimated by using a logistic regression model . all the prognostic valuables with p < 0.05 in a univariate logistic regression analysis were entered into a multivariate logistic regression analysis in order to determine independent predictors . a p value of < 0.05 was considered statistically significant . all analyses were performed with the spss 20.0 software package ( spss , chicago , il , usa ) . the mean age of the patients was 57.9 13.3 years ( range 1892 years ) . most of the patients ( 59.1% ) were aged between 40 and 65 years , 222 were men ( 40.0% ) and 333 were women ( 60.0% ) . the prevalence of current smokers among female patients ( 18.6% ) was significantly ( p < 0.001 ) lower than that among male patients ( 37.8% ) . the prevalence of proteinuria was 67.2% and considerably higher in male than in female patients ( p < 0.001 ) . among all patients , 14.9% had a family history of ischemic heart disease , 14.8% had family history of diabetes , and 53.4% had a family history of hypertension . of these patients , 26.3% were current smokers , 33.7% had a bmi of 25 , 78.2% had uncontrolled bp , and 59.2% had regular physical exercise . fequency distribution of sociodemographic characteristics of study participants ( n=555 ) the mean age of patients in the proteinuria and non - proteinuria groups was 62.0 13.6 years and 57.7 13.4 years , respectively . the male - to - female ratio in the proteinuria group was 1.15 and that in the nonproteinuria group was 2.81 . smokers had higher rates of proteinuria than non - smokers 30.7% vs. 17.5% ( p = 0.03 ) . patients with proteinuria had substantially increased levels of both systolic bp and diastolic bp ( 155.0 24.8 vs.146.2 24.0 mm hg , and 94.6 13.8 vs. 88.6 10.8 mm hg ; p < 0.001 , respectively ) . the fasting blood glucose levels were 115.1 34.9 versus 104.5 23.7 ; p = 0.001 ) . the prevalence of proteinuria was significantly higher among diabetic patients than non - diabetic patients ( 14.7% vs. 3.8% ; p < 0.001 ) . patients with myocardial infarction had significantly higher proteinuria levels than those without myocardial infarction ( 25.7% vs.13.2% ; p = 0.001 ) . proteinuria was more common in patients with heart failure ( 15.2% vs. 6.6% ; p = 0.04 ) . physical activity was inversely associated with proteinuria ( 45.0% vs. 25.9% ; p < 0.001 ) . there was also a considerable difference with regard to use of angiotensin - receptor blockers ( arbs ) , angiotensin - converting enzyme ( ace ) inhibitors , calcium channel blockers ( ccbs ) , and beta blockers ( bbs ) between patients with and those without proteinuria ( table 2 ) . comparison of factors associated with proteinuria among hypertensive patients multivariate logistic regression analysis was used to determine the association between proteinuria and smoking , heart failure , myocardial infarction , diabetes mellitus , stroke , hypercholesterolemia , and regular physical activity . the odds of proteinuria among smokers were 1.88 times greater than those in non - smokers ( 95%ci 1.102.95 ) . patients with heart failure had a more than two - fold increase in the odds of proteinuria than those without heart failure ( 95%ci 1.134.45 ) . patients aged 65 years showed a 1.02 times increase of in the odds of proteinuria than those < 65 years ( 95%ci 1.001.04 ) . the odds of proteinuria among diabetic patients were 3.41 times higher than those in patients without diabetes ( 95%ci 1.497.80 ) . myocardial infarction , regular physical activity and diabetes mellitus were other variables that had a significant association with proteinuria in the univariate logistic regression model , however , in the multivariate model , this association was not significant ( table 3 ) . there has been a lack of data about the prevalence of proteinuria in afghanistan , which is important information on the evaluation of hypertensive patients . the presence of proteinuria helps in identifying patients who are at high risk and need to receive aggressive treatment for risk factors . the prevalence of proteinuria among patients with hypertension in the northern part of afghanistan was 67.2% . a similar prevalence , 67.8% was reported in morocco , as part of an i - search ( survey for evaluating microalbuminuria routinely by cardiologists in patients with hypertension ) study , in which the data were collected from 40 cardiology centers with a population of 476 patients . in another i - search study performed in turkey in an international multi - center the study was designed to evaluate the frequency of microalbuminuria , which was observed in a large number of outpatient clinics , in a total of 21,050 patients that were receiving treatment for hypertension in 26 countries . however , in our study , the prevalence of proteinuria was higher than that seen in another study from a tertiary hospital in uganda ( 39.5% ) . these differences are most likely associated with the different criteria used in patient selection , such as age , severity of hypertension , race , and coexisting cardiovascular risk factors ( e.g. , diabetes mellitus , renal disease , dyslipidemia , and obesity ) , as well as the technique used for proteinuria determination . moreover , in a study conducted in 26 countries , the highest rates of microalbuminuria were observed in asia . the prevalence of smoking among female patients was lower than that among male patients . a similar this low prevalence of smoking among female patients may due to social factors that discourage smoking in women . in our study , we found a significant association between proteinuria and the male which is comparable with the results of others studies . this discrepancy is most probably due to a combination of risk factors ( i.e. , diabetes , smoking , and obesity ) and the fact that ischemic heart disease is more frequent in men than in women . in the present investigation , we found the association between high systolic bp and diastolic bp and proteinuria . a similar result was obtained by ya - pan et al . who found that an increase in systolic bp and diastolic bp was related to a prevalence of proteinuria , however , al - saffar et al . the likely reason might be due to a substantial reduction in renal perfusion , which leads to proteinuria . according to our study there was a substantial statistically difference between proteinuria and patient age , which is in agreement with other studies . this could be explained by relevant studies that found advancing age to be a risk factor for a higher prevalence of proteinuria . therefore , the early detection of proteinuria and screening of hypertensive patients might prevent complication such as renal and cardiovascular diseases . the prevalence of proteinuria is higher in smoking hypertensive patients than in those who do not smoke . this could be explained by the presence of endothelial dysfunction , which involves an imbalance between the contracting and relaxing substances produced by the endothelium . for example , the plasma concentration of endothelin-14 is higher in smokers than in nonsmokers . the present study has shown an independent relationship between proteinuria and the presence of concomitant diseases such as diabetes , congestive heart failure , and myocardial infarction . this observation is in agreement with other studies that found a relationship between albuminuria and cardiovascular risk . this relationship could be explained by other relevant studies in which microalbuminuria was found to indicate a generalized dysfunction of the vascular endothelium with permeability changes , which leads to a leakage of albumin through the glomerular membrane . furthermore , the finding that the prevalence of proteinuria was higher among patients with diabetes is most likely attributable to the fact that these patients had diabetes nephropathy . the result of our study also showed that physical activity decreased the risk of proteinuria in hypertensive patients . this association has been previously demonstrated by reports that showing physical activity was inversely associated with microalbuminuria . physical activity has protective effects on the vascular endothelium among patients with cardiac diseases . we found a significant association between the use of ccbs and proteinuria risk in the present study . this can be explained by the dilating effect of ccbs on the efferent glomerular arterioles . the present study also shows that the use of bbs is associated with proteinuria . however , kozan et al . could not find a significant association between the use of bbs and microalbuminuria risk . the reason for this association was illustrated by other observations in which different mechanisms were identified to explain the beneficial effect of ace inhibitors and arbs on proteinuria . second , this group of drugs can reduce the permeability of the basement membrane of the glomerulus . both hemodynamic and non - hemodynamic effects of ace inhibitors and arbs on renal activity lead to a reduction in proteinuria among patients with hypertension . the samples consisted of patients who were referred to an outpatient clinic for treatment . in addition , the data source was a single center and the study design was an observational analysis . patients were not randomly selected in our study ; therefore , there might be a selection bias . furthermore , the examination of proteinuria by using our data did not demonstrate the proportion of patients who had positive or negative results based on retesting . moreover , concerning the study sample , the patients were selected on a convenience basis . finally , the study only included the uzbek - turkmen ethnic group and the findings may not be generalizable to the general population . we conclude that our results confirm the presence of a high incidence of proteinuria among hypertensive outpatients in a clinic in andkhoy , afghanistan , especially in those with a high cardiovascular risk . therefore , more frequent screening for proteinuria might be imperative for the evaluation this project was supported in part , by the non - profit organization epidemiological and clinical research information network ( ecrin ) . we would like to thank dr.mirwais rabi , and dr.ahmad arsalam karimi for their cooperation for this study . the vast majority of cancers in the oral cavity are squamous cell carcinomas ( scc)its evolution is influenced by host immune response cells ( e.g. , cd8 t , cd4 t , natural killer cells - nk , dendritic cells - dc , macrophages , and eosinophils ) . the eosinophils are considered as destructive effector leukocytes with cytotoxic activities mainly implicated in parasitic infections ( e.g. , helminthic infections ) and allergic diseases ( e.g. , bronchial asthma , allergic dermatitis , etc . ) . however , studies have shown that they can also be involved in tissue remodelling and in innate and acquired immunity response modulation [ 2 , 3 ] ( figure 1 ) . under diverse stimuli ( e.g. , infections , tumours , etc . ) , the eosinophils are able to release different substances , such as , eosinophil cationic protein ( ecp ) , major basic protein ( mbp ) , eosinophil peroxidise ( epo ) , eosinophil - derived neurotoxin ( edn ) , il-1 , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , il-12 , il-13 , il-18 , interferon ( inf)- , tumor necrosis factor ( tnf)- , transforming growth factor ( tgf)- , tgf- , chemokines ( rantes , endotaxin-1 ) , platelet - activating factor ( paf ) , leukotriene c4 ( ltc4 ) , neuromediators , and indoleamine 2,3-dioxygenase ( ido ) [ 4 , 5 ] . these substances may cause cell death and induction of inflammatory symptoms as well as contribute to tumour progression or regulation . furthermore , the eosinophils present membrane receptors ( e.g. , il-1 , 3 , 4 , 5 , 8 , 10 , 12 , 13 , granulocyte monocyte colony - stimulating factor gm - csf , ifn- , tnf- , and macrophage inflammatory protein-1 ) that confer a survival and recruitment capacity of eosinophils themselves . in oral scc , several studies have shown that eosinophils can be associated with an improved prognosis , but there are other studies however , showing their association with a poor prognosis as well [ 6 , 7 ] . in this short review , we summarize briefly the role of the eosinophils in the general context of immunoregulation and its relation with oral squamous cell carcinoma . under a specific stimulus , the nave cd4 t and cd8 t cell can be differentiated , respectively , into th2 and tc2 cells and secrete mainly cytokines involved with humoral immunity such as il-4 , il-5 , il-10 , and il-13 [ 8 , 9 ] . the initial recruitment and activation of eosinophils towards the tumour microenvironment is principally related with th2 response , although necrotic cells can stimulate both the migration and the activation of eosinophils [ 10 , 11 ] . according to the literature , th2 response may eliminate the cancer on the dependence of eosinophils and macrophages [ 8 , 1214 ] . il-4 and il-13 are potent inducers of eotaxin chemokines that can explain the eosinophilia associated with th2 responses . in oral squamous cell carcinoma , the eotaxin expressed by tumour cells and eosinophils were involved in the mechanisms of eosinophil chemotaxis to the tumour . . il-13 has also been involved with the antitumour immune response mediated mainly by neutrophils ( gr-1 ) and macrophages ( mac-3 ) . however , il-13 can also inhibit the ifn- secretion and cd8 cytotoxic t lymphocyte ( ctl ) activity and compromise the anti - tumour immunity response . studies have also shown that eosinophils can process and present histocompatibility complex ii ( mhc - class ii ) molecules and polarize the th2 response . upon stimulation with ifn- , il-3 , and gm - csf , human eosinophilc cell line differentiated with dibutyryl cyclic amp ( deol-1 ) and human peripheral blood ( pb ) eosinophils were able to respond to the lymphoid chemokines ( e.g. , ccl11 , ccl21 , and ccl25 ) . in addition , cytokine - stimulated deol-1 cells expressed human leukocyte antigen ( hla)-dr and costimulatory molecules such as cd80 , cd86 and cd40 . therefore , eosinophils can migrate to the lymphoid chemokine microenvironment and express antigen - presenting cells ( apcs)- related costimulatory molecules . however , some experimental models have suggested that eosinophils are inefficient antigen - presenting cells when compared to macrophages or dendritic cells . the eosinophil - derived neurotoxin stored in eosinophils granules is able to induce dc maturation and activation through up - regulation of mitogen - activated protein ( map ) kinases , nuclear factor - kappa b ( nf kappa b ) and cd83 , cd86 costimulatory molecules , and mhc class ii expression [ 27 , 28 ] . edn - treated human dcs stimulated th2 immune response via toll - like receptor ( tlr)2-myd88 signal . they may express ifn- as well as il-4 , il-5 and il-10 suggesting a subpopulation of human eosinophils that expresses th1 or th2 cytokines , respectively . it has been shown that not only the th1 response is able to eradicate the tumour through the cellular immunity response but also th2 response via tumour necrosis . contrarily , eosinophils are capable to downregulate the antitumour immunity , through mainly il-10 and ido production . il10 is a potent inhibitor of mhc complex and cd80 and cd86 expression on dc as well as it suppresses the dc differentiation . contrarily , il-10 can play a role in the b cell activation and survival . through indoleamine this enzyme catalyzes the amino acid tryptophan to kynurenine which is able to cause cycle arrest and apoptosis from uncommitted cd4 t cells as well as maintenance of th2 response . the ido was correlated with the poor prognosis in the non - small cell lung cancer . it has been shown that the eosinophils are capable of producing various substances ( e.g. , vascular endothelial growth factor - vegf , fibroblast growth factor - fgf , tnf- , gm - csf , nerve growth factor - ngf , tgf- , and il-8 ) that can promote angiogenesis and produce collagenous fibers . in head and neck scc , it has been shown that the th1 response is mainly associated with a better prognosis than those with the th2 response [ 38 , 39 ] . according to argarwal and colleagues , early stage of oral scc expressed mainly inf- and il-2 genes ( th1 responses ) , whereas the advanced stage tumours presented il-4 and il-10 expression ( th2 response ) . in addition , advancing lesions of the tongue squamous cell carcinoma , induced by 4-nitroquinoline-1-oxide , have downregulated th1-type and upregulated th2-type cytokine production . regarding studies with eosinophils in the blood , the presence of eosinophils and th2 cells can be related with tumour progression and poor prognosis [ 43 , 44 ] . peripheral blood analyses of intraoral squamous cell carcinoma with a history of tobacco use presented enhanced expression of th2 cytokine . however , patients with malignant disease ( e.g. , acute lymphoblastic leukemia , acute myelogenous leukemia ) that underwent stem cell transplantation or patients with cervical cancer treated with whole - pelvic irradiation presented increased blood eosinophilia and better overall survival . tumour - associated tissue eosinophilia ( tate ) has been reported in diverse sites [ 14 , 35 , 4753 ] including the head and neck region [ 6 , 7 , 5465 ] ( figure 2 ) . in the head and neck region , some studies have shown that the tate has a favourable prognosis , suggesting that eosinophils may play a protective role against epithelial tumours [ 6 , 55 , 60 , 62 ] . other studies however , suggest that eosinophils may play a role in promoting epithelial tumour growth accounting for a poor prognosis [ 7 , 57 , 58 ] or even no effect on tumour evolution [ 54 , 56 , 59 , 63 ] . with regard to good prognosis , it has been shown that oral scc patients with tate presented higher overall survival and less incidence of distant metastasis in head and neck tumours . nevertheless , tadbir and colleagues have shown that tate was not associated with vascular , perineural , muscle invasion , and locoregional metastasis in scc of the oral cavity . similar results were observed in nasopharyngeal carcinoma and oral ssc where the eosinophils were not associated with local recurrence , distant metastases , or survival . in another study of laryngeal squamous cell carcinoma , tate was associated only with the age - tate - positive patients presented between 50 and 60 years of age , whereas the tate - negative patients were between 60 and 70 years of age . according to oliveira and colleagues and cormier and colleagues , the presence of eosinophis in damaged striated muscular fibers of oral cancer or in capsule of b16-f10 melanoma cell - derived tumours , respectively , could be related with tissue remodelling . on the other hand , the presence of eosinophils has also been related with a poor prognosis . in oral scc , eosinophilic infiltration and hla - dr expression in tumour cells were related to unfavourable prognosis . in the experimental studies of oral carcinoma , the depletion of tate with anti - il-5 mab was associated with a delayed development of the tumours . according to falconieri and colleagues and oliveira and colleagues , the eosinophil presence in the scc of the oral cavity similar results have been observed in head and neck carcinoma [ 57 , 58 ] and cervix where eosinophils infiltrate has increased the suspicion of invasion . the presence of > 3 eosinophils / high - power field ( hpf ) , 5/hpf , and 10/10 hpf in cervical incisional biopy and excisional specimens were associated with invasion . according to dorta and colleagues , the prognosis related with tate is controversial due to the different methodology utilized to count tumour - associated tissue eosinophila , thereby prejudicing a comparison of the results . for this reason alkhabuli and high suggested the counting of eosinophils through a density method , which utilizes the highest density of eosinophils per surface area , in preference to the classic method that counts eosinophils / hpf . in general , the presence as well as the state of activation of immunologic cells plays an important role in tumour cell progression . further studies on eosinophils and their state of activation are necessary in order to elucidate these findings .

abstractproteinuria in hypertension is an early marker of renal disease and a predictor for the progression of end stage renal disease , and cardiovascular diseases . this study was designed to determine the prevalence of proteinuria and its association with cardiovascular risk factors among adult hypertensive patients in afghanistan . five hundred fifty - five patients with a high blood pressure recorded in an outpatient clinic in andkhoy , afghanistan from december 2014 to may 2015 , were included in this study . data obtained from each patient , included demographic characteristics , body mass index , blood pressure patterns , cardiovascular history , cardiovascular risk factors , comorbidity , and current drug - therapy . dipstick screening for proteinuria was performed with reagent test strips . the mean age of the patients was 57.9 13.3 years , and a female predominance was observed ( n = 333 , 60% ) . the prevalence of proteinuria was 67.2% . the predictors of proteinuria were found to be age 65 years ( odds ratio [ or ] 1.02 , 95% confidence interval [ ci ] 1.001.04 ) , smoking ( or 1.88 , 95% ci 1.173.02 ) , heart failure ( or 2.23 , 95% ci 1.134.41 ) , and diabetes mellitus ( or 3.41 , 95% ci 1.497.81 ) . in conclusion , this study shows that proteinuria is highly prevalent among hypertensive outpatients in an outpatient clinic in andkhoy , afghanistan , especially in those with high cardiovascular risk . the eosinophil cell has been related as a prognostic indicator for cancers . however , its exact function in tumour behaviour is still not clearly defined . in the oral cavity the presence of eosinophils can be a favourable prognostic indicator as well as it may be associated with a poor prognosis . in this short review , we briefly summarize the role of the eosinophils in the general context of immunoregulation and its relation to oral squamous cell carcinoma .

the kcps is a prospective cohort study of korean government employees , public and private school teachers , and their dependents who were insured by the korean medical insurance corporation , the former national health insurance corporation ( 16,17 ) . the cohort includes 1,329,525 koreans ( 846,907 men and 482,618 women ) aged 3095 years ; 784,870 ( 59% ) subjects were enrolled in 1992 , 367,903 ( 27.7% ) were enrolled in 1993 , 98,417 ( 7.4% ) were enrolled in 1994 , and 78,335 ( 5.9% ) were enrolled in 1995 . follow - up began on 1 january 1993 , so 904 people enrolled in 1992 and who died in that year were excluded . to avoid confounding of the association between fasting serum glucose and the risk of death by preexisting disease , 88,420 who reported having cvd , liver disease , cancer , respiratory disease , and diabetes diagnosed at or before the initial study visit were excluded . in addition , 42,817 people with missing information on fasting serum glucose , total cholesterol , systolic blood pressure , and alcohol intake and those with extremely low bmi ( < 16.0 kg / m ) , or those with exceptionally short stature ( < 130 cm ) were also excluded . people insured by the korean medical insurance corporation were required to have biennial medical examinations conducted at designated hospitals or clinics nationwide by medical staff following guidelines provided by the korean medical insurance corporation . participants were asked to provide their medical history and to respond to a lifestyle questionnaire that included items on smoking , alcohol drinking , and performance of regular exercise . the examination included height , weight , and blood pressure measurements , urinalysis , blood cell count , and routine blood chemistries after overnight fasting . incident events were determined from diagnoses on the discharge summaries of inpatient hospital records . in korea , certified medical chart recorders review and abstract the medical chart and assign discharge diagnoses in a standardized form using the international classification of diseases , 10th revision ( world health organization 1992 ) . vital status and cause of death were determined from computerized searches of death certificate data from the korean national statistical office . study outcomes were defined as hospitalization or mortality attributable to ischemic heart disease ( international classification of diseases , 10th revision , codes i20i25 ) , stroke ( codes i60i69 ) , and atherosclerotic cvd , which included ischemic heart disease ( codes i20i25 ) , stroke ( codes i60i69 ) , hypertensive heart disease ( codes i10i15 ) , other forms of heart disease likely related to atherosclerosis ( codes i44i52 ) , disease of arteries ( codes i70i74 ) , and other sudden death with cause unknown ( code r96 ) . cardiovascular outcomes included hemorrhagic stroke ( i60i62 ) and ischemic stroke ( i63i66 ) . for those who had more than one event during follow - up an ischemic heart disease event validation study was conducted in collaboration with the korean society of cardiology through the formation of the event validation committee ( july 2008may 2009 ) . for 673 participants with an ischemic heart disease event , review of individual hospital records showed that 73% of myocardial infarction diagnoses were valid ( 18 ) . proportional hazards models were used to evaluate the association between baseline fasting serum glucose levels and atherosclerotic cvd . fasting serum glucose levels were categorized as < 70 , 7084 , 8599 , 100109 , 110125 , 126139 , and 140 mg / dl . we used the group with fasting glucose level of 8599 mg / dl as the reference category . all analyses were conducted separately for men and women and were adjusted for the following covariates : age at enrollment ; bmi ; systolic blood pressure ; total cholesterol ; alcohol intake ( five categories based on grams consumed per day : 0 , 124 g , 2549 g , 5099 g , and 100 g ) ; participation in regular physical activity ( yes or no ) and smoking status ( never smoker , former smoker , or current smoker ) ; and the number of cigarettes smoked daily among current smokers ( 19 , 1019 , and 20 ) . for more detailed analyses of the dose - response trends , we also used restricted quadratic spline models with knots at fasting glucose plasma levels of 70 , 85 , 100 , 110 , 126 , and 140 mg / dl . the kcps is a prospective cohort study of korean government employees , public and private school teachers , and their dependents who were insured by the korean medical insurance corporation , the former national health insurance corporation ( 16,17 ) . the cohort includes 1,329,525 koreans ( 846,907 men and 482,618 women ) aged 3095 years ; 784,870 ( 59% ) subjects were enrolled in 1992 , 367,903 ( 27.7% ) were enrolled in 1993 , 98,417 ( 7.4% ) were enrolled in 1994 , and 78,335 ( 5.9% ) were enrolled in 1995 . follow - up began on 1 january 1993 , so 904 people enrolled in 1992 and who died in that year were excluded . to avoid confounding of the association between fasting serum glucose and the risk of death by preexisting disease , 88,420 who reported having cvd , liver disease , cancer , respiratory disease , and diabetes diagnosed at or before the initial study visit were excluded . in addition , 42,817 people with missing information on fasting serum glucose , total cholesterol , systolic blood pressure , and alcohol intake and those with extremely low bmi ( < 16.0 kg / m ) , or those with exceptionally short stature ( < 130 cm ) were also excluded . people insured by the korean medical insurance corporation were required to have biennial medical examinations conducted at designated hospitals or clinics nationwide by medical staff following guidelines provided by the korean medical insurance corporation . participants were asked to provide their medical history and to respond to a lifestyle questionnaire that included items on smoking , alcohol drinking , and performance of regular exercise . the examination included height , weight , and blood pressure measurements , urinalysis , blood cell count , and routine blood chemistries after overnight fasting . incident events were determined from diagnoses on the discharge summaries of inpatient hospital records . in korea , certified medical chart recorders review and abstract the medical chart and assign discharge diagnoses in a standardized form using the international classification of diseases , 10th revision ( world health organization 1992 ) . vital status and cause of death were determined from computerized searches of death certificate data from the korean national statistical office . study outcomes were defined as hospitalization or mortality attributable to ischemic heart disease ( international classification of diseases , 10th revision , codes i20i25 ) , stroke ( codes i60i69 ) , and atherosclerotic cvd , which included ischemic heart disease ( codes i20i25 ) , stroke ( codes i60i69 ) , hypertensive heart disease ( codes i10i15 ) , other forms of heart disease likely related to atherosclerosis ( codes i44i52 ) , disease of arteries ( codes i70i74 ) , and other sudden death with cause unknown ( code r96 ) . cardiovascular outcomes included hemorrhagic stroke ( i60i62 ) and ischemic stroke ( i63i66 ) . for those who had more than one event during follow - up , we used only the first event for this analysis . an ischemic heart disease event validation study was conducted in collaboration with the korean society of cardiology through the formation of the event validation committee ( july 2008may 2009 ) . for 673 participants with an ischemic heart disease event , review of individual hospital records proportional hazards models were used to evaluate the association between baseline fasting serum glucose levels and atherosclerotic cvd . the assumption of proportional hazards was tested and met . fasting serum glucose levels were categorized as < 70 , 7084 , 8599 , 100109 , 110125 , 126139 , and 140 mg / dl . we used the group with fasting glucose level of 8599 mg / dl as the reference category . all analyses were conducted separately for men and women and were adjusted for the following covariates : age at enrollment ; bmi ; systolic blood pressure ; total cholesterol ; alcohol intake ( five categories based on grams consumed per day : 0 , 124 g , 2549 g , 5099 g , and 100 g ) ; participation in regular physical activity ( yes or no ) and smoking status ( never smoker , former smoker , or current smoker ) ; and the number of cigarettes smoked daily among current smokers ( 19 , 1019 , and 20 ) . for more detailed analyses of the dose - response trends , we also used restricted quadratic spline models with knots at fasting glucose plasma levels of 70 , 85 , 100 , 110 , 126 , and 140 mg / dl . on enrollment , the average age of study participants was 45.0 years for men and 49.4 years for women , and the average bmi was 23.2 kg / m for both sexes . the average fasting glucose values were 91.1 mg / dl and 94.4 mg / dl in men and women , respectively . the percentages of study participants with fasting serum glucose < 100 , 100125 , and 126 mg / dl were 76.8 , 19.3 , and 3.9% in men and 82.0 , 14.8 , and 3.2% in women . among men , 60.2% were current smokers and only 20.8% were never smokers ; among women , the corresponding figures were 4.1 and 93.9% , respectively . in the cross - sectional baseline data , men and women with higher fasting glucose were more likely to be older , to have a higher bmi , and to have higher systolic and diastolic blood pressure and total cholesterol levels ( supplementary tables 1 and 2 ) . participants in higher fasting glucose categories were also more likely to report that they were physically active . in men , the prevalence of current smoking was inversely , but weakly , associated with fasting glucose level , but positively and also weakly positively associated with glucose level in women . we separately assessed the association of fasting serum glucose for risk of various cvd outcomes in men and women , finding similar patterns of association by sex for fatal and nonfatal events combined ( tables 1 and 2 ) . the risks for the various cvd outcomes were lowest in both sexes for those with serum glucose in the range of 70109 mg / dl , and increased above and below this range . fasting glucose levels < 70 mg / dl were associated with slightly increased risk of atherosclerotic cvd in men ( hazard ratio [ hr ] 1.04 , 95% ci 1.011.08 ) and in women ( hr 1.06 , 1.021.10 ) . fasting glucose levels < 70 mg / dl were associated with increased risk of all stroke in men ( hr 1.06 , 1.011.11 ) in women ( hr 1.11 , 1.051.17 ) . for categories > 100109 mg / dl group , the hrs increased progressively with increasing serum glucose , except for hemorrhagic stroke . the results were similar after exclusion of the first 5 years of follow - up ( data not shown ) . fasting serum glucose levels at enrollment and risk of cardiovascular diseases in male participants of the korean cancer prevention study 19932010 fasting serum glucose levels at enrollment and risk of cardiovascular diseases in female participants of the korean cancer prevention study 19932010 for cardiac outcomes and thrombotic stroke , the general patterns of association were quite comparable . for participants having a glucose level in the range of 110125 mg / dl the hr was increased by 1020% , and those in the highest category experienced a doubling or more of the risk of having a cvd event . in restricted quadratic spline models , the risks of cardiac outcomes and thrombotic stroke were lowest at fasting serum glucose levels of 90 mg / dl and increased sharply above these levels in both men and women ( figs . 1 and 2 ) . hrs for cardiovascular outcomes by fasting serum glucose levels in male participants of the kcps . to convert glucose from mg / dl to mmol / l , hrs for cardiovascular outcomes by fasting serum glucose levels in female participants of the kcps . to convert glucose from mg / dl to mmol / l , we repeated the analyses for fatal and nonfatal events separately ( supplementary tables 36 ) . for men , the results for the fatal and nonfatal events were generally comparable . for women , the risks associated with low blood glucose were greater for fatal compared with nonfatal events . we also assessed potential effect modification of the risk for all cvd outcomes by other risk factors , age , and sex . in general , we found little indication of substantial effect modification ( supplementary table 7 ) . there were significant overall interactions for hypertension ( present versus absent ) and age ( younger than 55 years versus 55 years or older ) , but the differences in hrs were small . for both men and women , the hrs changed in a similar pattern with fasting glucose concentration across the two strata of age . in a large cohort of korean men and women , we found that fasting glucose level was associated with higher risk for major cvd outcomes , increasing from a level of 90 mg / dl after controlling for other risk factors . the dose - response curves showed progressive increments in the hrs from this value at both higher and lower levels ; the increased risk was greatest for stroke . the patterns of association were similar in men and women , but the associations were stronger in women . experimental studies show that abnormal glucose metabolism impairs normal endothelial function , accelerates atherosclerotic plaque formation , and contributes to plaque rupture and thrombosis . epidemiological studies provide complementary evidence . in the rotterdam study , among elderly participants with a fasting blood glucose < 110 mg / dl and without diabetes , those with higher blood glucose levels had higher levels of arterial stiffness ( 20 ) . the cathay study found that higher levels of glycemia ( 102124 mg / dl ) were associated with arterial endothelial dysfunction and intima - media thickening ( 21 ) . in a biomarker study in italy , a number of cvd biomarkers showed positive dose - response relationships with fasting glucose across three strata : < 100 ; 100109 ; and 110125 mg / dl ( 22 ) . our study adds to the increasing evidence that ifg is an independent risk factor for incident cvd , including ischemic heart disease and stroke ( 7 ) . in addition , the effects of other cvd risk factors may be enhanced by abnormal glucose metabolism ( 2325 ) . in 1997 , the american diabetes association expert committee introduced the ifg category , defined at the time as fasting plasma glucose level of 110 to 125 mg / ml or a 2-h value on the oral glucose tolerance test of 140199 mg / dl ( 26 ) . ifg is not a clinical entity in itself but is a risk category for development of diabetes and cvd ( 10 ) . in 2003 , the american diabetes association expert committee lowered the cut - point for ifg to 100 mg / dl to align the proportion of the population that would be included in the category using fasting glucose and the oral glucose tolerance test , and to indicate that glucose levels < 110 mg / dl were predictive of diabetes development ( 26,27 ) . although previous studies also have identified an increased risk of cvd associated with ifg ( 100125 mg / dl ) ( 3,5,28,29 ) , the size of kcps provides a much more precise characterization of the up - turn in risk in relation to blood glucose level , placing it at 90 mg / dl . as a consequence , our findings support lowering the current american diabetes association ifg cut - off of 100 mg / dl , and they also open the possibility that low fasting glucose levels may identify people at increased risk for cvd . severe hypoglycemia in diabetes is known to increase risks of vascular events ( 3032 ) . low fasting glucose levels in the nondiabetic population were associated with increased mortality in several studies ( 5,15 ) . in our study , the lowest risk for cvd mortality occurred in the interval of 8599 mg / dl , with a nadir at 90 mg / dl , suggesting that the range of fasting glucose levels associated with the lowest risk for cvd is narrow . in a prospective cohort study of > 40,000 people , the lowest total mortality was associated with fasting plasma glucose of 79109 mg / dl ( 15 ) . in the study by balkau et al . ( 33 ) , the interval of lowest total mortality was 94103 mg / dl , and in the decode study it was 8190 mg / dl ( 34 ) . the pooled analysis performed by the emerging risk factors collaboration described a nonlinear relationship between fasting glucose and coronary heart disease that had the same configuration as in the kcps analysis ( 3 ) . in the atherosclerosis risk in communities ( aric ) study , selvin et al . ( 35 ) found a j - shape relationship of glycated hemoglobin with risk for death attributable to coronary heart disease . fasting blood glucose level < 100 mg / dl was not associated with coronary heart disease death ( 35 ) . because different studies have used different categories for fasting glucose , further and perhaps pooled analyses of data from large studies should be performed using flexible models for dose - response analysis to confirm our findings of a narrow range of fasting glucose levels with lowest mortality . the basis of the association between low fasting glucose levels and risk for cvd in people without diabetes is unclear . wei et al . ( 15 ) hypothesized that long - term exposure to low fasting plasma glucose may serve as a risk factor for cvd mortality , perhaps through abnormal cardiac activity and thrombosis , particularly in patients with atherosclerosis . ( 36 ) reported a j - shape relationship between fasting plasma glucose and incident ischemic cerebrovascular events in patients with preexisting atherothrombotic disease and suggested that hypoglycemia or rapid changes in plasma glucose may lead to elevations of counter - regulatory hormones , such as epinephrine and norepinephrine , and these increases induce vasoconstriction and platelet aggregation ( 36 ) . in a pooled analysis of data from 97 cohorts involving people without vascular disease on enrollment , an increase in risk for vascular disease death was observed in those with blood glucose level < 70 mg / dl on enrollment ( 7 ) . because cvd risk factors and development of cvd are usually associated with metabolic abnormalities that increase fasting plasma glucose , additional studies need to be conducted to understand if low fasting plasma glucose levels are a consequence of impending disease ( i.e. , reverse causation ) or if they have a role in precipitating acute cvd events . limitations of our findings need to be considered . first , single fasting glucose measurements are subject to substantial within - person variation and we did not perform 2-h glucose tolerance testing . measurement error and between - study variability may have attenuated our results , adding to the identification of a narrow range of fasting glucose levels for optimal survival . additionally , we measured serum glucose , not the currently recommended plasma glucose ( 37,38 ) . however , because all measurements were of serum glucose , our findings should not be biased . second , we lacked information on the evolution of fasting glucose levels and incidence of diabetes during follow - up , and we can not identify if the increased risk associated with ifg depends on future evolution to diabetes or on other pathways . third , we lacked data on other metabolic abnormalities associated with overweight and obesity and fat distribution . fourth , the outcomes were obtained from admission records and death certificates . because the outpatient records were not included , the incidence of cvd morbidity in our study may be lower than actual incidence . with regard to generalizability of findings , the kcps population differs from those of other investigations in several ways . of course , participants were asian and the population was also leaner than those in the other studies of blood glucose and cvd risk . fasting glucose is readily and widely measured for early detection and prevention of diabetes . beyond risk for diabetes , it also conveys information about cvd risk . additionally , the findings suggest that the optimal glucose level may be below the current cut - offs used to identify people at risk for cvd . health care providers should recognize the j - shape relationship between fasting blood glucose and cvd risk in interpreting and communicating clinical data . in october 2011 , the u.s . national science advisory board for biosecurity ( nsabb ) was asked to review two papers for their potential as dual - use research of concern ( durc ) . these papers contained results on the adaptation of the highly pathogenic avian influenza a / h5n1 virus to mammalian hosts such that it could be transmitted via respiratory droplets from animal to animal . we found that this work had great potential for harm or misuse and recommended that the general conclusions highlighting the novel outcome be published , but that the manuscripts not include the methodological and other details that could enable replication of the experiments by those who would seek to do harm the recommendation not to publish scientific results was highly unusual and the first such recommendation by the nsabb membership . we are primarily a group of actively practicing basic research scientists , and we have consistently advocated for open publication practices . as per our advisory nature to the u.s . there was agreement by nsabb voting members for these recommendations , though the rationale of individual members as they arrived at the same conclusions varied . we had to judge the beneficial attributes of these research results against their potential to cause harm . over the last 7 years , nsabb has studied the issues associated with dual - use research , including risk / benefit assessments , and developed principles and tools to guide the deliberative process . much of this has been formalized in a series of reports and recommendations that are available at a public website ( http://oba.od.nih.gov/biosecurity/biosecurity.html ) . despite this experience and carefully crafted guidance , there are points in the deliberations where uncertainties and even contradictory information necessitate subjective decisions . is there a clear and bright line to be crossed or is this a more nebulous and fuzzy region of yes or no for this research ? i will present only my personal rationale and how i came to the strong conclusion that this work had the potential to be very dangerous and that its communication should be restricted at this time . i heard from members of the influenza research community and reviewed the world health organization ( who ) data indicating that this avian virus had a very high mortality rate in humans . while the influenza a / h5n1 virus rarely infects humans , when it does it causes catastrophic disease . we are all aware of the rapid global spread of human - adapted influenza both on a yearly basis and during less common pandemics . the documented devastation of the 1918 influenza pandemic , even with its lower mortality rate , was a testament to the powerful potential of influenza . the thought of combining the high human mortality of influenza a / h5n1 with a highly transmissible human - adapted phenotype was sobering . a pandemic by such a pathogen could reasonably be concluded to cause such devastation that it should be prevented at all costs . i carefully considered how restricting the information would compromise scientific research progress and even how it would hinder public health efforts to prevent such a horrific pandemic . i know from firsthand experience that the free flow of information is part of the best and most productive research endeavors and that any restrictions burden the progress . the conclusion that this virus could be adapted to mammal - to - mammal respiratory transmission was , in my mind , the foremost beneficial part of the research . with this firm conclusion in hand , policy makers , granting agencies , public health officials , and vaccine and drug developers should have both the motivation and a compelling argument to move forward to improve our influenza - fighting infrastructure . the details of the research , on the other hand , would add little to this short - term effort and could enable someone to replicate the work in a short period of time . the short - term negative consequences of restricting experimental details seemed small in contrast to the large consequences of facilitating the replication of these experiments by someone with nefarious intent . current public health surveillance and public health responses would be enhanced little by these details . this comes not only from my own professional experience in globally tracking dangerous pathogens but also from personally watching the 2009 h1n1 influenza pandemic spread globally . it was impossible to contain , and i believe that the same would be true for an h5n1 influenza pandemic . we were lucky in that the h1n1 virus has low virulence , but the best current data suggest that this would not be the case for the h5n1 virus . publishing a detailed experimental protocol on how to produce a highly transmissible h5n1 virus in a highly regarded scientific journal is a very bad idea . since our recommendations were announced in mid - december , there has been considerable response from scientists , policy makers , funding agencies , and global health organizations . there have been criticisms that restriction of the publications was insufficient and that even performing such experiments should be restricted . the debate has touched upon both biosafety and biosecurity aspects , with some calling for the destruction of the virus or for moving all such research to the highest safety level , biosafety level 4 ( bsl-4 ) . the nsabb has not yet offered specific recommendations concerning these statements , and my personal opinions are relatively unimportant . what is gratifying and essential is that the debate is occurring ; it is occurring on an international stage , and it is occurring rapidly . in the midst of nsabb deliberations and formulation of our recommendations , the need for a global debate to develop policy has always been in our discussions . why should the nsabb be telling the world what to do ? why has not the world already had these discussions and debates ? how could the nsabb stimulate the process such that global leaders in science , policy , and public health engage in a broad - based conversation on these issues ? the specific nsabb recommendations seem to have been accepted and are being implemented by two research groups and two scientific journals ; more importantly , the research issue of adapting an avian virus to mammals , potentially humans , is a topic that is being widely discussed . the who has agreed to participate and facilitate in policy development . and the u.s . research and public policy will be developed from this global engagement process , a process that should increase the public s confidence in the scientific endeavor , in scientists ethical behavior , and in the transparency that a free research environment embraces .

objectivealthough diabetes increases the risk of cardiovascular disease ( cvd ) and mortality , the dose - response relationship between fasting glucose levels below those diagnostic of diabetes with cardiovascular events has not been well characterized.research design and methodsa prospective cohort study of more than one million koreans was conducted with a mean follow - up of 16 years . a total of 1,197,384 korean adults with no specific medical conditions diagnosed were classified by baseline fasting serum glucose level . associations of fasting glucose level with cvd incidence and mortality , stroke incidence and mortality , and all - cause mortality were analyzed using multivariate proportional hazards regression.resultsthe relationships between fasting glucose levels and cvd risks generally followed j - shape curves , with lowest risk in the glucose range of 8599 mg / dl . as fasting glucose levels increased to > 100 mg / dl , risks for cvd , ischemic heart disease , myocardial infarction , and thrombotic stroke progressively increased , but risk for hemorrhagic stroke did not . fasting glucose levels < 70 mg / dl were associated with increased risk of all stroke ( hazard ratio 1.06 , 95% ci 1.011.11 ) in men and ( hazard ratio 1.11 , 1.051.17 ) in women.conclusionsboth low glucose level and impaired fasting glucose should be considered as predictors of risk for stroke and coronary heart disease . the fasting glucose level associated with the lowest cardiovascular risk may be in a narrow range . abstractthe national science advisory board for biosecurity ( nsabb ) has recommended that two scientific papers concerning the laboratory adaptation of avian h5n1 influenza virus to mammal - to - mammal respiratory transmission restrict their content to prevent others from replicating their work . after hearing from experts in the field of influenza research and public health , the benefits of the research were deemed less important than the potential negative consequences . the evaluation followed established nsabb procedures and prior policy recommendations for identifying dual use research of concern ( durc ) . this recommendation was received by the united states government , endorsed and forwarded to the research teams and scientific journals involved with the publications .

pneumococcal infections are especially dangerous for children and adults with immunodeficiencies , such as hiv , or with illnesses such as chronic cardiac disease and diabetes.1 pneumococcal vaccination has therefore been recommended for persons at high risk of severe illness or complications . asthma was first identified as an independent risk factor for pneumococcal disease ( pd ) in 2005 when talbot et al2 reported that asthma was associated with a 2.4-fold increased odds of pd among persons 249 years old enrolled in tennessee s medicaid program between 1994 and 2002 . several studies conducted since then have provided additional evidence that asthma is associated with an increased risk of pd in adults,35 but no study has provided convincing evidence that asthma is associated with an increased risk of pd in children.6 currently , pneumococcal vaccination is recommended for asthmatic adults , but it is not specifically recommended for asthmatic children . asthma is the most common chronic disease in children,7 and the prevalence of asthma is increasing worldwide.812 as the prevalence of asthma increases , so does the importance of understanding whether children with asthma are at increased risk of pd . the aim of the current study was to evaluate the association between asthma and the development of pd among danish children born between 1994 and 2007 . the study population included all singleton live births in denmark from january 1 , 1994 through december 31 , 2007 . routine childhood pneumococcal vaccination with 7-valent pneumococcal conjugate vaccine ( pcv7 ) did not begin in denmark until october 2007,13 so few children in the study population were expected to be vaccinated . we used the unique civil personal registry number assigned to all danish citizens at birth and to residents upon immigration , which has been used in public records since 1968 , to identify all live births from the danish civil registration system . this continually updated national registration system includes information about date and place of birth , immigration , sex , marital status , citizenship , emigration , and vital status.14 all study children were followed from birth until the diagnosis of pd , removal from the danish civil registration system due to any cause , or december 31 , 2007 , whichever came first . the danish data protection agency provided permission to use these data ( record number : 1 - 16 - 02 - 1 - 08 ) . because this study was based on data extracted from registries , it was exempt from human subjects review , and members of the study population did not have to provide informed consent . to ascertain pd , we used each subject s unique civil personal registry number to link their danish civil registration system data to the danish national registry of patients . the danish national registry of patients began in 1977 and includes inpatient diagnoses made at nonpsychiatric hospitals and , beginning in 1995 , diagnoses made at outpatient specialist clinics and emergency room visits.15 we used the danish version of international classification of diseases ( icd)-10 codes g00.1 , a40.3 , and j13.9 to ascertain pneumococcal meningitis , pneumococcal septicemia , and pneumococcal pneumonia , respectively . diagnosis of pneumococcal pneumonia using these icd codes was validated in a prior study in which 64% of icd-10-identified cases had microbiologic , radiologic , and clinical evidence consistent with pd , and the remaining 36% were classified as having probable pd.16 we used icd-10 codes ( j45 and j46 ) recorded in the danish national registry of patients to ascertain asthma . registry - based asthma diagnoses have been previously validated in denmark and determined to be of high quality.17,18 we classified study subjects as having asthma if they had at least one diagnosis code indicating asthma hospitalization , emergency , or outpatient visits at any time before the diagnosis of pd , or before the end of follow - up for children who did not develop pd . information obtained from the danish medical birth registry included place of birth , gestational age , fetal presentation , mode of delivery , birth weight , 5-minute apgar score , maternal place of birth , maternal age at the time of delivery , maternal cohabitation status , and maternal parity.19 maternal smoking status during pregnancy was based on self - report during the first antenatal visit and was classified as yes or no , and presence or absence of congenital malformations or selected underlying comorbidities was ascertained using icd-10 codes recorded in the danish national registry of patients ( table s1 ) . children accumulated unexposed person - time from birth until an asthma diagnosis , if any , and exposed person - time thereafter . we calculated the frequency and proportion of children with and without asthma within categories of demographic variables and birth outcomes , as well as age - specific incidence rates of pd . we used poisson regression to estimate crude and adjusted incidence rate ratios ( irrs ) and 95% confidence intervals ( cis ) associating childhood asthma and pd . we included covariates that changed the crude association between asthma and pd by more than 10% in adjusted models ; child s sex was retained in the adjusted models regardless of its impact on the unadjusted measures . because underlying comorbidity is known to substantially increase the risk of pd in children,20 and because it was strongly associated with childhood asthma exposure in our study population , we evaluated the impact of comorbidity on the association between asthma and pd in stratified analyses . the impact of congenital malformations was also assessed . for these analyses , we used poisson regression to calculate crude and adjusted irrs and 95% cis stratified by the presence or absence of congenital malformations or selected underlying comorbidities . we further evaluated any observed effect measure modification for evidence of biologic interaction using standard measures ( supplementary materials).21,22 we conducted all statistical analyses using sas / stat software , version 9.2 ( sas institute inc . , cary , nc , usa).23 the study population included all singleton live births in denmark from january 1 , 1994 through december 31 , 2007 . routine childhood pneumococcal vaccination with 7-valent pneumococcal conjugate vaccine ( pcv7 ) did not begin in denmark until october 2007,13 so few children in the study population were expected to be vaccinated . we used the unique civil personal registry number assigned to all danish citizens at birth and to residents upon immigration , which has been used in public records since 1968 , to identify all live births from the danish civil registration system . this continually updated national registration system includes information about date and place of birth , immigration , sex , marital status , citizenship , emigration , and vital status.14 all study children were followed from birth until the diagnosis of pd , removal from the danish civil registration system due to any cause , or december 31 , 2007 , whichever came first . the danish data protection agency provided permission to use these data ( record number : 1 - 16 - 02 - 1 - 08 ) . because this study was based on data extracted from registries , it was exempt from human subjects review , and members of the study population did not have to provide informed consent . to ascertain pd , we used each subject s unique civil personal registry number to link their danish civil registration system data to the danish national registry of patients . the danish national registry of patients began in 1977 and includes inpatient diagnoses made at nonpsychiatric hospitals and , beginning in 1995 , diagnoses made at outpatient specialist clinics and emergency room visits.15 we used the danish version of international classification of diseases ( icd)-10 codes g00.1 , a40.3 , and j13.9 to ascertain pneumococcal meningitis , pneumococcal septicemia , and pneumococcal pneumonia , respectively . diagnosis of pneumococcal pneumonia using these icd codes was validated in a prior study in which 64% of icd-10-identified cases had microbiologic , radiologic , and clinical evidence consistent with pd , and the remaining 36% were classified as having probable pd.16 we used icd-10 codes ( j45 and j46 ) recorded in the danish national registry of patients to ascertain asthma . registry - based asthma diagnoses have been previously validated in denmark and determined to be of high quality.17,18 we classified study subjects as having asthma if they had at least one diagnosis code indicating asthma hospitalization , emergency , or outpatient visits at any time before the diagnosis of pd , or before the end of follow - up for children who did not develop pd . information obtained from the danish medical birth registry included place of birth , gestational age , fetal presentation , mode of delivery , birth weight , 5-minute apgar score , maternal place of birth , maternal age at the time of delivery , maternal cohabitation status , and maternal parity.19 maternal smoking status during pregnancy was based on self - report during the first antenatal visit and was classified as yes or no , and presence or absence of congenital malformations or selected underlying comorbidities was ascertained using icd-10 codes recorded in the danish national registry of patients ( table s1 ) . children accumulated unexposed person - time from birth until an asthma diagnosis , if any , and exposed person - time thereafter . we calculated the frequency and proportion of children with and without asthma within categories of demographic variables and birth outcomes , as well as age - specific incidence rates of pd . we used poisson regression to estimate crude and adjusted incidence rate ratios ( irrs ) and 95% confidence intervals ( cis ) associating childhood asthma and pd . we included covariates that changed the crude association between asthma and pd by more than 10% in adjusted models ; child s sex was retained in the adjusted models regardless of its impact on the unadjusted measures . because underlying comorbidity is known to substantially increase the risk of pd in children,20 and because it was strongly associated with childhood asthma exposure in our study population , we evaluated the impact of comorbidity on the association between asthma and pd in stratified analyses . the impact of congenital malformations was also assessed . for these analyses , we used poisson regression to calculate crude and adjusted irrs and 95% cis stratified by the presence or absence of congenital malformations or selected underlying comorbidities . we further evaluated any observed effect measure modification for evidence of biologic interaction using standard measures ( supplementary materials).21,22 we conducted all statistical analyses using sas / stat software , version 9.2 ( sas institute inc . , cary , nc , usa).23 there were 890,681 singleton live births during the study period . after excluding 2,026 records , records were excluded if a child had 0 days of follow - up ( number [ n ] = 1,228 ) , birth weight < 500 g ( n=90 ) , gestational age < 25 completed weeks or > 45 completed weeks ( n=510 and n=5 , respectively ) , implausible gestational age and birth weight combinations ( n=70 ) , or if they met < 1 exclusion criteria ( n=123 ) . the mean and median follow - up periods were both 8.0 years ( interquartile range : 4.411.6 years ) . among the 888,655 children in the study population , 6.0% ( n=53,024 ) received an asthma diagnosis before the end of follow - up . the mean age of asthma diagnosis was 31 months ( standard deviation : 31.0 months ) , and the median was 18.7 months ( interquartile range : 10.739.6 months ) . a total of 6,641 children had a recorded comorbidity ; cardiac disease was the most common comorbidity ( 32.8% ) , followed by renal disease ( 21.5% ) and type 1 diabetes ( 20.3% ) . compared with children without asthma , children with an asthma diagnosis were more likely to be male , to have been born preterm ( < 37 weeks ) , to have a low birth weight ( < 2,500 g ) , and to be born to a mother who reported smoking at the first prenatal visit ( table 1 ) . in addition , asthmatic children had more congenital malformations ( 6.7% versus 4.4% , respectively ) and selected underlying comorbidities ( 2.4% versus 0.6% , respectively ) compared with nonasthmatic children . a total of 2,253 children were diagnosed with pd during the follow - up period , and most of them were admitted as inpatients ( 96.3% ) . pneumonia accounted for the majority of cases ( 72.9% ) , followed by septicemia ( 14.6% ) and meningitis ( 12.5% ) . most pd diagnoses occurred between 6 months and 24 months after birth ( n=1,180 ; 52.7% ) and the fewest occurred at more than 60 months after birth ( n=255 ; 11.3% ) . there was no trend toward increasing or decreasing incidence throughout the study s calendar period . the rate of pd was highest among children 6 to < 24 months old ( 91.2 cases per 100,000 child - years ) followed by children 0 to < 6 months old ( 78.8 cases per 100,000 child - years ) and children 24 to < 60 months old ( 21.5 cases per 100,000 child - years ) . children in the oldest age group had the lowest pd rates ( 8.0 cases per 100,000 child - years ) . pd incidence rates in asthmatic children were consistently higher than in nonasthmatic children , with the exception of children aged 0 to < 6 months . the unadjusted irr associating asthma with incident pd among all children was 2.4 ( 95% ci : 2.1 , 2.6 ) . age - specific measures of association were confounded by year of birth , birth weight , congenital malformations , and underlying comorbidities . the adjusted irr associating asthma with incident pd among all children was 2.2 ( 95% ci : 2.0 , 2.5 ) ; in order of increasing age strata , the adjusted irrs were 0.4 ( 95% ci : 0.2 , 0.8 ) in children 0 to < 6 months old , 2.1 ( 95% ci : 1.8 , 2.5 ) in children 6 to < 24 months old , 4.1 ( 95% ci : 3.3 , 5.1 ) in children 24 to < 60 months old , and 2.3 ( 95% ci : 1.6 , 3.2 ) in children 60 months old ( table 2 ) . restricting the study population to children without selected comorbidities or malformations did not substantially change the irrs associating asthma with incident pd in any age strata . restricting the study population to children who did have selected comorbidities or malformations , however , revealed that comorbidity was an effect modifier of the association between asthma and pd in children aged 24 to < 60 months and 60 months ( table 3 ) . evaluation of biologic interaction between asthma and comorbidity on the incidence of pd in these age groups showed that the rate of pd was greater among those with a recorded diagnosis of both asthma and comorbidity compared with the rate that would be expected based on the independent effects of asthma or comorbidity alone . figure 1 shows that among children aged 24 to < 60 months old , the unadjusted incidence rate of pd in children with both asthma and comorbidity was 7.5 times the rate in children with asthma alone , and that after adjusting for confounding variables , 55% ( 95% ci : 31 , 79 ) of pd cases among asthmatic children could be attributed to the presence of both asthma and comorbidity at the same time . among children aged 60 months old , the unadjusted irr of pd in children with both asthma and comorbidity was 14 times the rate in children with asthma alone , and the adjusted percentage of pd cases attributable to biologic interaction increased to 73% ( 95% ci : 52 , 93 ) . there were 890,681 singleton live births during the study period . after excluding 2,026 records , records were excluded if a child had 0 days of follow - up ( number [ n ] = 1,228 ) , birth weight < 500 g ( n=90 ) , gestational age < 25 completed weeks or > 45 completed weeks ( n=510 and n=5 , respectively ) , implausible gestational age and birth weight combinations ( n=70 ) , or if they met < 1 exclusion criteria ( n=123 ) . the mean and median follow - up periods were both 8.0 years ( interquartile range : 4.411.6 years ) . among the 888,655 children in the study population , 6.0% ( n=53,024 ) received an asthma diagnosis before the end of follow - up . the mean age of asthma diagnosis was 31 months ( standard deviation : 31.0 months ) , and the median was 18.7 months ( interquartile range : 10.739.6 months ) . a total of 6,641 children had a recorded comorbidity ; cardiac disease was the most common comorbidity ( 32.8% ) , followed by renal disease ( 21.5% ) and type 1 diabetes ( 20.3% ) . compared with children without asthma , children with an asthma diagnosis were more likely to be male , to have been born preterm ( < 37 weeks ) , to have a low birth weight ( < 2,500 g ) , and to be born to a mother who reported smoking at the first prenatal visit ( table 1 ) . in addition , asthmatic children had more congenital malformations ( 6.7% versus 4.4% , respectively ) and selected underlying comorbidities ( 2.4% versus 0.6% , respectively ) compared with nonasthmatic children . a total of 2,253 children were diagnosed with pd during the follow - up period , and most of them were admitted as inpatients ( 96.3% ) . pneumonia accounted for the majority of cases ( 72.9% ) , followed by septicemia ( 14.6% ) and meningitis ( 12.5% ) . most pd diagnoses occurred between 6 months and 24 months after birth ( n=1,180 ; 52.7% ) and the fewest occurred at more than 60 months after birth ( n=255 ; 11.3% ) . there was no trend toward increasing or decreasing incidence throughout the study s calendar period . the rate of pd was highest among children 6 to < 24 months old ( 91.2 cases per 100,000 child - years ) followed by children 0 to < 6 months old ( 78.8 cases per 100,000 child - years ) and children 24 to < 60 months old ( 21.5 cases per 100,000 child - years ) . children in the oldest age group had the lowest pd rates ( 8.0 cases per 100,000 child - years ) . pd incidence rates in asthmatic children were consistently higher than in nonasthmatic children , with the exception of children aged 0 to < 6 months . the unadjusted irr associating asthma with incident pd among all children was 2.4 ( 95% ci : 2.1 , 2.6 ) . age - specific measures of association were confounded by year of birth , birth weight , congenital malformations , and underlying comorbidities . the adjusted irr associating asthma with incident pd among all children was 2.2 ( 95% ci : 2.0 , 2.5 ) ; in order of increasing age strata , the adjusted irrs were 0.4 ( 95% ci : 0.2 , 0.8 ) in children 0 to < 6 months old , 2.1 ( 95% ci : 1.8 , 2.5 ) in children 6 to < 24 months old , 4.1 ( 95% ci : 3.3 , 5.1 ) in children 24 to < 60 months old , and 2.3 ( 95% ci : 1.6 , 3.2 ) in children 60 months old ( table 2 ) . restricting the study population to children without selected comorbidities or malformations did not substantially change the irrs associating asthma with incident pd in any age strata . restricting the study population to children who did have selected comorbidities or malformations , however , revealed that comorbidity was an effect modifier of the association between asthma and pd in children aged 24 to < 60 months and 60 months ( table 3 ) . evaluation of biologic interaction between asthma and comorbidity on the incidence of pd in these age groups showed that the rate of pd was greater among those with a recorded diagnosis of both asthma and comorbidity compared with the rate that would be expected based on the independent effects of asthma or comorbidity alone . figure 1 shows that among children aged 24 to < 60 months old , the unadjusted incidence rate of pd in children with both asthma and comorbidity was 7.5 times the rate in children with asthma alone , and that after adjusting for confounding variables , 55% ( 95% ci : 31 , 79 ) of pd cases among asthmatic children could be attributed to the presence of both asthma and comorbidity at the same time . among children aged 60 months old , the unadjusted irr of pd in children with both asthma and comorbidity was 14 times the rate in children with asthma alone , and the adjusted percentage of pd cases attributable to biologic interaction increased to 73% ( 95% ci : 52 , 93 ) . this study provides evidence that asthma is an important risk factor for the development of pd in children , and it provides new insight about a potentially meaningful interaction between asthma and comorbidity on the risk of pd . consistent with data presented by talbot et al,2 we observed a twofold increased rate of pd among all children < 18 years old following a childhood asthma diagnosis compared with person - time before an asthma diagnosis or in children who never had an asthma diagnosis after adjusting for confounding factors . the highest rate ratio occurred among children 24 to < 60 months old , which was twice as large as the rate ratios observed among children 6 to < 24 months old and 60 months old . we also observed a rate ratio less than 1 among children 0 to < 6 months old , but emphasize that we do not believe that this indicates that asthma is protective against pd in this age group . instead , we suspect that this observation is due to the difficulty of reliably diagnosing asthma in very young children.24 the evaluation of a biological interaction between childhood asthma and comorbidity on the incidence of pd revealed that the combined effect of these two exposures was synergistic in older children , and that a high proportion ( 55% in children aged 24 to < 60 months , and 73% in children aged 60 months ) of the pd incidence among asthmatic children with comorbidities was attributable to this interaction . no synergy between childhood asthma and comorbidity was observed in children less than 2 years old . these results suggest that children more than 2 years old who have underlying comorbidities are more sensitive to the effect of asthma on pd than children more than 2 years old without comorbidities . juhn et al3 also identified comorbidity as an effect modifier of the association between asthma and pd among minnesota adults . they reported that the odds ratio ( or ) of the effect of asthma on pd among adults with high - risk conditions was lower ( or : 1.2 ; p=0.86 ) than the or among those without high - risk conditions ( or : 2.9 ; p=0.04 ) . similar results were observed in our study in children between 2 years and 5 years old : the association between asthma and incident pd was lower among children with underlying comorbid conditions compared with children without underlying illnesses ( adjusted irr : 2.9 versus 4.2 , respectively ) . the lower rate ratios among children with comorbid conditions probably results from their higher risk of pd , which is sometimes called modification by the baseline risk . juhn et al3 did not assess for biological interaction on the additive scale in their study , and instead reported that there was no statistical interaction between asthma and illnesses based on the addition of an interaction term in a log - linear multivariate model . departure from additivity is a better measure of biologic interaction , and a lack of statistical interaction when measured as an interaction term in a log - linear multiplicative multivariate model can easily be mistaken for a lack of biologic interaction.22 several investigators have identified potential biologic mechanisms that may explain how asthma increases the risk of pd . two studies have identified associations between asthma and increased carriage of streptococcus pneumoniae in the nasopharynx,25,26 suggesting that children with asthma may be at increased risk of pd because they are more likely to be colonized with pneumococci . other proposed mechanisms include asthma - induced pathologic alterations that can impair clearance of pathogenic bacteria from the airway27,28 and chronic airway inflammation leading to impaired respiratory immunity.29,30 several limitations should be considered when interpreting the results from this study . first , the use of registry - based icd-10 codes to identify children with asthma is likely to result in some misclassification . underascertainment of asthma is possible if less severe cases of asthma were missed , most likely due to diagnosis or treatment only by a general practitioner . such underascertainment would bias the irrs describing the association between asthma and pd toward the null because some exposed children would be misclassified as unexposed . overascertainment is also possible if some children with wheezing due to other causes , for example respiratory syncytial virus , were classified as having asthma . although one way to increase sensitivity and specificity of asthma exposure would have been to incorporate the use of prescription asthma medications into a classification scheme for asthma exposure , we did not have access to these data for this study . we were also unable to include a mechanism by which to reclassify asthma - exposed children as unexposed if they grew out of an asthma diagnosis . although imperfect sensitivity and specificity of exposure classification is possible , several studies that have recently evaluated the quality of icd-10-based asthma diagnoses in the danish national registry of patients have found the diagnosis codes to be accurate.17,18 one study that used 3,550 medical records as the gold standard to validate icd-10 inpatient asthma diagnoses recorded in the danish national registry of patients reported 90% sensitivity and 99% specificity of asthma diagnoses among children aged 614 years old.17 another study reported 44% sensitivity and 98% specificity of asthma diagnoses recorded among 18-year - old men reporting for mandatory medical evaluation at the danish military draft board . the authors of this study subsequently demonstrated that the level of nondifferential asthma misclas - sification present in the danish national registry of patients was not sufficient to nullify the association between asthma and various skin cancers that they examined.18 we observed a 6% prevalence of asthma in our study population , which is less than an estimated asthma prevalence of 10%12% based on questionnaire data collected from parents of danish children aged 517 years.31,32 to determine the potential impact of imperfect sensitivity and specificity of exposure ascertainment on our results , we performed a bias analysis by calculating the irr that would have been observed if asthma ascertainment only has a sensitivity equal to 50% and a specificity equal to 97% . this sensitivity is consistent with the differences between the asthma prevalence recorded in this study and that recorded in published reports , and this specificity was the minimum specificity that resulted in no negative cell frequency in the corrected table . we assumed that exposure misclassification was nondifferential and independent for cases and noncases , as the data in our study were prospectively collected . the results from this analysis indicated a minimal impact on the age - specific irrs : the unadjusted irr in children aged 6 to < 24 months old would increase from 2.3 to 2.4 and the irr among children 24 to < 60 months old would increase from 4.8 to 5.0 ; the unadjusted irrs among children 0 to < 6 months old and 60 months old would not change . second , the exclusive use of icd-10 codes to identify pd cases creates the potential for misclassification . some pd cases could have been missed if the resulting illness was mild , if cultures were falsely negative , or due to recordkeeping errors . however , because pd is a serious disease typically requiring medical treatment , icd-10 codes are likely to capture the most important and costly infections . if underascertainment of pd did occur , it was likely to be nondifferential due to the prospective nature of the data which is , in turn , expected to produce unbiased ratio effect estimates33 in the absence of false positives . it is , however , possible that some cases could have been falsely attributed to pd when in fact they were caused by other bacterial infections . although we did not independently verify case status in this study , discharge diagnoses of pd have been found to have high specificity in validation studies conducted by other investigators.16,34 third , although we were able to collect extensive information about pregnancy- and birth - related characteristics , we were not able to capture complete information about some social factors associated with pd . misclassification of exposure to secondary tobacco smoke35 may have occurred in this study because information about smoking was only available from mothers at the beginning of pregnancy , and no information was available from fathers or other childcare providers . despite these limitations , the current study is an important contribution to the current knowledge of the association between asthma and pd . the evidence presented here indicates that asthmatic children are more likely to develop pd compared with nonasthmatic children , thereby providing support for the addition of asthma to the list of pneumococcal vaccine - eligible conditions for older children . these results also indicate that children who have asthma and another underlying comorbidity may be at especially high risk of pd , and should be carefully assessed in the clinic when presenting with bacterial illnesses . effect measure modification observed in stratified analyses was further evaluated for evidence of biologic interaction by using three standard measures1,2 to determine whether the independent effects of asthma and comorbidity summed to the total effect of both factors together . first , the interaction contrast ( ic ) between asthma and comorbidity was calculated by applying the following formula to crude pneumococcal disease ( pd ) incidence rates per 100,000 person years : ic = re+c+re+crec++rec(1)where r represents the rate of disease , e represents exposure to asthma , and c is the modifying covariate which , in this case , was comorbidity . the ic represents the number of cases of disease ( per 100,000 child - years ) that can not be accounted for by baseline factors among children without asthma or comorbidity , asthma only , or comorbidity only , and is therefore presumed to be attributable to a biological interaction between asthma and comorbidity . next , the interaction contrast ratio ( icr ) and 95% confidence interval around the icr was calculated to quantify the excess rate when both asthma and comorbidity were present at the same time relative to the baseline rate of disease that occurred when neither were present , while adjusting for important confounders of the association between asthma and pd:3 icr = icrec=irre+c+irre+cirrec++1(2 ) the icrs in our study were adjusted for sex , birth weight , child year of birth , and congenital malformation . we then used the icr ( to account for confounders ) to calculate the attributable proportion due to interaction ( ap ) , which quantifies the proportion of disease among exposed persons attributable to the interaction between an exposure and a modifying covariate . the ap was calculated by dividing the icr by the irr , comparing children with asthma and comorbidity to children without either of these:3 ap = icrirre+c+=irre+c+irre+cirrec++1irre+c+(3 ) icd-10 codes used to identify asthma , pneumococcal disease , and comorbidities from danish registries abbreviation : icd , international classification of diseases . effect measure modification observed in stratified analyses was further evaluated for evidence of biologic interaction by using three standard measures1,2 to determine whether the independent effects of asthma and comorbidity summed to the total effect of both factors together . first , the interaction contrast ( ic ) between asthma and comorbidity was calculated by applying the following formula to crude pneumococcal disease ( pd ) incidence rates per 100,000 person years : ic = re+c+re+crec++rec(1)where r represents the rate of disease , e represents exposure to asthma , and c is the modifying covariate which , in this case , was comorbidity . the ic represents the number of cases of disease ( per 100,000 child - years ) that can not be accounted for by baseline factors among children without asthma or comorbidity , asthma only , or comorbidity only , and is therefore presumed to be attributable to a biological interaction between asthma and comorbidity . next , the interaction contrast ratio ( icr ) and 95% confidence interval around the icr was calculated to quantify the excess rate when both asthma and comorbidity were present at the same time relative to the baseline rate of disease that occurred when neither were present , while adjusting for important confounders of the association between asthma and pd:3 icr = icrec=irre+c+irre+cirrec++1(2 ) the icrs in our study were adjusted for sex , birth weight , child year of birth , and congenital malformation . we then used the icr ( to account for confounders ) to calculate the attributable proportion due to interaction ( ap ) , which quantifies the proportion of disease among exposed persons attributable to the interaction between an exposure and a modifying covariate . the ap was calculated by dividing the icr by the irr , comparing children with asthma and comorbidity to children without either of these:3 ap = icrirre+c+=irre+c+irre+cirrec++1irre+c+(3 ) icd-10 codes used to identify asthma , pneumococcal disease , and comorbidities from danish registries abbreviation : icd , international classification of diseases . according to protocol analysis of co - variance reduced - antigen - content diphtheria tetanus and acellular pertussis vaccine filamentous hemagglutinin geometric mean concentration serious adverse event total vaccinated cohort pertussis is a highly infectious disease and remains an important worldwide public health problem , even in countries with sustained high vaccination coverage . despite established infant immunization programmes , pertussis continues to circulate , predominantly due to waning immunity beyond childhood and disease transmission from adolescents and adults to vulnerable infants . the need to maintain antibody levels against pertussis beyond childhood , through booster vaccines is therefore increasingly recognized . reduced - antigen - content combined diphtheria - tetanus - acellular pertussis ( dtpa ) vaccines , such as boostrix [ dtpa ; glaxosmithkline ( gsk ) vaccines ] , have been specifically developed to immunize older children from the age of 4 years , adolescents and adults . boostrix , which was first licensed in 1999 , is currently available in over 70 countries and has a well - established immunogenicity and tolerability profile in populations ranging from school age to the elderly . traditionally , boostrix has been available as a single - dose vial or a prefilled disposable syringe where the tip - cap and plunger stopper contained methylester w1883 . however , following the discontinuation of w1883 production by the manufacturer ( west ) , the syringe presentation has recently been replaced using prefilled syringes from a different manufacturer , wherein the tip - cap component contains fm27 ( a latex - free non - cytotoxic rubber compound ) and the plunger stopper component contains fm457 ( an ultra - low extractable bromobutyl compound ) . although this presentation change has been approved on the basis of a technical variation , this non - inferiority study was conducted to evaluate the impact on safety and immunogenicity , if any , due to the change in the material used in the rubber plunger of the prefilled syringe as required by a regulatory agency ; it compared the immunogenicity and safety of dtpa vaccine injected using the old and new syringe presentations , and thereby support the change with clinical data . this phase iv , randomized , single - blind , parallel group study ( nct01362322 ) , funded by glaxosmithkline biologicals sa , was conducted across 3 centers in chile and mexico between july 2011 and september 2012 . francisco i madero pte s / n y dr e aguirre pequeno , col mitras centro , monterrey , mexico ; subcomite de etica en investigacion , hospital general de ecatepec las america , estado de mexico ; comit de tica en investigacin , facultad de medicina , pontificia universidad catlica de chile ; comit tico cientfico del servicio de salud metropolitano central , santiago ; institute of public health chile ] and adhered to the declaration of helsinki and good clinical practice guidelines . written , informed consent was obtained from parents / guardians and assent from subjects before enrolment . healthy adolescents aged 1015 years who had received 5 or 6 previous doses of dt(p)/dt(pa ) vaccine were randomized ( 1:1 ) to receive dtpa booster via the new ( dtpa - new ) or previous ( dtpa - previous ) syringe presentations . due to visual differences in the presentation of the 2 syringes , this study was conducted in a single - blind manner . each 0.5 ml dtpa vaccine dose contained 2 iu diphtheria toxoid , 20 iu tetanus toxoid , 8 g pertussis toxin ( pt ) , 8 g filamentous hemagglutinin ( fha ) and 2.5 g pertactin ( prn ) . the vaccine was supplied in 2 prefilled syringe presentations : dtpa - previous group had syringes with w1833 tip - caps and plunger stoppers ( lot . no : dc37a005b and expiry date : 31 aug 2013 ) ; dtpa - new group had syringes with fm27 tip - caps and fm457 plunger stoppers ( lot . a single booster dtpa dose was injected intramuscularly into the deltoid region of the non - dominant arm , using a needle 2.54 cm length and 2225 gauge . blood samples ( 5 ml ) were collected from all subjects before , and one month post - booster dosing . antibodies against diphtheria , tetanus and pertussis antigens were measured using standard enzyme - linked immunosorbent assay ( elisa ) . seroprotection against diphtheria and tetanus antigens was defined as an antibody concentration 0.1 iu / ml . a booster response to diphtheria and tetanus antigens was defined as antibody concentrations 4-fold the assay cut - off in initially seronegative subjects or 4-fold increase in pre - vaccination antibody concentrations in initially seropositive subjects . seropositivity against pertussis antigens was defined as an antibody concentration 5 el.u / ml per antigen . a booster response to these antigens was defined as antibody concentrations 4-fold the assay cut - off in initially seronegative subjects ; a 4-fold increase in pre - vaccination antibody concentrations in initially seropositive subjects ( pre - vaccination concentrations 5 to < 20 el.u / ml ) or 2-fold increase in pre - vaccination antibody concentrations in initially seropositive subjects with pre - vaccination concentrations 20 el.u / ml . diary cards were used to assess solicited local ( injection site pain , redness and swelling ) and general ( fatigue , headache , fever [ axillary temperature 37.5c ] and gastrointestinal [ gi ] symptoms ) adverse events for 4 days ( day 03 ) after vaccination . the intensity of symptoms was graded on a 3-point scale : grade 3 redness and swelling : diameter > 50 mm ; grade 3 fever : axillary temperature > 39.0c . for all other symptoms large injection site reactions ( defined as swelling with a diameter > 100 mm , noticeable diffuse swelling or noticeable increase of limb circumference ) were evaluated by the investigator . all other symptoms , including serious adverse events ( saes ) occurring within 31 days of vaccination were recorded . the primary objective of the study was to demonstrate that dtpa - new is non - inferior to dtpa - previous , in terms of immune response to all vaccine antigens , one month after booster vaccination . the criteria for evaluation was that the upper limit ( ul ) of the 95% confidence interval ( ci ) on the gmc ratios [ dtpa - previous over dtpa - new ] for anti - diphtheria , anti - tetanus , anti - pt , anti - fha and anti - prn antibodies was 1.5 ( clinical limit for non - inferiority ) . the 95% cis for the gmc ratio of the 2 study groups was computed using an analysis of co - variance ( ancova ) model including the group and number of previous dt doses ( 5 or 6 ) as fixed effects and the log - transformed pre - vaccination concentration as co - variable . with a minimum of 600 evaluable subjects , the study had 94% power ( bonferroni adjustment of ) to achieve the primary objective . assuming a dropout rate of around 10% , a total of 670 subjects ( 335 subjects in each group ) were to be randomized to ensure a sufficient number of evaluable subjects were available for inclusion in the atp cohort for analysis of immunogenicity . secondary objectives included the evaluation of seroprotection / seropositivity rates , booster response and safety analysis one month after booster vaccination . the primary analysis of immunogenicity was performed on the according - to - protocol ( atp ) cohort , comprising vaccinated subjects who met the eligibility criteria , complied with protocol - defined procedures and for whom immunogenicity data were available . the analysis of safety was performed on the total vaccinated cohort ( tvc ) , which comprised all study participants for whom safety data were available . the safety results are described . of 671 subjects enrolled in the current study [ 376 at pontificia universidad , catolica de chile , santiago ; 93 at hospital universitario , de la uanl , monterrey ; mexico and 202 at hospital general de ecatepec las americas , estado de mexico , mexico ] , 335 received dtpa - new and 336 received dtpa - previous and were included in the tvc . one subject from the dtpa - previous group was eliminated from the atp cohort for safety after receiving a vaccine forbidden in protocol . fourteen subjects were eliminated from the atp cohort in the dtpa - new group due to non - compliance with blood sampling ( 8) and missing serological data ( 6 ) ; 16 were eliminated from the atp cohort in the dtpa - previous group due to protocol violation ( 1 ) , non - compliance with blood sampling ( 8) and missing serological data ( 7 ) . the atp cohort for immunogenicity therefore included 321 and 319 subjects in the dtpa - new and dtpa - previous groups , respectively . the mean age of the subjects in the atp cohort for immunogenicity was 11.9 years ( standard deviation 1.61 ) ; 50.6% subjects were of hispanic origin and 53.0% were female . before booster vaccination , 88.5% subjects in both groups were seroprotected against diphtheria ; 96.9% subjects in both groups were seroprotected against tetanus ( table 1 ) . at least 54.7% were seropositive against pertussis antibodies before the booster dose in both groups ( table 1 ) . table 1.seroprotection/seropositivity rates and gmcs one month after booster vaccination ( atp cohort for immunogenicity)dtpa - newdtpa - previousantibodytimingnnseroprotection ( 95% ci)gmc(95% ci)nnseroprotection ( 95% ci)gmc(95% ci)anti - diphtheriapre32128488.5 ( 84.591.8)0.472 ( 0.4030.553)31928689.7 ( 85.892.8)0.456 ( 0.3920.530)post32132099.7 ( 98.3100)6.784 ( 6.1787.450)319319100 ( 98.9100)6.493 ( 5.9157.128)anti - tetanuspre32131196.9 ( 94.398.5)0.956 ( 0.8351.095)31931498.4 ( 96.499.5)0.899 ( 0.7891.026)post321321100 ( 98.9100)18.937 ( 17.31320.713)319319100 ( 98.9100)18.515 ( 16.85120.342)dtpa - newdtpa - previousantibodytimingnnseropositivity ( 95% ci)gmc(95% ci)nnseropositivity ( 95% ci)gmc ( 95% ci)anti - ptpre32017554.7 ( 49.160.2)7.5 ( 6.68.7)31917554.9 ( 49.260.4)7.2 ( 6.38.2)post31831699.4 ( 97.799.9)140.2 ( 126.0156.1)31831599.1 ( 97.399.8)125.9 ( 112.7140.7)anti - fhapre31631098.1 ( 95.999.3)48.9 ( 43.355.2)31531098.4 ( 96.399.5)49.4 ( 43.656.0)post319319100 ( 98.9100)1080.2 ( 995.21172.5)319319100 ( 98.9100)1013.7 ( 940.01093.2)anti - prnpre32126983.8 ( 79.387.7)14.0 ( 12.315.9)31927285.3 ( 80.989.0)13.4 ( 11.915.0)post321321100 ( 98.9100)652.4 ( 572.1743.9)318318100 ( 98.8100)619.2 ( 546.0702.2)dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation.n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval ; d , diphtheria ; t , tetanus ; pt , pertussis toxin ; fha , filamentous hemagglutinin ; prn , pertactin iu , international unit ; el.u , elisa unit ; gmc , geometric mean concentration calculated on all subjects.*seroprotection = anti - diphtheria and anti - tetanus antibody concentration 0.1 iu / ml.**seropositive = anti - pt , anti - fha and anti - prn antibodies 5 eu / ml . seroprotection / seropositivity rates and gmcs one month after booster vaccination ( atp cohort for immunogenicity ) dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation . n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval ; d , diphtheria ; t , tetanus ; pt , pertussis toxin ; fha , filamentous hemagglutinin ; prn , pertactin iu , international unit ; el.u , elisa unit ; gmc , geometric mean concentration calculated on all subjects . seroprotection = anti - diphtheria and anti - tetanus antibody concentration 0.1 iu / ml . seropositive = anti - pt , anti - fha and anti - prn antibodies 5 eu / ml . one month after booster vaccination , 99.7% subjects in both groups were seroprotected against diphtheria antigens ; all subjects were seroprotected against tetanus antigens ; 99.4% were seropositive against the pertussis antigens ( table 1 ) . as the uls of the 95% ci for the gmc ratios ( dtpa - previous / dtpa - new ) for all antigens were 1.5 ( table 2 ) , non - inferiority of dtpa injected via the new syringe presentation ( dtpa - new ) against the previously used syringe presentation ( dtpa - previous ) table 2.adjusted gmc ratios between groups ( dtpa - previous divided by dtpa - new ) one month after booster vaccination ( atp cohort for immunogenicity)dtpa - previousdtpa - newadjusted gmc ratio ( dtpa - pre group/ dtpa - new group)antibodynadjusted gmcnadjusted gmcvalue ( 95% ci)anti - diphtheria3196.5213216.7650.96 ( 0.851.09)anti - tetanus31918.67232119.1710.97 ( 0.861.10)anti - pt318128.340317138.8320.92 ( 0.821.04)anti - fha3151013.1673141096.8270.92 ( 0.831.03)anti - prn318634.592321645.5040.98 ( 0.851.13)dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation.n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval for the adjusted gmc ratio [ ancova model : adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) pooled variance ] ) ; ll = lower limit , ul = upper limit.adjusted gmc = geometric mean antibody concentration obtained from an ancova model adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) . adjusted gmc ratios between groups ( dtpa - previous divided by dtpa - new ) one month after booster vaccination ( atp cohort for immunogenicity ) dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation . n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval for the adjusted gmc ratio [ ancova model : adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) pooled variance ] ) ; ll = lower limit , ul = upper limit . adjusted gmc = geometric mean antibody concentration obtained from an ancova model adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) . irrespective of which different syringe presentation was used for vaccine delivery , robust immune responses were observed and booster response rates for all antigens ranged from 79.0% to 99.7% in the 2 study groups ( data not shown ) . during the 4-day post - vaccination follow - up period , 78.8% and 83.0% subjects reported at least one symptom in the dtpa - new and dtpa - previous groups , respectively . injection site pain was the most commonly reported solicited local symptom in 71.8% ( 7.3% grade 3 ) and 75.4% ( 6.1% grade 3 ) of the subjects in the dtpa - new and dtpa - previous groups , respectively . headache , which was reported by 26.7% and 32.8% of the subjects , and fatigue , reported by 25.2% and 26.1% of the subjects , were the most frequently reported solicited general symptoms in the dtpa - new and dtpa - previous groups , respectively ( fig . 1 ) . figure 1.incidence of solicited local and general symptoms reported during the 4-day post - vaccination follow - up period ( total vaccinated cohort ) . incidence of solicited local and general symptoms reported during the 4-day post - vaccination follow - up period ( total vaccinated cohort ) . during the 31-day post - vaccination follow - up period , at least one unsolicited symptom was reported in 13.1% ( 2.4% grade 3 ) and 13.4% ( 2.7% grade 3 ) subjects in the dtpa - new and dtpa - previous groups , respectively . one subject in the dtpa - new group suffered accidental injury 13 days after vaccination ; this sae was considered to be unrelated to vaccination . have been distributed [ data on file ] and the immunogenicity , reactogenicity and safety of the vaccine has been well established in clinical trials and routine practice across a broad age range . owing to a recent technical change in the boostrix syringe presentation , this study was undertaken to compare the immunogenicity and safety of the new and previous syringe presentations , which differed in the nature of the compounds present in the tip - caps and plunger stoppers . in this study , the dtpa vaccine was immunogenic regardless of which syringe presentation was used to administer the vaccine . the results were consistent with previous reports in adolescents and non - inferiority of the new presentation compared to the old presentation was demonstrated . dtpa administered using the new dtpa syringe presentation was also well tolerated and the incidence and nature of adverse events were similar irrespective of the syringe presentation and comparable with previous studies . large swelling reactions , which can be associated with repeated booster doses of dtpa vaccines , were not observed in either study group . due to the resurgence of pertussis in adolescents and adults , the need to maintain antibody levels against pertussis beyond childhood , through booster vaccines although limited by single - blind design and inconsistent vaccination history [ subjects having received either 5 or 6 previous dt(p)/dt(pa ) vaccine doses ] , we demonstrated that a single dtpa booster dose was highly immunogenic and well tolerated in healthy adolescents in chile and mexico , irrespective of which syringe presentation was employed . in conclusion , clinical data from this study support the technical data and the use of the new syringe presentation with fm27 tip - caps and fm457 plunger stoppers to deliver the dtpa vaccine . hhh , sk , gj , kh , yc and al are employees of glaxosmithkline vaccines and hhh , gj , kh and al declare having glaxosmithkline stocks . ka has received grants , personal fees and non - financial support from glaxosmithkline and np - r has received research support from glaxosmithkline . glaxosmithkline vaccines was involved in all stages of the study conduct and analysis ; and also took charge of all costs associated with developing and publishing the manuscript .

backgroundalthough asthma has recently been established as a risk factor for pneumococcal disease ( pd ) , few studies have specifically evaluated this association in children.methodswe conducted a nation - wide population - based cohort study of the effect of asthma on childhood pd among all singleton live births in denmark from 1994 to 2007 , before the introduction of the 7-valent pneumococcal conjugate vaccine . all data were abstracted from danish medical registries . because underlying comorbidity substantially increases the pd risk in children , standard methods were used to assess the evidence of biologic interaction between comorbidity and asthma on the risk of pd.resultsthere were 2,253 cases of childhood pd among 888,655 children born in denmark from 1994 to 2007 . the adjusted incidence rate ratio of the effect of asthma on childhood pd was 2.2 ( 95% confidence interval [ ci ] : 2.0 , 2.5 ) . age - stratified incidence rate ratios were 2.1 ( 95% ci : 1.8 , 2.9 ) in children 6 months to < 24 months , 4.1 ( 95% ci : 3.3 , 5.1 ) in children 24 months to < 60 months , and 2.3 ( 95% ci : 1.6 , 3.2 ) in children 60 months . evaluation of the biologic interaction between asthma and comorbidity in older children revealed that 55% ( 24 months to < 60 months ) to 73% ( 60 months ) of cases among asthma - exposed children can be accounted for by the interaction between asthma and comorbidity.conclusionthese results confirm that asthma is an important risk factor for pd in children and suggest that children with underlying comorbidities are more sensitive to the effect of asthma on pd than children without comorbidities . reduced - antigen - content diphtheria - tetanus - acellular pertussis ( dtpa ) vaccine , boostrix , is indicated for booster vaccination of children , adolescents and adults . the original prefilled disposable dtpa syringe presentation was recently replaced by another prefilled - syringe presentation with latex - free tip - caps and plunger - stoppers . 671 healthy adolescents aged 1015 years who had previously received 5 or 6 previous dt(p)/dt(pa ) vaccine doses , were randomized ( 1:1 ) to receive dtpa booster , injected using the new ( dtpa - new ) or previous syringe ( dtpa - previous ) presentations . immunogenicity was assessed before and 1-month post - booster vaccination ; safety / reactogenicity were assessed during 31-days post - vaccination . non - inferiority of dtpa - new versus dtpa - previous was demonstrated for all antigens ( uls 95% cis for gmc ratios ranged between 1.03 - 1.13 ) . 1-month post - booster , immune responses were in similar ranges for all antigens with both syringe presentations . dtpa delivered using either syringe presentation was well - tolerated . these clinical results complement the technical data and support the use of the new syringe presentation to deliver the dtpa vaccine .

to identify the possible barriers to the implementation of pharmaceutical care among community and hospital pharmacists in enugu state using nsukka and enugu metropolis as a case study . the questionnaires were distributed to community and hospital pharmacists from designated areas during one of their quarterly meeting and their practice sites in 2009 . eighty completed questionnaires were collected with 22.8% from community pharmacists , and 77.2% from hospital pharmacists . the important barriers identified were lack of space , enough personnel in pharmacy to handle routine technical tasks , time , need for too much effort , and need for payment for services . the opinions on barriers to pharmaceutical care of pharmacists from community and hospital practice areas in these two metropolises of enugu state are majorly lack of time , space and routine technical task personnel . we retrospectively evaluated 128 consecutive young adults who were under 40 years of age and who had undergone a ccta evaluation with 64-slice multidetector ct ( mdct ) as a screening test for detecting cad during a general health evaluation from january 2006 to may 2008 . we excluded 16 subjects who had chest pain or discomfort ( n = 15 ) and those with a history of acute coronary syndrome ( n = 1 ) . none of the subjects were excluded due to nondiagnostic ccta image quality . as a result , 112 asymptomatic young adults ( 90 men and 22 women ) were finally enrolled . the mean age of the study population was 35.6 3.7 years ( range : 28 - 40 years ) . all the subjects had conventional coronary risk factors such as diabetes mellitus ( n = 8) , cigarette smoking ( n = 54 ) , hypertension ( n = 36 ) or obesity ( n = 62 ) . there were 77 subjects with a low cad risk , 27 with a moderate risk and eight with a high risk . a single oral dose of 20 mg of a -blocker ( propranolol [ pranol ; daewoong , korea ] ) was administered one hour before mdct for the patients with a heart rate of > 65 beats per minute ( bpm ) . for those with a heart rate that was still greater than 65 bpm , an additional 20 mg of oral propranolol was administered one hour after the administration of the first dose of oral -blocker . ccta was not performed for patients with heart rates more than 70 bpm after repeated drug administration or for those with an intermittent arrhythmia . coronary vasodilatation was achieved by administering 0.6 mg nitroglycerin ( myung moon , seoul , korea ) sublingually before the ccta to obtain the maximum coronary artery opacification . all the examinations were performed using a 64-slice mdct ( somatom sensation 64 , siemens medical solutions , germany ) and the following scan parameters were used : tube voltage 120 kvp , tube current 750 effective mas , detector collimation 64 0.6 mm , gantry rotation 370 msec and pitch 0.24 . a bolus of 70 ml iopamidol ( 370 mg of iodine per milliliter ; iopamiro 370 ; bracco , milan , italy ) was injected intravenously into an antecubital vein at a flow rate of 4 ml / sec , and this was followed by a 40 - 50 ml saline chaser using the bolus tracking technique . the images were initially reconstructed at the mid - diastolic phase ( 60 - 70% of the rr interval ) of the cardiac cycle , and they were then transferred to a computed workstation ( leonardo , siemens medical solutions , germany ) . all the scans were retrospectively analyzed on a three - dimensional workstation by two radiologists ( 14 and 7 years of experience in chest ct , respectively ) , using the multiplanar reformation technique , the maximum intensity projection technique and the volume - rendering technique with a short - axis and the two - chamber and four - chamber views . the coronary artery tree was segmented according to the modified american heart association classification ( 15 segments ) , and these segments were subsequently investigated for the presence and characteristics of coronary plaques ( 6 ) . the degree of stenosis was classified as significant if the patient had more than a 75% area stenosis on the cross - sectional images or more than a 50% diameter stenosis on the longitudinal images . coronary plaques were classified as non - calcified ( plaques without visible calcification ) , mixed ( plaques with non - calcified and calcified components ) or calcified plaques ( completely calcified plaques ) , according to the calcified component of the plaques seen on ccta . plaque densities of more than 130 hu ( hounsfield unit ) on the native scans were classified as calcified ( fig . changes in the coronary artery diameter were measured to determine the remodeling index ( ri ) . the maximum outer diameter at the lesion and at the proximal reference site was measured to calculate the ri ( i.e. , ri = [ maximum outer diameter at the plaque site - maximum outer diameter at the reference vessel ] / maximum outer diameter at the reference vessel ) . positive remodeling was defined as an ri > 0 and negative remodeling as an ri < 0 . the degree of remodeling was calculated by percentage ( i.e. , mean values standard deviations ) . measurements of the vascular diameter were obtained from the manually traced maximal diameters taken from the longitudinal source images ( fig . all the study related data , including the demographics , symptoms , medical histories and laboratory results , was systemically acquired from the hospital database and a chart review . the conventional coronary risk factors such as obesity , cigarette smoking , hypertension , hypercholesterolemia and diabetes mellitus were assessed , and the serum biomarkers such as homocysteine , c - reactive protein and triglyceride were measured . the framingham risk scores , as used by the national cholesterol education program ( ncep ) guidelines , were also calculated ( 7 ) . all the subjects were assigned to one of three different risk groups according to the revised ncep guidelines : the high - risk group ( cad risk equivalents or a 10-year risk > 20% ) , the moderate - risk group ( more than two risk factors and a 10-year risk 20% ) and the low - risk group ( 0 - 1 risk factors ) . of the 112 subjects , 109 underwent abdominal ultrasonography as part of a general health evaluation within one week of ccta , and the presence or absence of a fatty liver was determined . the degree of fatty liver on ultrasonography was classified as follows : 1 ) a mild , minimal diffuse increase of the hepatic echogenicity with normal visualization of the diaphragm and intrahepatic vessel borders ; 2 ) a moderate , moderate diffuse increase of the hepatic echogenicity with slightly impaired visualization of the intrahepatic vessels and diaphragm ; 3 ) a severe , marked increase of the echogenicity with poor visualization or nonvisualization of the hepatic vessels and diaphragm . we also compared the patients with cad to the normal subjects with respect to the coronary risk factors and a fatty liver . the clinical follow - up data ( range : 10 - 39 months ) was obtained from the patients who exhibited cad on ccta , based on the cardiac events , the treatment and the laboratory findings . all the statistical analyses were performed using spss version 17.0 for windows ( spss inc . , the differences between categorical variables were analyzed using chi - square or fisher 's exact tests , and the differences between continuous variables were analyzed using the unpaired student 's t - test or mann - whitney test , as appropriate . we retrospectively evaluated 128 consecutive young adults who were under 40 years of age and who had undergone a ccta evaluation with 64-slice multidetector ct ( mdct ) as a screening test for detecting cad during a general health evaluation from january 2006 to may 2008 . we excluded 16 subjects who had chest pain or discomfort ( n = 15 ) and those with a history of acute coronary syndrome ( n = 1 ) . none of the subjects were excluded due to nondiagnostic ccta image quality . as a result , 112 asymptomatic young adults ( 90 men and 22 women ) were finally enrolled . the mean age of the study population was 35.6 3.7 years ( range : 28 - 40 years ) . all the subjects had conventional coronary risk factors such as diabetes mellitus ( n = 8) , cigarette smoking ( n = 54 ) , hypertension ( n = 36 ) or obesity ( n = 62 ) . there were 77 subjects with a low cad risk , 27 with a moderate risk and eight with a high risk . a single oral dose of 20 mg of a -blocker ( propranolol [ pranol ; daewoong , korea ] ) was administered one hour before mdct for the patients with a heart rate of > 65 beats per minute ( bpm ) . for those with a heart rate that was still greater than 65 bpm , an additional 20 mg of oral propranolol was administered one hour after the administration of the first dose of oral -blocker . ccta was not performed for patients with heart rates more than 70 bpm after repeated drug administration or for those with an intermittent arrhythmia . coronary vasodilatation was achieved by administering 0.6 mg nitroglycerin ( myung moon , seoul , korea ) sublingually before the ccta to obtain the maximum coronary artery opacification . all the examinations were performed using a 64-slice mdct ( somatom sensation 64 , siemens medical solutions , germany ) and the following scan parameters were used : tube voltage 120 kvp , tube current 750 effective mas , detector collimation 64 0.6 mm , gantry rotation 370 msec and pitch 0.24 . retrospective electrocardiogram ( ecg ) gating and ecg - gated dose modulation were used . a bolus of 70 ml iopamidol ( 370 mg of iodine per milliliter ; iopamiro 370 ; bracco , milan , italy ) was injected intravenously into an antecubital vein at a flow rate of 4 ml / sec , and this was followed by a 40 - 50 ml saline chaser using the bolus tracking technique . the images were initially reconstructed at the mid - diastolic phase ( 60 - 70% of the rr interval ) of the cardiac cycle , and they were then transferred to a computed workstation ( leonardo , siemens medical solutions , germany ) . all the scans were retrospectively analyzed on a three - dimensional workstation by two radiologists ( 14 and 7 years of experience in chest ct , respectively ) , using the multiplanar reformation technique , the maximum intensity projection technique and the volume - rendering technique with a short - axis and the two - chamber and four - chamber views . the coronary artery tree was segmented according to the modified american heart association classification ( 15 segments ) , and these segments were subsequently investigated for the presence and characteristics of coronary plaques ( 6 ) . the degree of stenosis was classified as significant if the patient had more than a 75% area stenosis on the cross - sectional images or more than a 50% diameter stenosis on the longitudinal images . coronary plaques were classified as non - calcified ( plaques without visible calcification ) , mixed ( plaques with non - calcified and calcified components ) or calcified plaques ( completely calcified plaques ) , according to the calcified component of the plaques seen on ccta . plaque densities of more than 130 hu ( hounsfield unit ) on the native scans were classified as calcified ( fig . 1 ) . changes in the coronary artery diameter were measured to determine the remodeling index ( ri ) . the maximum outer diameter at the lesion and at the proximal reference site was measured to calculate the ri ( i.e. , ri = [ maximum outer diameter at the plaque site - maximum outer diameter at the reference vessel ] / maximum outer diameter at the reference vessel ) . positive remodeling was defined as an ri > 0 and negative remodeling as an ri < 0 . the degree of remodeling was calculated by percentage ( i.e. , mean values standard deviations ) . measurements of the vascular diameter were obtained from the manually traced maximal diameters taken from the longitudinal source images ( fig . all the study related data , including the demographics , symptoms , medical histories and laboratory results , was systemically acquired from the hospital database and a chart review . the conventional coronary risk factors such as obesity , cigarette smoking , hypertension , hypercholesterolemia and diabetes mellitus were assessed , and the serum biomarkers such as homocysteine , c - reactive protein and triglyceride were measured . the framingham risk scores , as used by the national cholesterol education program ( ncep ) guidelines , were also calculated ( 7 ) . all the subjects were assigned to one of three different risk groups according to the revised ncep guidelines : the high - risk group ( cad risk equivalents or a 10-year risk > 20% ) , the moderate - risk group ( more than two risk factors and a 10-year risk 20% ) and the low - risk group ( 0 - 1 risk factors ) . of the 112 subjects , 109 underwent abdominal ultrasonography as part of a general health evaluation within one week of ccta , and the presence or absence of a fatty liver was determined . the degree of fatty liver on ultrasonography was classified as follows : 1 ) a mild , minimal diffuse increase of the hepatic echogenicity with normal visualization of the diaphragm and intrahepatic vessel borders ; 2 ) a moderate , moderate diffuse increase of the hepatic echogenicity with slightly impaired visualization of the intrahepatic vessels and diaphragm ; 3 ) a severe , marked increase of the echogenicity with poor visualization or nonvisualization of the hepatic vessels and diaphragm . we also compared the patients with cad to the normal subjects with respect to the coronary risk factors and a fatty liver . the clinical follow - up data ( range : 10 - 39 months ) was obtained from the patients who exhibited cad on ccta , based on the cardiac events , the treatment and the laboratory findings . all the statistical analyses were performed using spss version 17.0 for windows ( spss inc . , the differences between categorical variables were analyzed using chi - square or fisher 's exact tests , and the differences between continuous variables were analyzed using the unpaired student 's t - test or mann - whitney test , as appropriate . atheromatous plaques were noted in 15 segments in 12 of 112 subjects ( 11% ) . nine patients had single - vessel disease , and three had two - vessel disease . there were 11 men and one woman with an age range from 31 to 40 years ( mean age : 36.8 2.6 years ) with atheromatous plaques . the patient population included four of 77 subjects ( 5% ) with a low cad risk , six of 27 ( 22% ) with a moderate risk and two of eight ( 25% ) with a high cad risk . the prevalence of cad was significantly higher in the moderate cad risk group than that in the low risk group ( p = 0.018 ) ; however , there was no significant difference between the moderate and high risk groups ( p = 0.604 ) ( fig . the presence of obesity , hypertension , hypercholesterolemia , high triglycerides and fatty liver was significantly higher in the patients with cad than that in the normal individuals . there also tended to be a higher incidence of cigarette smoking , high ldl ( low - density lipoprotein ) cholesterol , low hdl ( high - density lipoprotein ) cholesterol , diabetes mellitus and high c - reactive protein in the patients with cad ; however , any statistical significance was not identified ( table 1 ) . the location and degree of stenosis as well as the type of plaque are shown in table 2 . the coronary plaques were located in the left anterior descending ( lad ) ( n = 11 , 73% ) and the left main ( lm ) ( n = 4 , 27% ) arteries , with the most common location being the proximal lad ( n = 9 , 60% ) ( fig . the types of plaque included non - calcified ( n = 4 , 27% ) , mixed ( n = 7 , 47% ) and calcified ( n = 4 , 27% ) . the changes in the vascular diameter at the cad site were measured in four coronary segments with noncalcified plaque and in seven with mixed plaques . this measure was not evaluated in segments with calcified plaques because the exact measurement of the vascular diameter was unavailable due to beam - hardening and blooming artifacts . positive remodeling was identified in all the segments with noncalcified or mixed plaques , and the degree of positive remodeling tended to be higher in the patients with noncalcified plaques than in those with mixed plaques ( table 3 ) ; however , there was no statistically significant difference between the two groups ( p = 0.471 ) . twelve patients with cad have been free of cardiac events 10 - 39 months after ccta . four of the five patients with hypercholesterolemia started antihypercholesterolemic medication , based on the results of the ccta , and they showed improvement of their hypercholesterolemia on the follow - up tests . early detection of cad provides the opportunity to initiate interventions that will stabilize existing lesions , including ldl - lowering drug therapy ( statins ) , smoking cessation intervention for cigarette smokers , blood pressure lowering for persons with hypertension , and lifestyle intervention for the physically inactive and obese . such interventions can reduce the risk of developing acute coronary syndromes later in life . the current approach for managing the asymptomatic individuals who are at risk for cad is based on traditional clinical risk assessments such as the framingham risk score or the ncep guidelines ( 7 ) . however , there is growing evidence that these traditional risk assessment tools , which are based on risk factor analysis , have substantial limitations when they are used to guide individual therapy ( 8 , 9 ) . in a recently published study by choi et al . ( 10 ) in which cad was evaluated using ccta in 1,000 middle - aged asymptomatic adults ( mean age : 50 years ) , cad was noted in 215 subjects ( 22% ) . this rate is higher than that observed in earlier studies ( 3 - 5% ) , and the results were confirmed by coronary angiography in the asymptomatic individuals ( 11 - 13 ) . to date , there is a paucity of data regarding the prevalence of atherosclerotic plaques as detected on ccta in asymptomatic young adults , and this is largely because these asymptomatic young adults typically do not undergo medical evaluations for cad . in this study that enrolled asymptomatic young adults ( i.e. , < 40 years of age ) who were at risk for cad , coronary plaques were demonstrated in 11% of the subjects , and the prevalence of cad was significantly higher in the subjects with moderate ( 22% ) or high ( 25% ) risk than in those subjects with low risk ( 5% ) . positive remodeling was noted in all the coronary segments that exhibited noncalcified or mixed plaques with non - significant stenosis . this finding may indicate an important cause for underestimating cad on coronary angiography and it may explain the acute coronary syndrome of young patients with angiographically normal coronary arteries ( 14 - 16 ) . positive remodeling occurs in the early stages of coronary plaque development , and it is well known that positive remodeling is related to plaque instability , suggesting it is more prone to rupture and erosion with subsequent coronary events ( 17 - 19 ) . nakamura et al . ( 19 ) reported that positive remodeling was observed more frequently in patients with acute coronary syndrome ( 78 - 82% ) than in patients with stable cad ( 33 - 40% ) , and a significantly higher degree of positive remodeling was noted in patients with acute myocardial infarction ( remodeling index , 1.26 0.15 ) than in elderly patients with unstable angina pectoris ( 1.11 0.10 ) or stable angina pectoris ( 0.94 0.11 ) ( mean age , 63 10 years ) . tanaka et al . ( 18 ) demonstrated that all lipid core plaques showed positive vascular remodeling , which is in contrast to the severely calcified plaques that did not show positive remodeling . furthermore , it is well known from angiographic studies that most myocardial infarctions occur at sites that previously caused only mild to moderate luminal stenosis ( 20 , 21 ) . taken together , these studies demonstrate the clinical significance and importance of early plaques with mild stenosis . choi et al . ( 10 ) also reported that 85% of cad presented at the lad . in our study , plaques were not observed in the left circumflex artery , the peripheral branches of the coronary arteries or the diagonal branches . this may have resulted from the limited temporal resolution of ccta , which would underestimate the small branches of the coronary arteries , or it may also result from a lower prevalence of plaques in those segments in patients with early - stage cad . the coronary calcium score is intimately associated with the total plaque burden ( 22 ) and it is a strong predictor of coronary events , and the coronary calcium score is independent of the traditional coronary risk factors or risk - factor scores ( 23 ) . however , it sometimes fails to predict acute coronary events or significant stenosis because a significant numbers of vulnerable plaques tend to be predominantly noncalcified and nonstenotic lesions ( 20 ) . the absence of coronary calcification has been described in 1% of the male patients with significant coronary stenosis ( 24 ) and in 4% of the patients suffering from an unheralded myocardial infarction ( 25 ) . in our study , 27% of the patients with cad had only noncalcified plaques , which is slightly higher than the 19% reported by choi et al . this may be linked to the observation that the plaques in young patients are probably in the early stages , so they have less calcification than those of older individuals . therefore , the coronary calcium score may underestimate cad in young adults to a greater degree than that in older individuals . obesity , smoking , hypertension and hypercholesterolemia are well known risk factors for cad , and their incidences were significantly higher in the patients with cad than that in the normal subjects of our study . in a recent report , fatty liver was demonstrated to be associated with the risk of cad and early atherosclerosis in middle - aged individuals ( 26 ) . in the current study , we were able to evaluate the incidence of fatty liver because almost all the subjects underwent abdominal ultrasonography and ccta as a part of a general health evaluation . as such , we also found that a fatty liver was significantly more prevalent in the patients with cad ( 83% ) than that in the normal individuals ( 39% ) . the study population was small , it included only koreans living in urban areas and all the subjects were self - referred . therefore , the actual prevalence of cad in the young asymptomatic subjects in various ethnic groups and geographic regions may differ from our results , although this was evaluated in relation to the ncep cad risk categories . in conclusion , the prevalence of occult cad was not negligible in asymptomatic young adults , and the prevalence of cad was significantly higher in those subjects with moderate to high risks than in those with low risk . additionally , most of the patients had single - vessel disease , plaques of various composition , non - significant stenosis and positive vascular remodeling and one fourth of the patients had non - calcified plaques . finally , the most common location for plaques was the proximal lad . this study suggests the importance of management and risk factor modification for the asymptomatic young adults who have a moderate or high risk for cad .

objective : to identify the possible barriers to the implementation of pharmaceutical care among community and hospital pharmacists in enugu state using nsukka and enugu metropolis as a case study.method:a semi structured questionnaire was designed to carry out a cross sectional descriptive study . the questionnaires were distributed to community and hospital pharmacists from designated areas during one of their quarterly meeting and their practice sites in 2009.results:eighty completed questionnaires were collected with 22.8% from community pharmacists , and 77.2% from hospital pharmacists . the important barriers identified were lack of space , enough personnel in pharmacy to handle routine technical tasks , time , need for too much effort , and need for payment for services.conclusion:the opinions on barriers to pharmaceutical care of pharmacists from community and hospital practice areas in these two metropolises of enugu state are majorly lack of time , space and routine technical task personnel . objectivewe aimed at evaluating the prevalence and ct characteristics of occult coronary artery disease ( cad ) in young korean adults under 40 years of age by performing coronary ct angiography ( ccta).materials and methodswe retrospectively enrolled 112 consecutive asymptomatic subjects ( 90 men , mean age : 35.6 3.7 years ) who underwent ccta as part of a general health evaluation . we classified the subjects into three national cholesterol education program risk categories and we assessed the plaque characteristics on ccta according to the number of involved vessels , the location and type of plaques and vascular remodeling.resultstwelve individuals had cad ( 11% , 11 men ) . the prevalence of cad was significantly higher in the subgroups with moderate ( 22% ) or high ( 25% ) risk than that in the low risk subgroup ( 5% ) ( p < 0.05 ) . nine patients had single - vessel disease and three patients had two - vessel disease . the most common location for plaque was the proximal left anterior descending coronary artery ( 60% ) . all the patients had non - significant stenosis and plaque , including the non - calcified ( 27% ) , mixed ( 47% ) and calcified ( 27% ) types . positive vascular remodeling was identified in all the patients with non - calcified or mixed plaques.conclusionthe prevalence of occult cad was not negligible in the asymptomatic young adults with moderate to high risk , and this suggests the importance of management and risk factor modification in this population . all the patients had non - significant stenosis , and one fourth of the plaques did not show calcification .

several risk factors such as contact lens wear , trauma , ocular surface disease , ocular surgery , and systemic disease have been reported to predispose patients to corneal infections . management of microbial keratitis commonly involves obtaining corneal scrapings for microbiological studies , and then , empiric broad - spectrum treatment is typically initiated before culture results are available . appropriate empiric therapy is selected by practitioners on the basis of epidemiological information such as predisposing factors and the spectrum of causative organisms . for example , trauma is a common risk factor for fungal keratitis in developing agricultural countries , whereas contact lens wear is the main risk factor for bacterial keratitis in developed countries . thus , it is essential to establish related information on microbial keratitis , including patient - specific risk factors and the most likely causative organisms , which would facilitate the development of effective strategies for the prevention , diagnosis , and treatment of microbial keratitis . in taiwan , a study from a university hospital reported that contact lens - related pseudomonas keratitis was the most common form of microbial keratitis from 1992 to 2001 . conducting periodic surveys of infectious keratitis is crucial for updating the local information for reference by clinicians because epidemiologic patterns may change over time . therefore , the present study collected data on the predisposing factors , clinical manifestations , spectrum of microorganisms , and treatments for patients with microbial keratitis who were admitted to and treated at chang gung memorial hospital ( cgmh ) , a major teaching hospital in northern taiwan , from 2003 to 2012 . this study was conducted as a retrospective , cross sectional design , and in accordance with the declaration of helsinki and was approved by the institutional research ethics board of cgmh , taiwan ( irb102 - 4073b ) . consent was waived because of the retrospective design of the project and the anonymous analysis of the data . we retrospectively reviewed the medical records of 558 patients with infectious keratitis who were admitted to cgmh from january 1 , 2003 to december 31 , 2012 . we included patients with negative culture results if they presented with an epithelial defect and stromal infiltrate and responded favorably to antimicrobial treatment . admission criteria were primarily severe ( ie , potentially sight - threatening ) keratitis and the need for intensive topical antimicrobials . information on age , sex , predisposing factors , clinical features , microbiological results , treatments , and visual acuity ( if recorded ) was collected . we defined an ulcer as being central if it encroached within 2 mm of fixation , peripheral if it involved a zone within 2 mm from the limbus , and paracentral if it was between the central and peripheral zone . corneal ulcers were defined as being small ( < 2 mm ) , medium ( 26 mm ) , or large ( > 6 mm ) . corneal scrapings were obtained using a surgical blade and directly inoculated into blood agar , chocolate agar , modified sabouraud agar , lowenstein corneal scrapings of patients with clinical characteristics suggestive of acanthamoeba keratitis were inoculated into nonnutrient agar seeded with escherichia coli . the various media were routinely incubated for a week or longer depending on the media before the final culture result was obtained . a positive culture was defined as growth of at least 3 colonies along the line of inoculation on one solid medium on the basis of the criteria in a previous study . before the result of corneal ulcer culture could be obtained , levofloxacin ( 0.5% ) alone or a combination of 2 fortified antibiotics ( 25 mg / ml of cefazolin and 25 mg / ml of amikacin ) was administered topically once per hour . topical natamycin and amphotericin b ( 0.1% ) were applied hourly for mold and yeast infection , respectively . topical polyhexamethylene biguanide ( 0.02% ) was applied for acanthamoeba keratitis . for data presentation , we arbitrarily divided the study years into 2 periods ; the first half was from 2003 to 2007 , and the second half was from 2008 to 2012 . mantel - haenszel linear - by - linear association test was used to detect the trends over the 10-year period . categorical variables were analyzed using a test ; continuous variables were analyzed using analysis of variance ( anova ) . simple linear regression for univariate analysis was used to identify the factors associated with hospital stay . all statistical analyses were performed using spss software , version 22 ( ibm , armonk , ny ) . this study was conducted as a retrospective , cross sectional design , and in accordance with the declaration of helsinki and was approved by the institutional research ethics board of cgmh , taiwan ( irb102 - 4073b ) . consent was waived because of the retrospective design of the project and the anonymous analysis of the data . we retrospectively reviewed the medical records of 558 patients with infectious keratitis who were admitted to cgmh from january 1 , 2003 to december 31 , 2012 . we included patients with negative culture results if they presented with an epithelial defect and stromal infiltrate and responded favorably to antimicrobial treatment . admission criteria were primarily severe ( ie , potentially sight - threatening ) keratitis and the need for intensive topical antimicrobials . information on age , sex , predisposing factors , clinical features , microbiological results , treatments , and visual acuity ( if recorded ) was collected . we defined an ulcer as being central if it encroached within 2 mm of fixation , peripheral if it involved a zone within 2 mm from the limbus , and paracentral if it was between the central and peripheral zone . corneal ulcers were defined as being small ( < 2 mm ) , medium ( 26 mm ) , or large ( > 6 mm ) . corneal scrapings were obtained using a surgical blade and directly inoculated into blood agar , chocolate agar , modified sabouraud agar , lowenstein corneal scrapings of patients with clinical characteristics suggestive of acanthamoeba keratitis were inoculated into nonnutrient agar seeded with escherichia coli . the various media were routinely incubated for a week or longer depending on the media before the final culture result was obtained . a positive culture was defined as growth of at least 3 colonies along the line of inoculation on one solid medium on the basis of the criteria in a previous study . before the result of corneal ulcer culture could be obtained , levofloxacin ( 0.5% ) alone or a combination of 2 fortified antibiotics ( 25 mg / ml of cefazolin and 25 mg / ml of amikacin ) was administered topically once per hour . topical natamycin and amphotericin b ( 0.1% ) were applied hourly for mold and yeast infection , respectively . , we arbitrarily divided the study years into 2 periods ; the first half was from 2003 to 2007 , and the second half was from 2008 to 2012 . mantel - haenszel linear - by - linear association test was used to detect the trends over the 10-year period . categorical variables were analyzed using a test ; continuous variables were analyzed using analysis of variance ( anova ) . simple linear regression for univariate analysis was used to identify the factors associated with hospital stay . all statistical analyses were performed using spss software , version 22 ( ibm , armonk , ny ) . during the 10-year study period , 558 patients were included in this study , of which 285 ( 51.1% ) and 273 ( 48.9% ) were male and female , respectively ( table 1 ) . the mean age of patients was 50.3 22.7 years ( range , 2100 yr ) . the right eye was involved in 287 patients and the left eye was involved in 271 patients . the corneal ulcer was small in 192 eyes ( 37.7% ) , medium in 267 eyes ( 52.5% ) , and large in 50 eyes ( 9.8% ) . the location of the corneal ulcer was central in 238 eyes ( 47.1% ) , paracentral in 206 eyes ( 40.8% ) , and peripheral in 61 eyes ( 12.1% ) . the presence of hypopyon was noted in 153 eyes ( 33.8% ) . except for laterality , trends for demographics and clinical features were not statistically significant . demographics and clinical features of microbial keratitis risk factors for microbial keratitis were identified in 426 patients ( 76.3% ) . the most common risk factor was contact lens wear ( 31.4% ) , followed by systemic and ocular diseases ( 26.3% ) , trauma ( 23.5% ) , and previous ocular surgery ( 12.7% ) ( table 2 ) . a total of 54 patients ( 12.7% ) had at least 2 predisposing factors for corneal ulcers . materials causing ocular trauma included plant ( n = 24 ) , iron ( n = 16 ) , mud ( n = 5 ) , chemical ( n = 4 ) , wood ( n = 3 ) , and unidentified sources ( n = 48 ) . lagophthalmos ( n = 16 ) and dry eye ( n = 14 ) were the most common ocular surface diseases . the trend test showed that during the 10-year study period , the proportion of patients wearing contact lenses and with trauma increased ( p = 0.011 and p = 0.035 , respectively ) , and the rate of previous ocular surgery decreased ( p = 0.027 ) . predisposing factors for microbial keratitis positive culture results were obtained in 353 patients ( 63.3% ) . two hundred thirty - eight ( 42.7% ) patients were administered topical antibiotics before referral , and 88 patients had negative culture results . regarding isolates , 210 bacterial isolates ( 59.9% ) , 62 fungal isolates ( 17.6% ) , 8 nontuberculous mycobacteria ( ntm ) isolates ( 2.3% ) , and 2 acanthamoeba isolates ( 0.6% ) were identified ( table 3 ) . seventy - one patients had polymicrobial infections ( 20.1% ) . among bacterial isolates , gram - negative bacteria ( 37.4% ) were more common than gram - positive bacteria ( 22.1% ) . the most commonly isolated bacterium was pseudomonas aeruginosa ( 28% ) , followed by coagulase - negative staphylococcus ( cns , 5.4% ) and staphylococcus aureus ( 4.5% ) . the percentage of serratia marcescens decreased significantly ( p = 0.033 ) ; the identified organisms did not change over 10 years . isolated organisms in microbial keratitis table 4 lists isolated organisms from patients with different risk factors for microbial keratitis . gram - negative bacteria , particularly p. aeruginosa , mainly accounted for contact lens - related keratitis ( 52.9% ) . keratitis associated with ocular and systemic diseases was mainly caused by bacteria ( 33.8% and 29.4% for gram - positive and gram - negative bacteria , respectively ) . risk factors versus isolated organisms in microbial keratitis all patients were initially treated with empiric antimicrobials , which were adjusted on the basis of the clinical response and the results of the drug susceptibility test . medical treatment was successful for patients with gram - positive bacterial infections ( 62.8% ) , gram - negative bacterial infections ( 78% ) , ntm infections ( 75% ) , acanthamoeba infections ( 100% ) , and polymicrobial infections ( 74.6% ) ( table 5 ) . surgical procedures included amniotic membrane transplantation , patch graft , lamellar keratectomy , penetrating keratoplasty , and evisceration . one patient with fungal keratitis , 2 patients with pseudomonal keratitis , and 3 patients with polymicrobial infections underwent evisceration to eradicate the infections . treatment for microbial keratitis the mean hospital stay was 13.7 11.5 days . longer hospital stay was correlated with previous steroid use , ocular and systemic diseases , longer interval between symptom presentation and admission , previous ocular surgery , large ulcer size , fungal infection , ntm infection , poor visual acuity at presentation , old age , and surgery during admission ( all p < 0.05 by simple linear regression ) . in multiple linear stepwise regression analysis , 4 factors including previous ocular surgery , large ulcer size , ntm infection , and surgery during admission were associated with longer hospital stays ( table 6 ) . during the 10-year study period , 558 patients were included in this study , of which 285 ( 51.1% ) and 273 ( 48.9% ) were male and female , respectively ( table 1 ) . the mean age of patients was 50.3 22.7 years ( range , 2100 yr ) . the right eye was involved in 287 patients and the left eye was involved in 271 patients . the corneal ulcer was small in 192 eyes ( 37.7% ) , medium in 267 eyes ( 52.5% ) , and large in 50 eyes ( 9.8% ) . the location of the corneal ulcer was central in 238 eyes ( 47.1% ) , paracentral in 206 eyes ( 40.8% ) , and peripheral in 61 eyes ( 12.1% ) . the presence of hypopyon was noted in 153 eyes ( 33.8% ) . except for laterality , trends for demographics and clinical features were not statistically significant the most common risk factor was contact lens wear ( 31.4% ) , followed by systemic and ocular diseases ( 26.3% ) , trauma ( 23.5% ) , and previous ocular surgery ( 12.7% ) ( table 2 ) . a total of 54 patients ( 12.7% ) had at least 2 predisposing factors for corneal ulcers . materials causing ocular trauma included plant ( n = 24 ) , iron ( n = 16 ) , mud ( n = 5 ) , chemical ( n = 4 ) , wood ( n = 3 ) , and unidentified sources ( n = 48 ) . lagophthalmos ( n = 16 ) and dry eye ( n = 14 ) were the most common ocular surface diseases . the trend test showed that during the 10-year study period , the proportion of patients wearing contact lenses and with trauma increased ( p = 0.011 and p = 0.035 , respectively ) , and the rate of previous ocular surgery decreased ( p = 0.027 ) . two hundred thirty - eight ( 42.7% ) patients were administered topical antibiotics before referral , and 88 patients had negative culture results . regarding isolates , 210 bacterial isolates ( 59.9% ) , 62 fungal isolates ( 17.6% ) , 8 nontuberculous mycobacteria ( ntm ) isolates ( 2.3% ) , and 2 acanthamoeba isolates ( 0.6% ) were identified ( table 3 ) . seventy - one patients had polymicrobial infections ( 20.1% ) . among bacterial isolates , gram - negative bacteria ( 37.4% ) were more common than gram - positive bacteria ( 22.1% ) . the most commonly isolated bacterium was pseudomonas aeruginosa ( 28% ) , followed by coagulase - negative staphylococcus ( cns , 5.4% ) and staphylococcus aureus ( 4.5% ) . the percentage of serratia marcescens decreased significantly ( p = 0.033 ) ; the identified organisms did not change over 10 years . isolated organisms in microbial keratitis table 4 lists isolated organisms from patients with different risk factors for microbial keratitis . gram - negative bacteria , particularly p. aeruginosa , mainly accounted for contact lens - related keratitis ( 52.9% ) . keratitis associated with ocular and systemic diseases was mainly caused by bacteria ( 33.8% and 29.4% for gram - positive and gram - negative bacteria , respectively ) . all patients were initially treated with empiric antimicrobials , which were adjusted on the basis of the clinical response and the results of the drug susceptibility test . medical treatment was successful for patients with gram - positive bacterial infections ( 62.8% ) , gram - negative bacterial infections ( 78% ) , ntm infections ( 75% ) , acanthamoeba infections ( 100% ) , and polymicrobial infections ( 74.6% ) ( table 5 ) . surgical procedures included amniotic membrane transplantation , patch graft , lamellar keratectomy , penetrating keratoplasty , and evisceration . one patient with fungal keratitis , 2 patients with pseudomonal keratitis , and 3 patients with polymicrobial infections underwent evisceration to eradicate the infections . treatment for microbial keratitis the mean hospital stay was 13.7 11.5 days . longer hospital stay was correlated with previous steroid use , ocular and systemic diseases , longer interval between symptom presentation and admission , previous ocular surgery , large ulcer size , fungal infection , ntm infection , poor visual acuity at presentation , old age , and surgery during admission ( all p < 0.05 by simple linear regression ) . in multiple linear stepwise regression analysis , 4 factors including previous ocular surgery , large ulcer size , ntm infection , and surgery during admission were associated with longer hospital stays ( table 6 ) . severe infectious keratitis is a leading cause of corneal blindness . optimal clinical practice for the prevention and treatment of microbial keratitis should account for patient - specific risk factors and possible causative organisms in different regions . in this study , we focused on patients admitted with microbial keratitis ; first , we ensured that only patients with severe infection leading to admission were included in this study , and second , we used the same ( or as similar as possible ) criteria as used in a previous report conducted at another university hospital in northern taiwan between 1992 and 2001 to provide updated regional epidemiological information . our findings showed that contact lens wear remained the leading risk factor for inpatient microbial keratitis , and the trend increased significantly over the 10-year study period ; p. aeruginosa was the most common causative organism . in the current study , risk factors for microbial keratitis from 2003 to 2012 were contact lens wear , ocular and systemic diseases , trauma , and previous ocular surgery , in descending order , which was similar to the results of a previous taiwanese report . in taiwan , the leading risk factor for microbial keratitis was still contact lens wear , which was also reported in the united states , western europe australia , and hong kong . rattanatam et al found that the number of patients with contact lens - related microbial keratitis decreased in their hospital , suggesting that a higher number of such patients were treated in the community after the introduction of fluoroquinolones . by contrast , our study demonstrated an increasing trend in the rate of contact lens - related microbial keratitis in our hospital over the 10-year study period ( p = 0.011 ) . in a prospective , population - based study of contact lens - related microbial keratitis in australia , risk factors for infections included overnight use , poor storage case hygiene , smoking , internet purchase of contact lenses , less than 6 months wear experience , and higher socioeconomic class ; however , new lens types did not reduce the incidence of infection . because of the retrospective design of this study , it was difficult to correlate contact lens wearing modalities , hygiene , and other factors with infections ; however , approximately 35.8% of patients had overnight use in our study . wearing contact lenses is popular in taiwan , which is a country with high prevalence of refractive errors , and contact lens - related microbial keratitis has become a public health concern . in 2012 , corneal health for care network to advocate the 3 c 's for contact lens wearers : consulting a physician on how to fit contact lenses correctly ( correct ) , cleaning and maintaining contact lens ( care ) , and receiving regular health check - ups ( check ) . this campaign is anticipated to facilitate decreases in the rate of contact lens - related microbial keratitis in taiwan . in this study , the most common causative organisms were bacteria , followed by fungi ; gram - negative bacteria were more common than gram - positive bacteria , and p. aeruginosa was the most commonly identified isolate . the spectrum of microorganisms accounting for microbial keratitis differ depending on geographic location , climate , and etiology . for example , gram - positive bacteria are predominant in temperate climate regions , whereas gram - negative bacteria and fungi are prevalent in tropical regions;pseudomonas species are associated with contact lens - related infections , whereas fungi are related to trauma caused by plants . the predominance of p. aeruginosa infection in taiwanese studies may reflect both the geographic prevalence of the microorganism and contact lens - related keratitis . fungi are also crucial causative organisms of microbial keratitis in taiwan ; similarly , this might be due to the geography and climate , and injury caused by plant materials . notably , 4 of 5 previous studies in europe and taiwan investigating microbiological findings of hospitalized patients with microbial keratitis have reported that gram - negative bacteria are the most common causative organism . this finding might suggest that microbial keratitis caused by gram - negative bacteria , particularly pseudomonas species , tends to be severe and progresses rapidly , thus requiring admission . in the current study , medical treatment was successful in 69.5% of patients ; more than half of the patients with fungal keratitis required additional surgical interventions to control infections . medical treatment of fungal keratitis , particularly deep - seated infections , is often unsatisfactory because of delayed diagnosis , inadequate drug penetration , and slow response to therapy . surgical intervention to remove infectious elements and necrotic tissue may increase drug penetration and shorten the clinical course . however , therapeutic corneal transplantation and even destructive surgery may be indicated for severe keratitis with a poor response to medical therapy or when severe complications supervene . long hospital stay also has effects on financial resources , staffing , and turnover rate of beds in the public health service . in this study , the mean hospital stay was 13.7 days , and longer hospital stays were associated with previous ocular surgery , large ulcer size , ntm infection , and surgery during admission . a study of hospitalized patients with infectious keratitis in new zealand reported that longer hospital stay was associated with the presence of hypopyon , larger ulcers , previous ocular surgery , and poor visual acuity . as expected , large ulcers or surgery during admission prolonged hospital stay . older age and longer duration from symptom onset to diagnosis were noted in patients with previous ocular surgery ( data not shown ) , which might explain the relationship between longer hospital stay and previous ocular surgery . ntm has a relatively slow growth rate , and the infection can mimic that caused by other pathogens . thus , the delay in diagnosis and treatment of ntm infections might prolong hospital stay . morbidities caused by microbial keratitis can be assessed on the basis of surgical intervention , hospital stay , and visual loss . in this study , we did not analyze the predictors for poor visual outcome in microbial keratitis because of visual assessments with variable follow - up intervals . however , the study in new zealand reported that longer hospital stays were associated with poor visual acuity both at presentation and final assessment . we included cases with both positive and negative culture results , and we could not exclude the possibility that cultured isolates were contaminated . because our patients were admitted to a tertiary referral hospital in taiwan , the results can not be generalized . in conclusion , p. aeruginosa was the most common causative organism , and contact lens wear was the most common risk factor for microbial keratitis ; there was a significant increase in the percentage of contact lens - related keratitis during the 10-year study period in taiwan . the majority of patients with microbial keratitis were cured through medical treatment , but a high proportion of patients with fungal keratitis required surgical interventions . microbial keratitis has the potential to cause devastating visual impairment and major costs to the public health system ; our findings provide updated information and facilitate future prevention and treatment of microbial keratitis in taiwan . the human rights act of 1977 followed by the privacy act of 1982 were the first legislative acts to protect personal information across the canadian public sector ( peekhaus 2008 ) . in 1987 , at a time when the hiv epidemic was gaining momentum across canada , the freedom of information and protection of privacy act was the first legislation to outline principles of conscientious and cautious handling of phi that institutions were responsible to adhere to ( cavoukian 1990 ) . what followed was legislation that extended the protection of personal information in the private sector in canada , and in 2001 , the personal information protection and electronic documents act ( pipeda ) came into effect ( canadian hiv / aids legal network 2004 ; peekhaus 2008 ) . since 1997 , provinces have adopted privacy legislation that specifically applies to healthcare providers regardless of whether they are engaged in commercial activities ( peekhaus 2008 ) , and , currently , nine provinces have specific laws that protect phi and impose obligations on healthcare providers to protect that information ( canadian hiv / aids legal network 2014 ) . the personal health information protection act ( phipa ) was enacted in 2004 and governs the collection , use and disclosure of phi within the ontario health sector and aims to keep phi confidential and secure while allowing for the effective delivery of healthcare and the effective operation of the healthcare system ( beardwood and kerr 2004 , 2005 ; cavoukian 2008 ; oipc n.d . ) . phi is broadly defined under phipa as identifying information about an individual in oral or recorded form that could be used to identify a specific individual , for example , the physical and/or mental health of the individual ( including the health history of the individual 's family ) , the provision of healthcare to the individual , payments or eligibility for healthcare , and the individual 's health number ( beardwood and kerr 2004 ) ; hiv status is included under this definition ( canadian hiv / aids legal network 2004 ) . under phipa , persons and organizations that provide healthcare are collectively known as health information custodians , or hics ( oipc n.d . ; peekhaus 2008 ) as they have custody or control of personal health information in connection with performing their duties or work ( beardwood and kerr 2004 : 63 ) . whether individuals are hics or agents of a hic , their obligation to abide by phipa and ensure the security , confidentiality , accuracy and integrity of phi in their custody is the same ( fletcher 2014 ; peekhaus 2008 ) . phipa requires that hics take reasonable steps to ensure phi is protected against theft , loss and unauthorized use or disclosure regardless of the type of records being used ( beardwood and kerr 2005 ; cavoukian and rossos 2009 ) . consent , disclosure and circle of care are key constructs outlined in phipa that have important implications on the activities and decision - making of hics with regard to phi . hics may imply consent for the collection , use and disclosure of phi for the delivery of healthcare services ( cavoukian 2008 ) , for example , consent is implied if a patient accepts a referral and shows up for care ( fletcher 2014 ) . with regard to disclosure , under phipa , phi may only be disclosed by hics if the individual consents or if phipa specifically permits the disclosure without consent ( oipc n.d . ) . phipa was specifically designed so that it would not prevent a barrier to the disclosure of personal health information among healthcare providers , hics are permitted to disclose phi for the purposes of providing or assisting in providing care on the basis of implied or assumed implied consent ( canadian hiv / aids legal network 2014 ; cavoukian and rossos 2009 ) . a patient 's express consent , that is verbal or written consent , is not required to share information within the circle of care to other healthcare providers ( canadian hiv / aids legal network 2014 ) . at the same time , phipa permits disclosure of phi without implied consent in a number of vague and unspecified circumstances including providing healthcare ( as an individual healthcare provider or as a facility ) ; managing risks and error ( beardwood and kerr 2004 ) ; planning and management of the health system ; and analysis of the health system , etc . the assumption of implied consent is no longer true , however , when the hic is aware that the individual wishes to withhold or withdraw their consent . furthermore , when phi is disclosed to a non - hic outside of the circle of care , or for purposes other than delivery of healthcare , express consent is required ( beardwood and kerr 2004 ; cavoukian 2008 ) . implied consent and disclosure within the circle of care for the purposes of providing or assisting in the provision of healthcare is arguably the most significant provision in phipa yet it is buried in a sub - section of the legislation ( beardwood and kerr 2004 : 65 ) . permitting disclosure of phi in a variety of circumstances for the broad purposes of providing healthcare vis - - vis a focus on implied consent means that hics need to use little or no effort to comply with the requirements of phipa ( beardwood and kerr 2004 : 67 ) . therefore , while it is essential that people who use health services trust that their privacy will be protected , at the same time , the delivery of high quality healthcare depends on the availability of accurate and complete health information phipa attempts to strike a balance between protecting privacy and facilitating care delivery ( fletcher 2014 ) . although identifying information in oral form falls under the category of phi ( beardwood and kerr 2004 ) , it appears that sharing information verbally amongst hics and to non - hics is not the central focus of phipa nor is it explicitly mentioned in the legislation compared to other forms of information sharing practices . instead , phipa focuses on health records , in particular paper and electronic records ( cavoukian and rossos 2009 ) . furthermore , phipa does not lend itself to how information is currently exchanged in healthcare institutions with the advent of new technologies and increasing use of e - mail and digital interfaces to facilitate communication between and amongst hics ( fletcher 2014 ) . although healthcare providers may mention hiv status in the process of delivering care to the patient , it may result in disclosure of phi to a non - hic or someone outside of the circle of care , for example , hospital visitors . healthcare providers may use hiv status as a label , reminder , and/or communication tool , both for themselves and within the healthcare team , to prevent risks and error in the process of delivering care ( beardwood and kerr 2004 ) . employing hiv in this way may help those providing care to ensure appropriate checks and balances within a woman 's care plan ( gagnon 2014 , 2015 ) . healthcare providers may also assume hospital visitors are aware of one 's hiv status , especially if the patient does not explicitly discuss with them the importance of keeping it private . regardless of whether a patient articulates if it is safe to disclose their hiv status is not the point ; when hiv status is disclosed without a person 's express consent , the attempt to strike a balance between maintaining privacy while delivering high quality healthcare is not achieved . what mechanisms are in place for service users to take action in the event that their privacy is breached ? how would management of the institution in which care is provided respond to this situation and take reasonable steps to right this wrong ? there are many consequences when privacy is not respected including reputational consequences and changes in therapeutic relationships with healthcare providers ( fletcher 2014 ) . most privacy breaches are avoidable , even if conducted by well - intentioned healthcare providers ; regardless of the intention behind the behaviour , the effect and consequences are the same ( fletcher 2014 ) and hics who willfully collect , use or disclose phi in contravention of phipa can be found liable ( beardwood and kerr 2005 ) . at a provincial level , enforcement of phipa individuals have up to one year to file a complaint concerning a breach of privacy under phipa ( canadian hiv / aids legal network 2012 ) . enforcement involves an adversarial system whereby the commissioner , viewed as an impartial adjudicator , has discretion to determine the course of action including initiating a review or ordering a hic to modify , cease or implement a particular information practice ( beardwood and kerr 2005 ) . patients have the choice to withdraw their consent for the use and disclosure of phi to other healthcare providers who deliver care , but the details of how this would be achieved , as well as how the hic would monitor patient consent , are unclear . how policies and practices that align with phipa are disseminated , interpreted and enforced by the hic and its agents , especially within a fast - paced and overburdened healthcare system , presents a whole different set of challenges . the bottom line is that healthcare institutions implicated under phipa do not do an optimal job to keep phi private . patients are encouraged to stand up for and assert their rights to privacy ( canadian hiv / aids legal network 2014 ; fletcher 2014 ) , but is this a realistic expectation and outcome ? although disclosure of phi often occurs by well - intentioned healthcare providers , wlwh believe there will be serious repercussions if they call on healthcare providers who have disclosed their hiv status to account for their inappropriate behaviour ( ion et al . , many wlwh who experience disclosure of phi will not report the actions of their healthcare providers . is there something unique about having hiv that positions wlwh in a particular way while in hospital ? early in the hiv epidemic , there was virtually no privacy protection for those living with hiv because of fear , ignorance and aids hysteria within the healthcare system . despite significant advances in the clinical management of hiv and its evolution into a complex chronic illness ( scandlyn 2000 ; thompson et al . 2010 ) , wlwh around the world continue to face a number of health , social and legal challenges including access to hiv treatment , hiv - related stigma , discrimination and the criminalization of hiv ( debruyn 2004 ; greene et al . wlwh continue to report stigmatizing interactions with healthcare providers , for example , being treated differently in the labour and delivery unit because of their hiv status and the societal perception that wlwh should not be having children ( greene et al . the current experiences of wlwh may reflect a long and enduring history of fear and ignorance of hiv within the healthcare system . women 's experiences may also reflect an enduring lack of knowledge and awareness about hiv amongst healthcare providers who do not work in settings that specialize in hiv care . it is critical to consider the legislative frameworks pertaining to privacy through the lens of hiv because hiv has been classified as phi that is particularly sensitive ( cavoukian 1990 ; gostin 1995 ) . it is also apparent that healthcare practices within non - hiv - specific services have not kept pace with the evolution of hiv as a chronic condition . it is clear why wlwh continue to perceive hiv - related stigma when they access care , in particular , during pregnancy and early postpartum , as well as why a climate of fear , ignorance and stigma continues to surround the hiv epidemic across canada ( canadian hiv / aids legal network 2004 ) . although patients have every right to hold hics accountable , expecting health service users to stand up for their rights is not always optimal or possible , especially when they are ill , in hospital , are not comfortable speaking up for themselves , or feel powerless to do so ( fletcher 2014 ; greene et al . 2015 ; ion and elston 2015 ; ion et al . moreover , healthcare providers and trainees may not truly understand their accountability and duty of confidentiality requirements ( canadian hiv / aids legal network 2004 ) . power dynamics may be at play between wlwh and their healthcare providers within the healthcare system . the choice of wlwh not to respond to or hold healthcare providers accountable for disclosing their hiv status sheds some light on how power dynamics may flourish within healthcare systems and may not always position the patient at the centre of care . furthermore , expecting patients to express their privacy complaints to individual healthcare providers , then channel these complaints to the upper echelons of a healthcare corporation , as well as a provincial body like the office of the information and privacy commissioner , is a tall order . although hics may not do an optimal job to protect the privacy of patients ( fletcher 2014 ) , expecting patients to advocate for themselves when privacy concerns arise and submit complaints to an adversarial system after disclosure has occurred may not be a perfect solution . could the healthcare system be organized differently to decrease the chance of a privacy breach and to facilitate the appropriate collection , use and disclosure of phi , including hiv status ? a number of steps can be taken to optimize information practices to ensure the phi of wlwh is protected and remains confidential . these steps are relevant not only to wlwh , but all people living with hiv ( plwh ) and other patients who experience challenges related to privacy and confidentiality when navigating the healthcare system . any system changes must first be grounded in the perspective that , as a matter of public policy , the right to privacy is a fundamental human right for all plwh ( canadian hiv / aids legal network 2004 ) . privacy is essential to freedom and revolves around personal control and freedom of choice ( cavoukian 2014 ) . it must also be recognized that not only do plwh have a right to privacy regarding their phi , hics owe a duty to plwh to keep their phi confidential ( canadian hiv / aids legal network 2004 ) . confidentiality of phi is fundamental to the preservation of the ethical values of autonomy , dignity and respect for the individual ; patient confidentiality is not only an essential pre - condition to successful treatment it 's an issue of human dignity and respect the concept of privacy by design ( pbd ) offers a framework for ensuring that privacy is embedded directly into the design specifications of information technologies , business practices and operational processes ( cavoukian 2014 ) . pbd emphasizes service user privacy and the need to embed privacy as a default condition by transforming service user privacy issues from a pure policy or compliance issue into a business imperative ( cavoukian 2014 : 13 ) . pbd is focused on process rather than singular , technical outcomes and recognizes the need to introduce privacy principles during architecture planning , system design including networked infrastructure , and the development of operational procedures including work processes and management structures ( cavoukian 2014 ) . scholars have suggested a number of recommendations at the macro- , meso- and microlevels of policy and practice regarding how information practices could be optimized to ensure the appropriate collection , use and disclosure of phi . at the macro level of legislation , the canadian hiv / aids legal network has taken issue with the discretionary disclosure clauses inherent in health privacy legislation noting that these clauses fail to provide the level of privacy protection accorded to health information under the canadian charter of rights and freedoms ( canadian hiv / aids legal network 2004 ) . as such , the canadian hiv / aids legal network ( 2004 ) recommends that only in exceptional and circumscribed situations should a hic be permitted to disclose health information without the express and informed consent of plwh , rather than the vague and unspecified circumstances that are currently permitted . the legal network also recommends that hics be prohibited from disclosing any information that may reasonably reveal a person 's health information to family and friends without the person 's consent ( canadian hiv / aids legal network 2004 ) . at the meso level of systems , buffet and kosa ( 2006 ) have investigated how hics ensure that patient preferences regarding disclosure of phi are acted upon . the authors note that heath information network providers , for example , digital interfaces used to facilitate communication between hics and its agents , bear some responsibility for tracking and monitoring patient consent ( buffet and kosa 2006 ) . the authors propose a systematic consent management program , which they believe will minimize , if not eliminate , risk for the hic and health information network provider ( buffet and kosa 2006 ) . the consent management system relies on utilities and assigns a valuation to patient attitudes with regard to the handling of phi . the utilities are applied as part of a risk - based consent management framework and could be updated and reviewed each time a patient 's records are accessed . at any time a hic performs an action that involves a patient 's phi , an analysis would be conducted to help the hic determine whether to proceed based on the patient 's valuations and the likelihood that the patient 's preferences would be violated in the process ( buffet and kosa 2006 ) . the system for consent and risk management would assist hics to meet their legal obligations under phipa by managing a patient 's consent for releasing phi . scholars have also highlighted how training and professional development initiatives for healthcare providers and learners are important domains where the pbd framework could be enacted . for example , privacy may only be a small portion of orientation modules trainees are expected to complete ; these modules may present privacy in very theoretical terms and lack practical and applied elements ( fletcher 2014 ) . training programs could be redesigned to better prepare and orient healthcare providers and learners to privacy protection and duty of confidentiality . concrete examples of privacy breaches could be included as a way to apply knowledge to practice including the nuances of privacy in the context of hiv and other chronic and/or stigmatizing health conditions ( fletcher 2014 ) . health professional licensing bodies also need to educate their members about legal and ethical obligations regarding privacy and confidentiality . for example , just because discretionary disclosure clauses permit disclosure of phi under privacy legislation does not mean that the disclosure is necessary and valuable for the provision of care ( canadian hiv / aids legal network 2004 ) . regulatory bodies could make the protection of phi a performance metric for meeting licensing requirements . enhancing education , training and resources for healthcare providers within regulatory licensing bodies , as well as healthcare corporations , could result in important meso- and macro - level changes . the canadian hiv / aids legal network ( 2004 ) has offered recommendations regarding how the healthcare system , and its legislative and regulatory structures , could be better organized to protect the privacy of plwh . the legal network believes that the meso - level remedies currently in place for plwh whose privacy rights have been violated could be improved . the current adversarial system in ontario , for example , which investigates complaints brought forward by service users , could be made more accessible vis - - vis increasing the modes through which people could file a complaint such as audio or videotape in addition to written form , and eliminating fees associated with filing a complaint ( canadian hiv / aids legal network 2004 ) . accessibility could also be enhanced by increasing the public 's awareness of the system and improving transparency ; for example , the privacy commission of ontario could develop education programs to inform the public about the existence of and rights under privacy legislation including information about the complaint process and remedies ( canadian hiv / aids legal network 2004 ) . the legal network ( 2004 ) believes that remedies currently available to plwh whose privacy rights have been violated should also be strengthened ; for example , a system of deterrents should be implemented if hics improperly use and disclose phi including increased enforcement and compensation to patients . at the micro level of patient and provider , the legal network ( 2014a ) reminds us that healthcare providers should only ask questions that are relevant to providing care , for example , there is no need to ask about hiv status if that information is not required to examine or treat someone . plwh have often remarked about being asked how they acquired hiv during a routine clinical encounter ; when telling these stories they are always outraged and annoyed because healthcare providers , who they often have never met before , are driven more by their curiosity than delivering high quality , patient - centred care . asking relevant questions during a healthcare encounter is incredibly important for plwh and highlights one of the nuanced ways that the sensitivity of hiv can be considered in practice because it prioritizes the person 's privacy . healthcare providers should have a confidential , one - on - one conversation with their patients in advance so that a plan to maintain privacy and respect confidentiality is co - created and agreed to . how will we get there , especially considering that policies in the current political climate are considered within the micro - space of marginal incremental objectives , continually building out from the current situation ( lindblom 1959 ) ? at the same time , healthcare decisions are also influenced by economic , social , environmental and political forces ; policy makers and governments make decisions based on public opinion , electoral considerations , personal preferences and crisis management ( fafard 2008 ) . luckily , pbd has received global acceptance and endorsement by public and private sector privacy regulators around the world ( cavoukian 2014 ) . also , a number of scholars have spoken out to highlight public opinion regarding how governments handle phi ( peekhaus 2008 ) ; why hiv should be framed as health information that is particularly sensitive ( canadian hiv / aids legal network 2004 , 2012 , 2014 ) ; and how issues of privacy and hiv - related stigma complicate access to care and result in negative care experiences for plwh ( carter et al . forthcoming ; mccoy 2005 ; ohtn 2010 ; wong - wylie and jevne 1997 ) . as a result , community champions , social workers , researchers , activists and legal experts working in the hiv sector , as well as those concerned with privacy and its protection more broadly , are positioned with sufficient ammunition to enable a paradigm shift . the time is now to ensure that policy and practice decisions that affect privacy at the micro- , meso- and macro - levels are on the government 's agenda , and at the very least , are informed by evidence ( fafard 2008 ) , and grounded in the lived experiences and current realities of service users .

abstractwe conducted a retrospective , cross - sectional study to analyze predisposing factors , clinical features , and microbiological characteristics of patients with microbial keratitis hospitalized over 10 years.the medical records of 558 patients who were diagnosed with microbial keratitis and admitted to chang gung memorial hospital ( cgmh ) , a referral center in taiwan , from january 1 , 2003 to december 31 , 2012 were reviewed . demographics , predisposing factors , isolated organisms , treatment , and hospital stay were recorded . yearly trends were tested using a linear - by - linear association.contact lens wear was the most common predisposing factor ( 31.4% ) , followed by ocular and systemic diseases ( 26.3% ) and trauma ( 23.5% ) . contact lens - related infectious keratitis increased year by year ( p = 0.011 ) . pseudomonas aeruginosa was the most commonly isolated organism ( 28% ) , followed by fungi ( 17.6% ) and coagulase - negative staphylococcus ( 5.4% ) . except for serratia marcescens , the identified organisms did not change over 10 years . most bacterial infections were controlled using antimicrobial treatment , but more than half of patients with fungal keratitis required surgical interventions . the mean hospital stay was 13.7 11.5 days . previous ocular surgery , large ulcer size , nontuberculous myycobacteris infection , and surgery during admission were related to prolonged hospital stay.in taiwan , contact lens - related pseudomonal keratitis remained the most common cause of microbial keratitis in patients hospitalized from 2003 to 2012 . in the process of receiving perinatal care , women living with hiv ( wlwh ) in canada have experienced disclosure of their hiv status without their express consent . this disclosure often occurs by well - intentioned healthcare providers ; however , from the perspective of wlwh , it is a breach of confidentiality and leaves wlwh to manage the consequences . this paper is a critical review of the regulatory and legislative infrastructure that exists to protect the personal health information of wlwh in ontario and canada ; the recourse that wlwh have in the event that their confidentiality is breached ; and potential approaches that could be applied to organize the system differently to decrease the chance of a privacy breach and to facilitate appropriate collection , use and disclosure of personal health information .

in 2005 , cardiovascular disease ( cvd ) was the underlying cause of death in 864,480 of the approximately 2.5 million total deaths in the united states [ 1 , 2 ] . cvd is the leading cause of death and the most costly disease in america and is expected to increase in costs to $ 1.48 trillion by 2030 . although the focus has been to reduce the modifiable risk factors for cardiovascular disease ( such as lipid levels , diabetes , and sedentary lifestyle ) , the unmodifiable risk factor , aging , is a major risk factor for coronary disease , hypertension , congestive heart failure , and stroke . the number of the aged in the united states is projected to increase by more than 20% by the year 2030 . this growth in the elderly population is expected to significantly test our already overloaded health care system . although years of research have been conducted in regard to aging , we are still a long way from understanding the intricacies of age - related changes in human physiology , in particular the cardiovascular system . whether aging in animal models mimics many of the cardiovascular changes seen in humans is not well understood . in addition , the process of delineating the effect gender has on aging provides yet another variable to be considered . aging is believed by some to be a progressive disorder that decreases an organism 's ability to maintain reproductive capacity and normostasis . indeed a strong correlation exists between aging and higher incidence of several diseases including dementia , parkinson 's disease , diabetes , cancer , and alzheimer 's disease . in the cardiovascular system , aging is positively correlated with an increasing risk of cardiac problems including arrhythmias and is a major independent risk factor for cardiovascular - related morbidity and mortality . indeed , more than 70% of males and females over 75 years of age present some clinically evident cardiovascular disease ( cvd ) . cvd is the number one killer of women in western nations . among women , more than 200,000 of the 454,613 total cvd deaths occurred in those above 85 years of age . although cardiovascular risk increases with age in both sexes , the increase in age - associated risk appears to be sharper in women . why cvd risk may differ between men and women is not well understood but may be related to differences in age - associated cardiovascular function . whereas congestive heart failure in men is oftentimes due to systolic insufficiencies , congestive heart failure in women is often related to diastolic dysfunction [ 10 , 11 ] . the incidences of ischemia , cardiac failure , and cardiac rupture in addition to ventricular remodeling have also been shown to differ between genders with aging and often lead to worse outcomes in women [ 1215 ] . recent data suggests that premenopausal women have a decreased risk of cvd compared to men of comparable age [ 16 , 17 ] . this cardioprotective benefit appears to be lost over time as the risk of developing cardiovascular disease in postmenopausal women increases to a rate that is similar to that observed in men . the reason(s ) for this increase in cvd risk following menopause is currently unclear ; however , it is well known that early menopause is associated with an increased risk for coronary heart disease and cardiovascular disease death [ 1921 ] . this finding is thought to be related , at least in part , to diminished estrogen exposure . consistent with this notion , each year of increasing age at natural menopause has been found to be associated with a 2% increase in total cardiovascular mortality [ 22 , 23 ] . whether differences in estrogen alone or changes in estrogen along with other factors can fully explain the sex related differences in age - related cardiovascular function is unknown and requires further investigation . human aging research is limited due to cost , differences in lifestyle / history , and more importantly the time required for data collection as well as analysis of that particular system . importantly , there are a number of different animal models that can be used to acquire information on how aging affects the female cardiovascular system . primates are the closest in regard to female human aging due to the fact that they are the only species to undergo menstrual sloughing of the endometrial lining . nonetheless , it is important to note that female primates do not experience menopause . rats are widely used in aging research as they exhibit a relatively short lifespan and are genetically quite homogenous . among the various strains of rats , the wistar , f344 , and f344/brown norway are although rats do not experience menses , they do experience estrus cycling and ovarian aging . table 1 presents the stages of ovarian aging in female rats . reproductive maturity is reached at five months when there is an estrous cycle that lasts four to five days . aging rats exhibit periods of persistent estrous cycle which consists of elevated and constant levels of estradiol , low levels of progesterone , and lack of luteinizing hormone ( lh ) surges , in addition to ovulation [ 24 , 31 , 32 ] . ovarian decline occurs between six and eighteen months depending on the rodent strain and is characterized by low levels of estradiol and progesterone , with little or no developing follicles and increased prolactin secretion [ 24 , 31 , 32 ] . comparisons between aging research in female rats models and humans can be complicated due to the differences in the mechanisms of ovarian / hormone aging in human females and its potential impact on cardiovascular disease . though not fully understood , it is thought that the loss of the hormones estrogen and progesterone in aging human females is due to the decrease in the ovarian follicular reserve . conversely , aging female rats experience a persistent estrous cycle due to chronic anovulation which consist of pseudopregnant / disestrus estrogen levels as well as high progesterone levels from increased ovulation and the corpora lutea . therefore , reproductive senescence in female rats consists of alterations in the hypothalamic - pituitary axis while reproductive senescence in human females is classified as ovarian follicle depletion . due to these reasons and others , although estrogen secretion by the ovary promotes hypothalamic changes , the chronic administration of estrogen can damage neurons in the arcuate nucleus and medial basal hypothalamus . therefore , it can be important to remember that the surgical removal of the ovaries can lead to neurological changes that may affect other organs during the aging process . the pharmacological acceleration of ovarian aging using 4-vinylcyclohexene diepoxide ( vcd ) ( accelerated ovarian failure ( aof ) model ) or ovariectomy ( ovx ) procedures are often performed due to the lack of natural menopause in rat research models because they are believed to better mimic the hormone milieu seen in aging human females . vcd has been shown to target the plasma membrane of the primordial and primary follicles through direct inhibition of autophosphorylation of the oocyte - associated receptor , kit . kit acts as an antiapoptotic factor in primordial follicular survival [ 36 , 37 ] . although an effective model of chemical ovotoxicity limited research has been performed using female rat models [ 3841 ] and to date no published data exist regarding vcd and rat cardiovascular aging . vcd in juvenile ( 1 month ) and adult ( 3 months ) sprague - dawley rats depleted follicles but had no effect on the duration or onset of persistent estrus . however , fsh levels were significantly high in vcd treated animals with no change in cyclicity or serum levels of 17-estradiol . the absence of a human based clinical menopausal hormone profile in the aof model brings into question the ability to apply data derived from this model for translational relevant treatment of cardiovascular human based menopausal pathologies . an ovariectomy is the removal of the ovaries which induces a surgical menopause characterized by the cessation of estrogen and progesterone , as well as reduced production of testosterone . surgical menopause leads to more severe and sudden symptoms compared to that observed during the natural human menopause where ovaries produce lower levels of hormones over time . like that seen in menopausal human females , ovariectomy in rats also increases cardiovascular risk [ 42 , 43 ] . studies have found that the majority of human females who undergo natural menopause exhibit different age - associated cardiovascular alterations than those who underwent ovariectomy - induced menopause . elective bilateral removal of the ovaries at a young age is associated with an increased risk for cardiovascular disease and premature death . in addition to declines in well - being and sexual function , it is thought that elective oophorectomies are also associated with elevated risk of cognitive impairment , dementia , and parkinsonism . it is not known whether the increased risk to cardiovascular disease is due to alterations in hormones or the hypothalamic - pituitary axis . as expected , bilateral oophorectomy is associated with different hormonal alterations including changes in estrogen production and reduced levels of progesterone and testosterone , as well as increases in gonadotropins ( lh and follicle stimulating hormone ( fsh ) ) , compared to those that occur in human females who experience natural menopause [ 45 , 46 ] . in rats , ovariectomy causes alterations to heart structure and function which include increases in cardiac interstitial space , cardiac fibrosis , heart weight , and left ventricular weight . ovariectomized rats appear to exhibit increased evidence of oxidative stress and cardiac apoptosis [ 4751 ] . in rats , cytokine expression ( tumor necrosis factor - alpha ( tnf- ) and interleukin-1 beta ( il-1 ) ) and angiotensin converting enzyme ( ace ) and angiotensin ii type 1 receptor gene expression also appear to be increased following ovariectomy [ 48 , 50 ] . when estradiol treatment was given to ovariectomized rats , it prevented the reduction of cardiac contractility as well as the increase in apoptosis and cytokine expression [ 48 , 51 ] . research in other rats , namely , aging female dahl salt - sensitive rats , has shown that female rats are more likely to develop hypertension after ovariectomy . the researchers postulated that estrogen protects against the increased activity of the renin angiotensin system . however , in human females , research has shown that postmenopausal hypertension occurs approximately five to 10 years after menopause , not immediately after starting menopause . therefore , other age - related causes must occur independently of estrogen loss that promotes cvd in postmenopausal human females , as is shown by the fact that estrogen replacement therapy is not cardioprotective after menopause [ 53 , 54 ] . interestingly , both natural and surgically induced reproductive senescence rats exhibit increased fsh as well as decreased levels of estradiol and inhibin ( a / b ) . surgically induced reproductive senescence in rats has also been shown to alter dopamine receptor affinity in the heart . alterations in cardiac structure and function are not the only alterations found after ovariectomy . compared to aged controls , human females that underwent prophylactic bilateral salpingo - oophorectomy exhibit increased total and low - density lipoprotein cholesterol levels . in light of the fact that within human females natural menopause and ovariectomy results altered hormonal profiles and cardiovascular risk , the data suggest that age - associated cardiovascular disease in human females may be associated with both time dependent hormone deprivation and age - associated changes in structure , function , and protein signaling . of note is the fact that most studies have used ovariectomized female rats at young ages ( 612 weeks old ) to examine the effects of hormone deprivation on the cardiovascular system which can lead to unreliable results given that the cardiovascular system has not yet aged . the increased risk of cvd in human females may be due to differences in the type or magnitude of age - associated alterations in cardiac structure and function . olivetti and colleagues have demonstrated a small decrease in human heart weight with aging in males but not females . additionally , no significant change was noted with myocardium , left ventricular , or right ventricular free wall weight in the aging human female heart with no underlying cardiovascular pathology nor were age associated changes detected in proportion , shapes , size , or number of mononucleated and binucleated cardiac myocytes . given this information , it appears that natural aging in human females is not associated with cardiac remodeling during or after menopause in the absence of preexisting pathologies or loading abnormalities . although a limited number of studies have looked at how cardiac structure and function change with age in female rats , very few have directly investigated how sex may affect cardiac structure and function . go demonstrated that cardiovascular aging in male and female rats demonstrated significant gender differences in lv size and function . boluyt and colleagues investigated how aging may affect cardiac structure and function in female f344 rats using echocardiography . aging female rat cardiac structural changes included a dilatation of the left ventricle between 13 and 22 months of age . this dilatation was characterized by increases in posterior and septal wall thickness during diastole at 22 and 30 months of age . aging in the female f344 was also associated with increases in collagen content and collagen cross - linking . in addition , boluyt and colleagues demonstrated mild systolic dysfunction ( decline in left ventricle ejection fraction , fractional shortening , and velocity of circumferential fiber shortening ) in female 22-month - old animals when compared to young adults and that these changes preceded the development of mild diastolic dysfunction [ 61 , 63 ] . these authors suggested that this modest decline in systolic function was due , at least in part , to a shift in the amount of alpha and beta myosin heavy chains [ 61 , 62 ] , similar to what is seen in the human failing heart . additional work , perhaps using other rat models , is needed in order to truly distinguish if these alterations are due to increasing age or if they are simply specific to the f344 strain . fannin and colleagues found that increased age in the female f344xbn was associated with increases in oxidative stress and damage . age - related changes in cardiac structure were also found , such as increase in heart to body weight ratio , cardiomyocyte cross - sectional area , posterior wall thickening , and left ventricle chamber dilatation . further , increased age was also associated with diastolic dysfunction , alterations in heart rhythm intervals , and alternations in connexin 43 expression . the presence of age - associated cardiac structural changes in the f344 and f344xbn animal models calls into question the use of these strains for translational data , in light of the absence of age - associated change in human females reported by olivetti et al . . additionally , the menstrual cycle induced hormonal changes have been shown to cause cardiac functional fluctuations . this fact could indicate that the time at which functional measurements are taken may impact the results and must be a consideration in comparing all female derived cardiac functional data . in addition , aging is also associated with an increase in the percentage of incidence of atrial fibrillation [ 60 , 69 ] . in male rats , age - associated increases in cardiac fibrosis as well as the changes in gap junction morphology have been hypothesized to cause changes in cardiac conduction in addition to the incidence of arrhythmias and death [ 68 , 70 ] . as humans age , there is an increase in the ekg abnormalities which are associated with increased mortality . men tend to experience more atrial fibrillation , early repolarizations , brugada syndromes , and sudden cardiac death than women , while women are more likely to be at risk to develop long qt syndrome - based arrhythmias as well as bradycardia - induced torsades de pointes . although sex related differences in cardiac electrocardiogram parameters have been observed , electrocardiogram abnormalities seem to vary between studies . in men , there is an increase in qrs duration and sinus cycle length while women tended to a have a higher maximum heart rate [ 73 , 74 ] . electrocardiogram analysis in elderly men and women has demonstrated large or intermediate q waves , left axis deviation , negative t - waves , and complete right bundle branch block as well as atrial fibrillation / flutter . in aging men , the qrs complex has been found to be narrowed , while aged men and women have been shown to undergo a leftward shift of the qrs axis . another work has suggested that puberty in men is associated with a shortened qt interval which is not present in women and that aging in men demonstrates an increase in pr intervals and a prolongation of the qt interval . conversely , another research found that aging did not appear to alter ekg tracings in either men or women . similar alterations in electrocardiogram parameters have also been found in aging rats . in male wistar rats , the pr and corrected qt intervals alterations in aging electrocardiogram parameters have been shown to be rat strain dependent and could only be detected subsequently to heart failure as in male shhf / mcc - cp rat strain at 19 months of age . though not fully understood , these differences in gender associated gonadectomy induced arrhythmia frequency have been attributed to higher heart rates in male animals and longer qt intervals , as well as gender differences in ion channel expression . fannin and colleagues found that the arrhythmias were not significantly higher in older female f344xbn rats when compared with younger rats ; however , the study did find that valvular dysfunction was increased in the older rats when compared to the younger ones . the exact mechanisms of the gender associated reduction of arrhythmias in humans and the role estrogen may play are not fully understood but two potential indirect mechanisms have been proposed . one potential indirect mechanism may be related to the ability of estradiol to reduce the incidence of cardiac ischemia . other works have suggested that high levels of estradiol in female and male rats may directly reduce arrhythmias , possibly by causing slowing of the inward calcium current [ 80 , 81 ] . although multiple studies have investigated ekg changes in aging rats , these parameters have not been determined in the nia approved aging f344xbn rat model . however , according to the ich - s7b , the use of rats for the safety testing of drugs is not considered appropriate due to differences in the ionic mechanisms of repolarization . arrhythmia mechanisms , resting heart rate , ca homeostasis , and action potential configuration are all important differences that limit the translational relevance of rat generated cardiovascular data for human application . aging in the male rat heart is associated with the accumulation of oxidative damage to lipids and proteins as well as decline in mitochondrial enzymes . in addition , an increasing number of mutations in mitochondrial dna ( mtdna ) has been gradually observed during aging . the level of 8-ohdg mtdna adducts and deletions increase exponentially with age . in human muscle , liver , and brain tissue , complex iv and mitochondrial oxidative phosphorylation this decline in function is correlated with the accumulation of mtdna mutations , including deletions , and base substitutions . similarly , aged male rat hearts exhibit an increase in superoxide , 4-hydroxynonenal ( 4-hne ) , and nitrosative stress levels which appear to be highly correlated to increases in left ventricular thickness . the exact role that increased levels of oxidative stress may play in the development and progression of age - associated cardiovascular disease remains to be determined . compared to that observed in females , the hearts of aging male rats exhibited increases in protein carbonylation , advanced oxidation protein products , nitrotyrosine , nonprotein thiol , reduced glutathione , and iron levels . although aging is associated with increases in oxidative stress in both male and female rat hearts , female rat hearts exhibited lower mitochondrial hydrogen peroxide production , oxidative damage , and a greater mitochondria differentiation compared to that seen in male animals . it is thought that higher mitochondrial differentiation is a metabolic adaptation to increase energy efficiency as it is associated with their lower mitochondrial free radical production and oxidative damage . in addition to differences with sex , it is also likely that reactive oxygen species ( ros ) levels vary with animal strain as female wistar and f344 rats exhibit lower ros production and indices of oxidative damage than that seen in female sprague - dawley rats which may help to explain their greater mean lifespan [ 91 , 92 ] . the mitogen - activated protein kinase ( mapk ) cascades are evolutionary conserved serine / threonine protein kinases that regulate several important cellular functions including proliferation , differentiation , development , cell cycle , and cell death . the major mapk signaling pathways are the extracellular signal - regulated protein kinase cascade ( p44/42 ) , c - jun amino - terminal kinase / stress - activated protein kinase cascade ( jnk / sapk ) , and the p38 cascade . stimuli that include stress or injury typically activate the jnk and p38 mapk kinases , while the p44/42 mapks are stimulated by mitogenic and growth factors [ 94 , 95 ] . mapk signaling is involved in many of the age - associated physiological changes ( hypertrophy , oxidative stress , and apoptosis ) as well as other cardiac pathologies . works investigating mapk signaling in aging human hearts have demonstrated reduced p38 mapk activity / signaling in heart failure and impaired activation of mapk target genes following increase in oxidative stress [ 9698 ] . it is well recognized that increases in cellular stress cause the upregulation of the heat shock protein ( hsp ) . hsps range in size from 10 to 150 kda and function to prevent protein aggregation and assist in ensuring proper protein folding , as well as helping to mediate the translocation of damaged proteins . hsps are also involved in cardiac hypertrophy , in response to vascular wall injury , ischemic preconditioning , and aging . during aging , there is an accumulation of damaged or misfolded proteins which may cause a burden on maintaining proteostasis [ 102104 ] . an important function of heat shock proteins is to protect against age - related protein misfolding [ 103105 ] . in aged male f344 rats ( 24 months ) , it appears that aging decreases hsp70 upregulation following chronic exercise and heat stress [ 106108 ] . similar to the mapk proteins , the nuclear factor-b ( nf-b ) pathway is thought to play a role in cardiac remodeling , apoptosis , acute ischemia and reperfusion , and unstable angina , as well as heart failure in both humans and rodents [ 109112 ] . the nf-b family consists of rela ( p65 ) , c - rel ( rel ) , relb , nf-b1 ( p50 ) , and nf-b 2 ( p52 ) . figure 1 presents the nf-b signaling pathway . in the cytoplasm , the p50 and p65 are found as an inactive heterodimer due to the binding of the ib kinase inhibitory proteins ( ikk and ikk ) . cellular stimuli known to activate the nf-b pathway include ros , tnf- , il-1 , and bacterial lipopolysaccharides . it has been shown that the ibs must first become phosphorylated and degraded before p50/p65 activation can occur . the p50/p65 when phosphorylated ( activated ) then translocates into the nucleus to induce the transcription of chemokines ( il-8 , mcp-1 ) , cytokines ( tnf- , il-1 , il-2 , il-6 ) , adhesion molecules ( icam-1 , vcam-1 , e - selectin ) , acute phase proteins , antimicrobial peptides , secondary inflammatory enzymes ( cox-2 , inos , pla2 , mnsod ) , and antiapoptotic factors . upon termination of the nf-b stimuli , p50/p65 binds to new ibs and the complex it is thought that the mapk pathway works in concert with the nf-b pathway to increase transcription of inflammatory genes . extensively review the role of sex hormones in intracellular signaling mechanisms within the myocardial inflammation . although the effects of estrogen on mapk signaling have been studied in several pathological processes , for example , breast cancer , migraines , and polycystic ovarian syndrome , little is known regarding how this molecule affects mapk signaling in the heart . it is thought that differences in mapk activation between male and females may be regulated , at least in part , by the influence of estrogen [ 66 , 115 , 116 ] . estrogen has been shown to stimulate the activation of p44/42 mapk and jnk mapk in different model systems including cardiomyocytes , mammary cancer cells , pituitary tumor cells , tissue slices of the hippocampus , and endometriotic stromal cells [ 117119 ] . in males as well as ovariectomized females mapk signaling is decreased after acute ischemia [ 115 , 116 ] . in a model of cardiac pressure overload , estradiol treatment appeared to exhibit antihypertrophic effects by increasing the expression of atrial natriuretic peptide ( anp ) and inhibiting p38-mapk activation . in addition to the regulation of mapk proteins , there is evidence to suggest that gender also has an effect on the hsp response . in hearts that were not exposed to increased cellular stress , male hearts have half as much hsp72 expression compared to that observed in the female heart . estradiol treatment has also been found to increase hsp27 , hsp70 , hsp72 , and hsp90 expression in the heart [ 122124 ] . to our knowledge , no studies have looked at hsp27 , hsp70 , and hsp90 in the aging female heart . aging female and male hearts have shown increased apoptosis and inflammation , as well as age - associated gender differences in nf-b signaling . to our knowledge , only a few studies have investigated the changes in nf-b expression and activity in the aging female rat heart . apoptosis or programmed cell death is a highly conserved and regulated cell response to inhibit the abnormal proliferation of cells . cardiomyocytes are not capable of self - regeneration and are terminally differentiated but can undergo apoptosis , necrosis , or autophagy when unduly stressed . apoptosis is increased in many cardiovascular diseases such as dilated and ischemic cardiomyopathy , hypertrophic heart disease , and arrhythmias [ 126 , 128 , 129 ] . in rats , increased age has been shown to cause apoptotic susceptibility in the heart following oxidative injury [ 130 , 131 ] . aging in the male f344xbn heart is characterized by increases in the amount of cytochrome c , aif , bax , rate of permeability transition pore opening , and fragmented dna . the age - associated increase in apoptosis appears to be due not only to the activation of proapoptotic molecules but also to decreases in antiapoptotic nf-b , bcl - xl , and grx1 signaling . another work has demonstrated that the aging male f344xbn rat heart is characterized by increases in tdt - mediated dutp nick end labeling- ( tunel- ) positive nuclei , caspase-3 activation , caspase - dependent cleavage of alpha - fodrin , and diminished phosphorylation of protein kinase b / akt ( thr308 ) . the increase of apoptosis in the aging male f344xbn was highly correlated to age - associated increases in oxidative - nitrosative stress . though not demonstrating cause and effect , these results suggest that increased cellular ros and cardiomyocyte apoptosis may play a role in age - related cardiac remodeling . the incidence of cells undergoing apoptosis in the heart has been shown to differ with sex in humans and rats . in humans , a higher number of tunel positive myocytes are seen in young males compared to females , aging appears to increase cardiac apoptosis in male but not female ; however , there was no gender difference in apoptosis in male and female f344 rat [ 92 , 135 ] . structural changes found in the aging vasculature include an enlarged lumen , media - intimal thickening , irregularly shaped endothelial cells , migration and proliferation of vascular smooth muscle cells , increased deposition of extracellular matrix , increased expression of adhesion molecules , and alterations in the expression of metalloproteinases as well as cytokines [ 4 , 136138 ] . functional changes with age in the vasculature consist of impaired distensibility , increased stiffness , increased endothelial permeability , attenuation of 2-adrenoceptor vasodilator response to agonists , and diminished response to adrenergic receptor stimulation [ 4 , 139 ] . like humans , rats also appear to exhibit several age - associated changes in vascular structure and function including changes in intimal thickening , elevations in inflammation - associated molecule expression , and increased evidence of oxidative stress [ 4 , 140 ] . similar to that seen in other parts of the cardiovascular system , differences in aortic structure and function between genders have also been observed in the aging human and animals . in males , aortic wall and intimal - medial thickness are greater during aging than that observed in women ; however , stiffness was not different between men and women . similarly , there were no differences in distensibility in the aging aorta between men and women . although the human aging aorta did not show increases in aortic stiffness , it has been documented that the increased vascular stiffness with aging is more prominent in male than female animal models [ 143 , 144 ] . this may be due in part , to the lower total peripheral resistance and higher cardiac output seen in women compared to men of similar arterial pressure and age . abnormal proliferation and migration of vascular smooth muscle cells ( vsmcs ) play important roles in the pathophysiology of atherosclerotic diseases . previous studies have shown that vsmcs isolated from old animals replicate more actively than those obtained from young animals [ 145 , 146 ] . similarly , aging has been shown to be associated with an increased proliferative response of vsmcs after balloon angioplasty . endothelial dysfunction is considered to be a common and early feature of vascular disease and impaired endothelium - dependent relaxation has been demonstrated in several rat models of hypertension , experimental diabetes , atherosclerosis , and high salt diet , as well as aging . the factors regulating endothelial dysfunction are likely complex and may vary between models and with aging . nitric oxide ( no ) is a vasodilator that is produced by endothelial nitric oxide synthase ( enos ) that plays a crucial role in blood pressure regulation . no production is stimulated by shear stress , cyclic strain , acetylcholine , vascular endothelial growth factor , bradykinin , estrogen , sphingosine-1 phosphate ( s-1p ) , hydrogen peroxide , and angiotensin ii . it is thought that enos activity is regulated by enos phosphorylation at serine 1177 , serine 635 , and serine 617 by phosphatidylinositol 3-kinase ( pi3k)/akt , adenosine monophosphate - activated protein kinase ( ampk ) , and cyclic amp - dependent protein kinase signaling [ 149 , 150 ] . given its important role in regulating vascular function , it is not surprising that abnormalities in vascular no production are thought to contribute to the pathogenesis of atherosclerosis and hypertension [ 151 , 152 ] . advanced age has been found to be associated with impaired endothelial no synthesis and endothelial dysfunction [ 153 , 154 ] . the mechanisms responsible for age - associated alterations in no synthesis are not fully known but may include changes in activity or expression of enos , increased breakdown of no due to oxidative stress / oxidative injury , changes in antioxidant defense systems , and decreased availability of enos substrate , l - arginine , although the activity of enos is generally thought to be diminished with aging in the aorta . in addition to changes in enos activity , decreased no availability during aging can also be caused by oxidative stress and alterations in enos structure [ 155 , 156 ] . although estrogen ( 17- estradiol ) has been shown to activate enos , estrogen studies in rats have shown no effect or increased enos expression following estrogen treatment . conversely , in humans , estrogen replacement appears to be largely ineffective as a means of decreasing cvd risk [ 159 , 160 ] . in women , the incidence of vascular dysfunction is thought to be related , at least in part , to the cessation of ovarian hormone production . in vascular smooth muscle cells from young female wistar kyoto rats , estrogen inhibited vsmc proliferation following stimulation with fetal calf serum similar decreases in vsmc proliferation with estrogen treatment following cell stimulation by growth factors or mechanical stress have also been noted [ 161 , 163 ] . like estrogen , progesterone is also thought to inhibit the proliferation of aortic vsmc , most likely via its ability to inhibit dna synthesis . nonetheless , it should be noted that the effects of estrogen in vivo are likely more complex than those seen in cell culture . for example , in aortas from diabetic female rats , estrogen failed to reverse the impaired basal release of nitric oxide and the abnormal relaxation to histamine . the mapk signaling pathways function to regulate many processes in the aorta including vsmc proliferation , contraction , migration , differentiation , and cell survival [ 166168 ] . aging has also been shown to increase the activation ( phosphorylation ) of p44/42 and jnk mapks in the aorta while another work has shown that aging affects the ability of the aged aorta to activate p38 and jnk mapk signaling following mechanical loading . in aged animals the activation of p44/42 mapk in vascular smooth muscle was increased compared to young animals [ 145 , 171 ] . similar to that observed for the heart , age - associated changes in the expression / activity of nf-b were found in the vascular smooth muscle cells of the aorta . aging has been found to increase the sensitivity of nf-b to glucose in aortic vscm cells . another research has shown that vscm proliferation and nf-b activation stimulated by interleukin-1 were increased more in aged female rats than young female rats . in premenopausal women , receiving hormone treatment appeared to reduce nf-b activation suggesting that estradiol reduces nf-b activation . how estrogen may inhibit nf-b activity is currently unclear but may be related to increased ib levels or decreased levels of circulating tnf- . in summary , the loss of ovarian function during aging may influence cardiovascular structure and function . however , the fact that human females do not display cardiac hypertrophy or myocyte loss in the absence of loading abnormalities with aging may suggest the presence of estrogen prior to menopause acts to attenuate the cardiac response to preexisting underlying pathologies . due to the complexities of the aging process , more studies are needed to distinguish whether observed differences are due to aging alone , hormone deprivation , or a combination of both . in order to overcome the difficulties found in aging human research ( ethics , cost , and long lifespan ) , rat models have been used to provide great insight in investigating age - related changes in cardiovascular structure and function . however , additional research examining how aging may affect cardiac structure and function in rats is needed to determine whether the alterations seen in rats mimic the changes seen in humans . because of the systemic nature of reproductive senescence , the use of surgical practices to induce pseudomenopause or ovariotoxicity may be incomplete in the overall approach to female aging and cardiovascular changes . the global changes in the body with age may play a cumulative role in combination with the hormonal changes seen in reproductive senescence . aging - associated increases in oxidative stress can lead to alterations in the cardiac cell signaling which may lead to deleterious changes in the heart . because cardiac structure , function , signaling , and hormonal response appear to differ between male and female rats and humans with aging , care must be taken in evaluating current literature with regard to specific finding and gender . studies investigating the details of cardiac signaling with gender and aging are likely to lead to a better understanding of the mechanisms responsible for cardiovascular diseases in both male and females . these findings may prove critical in the application of current prophylactic practices and pharmaceutical interventions in patient outcomes . the mycotoxic nephropathy ( mn ) is a renal disorder caused by alimentary ingestion of secondary fungal metabolites possessing nephrotoxic properties and encountered in feeds / foods / forages made mainly from cereals or fibrous plants , and kept in storehouse conditions and increased humidity . since its discovering the disease has been described using various names : nephrosis provoked by moulds , chronic interstitial fibrosis of kidneys , chronic interstitial nephritis , etc . recently , the terms ochratoxicosis and mycotoxic nephropathy are the most frequently used ones for describing this nephropathy . during the last years this kidney disease has gained high publicity in some countries with well developed stock - breeding ( especially pig - breeding ) as denmark , sweden , bulgaria , etc . in order to prevent human exposure to various nephrotoxic mycotoxins , mainly ochratoxin a ( ota ) , via consuming the meat of animals with nephropathy , the timely diagnosis of disease during the meat inspection at slaughterhouses is very important . in such a way the exposure of humans to this very hazardous and relatively heat stable toxin from chicken / pigs meat can be prevented . there are some variances in the manifestation of the disease , especially in the clinicomorphological picture , which in a lot of the cases is influenced by the secondary bacterial infections as a result of the pronounced immunosuppression in the affected animals [ 2 , 3 , 4 , 5 , 6 ] . therefore , this review paper could contribute to timely diagnostics of mycotoxic nephropathy in farm animals ( mnfa ) as well as could help the organization of the prophylactic measures against this disease . in addition , some information is given how to make an evaluation of human exposure to ota on the base of the found concentrations of ota in human serum via calculating the daily ota intake of humans in endemic areas . although many nephrotoxic fungal compounds such as fumonisins are known , only ota and partly citrinin can be firmly associated with spontaneous cases of mn . the first and the most toxic compound discovered is ota , which was isolated from aspergillus ochraceus and chemiclly defined in 1965 by several south african scientists [ 811 ] . ota consists of a dihydroisocoumarin moiety linked through its 7-carboxyl group by an amide bond to one molecule of l- phenylalanine ( 7-carboxy-5-chloro-8-hydroxy-3 - 4-dihydro-3r - methylisocoumarin ) . citrinin was discovered in 1931 by hetherington and raistrick , who isolated the compound from several strains of penicillium citrinum . fungi that produce ota are widespread in nature and various foods or drinks [ 13 , 14 , 15 ] , but commonly contaminate stored grain . penicillium viridicatum , predominantly a storage fungus , is the mould that usually develops on grains at low temperatures and also is the major producer of ota in the colder areas of the world ( northern europe and canada ) . on the other hand aspergillus ochraceus probably is the most impotant producer of ota in tropical and semitropical areas [ 16 , 17 ] . however , the growth of a. ochraceus on natural substrates has not been associated with a natural occurrence of ota . not all isolates of p. viridicatum and a. ochraceus are ota producers , but some of them produce other toxic compounds , e.g. , citrinin , penicillic acid ( pa ) , hydroxyaspergillic acid and secalonic acid . a positive correlation has been observed between the frequency of spontaneous porcine nephropathy in bulgaria and the rate of ota in corresponding feed samples . however , the analysis for ota of various feed samples from farms with high frequency of spontaneous mycotoxic porcine nephropathy ( mpn ) revealed that the values were substantially low and ranged from 38 to 552 ( mean 207.1 65.14 ) ng / g for 1993 and from 42 to 427 ( mean 114.06 35.79 ) ng / g for 1994 respectively . sometimes the problem seemed to come from certain feed plants whose grains , collected during moist and rainly days , had not been properly dried . all farms supplied by these plants subsequently produced some pigs with nephropathy and growth depression , but after changing the certain suspected feeds the problems with poor growth of pigs disappeared . as a whole , the frequency and duration of the observed nephropathy in different batches of slaughtered pigs varied significantly ( from 12% up to 8090% frequency ) and have depended on the duration of feeding of various suspected feeds stored for a long time in poor conditions and at high humidity [ 1821 ] . it is important to mention that kidney damages in spontaneous mpn in bulgaria ( figure 1 ) are more similar to those in balkan endemic nephropathy ( ben ) in humans , than in danish mpn . for example , the fibrotic changes and contraction of kidneys in the end - stages of mpn in bulgaria are very comparable to those in ben in humans , whereas such changes are not seen in the classical description of mpn as made in denmark . in addition to the contraction of kidneys in later stages of bulgarian mpn and ben , there are various other similarities between ben in humans and bulgarian mpn as the low contamination levels of ota in feeds or foods and serum , neoplastic changes in kidneys ( pigs figure 2 ) or urinary tract ( humans ) , retention cyst formation in proximal tubules of kidneys , vascular lesions in kidneys , electron dense formations and myelin - like figures in mitochondria of epithelial cells ( figure 3 ) . however , these pathological changes have not been observed in classical mpn as described in denmark or elsewhere . this circumstance , in addition to low feed / food levels of ota in bulgarian mpn and ben , suggest a possible interaction between ota and other mycotoxins , which needs to be proved ( figure 3 ) . recently , it was found that pathomorphological changes , including electron - dense formation and myelin - like figures , induced in pig 's kidneys by combined exposure to such low contamination levels of ota together with pa , are more similar to those in spontaneous mpn in bulgaria than to classical danish mpn . all feed samples originating from farms with high frequency of spontaneous avian nephropathy in bulgaria were also positive for ota and similarly to pig farms , the levels of ota were substantially low ( the values ranged from 90.8 to 310 ng / g ( mean 196.2 ng / g 45.9 ) . the nephropathy in chickens was usually observed during the spring and summer , similarly to porcine nephropathy and in the same regions as well . the occurrence of nephropathy in different batches of slaughtered chickens varied from 12 per cent up to 90100 per cent . the continuance of this nephropathy , also differed widely from one month to about 56 months or even throughout a year . it is known that experimental pigs exposed to ota contaminated feed at levels of about 200 ppb develop only microscopic lesions after a period of 34 months [ 24 , 25 ] . the average contamination level of ota in bulgarian feeds for pigs in 1993 only exceeded 200 ppb ota . on the other hand , the low contamination levels of ota in pigs / chicks feeds suggest a possible synergism between ota and other compounds produced by the same ochratoxinogenic fungi . it is very likely that feeds contain other nephrotoxic mycotoxins or compounds which enhance the toxicity or produce a synergistic interaction with ota [ such as pa , fumonisin b1 ( fb1 ) , citrinin , viomelein , xanthomegnin etc ] . several years ago , it has been found that the toxicity of various strains of the same aspergillus ochraceus group is very different , independently of the capacity of ota production [ 22 , 26 , 27 ] . a potent synergistic effect was found between ota and pa , mycotoxins produced by the same ochratoxinogenic fungi , when the same mycotoxins were given simultaneously to pigs and chickens [ 22 , 2630 ] . the production of multiple toxins by a single organism , such as aspergillus ochraceus ( which produces ota and pa simultaneously ) , or by mixture of fungi presents a problem that has not been sufficiently investigated . pa was suspected to be carcinogenic and was found to have dna - attacking ability in the rec assay as well as to induce chromosome aberations . some data suggest that feed contamination levels of pa itself up to 4000 ppb have little toxicity ( mostly hepatotoxicity ) and do not adversely affect body or organ weights or biochemical parameters in chickens . only dna breaks in mammalian cell lines [ 36 , 37 ] and an inhibition of rat liver glutathione s - transferase activity in crude extracts in vitro as well as pa - induced hepatobiliary excretory dysfunction and liver hemorrhages have been found so far . the higher toxicity of ota in all above mentioned studies might be due to synergistic toxic effects between ota and pa , as has been reported in mice [ 41 , 42 ] and in poultry . previous studies have suggested an important role of the pancreatic enzyme carboxypeptidase a in the detoxification of ota in the small intestine [ 43 , 44 ] . parker and colleagues showed that pa inhibits carboxypeptidase activity in vitro and in vivo and such inhibition may significantly impair the primary detoxification of ota in the intestinal tract and so be partly responsible for the enhanced toxicity of ota observed in combination with pa [ 22 , 2630 ] . the pa - induced hepatobiliary excretory dysfunction may also result in decreasing of hepatobiliary excretion of ota . such synergism between ota and pa or other mycotoxins under field conditions may be responsible for the spontaneous mycotoxic nephropathy in bulgaria , which can be caused by relatively low contamination levels of ota in feed [ 18 , 21 , 23 ] . this could explain why the low levels of ota in bulgarian feeds for pigs [ 18 , 21 ] , chickens or humans have such high toxic effect on kidneys , when ingested with spontaneous contaminated feeds . the concentration of ota in feeding forages is probably more important than that of pa , because the higher contamination levels of pa ( 60000 ppb pa in combine with 1000 ppb ota ) have not been able to produce more significant toxic effects . this findings clearly suggest that the increase in ota - toxicity is probably due to impaired detoxification of ota via pa - inhibited carboxypeptidase activity in the intestinal tract . several years ago , stoev and collaborators induced macroscopic kidney damages in pigs fed on a mouldy diet containing low levels of ota ( 180 ppb ) in combination with pa for a 3 months feeding period , whereas microscopic lesions in kidneys in the same experiment were observed in pigs fed on a diet containing only 90 ppb ota in combination with pa . the observed changes were similar to those caused in pigs at the end of the 4-month period of exposure to higher levels ( markedly above 200 ppb ) of pure ota in feed . it is therefore important to investigate the effect of combined administration of ota and other mycotoxins , produced by the same ochratoxinogenic fungi as occur in the field . in combined administration of ota and pa much lower concentrations of ota ( about 100 ppb ) are enough for a significant toxic effect in chickens , expressed by degenerative changes in internal organs , by a depletion of cells in lymphoid tissue and decrease of lymphoid organ 's weight as well by known changes in various biochemical parameters . moreover , degenerative and weight changes in kidneys , liver and lymphoid organs as well as immunosuppression were seen recently in chickens at only 0.2 or 0.3 ppm ota in combination with pa [ 26 , 27 , 29 , 30 , 47 ] , whereas such changes had been seen in chickens exposed to significantly higher levels of pure ota ( about 4 ppm ) in feed [ 4851 ] . according to other authors a similar but less pronounced synergistic toxic effect has been observed at higher contamination levels of ota ( 1000 ppb ) . the mentioned above low experimental levels of ota correspond very well to the feed levels of ota found in spontaneous cases of mycotoxic avian nephropathy ( man ) in bulgaria ( 0.090.31 ppm ) , which suggests a possible multicausal nature of man in bulgaria . furthermore , to ascribe the source of ota to the a. ochraceus group of fungi is unwise because ochratoxinogenic forms are isolated so rarely [ 18 , 52 ] . therefore , it is already of great importance to carry out more mycological investigations in order to isolate the ochratoxinogenic fungi in bulgarian feeds . in addition , the overall concentration of ota in feed samples ( from 0.38 to 0.552 ppm ) were substantially lower than the 1 to 2 ppm required to reproduce mpn / man of severity similar to that observed in spontaneous cases [ 18 , 21 , 23 , 53 ] . it seems , therefore , that the mpn / man in bulgaria may have a multitoxin or multifactor etiology , because it can not be explained by the concentration of ota alone . the multimycotoxin etiology of mpn / man in bulgaria is recently confirmed as high contamination levels of pa ( 838,6 223,9 g / kg - 88% positives ) and fb1 ( 5564,1 584 g / kg - 96% positives ) in bulgarian feed samples from farms with mycotoxic porcine nephropathy were found , whereas the levels of ota ( 188,8 27,3 g / kg - 100% positives ) in the same feeds were consecutively lower ( unpublished personal data ) . a similar multimycotoxin etiology was also found for south african mpn as the same mixture of mycotoxins ( 67,8 39,2 g / kg ota - 83,3% positives ; 149,2 64,1 g / kg pa - 41,7% positives , 5046,2 1301 g / kg fb1 - 80% positives ) was found in south african feed samples from pig farms with nephropathy problems ( unpublished personal data ) . some mycological investigations of ota - contaminated feeds in bulgaria revealed the common presence of p. aurantiogriseum complex , which is a potent producer of pa . pa is produced by numerous species of penicillium ( especially p. aurantiogriseum ) and aspergillus at temperatures ranging from 4c to 30c , with the maximum rate of production occurring at about 25c . the production of pa decreased sharply at low oxygen concentrations , while fungal growth was not noticeably influenced . the rate of toxin coupling with -sh radicals increases with ph as well as in high temperature . the resulting complexes are much less toxic than the uncoupled molecules , resulting in actual detoxification . as a result , this toxin usually accumulates at relatively low temperatures during the winter at which detoxification is more reduced than toxin production . that could explain why bulgarian nephropathy in pigs / chickens was usually observed during the spring and summer it has recently been found that administration of p. polonicum extract ( not containing ota ) to rats can provoke profound and persistent histopathological damages such as apoptic and karyomegalic or mitotic changes in the nuclei of tubular epithelium in kidneys of rats , including dna - adducts formation . the same p. polonicum strain , which is a common food / feed spoilage mould in warm temperate latitudes , has been found as a frequent contaminant in bulgarian feeds , suspected of causing spontaneous porcine nephropathy [ 18 , 52 ] . it is known that the p. aurantiogriseum group ( including p. polonicum strain ) is a potent producer of pa , which can also provoke dna breaks in mammalian cell lines as has been reported . in such a way it could appear that the source of pa in bulgarian feeds can be different from the source of ota . also , the same changes ( apoptosis and karyomegaly in tubular epithelium ) , provoked by p. polonicum extract , could be induced by another unknown mycotoxin ; this would be of interest , because the same mycotoxin could be partly responsible for the nephrotoxic damages described in bulgarian nephropathy . moreover , the quiet apoptosis , induced by p. polonicum nephrotoxin , could be a possible model for the cryptic and clinically - silent onset of renal atrophy in the idiopathic balkan endemic nephropathy in humans . on the other hand , it has been reported that ota has a high potential to induce apoptosis and dna - adducts in vitro [ 61 , 62 ] and in rodent in vivo . also , rahimtula et al . showed that lipid peroxidation in kidney microsomes was enhanced by ota and produced malondialdehyde . in such a way , the extent to which ota may interact with other components of commercial chicken / pig rations or human food compounded from agricultural produce may also influence the significance of the relatively lower doses of ota that commercial chickens [ 23 , 27 ] , pigs [ 18 , 21 ] or humans may encounter in some feed / food . various different strategies can be employed to reduce human / animal exposure to ota or to reduce its toxic effects when it is fed to animals . the same include the use of management practices to prevent the production of ota by the storage fungi , the feeding of contaminated grain to animal species that are less susceptible to the toxic effects of ota , such as ruminants , modification of the diet to promote enhanced hydrolysis of ota in the gastrointestinal tract or reduced absorption , the use of a feeding regimen that can counteract the metabolic effects of ota , the use of various procedures that can destroy ota , such as physical and chemical methods for decontamination of commodities as well as the use of various feed additives , which can protect against the toxic effects of ota etc . . gastrointestinal microorganisms have a large effect on the disposition of ota because they promote the hydrolysis of ota to its nontoxic form ot. this is particularly important in ruminants and therefore they are less susceptible to ota - toxicity . in addition , the type of diet also affects the disposition of ota in the rumen [ 67 , 68 ] . ruminal fluid obtained from hay - fed animals ( ph 7.0 ) was able to hydrolyze ota to ochratoxin ( ot ) in vitro five times faster than ruminal fluid obtained from grain - fed animals ( ph 5.5 ) . disappearance of ota from the rumen and the corresponding formation of ot was also much faster for hay - fed than for grain - fed sheep . the bioavailability of ota in sheep fed grain was 4.3 times greater than that in sheep fed hay . thus , the rumen of the sheep has an important role in the detoxification of ota and the type of diet affects the rate and extent of this process . as a result , the bioavailability of ota , and probably the toxicity to ruminants , is decreased . in addition , the intestinal microflora of nonruminant animals , also affect the bioavailability of ota , because the microorganisms in the intestines and particularly in the caecum and large intestine are also responsible for the hydrolysis of ota to its nontoxic form ot . another way to prevent the production of ota is prevention of the growth of the storage fungi . the most important factors in safeguarding stored feedstuffs / foodstuffs from ota prodution by moulds are their moisture content ( water activity ) and temperature . large quantities of ota can be produced at intermediate and high ambient temperatures and high moisture contents by species belonging to the penicillium and aspergillus , whereas at lower ambient temperature the toxin can be produced by penicillium spp . only . in addition , the moulds in the corn when stored at 21% moisture produced a large quantity of ota ( about 3.6 ppm ) , whereas none of this toxin was found in corn stored at 16% moisture . these and some other studies indicate that fungi in cereal grains stored at moisture contents lower than 15% generally do not produce ota , which suggests that the moisture content in the cereal grains have to be decreased under 15% via various drying procedures before storage . storage at higher moisture levels requires that grain be maintained under anaerobic conditions , which prevent growth of fungi . otherwise , a combination of mould inhibitors and sterilization techniques , possibly coupled with the inoculation of nontoxigenic competitive microflora , have to be applied to prevent the growth of ota - producing fungi . chelack and collaborators [ 72 , 73 ] have used another way to control the growth of ota - producing fungi . they demonstrated that gamma or electron beam irradiation is a highly effective means of destroying spores from some ota - producing fungi as a. alutaceus [ 72 , 73 ] . various other types of radiation ( x - rays , ultraviolet light , microwaves ) in addition to -irradiation are also used as a means for detoxification of mycotoxins as well as to control the growth of fungi [ 74 , 75 ] . leitao and collaborators demonstrated that phosphine ( ph3 ) was effective at inhibiting both fungal growth and sterigmatocystin production by a. versicolor , and this could be considered as another specific way to control the growth of ota - producing fungi . for foods with ph values from 5 to 6 , such as the cereals or sorghum , the antimicrobial agent ( food additive ) that is able to prevent the growth and ota - production by aspergillus and penicillium species , is methyl paraben or potassium sorbate . small concentrations of these compounds are able completely to inhibite the growth by both genera of fungi and their ota - production . at ph 4.5 , as occurs in silage , fungal growth and ota - production was completely inhibited by 0.02% potassium sorbate , 0.7% methyl paraben , and 0.2% sodium propionate . the use of various procedures that can destroy ota , such as physical and chemical methods for decontamination of commodities can be considered as another important way for safely utilizing of feedstuffs . madsen and collaborators reported that treatment of ota - contaminated barley with 5% nh3 for 96 h at 70c or warming of grain to 105c in the presence of 0.5% naoh as well as autoclaving at 132c for 0.5 h can destroy the main part of ota in the barley , but such treatments are not practical and are not able effectively reduce the concentration of ota in grain . chelkowski and collaborators reported that treatment of ota - contaminated grain with ammonia reduced ota concentrations to undetectable levels . moreover , weight gains of chickens fed the ota - contaminated grain were markedly decreased , whereas there was no reduction in weight gains when the chickens were fed the ota - contaminated grain that had been ammoniated . they concluded that ammoniation of grain not only detoxifies several mycotoxins , including ota , but also inhibits mould growth . feeding studies have shown no toxic effects related to the ammoniation process , but there are some changes in the nutritional quality of the feed , such as a decrease in lysine and sulfur containing amino acids . in addition , adequate aeration after ammoniation is necessary for acceptance of the feed by animals . however , other than ammoniation , many of the techniques proposed to remove mycotoxins are currently perceived as impractical , ineffective , and/or potentially unsafe for largescale use . rotter and collaborators demonstrated that inoculation of barley with a lactobacillus species followed by ensiling ( decreasing of ph ) reduced the concentration of ota by approximately 50% . also , scott reported that ota is completely destroyed during malting of barley . according to laciakova and collaborators formic acid of 0.25% concentration degraded ota after 3 h exposure , propionic and sorbic acids in 1% concentration after 24 h exposure and benzoic acid in 0.5% concentration after 24 h exposure . several different approaches have been used to reduce ota absorption , including the use of hydrated sodium calcium aluminosilicate ( hscas ) , bentonite , charcoal , and cholestyramine . the addition of hscas ( 1% ) and bentonite ( 1% and 10% ) to a diet containing ota had no effect on ota concentration in swine blood , serum , tissues , and bile . the addition of 1% activated charcoal to the diet , in contrast , caused a slight decrease in the concentration of ota in swine blood , whereas 10% charcoal decreased the concentration of ota in blood , liver , kidney , spleen , and heart by 50% to 80% . however , the supplementation of ota - contaminated diet with activated charcoal was considered as an impractical method of reducing ota toxicity in chicks or pigs that were continuously consuming ota . cholestyramine , in contrast to the nonspecific absorbent discussed above , seems to be an effective absorbent of ota in the gastrointestinal tract of nonruminant animals . cholestyramine is a commercial anion exchange resin that has been shown to reduce blood ota concentrations by 50% , when it was included in 0.5% in a rat diet containing 1 ppm ota . in addition , the faecal excretion of ota was significantly increased in rats given a single oral dose of ota , which were fed on diet containing cholestyramine . another way to reduce ota - toxicity supposed a good understanding of the mechanisms of ota - toxicity : inhibition of phenylalanine metabolizing enzymes ( having in mind a phenylalanine moiety of ota ) , enhancing of lipid perooxidation , and inhibition of mitochondrial atp production . it is well known that the phenylalanine moiety of ota competitively inhibits at least two enzymes : phenylalanyl - trna synthetase and phenylalanine hydroxylase , resulting in reduced protein synthesis and altered rates of tyrosine production from phenylalanine . the addition of phenylalanine to cell cultures containing ota in vitro or coadministration of phenylalanine with ota [ 88 , 89 ] reduced ota - induced inhibition of protein synthesis . in vivo experiments show that injection of phenylalanine also prevented ota - induced immunosuppression in mice [ 90 , 91 , 92 ] and partially reduced teratogenesis in rats . the phenylalanine has to be used in 5:1 molar ratio towards ota , because it had been found that phenylalanine given in higher doses as 10:1 molar ratio , provides only a slight protection against ota , because of increasing the absorption of ota from the stomach and intestine . moreover , the higher supplementation of pure phenylalanine in chick diets contaminated with ota also tended to create an amino acid imbalance , which reduced b. w. gain and feed conversion efficiencies [ 95 , 96 ] . these studies suggest that little or no benefit is obtained by supplementing ota - contaminated diets with phenylalanine . the concentration of protein in the diet of growing chicks was also shown to ameliorate the toxicity of ota [ 97 , 98 ] . the consumption of high - protein diets ( 26% ) by growing chicks over a three - week period compared to a diet containing a lower concentration of protein ( 14% ) decreased the toxicity of 5 ppm ota in diet as indicated by rate of growth , mortality and relative organ weights of chicks [ 97 , 98 ] . this treatment , however , may not be practical because a large change in the protein concentration in diet may be costly and produces relatively small benefits the ascorbic acid supplementation ( 300 mg / kg ) of laying hen diet that contained 3 ppm ota has been reported to have a good protective effect against ota - action and can partially ameliorate ota - toxicity ( including the negative effect of ota on the eggs production and on the weight of the eggs ) [ 99 , 100 ] . the mechanism of the protective effect is not clear but it is supposed to affect the production of lipid peroxides by ota and to improve the membrane integrity of cell organelles , which is always affected by ota - action . the use of various feed additives can also protect against the toxic effects of ota and can reduce farm losses from a decrease of weight gain in stock chicks avoiding the rejecttion or condemnation of ota - contaminated feed [ 27 , 30 , 94 ] . sesame seed ( rich in proteins - about 20% and relatively rich in l- phenylalanine - about 4.3% ) , given in 8% to the feed , offered a cheaper agronomic additive to supply animals with phenylalanine and thereby to protect against toxicity of ota . it might also avoid the increase of ota - absorption from the stomach and intestine provoked by the pure phenylalanine in feeds . simultaneously , the increase of urinary excretion of ota in presence of phenylalanine could be preserved in a later stage . moreover , the sesame seed ( 8% to the feed ) increases the energy metabolism in animals , which usually is disturbed by ota . ( hofmann la roche , grenzach - wyhlen , austria ) is a polyenzyme complement produced by fungi trichoderma , which contains : cellulase , xylanase , endo - beta 1,3 - 1,4 glucanase , pectinase and amylase . roxazyme - g , given in concentration 0.2g / kg feed , was reported to improve digestive dissimilation of polysaccharides to easily assimilated substances , that could improve utilization of feed by increasing digestible energy production by 813% , thereby counteracting ota impairment of energy production in chicks . artichoke - extract is a complex containing compounds which might protect against ota intoxication [ 26 , 27 , 30 , 94 ] . the 5% total water - extract of artichoke ( cynara scolymus l ) , prepared as a steam infusion from dried leaves of artichoke and given to chicks in concentration 5 ml / kg.b.w . via the feed or water , usually contains various biologically active compounds : cynarine , flavonoids , cynaropicrin , dehydrocynaropicrin , grosheimin as well as a high content of calcium and ascorbic acid . it has been recommended as a diuretic agent in cardiac and renal insufficiency , and might accelerate the urinary route of excretion of ota [ 26 , 27 , 30 ] . moreover , the cynarine content in such extract stimulates metabolism of cholesterol , decreases serum urea and lipids , improves diuresis and increases biliary secretion , which probably augment the hepatobiliary route of excretion of ota ( ota is mainly eliminated via bile and urine ) and thereby has been found to protect against ota nephrotoxicity and hepatotoxicity [ 26 , 27 , 29 , 30 , 94 ] as well as against the immunosuppressive effect of ota [ 27 , 94 , 104 ] or against ota - induced anaemia . cynarine and flavonoids as well as some medical preparations prepared from artichoke - extract ( chophytol and chophytamine ) have a potent hepatoprotective effect against hepatotoxic damage [ 105107 ] . also , antipermeability and vasoconstrictive effects of water extract of artichoke could decrease oedema in various internal organs provoked by ota [ 18 , 27 , 29 , 30 , 94 ] . the high content of calcium and vitamin c in the steam extract probably has also a protective effect against ota - toxicity via improving membrane integrity of cell organelles and skeleton - strength of the chicks or via some other ways [ 27 , 29 , 94 ] , since some authors reported that ascorbic acid supplementation ( 300 mg / kg ) of laying hen diet that contained ota ( 3.0 mg / kg ) partially reduced ota - toxicity . rosallsat , which is another plant extract ( a polyextract of bulbus alii sativi and seminum rosai canina ) , was seen also to have a protective effect against ota - toxicity , when it was given per os in 0.6 ml per kg body mass daily as a supplement to the feed . the rosallsat is a new natural galenic phytosubstance of balanced polyvitamine , phytoncide and steroid - saponine composition - described completely in patent no 98915 bg / 1994 , which contains biological active compound allicin ( 12.08 mg / g ) as well as large quantities of vitamins a ( 0.0332 mg / g ) , f ( 0.8112 mg / g ) , e ( 0.1548 mg / g ) and furostanic sapogenine of c57h96o30 [ 26 , 29 , 30 ] . the allicin in that study was formed in advance and included in a natural oil extract in commercial lots stabilized with natural antioxidants . this is an advantage when compared with fresh or dried garlic cloves since thiosulphinate is hardly formed in the ph conditions of the stomach and intestine after eating fresh or dried garlic . the precise mechanism of protection of rosallsat against ota - toxicity has not been investigated in depth , but it is supposed that the biological active compound allicin and the high quantities of vitamins a , f and e in this plant extract probably are of great importance [ 26 , 29 , 30 ] . the rosallsat is found to inhibit lipid peroxidation ( unpublished personal data ) and in such a way prevents one of the mechanism of ota toxicity . it was found that the intensity of macroscopical and histopathological changes , the deviations in relative organs weight and body weight ( table 1 ) , the decrease of antibody titer ( table 2 ) , as well as the intensity of changes in various haematological and biochemical parameters ( table 3 ) were slighter in chicks treated with some antidotes ( 5% artichoke - extract , 0.02% roxazyme - g , 8% sesame seed , 0.0025% phenylalanine ) in addition to 5 ppm ota than in chicks only treated with 5 ppm ota . in chicks of groups treated with sesame seed or artichoke - extract the described changes , especially the antibody titer against newcastle disease , were more similar to those ones observed in chicks exposed to an approximately 5 times lower contamination levels of ota ( 1 ppm ) ( table 2 ) . moreover , it appeared that all of the mentioned above antidotes , used as supplements to the feeds , especially sesame seed and artichoke - extract , had a potent protective effect against ota - induced toxicity and could be used as a practical approach for safely utilizing of ota - contaminated feed . in such a way the rejection or condemnation of such feed will be avoided as well as there will be no need to eliminate ota from the feed , if its contamination levels are similar to these encountered in the practice ( normally upto 12 ppm ota ) . however , in the pigs which are more sensitive to ota , the levels of 1 ppm ota can be more dangerous [ 2 , 22 , 108 ] and this requires additional experimentation to clarify the antidote effect of the same additives in these animals . a protective effect against ota - induced decrease of total serum protein was found in chicks of all antidote - treated groups mentioned above , but a protection against the increase of serum glucose was only seen in groups treated with roxazyme - g and artichoke - extract . also , a protective effect against ota - indiced increase of serum creatinine and urea was seen in all antidote - treated groups especially on day 70 . in addition , the protective effect against the increase of serum values of uric acid and creatinine was strongest in the group treated with sesame seed ( table 3 ) . however , it appeared that the protective effect of phenylalanine was slighter than expected one in contrast to some of the other studied antidotes . the main reason for that could be the circumstance that phenylalanine was found to increase the absorption of ota from the stomach and intestine as well as the gastrointestinal transit of ota . this resulted in eightfold and fourfold higher levels of ota in serum and liver of mice , respectively , during the first 12 h , which could also increase its elimination . therefore , phenylalanine has to be given after changing the feed source and stopping ota - exposure of animals . the strong protection of roxazyme - g against ota - induced increase of serum glucose could be due to the improved energy metabolism , whereas the protection of artichoke - extract against that increase could be due to an improvement of diuresis . the protective effect of sesame seed and phenylalanine against ota - provoked immunosuppression in humoral immune response and changes in differential wbc count might be due to an improvement of protein synthesis , which usually is disturbed by ota and subsequently , an improvement of ota - induced delay of the division of the cells of the immune system . it is known that ota induces an increase of the weight of the organs taking part in its detoxification or elimination ( kidneys and liver ) as well as a decrease of the lymphoid organs weight and body weight , whereas some of the studied feed additives ( 5% artichoke - extract , 0.02% roxazyme - g , 8% sesame seed , 0.0025% phenylalanine ) significantly protects against these weight changes as the protective effect of sesame seed and roxazyme - g against these weight changes was stronger than the protective effect of artichoke - extract and phenylalanine . in other experiments [ 26 , 30 , 110 ] it was also confirmed that artichoke - extract has a significant protective effect against ota - toxicity . in the same studies , another plant extract rosallsat was also seen to protect against ota - toxicity , expressed by statistically significant protection against the ota - induced changes in serum levels of total protein , uric acid and cholesterol ( table 4 ) . the serum levels of uric acid , which were most significantly influenced of the quantity of ota in fed forages , usually give an exact assessment of the impairment of kidney function in chicks . the absence of a confident increase in that parameters in artichoke - extract and rosallsat treated groups suggests a slight impairment in kidney function in these groups and confirms the protective effects of these feed additives against ota - toxicity . on the other hand , the protective effect of artichoke - extract and rosallsat on the liver function may contribute to the higher cholesterol levels in the groups of chicks treated with the same antidotes . in addition , the toxicological investigations in the same experiments revealed that the contamination levels of ota in kidneys are lower in chicks treated with artichoke or rosallsat in addition to ota than in chicks only treated with the same feed level of ota . by 1990 , at least nine countries have regulations for ota levels or had proposed and enforced official limits for ota . regulatory control of nephrotoxic mycotoxins in animal feeds in european countries has been summarized by van egmond and later by boutrif and canet . on the other hand , european union decided on an official limit for ota in cereals designed for direct consuming of about 5 g / kg . the official limits for ota in cereals in some countries can be seen in various fao reports and commonly ranged between 2 g / kg ( switzerland ) and 20 g / kg ( czech republic ) , and rarely reached up to 50 g / kg ( uruguay ) . in countries with wide spreading of mpn and man residues of ota were often found in animal tissues samples during the meat inspection [ 18 , 23 , 114 , 115 , 116 ] . the contamination levels of ota in liver , muscles and fat tissues of pigs can be easily found using the following experimental calculations : by 1990 , at least nine countries have regulations for ota levels or had proposed and enforced official limits for ota . regulatory control of nephrotoxic mycotoxins in animal feeds in european countries has been summarized by van egmond . because of the absence of correlation between macroscopic changes and concentration of ota in pig kidneys it is very difficult to prevent the exposure of humans to the toxin from pork by toxicological investigations of pale or mottled kidneys as it is occurred in denmark . the practice in denmark of monitoring kidney tissues in only those pigs that have detectable kidney lesions could not provide a good basis for condemnation of carcases , because the intake of ota may have not been of sufficient duration to produce detectable lesions . therefore , the regulations in denmark , according to which all enlarged and mottled kidneys are investigated for residues of ota at slaughter time and all carcasses , whose kidneys contained ota levels above 10 g / kg are condemned are not very safe and clever , because macrscopic changes in kidneys can be found only after 13 months ota exposure via the feeds [ 22 , 118 ] . some investigations made by various authors revealed that at least 12 months are needed to develop characteristic macroscopical renal lesions in pigs exposed to natural or experimental ota contaminated barley [ 24 , 119 ] . unfortunately , characteristic for mpn macroscopic changes in kidneys can not be found after a shorter period of ota exposure via the feeds even with such high levels as 1000 ppb ota and animal products containing dangerous ota contamination levels could easily pass meat inspection at slaughterhouses . on the other hand the renal lesions in kidneys ( mottled appearance ) induced at an early age do not disappear when the pigs are fed on ota - free diet , which also indicates the lack of efficiency of such control system and the implementation of some unnecessary toxicological investigations , which could be avoided [ 21 , 22 ] . in spite of the toxicological investigations of such kidneys ota - contaminated pork may enter the human food chain and thus represents a potential public health hazard . since the toxin is relatively heat stable , a limited occurrence of ota might anticipated in prepared food from such animals . because of the assumption that mycotoxins and especially ota are involved in etiology of balkan endemic nephropathy [ 1 , 122124 ] the exposure of humans to this very hazardous toxin from pork or chicken meat ( by the way feed pork / chicken - food ) need to be prevented . a much better procedure for preventing the exposure of humans to the toxin from meat would be a toxicological analysis of a few blood samples of pigs / chicken from farms , suspected of having mn , several weeks ( in pigs ) or several days ( in chicken ) before slaughter and a change in the feed source for a week ( pigs ) or for 23 days ( chicken ) , if it is necessary . also , the period of feed deprivation of pigs / chicken before slaughter could be prolonged [ 21 , 23 ] . because of the short half - life of ota in pigs ( 72 120 hours ) and especially in chickens ( 4 hours ) its concentration in blood and various tissues quickly decreases after changing the feed source or after prolonging the period of feed deprivation before slaughtering . thus , the loss of condemnation of pig / chicken production would be prevented and a better procedure ( than toxicological invesigations of enlarged mottled kidneys accepted in denmark ) would be realized for preventing the exposure of humans to ota from meat [ 2123 , 114 ] . the preventive measures in already slaughtered chicks could include condemnation and removing of the kidneys and liver , where ota is accumulated . however , having in mind that a combined administration of ota and other mycotoxins such a pa , often occurrs in the practice and synergistic effects between ota and the same mycotoxins are reported , the lower contamination levels of ota in food / feed would be also very dangerous for human and animal health [ 22 , 30 ] . balkan endemic nephropathy ( ben ) in humans is another renal disorder having a similar pathological characteristics as mpn . it is most commonly observed in farmers at ages between 30 and 50 years , has oligosymptomatic clinical picture , silent onset and poor prognosis , progresses very slowly and manifests itself when the function of majority of nephrons have become impaired . at this time there is agreement that the disease represents an unusual type of chronic interstitial nephropathy of unknown etiology , although there is an assumption that mycotoxins are involved . the disease has a strict endemic character and is encountered in the rural populations in bulgaria , romania and ex - yugoslavia . the high humidity and the poor food storage conditions in endemic villages , the low living and hygienic standard as well as the consumption of home prepared food from own crops - bread , bean and others [ 123 , 127 ] correspond to the fact that the moulds producing mycotoxins contaminate mostly home produced grain feeds stored in bad conditions and high humidity , while the situation in the towns is markedly different - foodstuff supply is centralized and quality controlled ( that explain the family character of ben only in rural inhabitants ) . also , the focality and zonality ( characteristic for ben ) are typical for mycotoxicosis ( i. e. endemic dissemination ) and are determined mainly by the ecological conditions of the regions which are optimal for the development of the corresponding fungi and by the altitude . the seasonal and yearly variations in incidence and prevalence of the disease and ota - content in the human food as well as the positive correlation between the excess rainfall and the number of patients suffering from ben , who died during the following two years , probably because of additional intake of moulded food ( provoking new degenerative damages in kidneys and impairing the disease state ) , give additional support to the mycotoxin hypothesis of ben . however , the etiology of the disease is still unknown in spite of various etiological investigations including the search for heavy metals , trace elements , infectious agents and genetic factors as well as various bacteriological , toxicological , and hydrological investigations of water . nephrotoxic mycotoxin ota has been suggested as one of possible disease determinants of ben based on a comparison of morphological and functional kidney impairment between ota - induced mpn and ben . in endemic areas , ota has been found in human blood as well as in human food [ 123 , 126 , 130 , 131 , 132 ] and in animal feed in higher concentrations than in nonendemic areas or the countries without ben . it is important to mention that the feed levels of ota in animal feeds from endemic for mpn regions ( 38552 ppb ) are approximately the same as in the foods from endemic for ben regions ( 10890 ppb ) , which additionally suggest a possible common etiology . the mean concentration of ota in human blood in bulgarian - endemic areas ranges respectively from 20 g / l for 1986 to 27.2 g / l for 1989 [ 130 , 132 ] . these high concentrations suggest for a possible role of ota in a disease causation of ben . however , ota was detected in a concentration range of 5100 g / l only in 0.4 to 2.5% of a total 13,797 analysed blood samples from 14 endemic areas in slavonski brod ( croatia ) during 9-year ( 19811989 ) period and in a concentration range 250 g / l in 0.2 to 4.5% of a total 6,910 analysed serum samples from endemic village of kaniza over the 10-year ( 19851994 ) period . these concentrations of ota in human blood are significantly lower towards those ones in bulgaria , but correspond quite well to the prevalence of the disease ( ranged between 1% and 4.5% during the last 15 years ) and the incidennce of newly recorded ben - suffering patients ( ranged between 1.0 and 2.5 per thousand ) in the same endemic areas . the disease is also closely associated with a high frequency of carcinoma in the renal pelvis , ureter and urinary bladder [ 135 , 136 ] , which could be due to the carcenogenicity of nephrotoxic mycotoxin ota [ 125 , 137 , 138 ] , but the working group that evaluated the carcinogenicity of ota to humans could not draw any conclusion . interestingly , some tumors in kidneys and bladders from patients living in areas associated with balkan endemic nephropathy , have been found to contain dna adducts similar to those obtained from the kidneys of mice exposed to ota , which provides new evidence of the possible role of ota in the development of tumors of the urinary tract . moreover , 11 of 31 patients with urinary tract tumors in france exhibited a ota - specific dna adduct pattern similar to those found in bulgarians suffering from ben , ota - treated rodent and bulgarian pig suffering from nephropathy . it was found that changes in debrisoquine 4-hydroxylation in vivo can occur in the rat in response to mouldy food and patients suffering from ben and/or upper urothelial tumors were more frequently extensive metabolizers of debrisoquine ( db ) . a recent study in egypt revealed a positive correlation between ota and the end stage renal disease ( esrd ) or urothelial tumors in humans . patients with esrd as well as patients with nephrotic syndrom or urothelial tumors have significantly higher mean concentrations of ota in blood ( between 0.52 and 2.19 ng / ml ) and urine ( between 0.36 and 3.09 ng / ml ) than patients from control groups ( between 0 and 0.08 ng / ml in blood and between 0.01 and 0.26 ng / ml in urine ) . it is established that ota decreases natural killer cell activity by the specific inhibition of endogenous interferon levels . as natural killer cells are involved in the distribution of tumor cells , the ability of ota to modulate the activity of these cells might contribute to its capacity to induce renal and hepatic carcinomas . recently , it has been found that ota has a significantly longer plasma half - life in humans ( 33.55 days ) than in animals . a slow excretion of ota suggests a tendency to an increase of toxin levels in the serum as well as assumes a very constant rate of ota in human blood for a long period of time , even after repeated exposure to very low concentrations . in such way the humans are more influenced by the dose and less influenced by the frequency , duration and quantity ( if the quantity of the toxin ingested is comparatively low ) of ota exposure than other species , because in the humans ota has a very constant and continuous effect on the proximal tubules even in little amounts , because of the very low unbound ( free ) fraction of ota in blood . it can be supposed that only the free ota in the macroorganism has a toxic effect on the tissues as well that continuous persistence of ota in human blood and its slow excretion via kidneys may be influenced on the slow progression of ben . thus , consumption of food containing very low concentrations of ota over a long period of time may become toxicologically significant . a method is also presented for calculating the daily consumption of ota through various food products on the basis of the measured content in plasma , even though the toxicokinetic constants are unknown . the renal filtration rate of ota in humans , calculated from the glomerular filtration rate of inulin and the free fraction of ota is 0.67 ml / kg body weight per day . the kidney filtration has been found to correspond to the total plasma clearance of ota ( almost 100% ) in monkeys , but only to 10% in mice , rats and pigs . the equation : k0 = clp.cp/a shows the relationship between continuous intake ( k0 , ng / kg b. w. per day ) , the plasma clearance ( clp , ml / kg b. w. per day ) , the plasma concentration of ota ( cp , ng / ml ) and the bioavailability ( a , proportion of toxin absorbed - about 50% ) . . the same equation was used to calculate the daily intake of ota from the concentration of the toxin in plasma of humans in endemic regions in bulgaria . the daily intake of humans , calculated on the basis of the average plasma levels of ota in endemic area in bulgaria was found to be between 26.8 and 36.4 ng / kg b. w. as can be seen from table 5 [ 1 , 124 ] . it is important to mention , that this calculation should give an underestimate of the intake , since the value used for plasma clearance only involves glomerulal filtration . this underestimate could be as much as ten times , if the ota clearance in man is more similar to those in rat , mouse and pig than that in monkey . because of the increase of scientific reports on ota contamination in beverages and many kinds of food , the who / fao joint expert committee on food additives ( jecfa ) assessed the available information and proposed 112 ng / kg b.w . having in mind the strong carcinogenic effect of ota its ptwi was later decreased to 100 ng / kg b.w . , which corresponds to about 14 ng / kg body weight per day . it can be concluded that the average daily intakes in the endemic areas in bulgaria from 26.8 ng / kg b. w. for 1988 [ 1 , 124 ] , 36.4 for 1989 and 34.2 for 1990 respectively , exceeds strongly ptwi ( 100 ng / kg b. w. or 14 ng / kg b. w. per day ) , proposed by the jecfa . jecfa bases its calculation of the tolerable intake mainly on the nephrotoxicity of ota and does not address the question of the toxin 's carcenogenic effect . kuiper - goodman and scott , however , regard the carcinogenic effect as the most important effect and base their analysis on this . tolerable daily intakes ( tdis ) , depending on the method used and calculated on the base of carcinogenic effect of ota , range from 0.2 to 4,2 ng / kg b. w. it can be concluded that the maximum tolerable daily intake of ota is 5 ng / kg b. w. . it can be seen that the average daily intake of the endemic and in some extent nonendemic areas exceeds strongly the tdi calculated on the base of cancerogenic effect of ota . the performed calculations on tdis show that ota contamination of food and feed constitutes a public health problem of unknown dimensions . therefore , it is quite relevant to investigate to what extent ota is hazardous to humans . because of the high thermal stability of this mycotoxin , ordinary cooking does not eliminate it . recently , however , more and more evidences appeared , that exposure to more than one mycotoxin may be an important factor in etiology of ben and mpn . moreover , the synergism between ota and various other mycotoxins such a pa , citrinin or fb1 in field conditions may be responsible for an enhanced toxicity of ota [ 22 , 27 , 30 ] . due to the potent toxic and synergistic effects of ota and pa or citrinin [ 22 , 150 ] as well as between ota and fb1 [ 151 , 152 ] , simultaneous exposure to those mycotoxins might be an important factor for development of chronic renal diseases in animals and humans , especially after long - term exposure . the same mycotoxins were recently found in high contamination levels ( especially fb1 and pa ) in most of the feeds originated from farms with mycotoxic porcine or avian nephropathy in bulgaria ( unpublished personal data ) . because of that it is of great importance to investigate the combined effect between ota and other mycotoxins produced by the same species as it is occurred in field conditions .

cardiovascular disease is the leading cause of death in women in the united states . aging is a primary risk factor for the development of cardiovascular disease as well as cardiovascular - related morbidity and mortality . aging is a universal process that all humans undergo ; however , research in aging is limited by cost and time constraints . therefore , most research in aging has been done in primates and rodents ; however it is unknown how well the effects of aging in rat models translate into humans . to compound the complication of aging gender has also been indicated as a risk factor for various cardiovascular diseases . this review addresses the systemic pathophysiology of the cardiovascular system associated with aging and gender for aging research with regard to the applicability of rat derived data for translational application to human aging . various etiological factors contributing to the development of mycotoxic nephropathy in farm animals and humans are reviewed . the possible synergistic effect between ochratoxin a ( ota ) and other mycotoxins , as penicillic acid ( pa ) and fumonisin b1 ( fb1 ) , contributing to this nephropathy is also considered and discussed . the most convenient ways of prophylaxis and various preventive measures against ota contamination of feeds or foods are reviewed . a reference is made concerning the most successful methods of veterinary hygiene control in the slaughterhouses in order to prevent the entering of ota in commercial channels with a view to human health . the economic efficacy of these prophylactic procedures is also considered . an evaluation of human exposure to ota is made .

distal anterior cerebral artery aneurysms , also known as pericallosal artery ( pa ) aneurysms , are located on the anterior cerebral artery distal to the anterior communicating artery . these aneurysms represent approximately 6% of all intracranial aneurysms and 4% of those that rupture49 ) . furthermore , they were similarly represented in the international subarachnoid aneurysm trial ( isat ) study population , in which they accounted for 95 of the 2143 patients randomized ( 4.4%)14 ) . pa aneurysms present several problems for direct surgical clipping , that is , difficult exposure via an interhemispheric approach , especially in a swollen brain after subarachnoid hemorrhage ( sah ) , sacrifice of a bridging vein for adequate surgical exposure ( thus increasing the risk of postoperative morbidity ) , difficult controlling the parent artery , and the unfavorable orientation of the aneurysm fundus . as a consequence , surgical morbidity has been reported to be relatively high , with incidences ranging from 0 to 25%141221 ) . advances in endovascular techniques and devices have led to the rapid evolution of coil embolization of ruptured and unruptured intracranial aneurysms , and it has now become an efficient treatment technique that is comparable to surgical clipping . however , because of their distal location and the small diameter of the parent vessel , pa aneurysms pose a technical challenge for endovascular therapy and , as is the case for most bifurcation cerebral aneurysms , have a potential high recurrence rate18 ) . a search of the literature revealed relatively few studies have been performed on the endovascular treatment of ruptured pa aneurysms , and that the studies performed involved relatively small numbers of patient . accordingly , it is evident that the effectiveness and safety of the endovascular treatment of ruptured pa aneurysms have not been well - defined . here , we present 30 patients that were treated endovascularly for a ruptured pa aneurysm and describe their clinical and radiologic outcomes . we retrospectively analyzed the angiographic and clinical records of all patients that underwent endovascular treatment for a ruptured pa aneurysm at our institution from september 2003 to december 2013 . the treatment strategy for ruptured pa aneurysm at our institution was to attempt endovascular treatment as the first - line treatment . this strategy was applied to those accompanied by intraparenchymal hematoma ( ich ) , and surgical evacuation , if necessary , was performed in the immediately following coiling . however , microsurgery was used in the early years of this study because the technology for coiling of distally located aneurysms was in its infancy . therefore , surgical clipping was first attempt in 6 cases during the same period . there was no technical failure in both clipping and coiling groups . due to the retrospective nature , the present study is exempt in accord with the institutional review board standards of our institution . thirty ruptured pa aneurysms in 30 patients were treated endovascularly , and there were 10 males and 20 females of mean age 55.9 years ( range , 24 to 88 years ) . dissecting or fusiform aneurysms , aneurysms associated with a brain arteriovenous malformation , and traumatic or mycotic aneurysms were excluded . size of aneurysms and parent arteries were measured automatically and manually on two - dimen sional digital subtraction angiography working views . aneurysm size was defined as the maximum distance of the dome from the neck plane . aneurysm and neck sizes ranged from 2.5 to 10.4 mm ( mean 5.0 mm ) and from 0.9 to 6.1 mm ( mean 2.2 mm ) , respectively . 21 of the 30 saccular aneurysms were defined as wide - neck aneurysms ( a large neck size of 4 mm and/or a dome - to - neck ratio of 2 ) . of the 30 pa aneurysms , 23 ( 76.7% ) were located at the pericallosal - callosomarginal junction and 7 ( 23.3% ) at the pericallosal - frontopolar junction . fifteen pa aneurysms were on the left side , 13 on the right , and 2 at midline ( azygos artery ) . all patients were clinically assessed at admission using hunt and hess grades ; 12 patients ( 40.0% ) grade ii , 9 ( 30.0% ) grade iii , 6 ( 20.0% ) grade v , and 3 ( 10.0% ) grade iv . fifteen patients ( 50.0% ) had a concomitant ich that was managed conservatively in all but two cases that required surgical evacuation . in general , endovascular treatment was performed as soon as possible after sah , regardless of clinical condition . coiling of aneurysms was performed under local anesthesia , and intravenous bolus injection of standard heparin was not performed during the procedure . however , drip infusion of heparin was carried out employing catheter pressure infusion systems with continuous infusion of 1000 u of heparin per 1000 ml of saline . the aim of the coiling was to obtain a packing of the aneurysm as attenuated as possible . the remodeling techniques using a balloon or stent or multiple catheters were unavailable for the aneurysms treated during the early part of our series . however , we have recently favored these remodeling techniques to achieve higher postoperative occlusion rates . when an aneurysm could not be reached using standard procedures because of a distal location and instability of the microcatheter , the guiding catheter was placed more distal into the internal carotid artery . care was taken not to interfere with the flow of the carotid or parent artery . by using these means , all aneurysms were reached with a microcatheter . in the case of intraprocedural thromboembolic complication , various strategies for thrombolysis were applied , such as mechanical thrombolysis and intra - arterial or intravenous abciximab , or heparin infusion during or after the procedure . immediately after the procedure furthermore , all patients underwent a non - enhanced brain computed tomography ( ct ) scan to evaluate possible hemorrhagic complications . unless the thromboembolic complication occurred or a stent was used , further antiplatelet or anticoagulant medications were not administered . the indications for long - term antiplatelet therapy of acetylsalicylic acid and/or clopidogrel were ; the formation of a thrombus at the end of the coil or thromboembolic events , stent - assisted coiling , and coil protrusion into the parent artery . occlusion was classified as ; complete when no contrast filling of the aneurysmal sac and neck was observed , near - complete when contrast filling of the neck of the aneurysm was slow or partial when any degree of contrast filling was observed within the aneurysm sac . clinical results were assessed upon discharge from hospital or at last visit using the modified rankin scale ( mrs ) , as follows : 0 , no symptoms at all ; 1 , no significant disability ; 2 , slight disability ; 3 , moderate disability ; 4 , moderately severe disability ; 5 , severe disability ; and 6 , death . complications were defined as all adverse events related to the procedure , and these were studied retrospectively using medical and operative reports . early rebleeding was defined as expanding hemorrhage with worsening of the patient 's condition within 30 days after coiling , and diagnostic confirmation was made by ct when it showed an increased amount of hemorrhage compared with immediate postprocedural ct . univariate and multivariate logistic regression analyses were used to identify risk factors associated with periprocedural rebleeding or a poor clinical outcome . results are presented as odds ratios ( ors ) and 95% confidence intervals ( cis ) . variables showing a univariate association with a dependent risk factor ( p<0.10 ) were included in the multivariate logistic analysis . chicago , il , usa ) . statistical significance was accepted for p values of < 0.05 . we retrospectively analyzed the angiographic and clinical records of all patients that underwent endovascular treatment for a ruptured pa aneurysm at our institution from september 2003 to december 2013 . the treatment strategy for ruptured pa aneurysm at our institution was to attempt endovascular treatment as the first - line treatment . this strategy was applied to those accompanied by intraparenchymal hematoma ( ich ) , and surgical evacuation , if necessary , was performed in the immediately following coiling . however , microsurgery was used in the early years of this study because the technology for coiling of distally located aneurysms was in its infancy . therefore , surgical clipping was first attempt in 6 cases during the same period . there was no technical failure in both clipping and coiling groups . due to the retrospective nature , the present study is exempt in accord with the institutional review board standards of our institution . thirty ruptured pa aneurysms in 30 patients were treated endovascularly , and there were 10 males and 20 females of mean age 55.9 years ( range , 24 to 88 years ) . dissecting or fusiform aneurysms , aneurysms associated with a brain arteriovenous malformation , and traumatic or mycotic aneurysms were excluded . size of aneurysms and parent arteries were measured automatically and manually on two - dimen sional digital subtraction angiography working views . aneurysm size was defined as the maximum distance of the dome from the neck plane . aneurysm and neck sizes ranged from 2.5 to 10.4 mm ( mean 5.0 mm ) and from 0.9 to 6.1 mm ( mean 2.2 mm ) , respectively . 21 of the 30 saccular aneurysms were defined as wide - neck aneurysms ( a large neck size of 4 mm and/or a dome - to - neck ratio of 2 ) . of the 30 pa aneurysms , 23 ( 76.7% ) were located at the pericallosal - callosomarginal junction and 7 ( 23.3% ) at the pericallosal - frontopolar junction . fifteen pa aneurysms were on the left side , 13 on the right , and 2 at midline ( azygos artery ) . all patients were clinically assessed at admission using hunt and hess grades ; 12 patients ( 40.0% ) grade ii , 9 ( 30.0% ) grade iii , 6 ( 20.0% ) grade v , and 3 ( 10.0% ) grade iv . fifteen patients ( 50.0% ) had a concomitant ich that was managed conservatively in all but two cases that required surgical evacuation . in general , endovascular treatment was performed as soon as possible after sah , regardless of clinical condition . coiling of aneurysms was performed under local anesthesia , and intravenous bolus injection of standard heparin was not performed during the procedure . however , drip infusion of heparin was carried out employing catheter pressure infusion systems with continuous infusion of 1000 u of heparin per 1000 ml of saline . the aim of the coiling was to obtain a packing of the aneurysm as attenuated as possible . the remodeling techniques using a balloon or stent or multiple catheters were unavailable for the aneurysms treated during the early part of our series . however , we have recently favored these remodeling techniques to achieve higher postoperative occlusion rates . when an aneurysm could not be reached using standard procedures because of a distal location and instability of the microcatheter , the guiding catheter was placed more distal into the internal carotid artery . care was taken not to interfere with the flow of the carotid or parent artery . by using these means , all aneurysms were reached with a microcatheter . in the case of intraprocedural thromboembolic complication , various strategies for thrombolysis were applied , such as mechanical thrombolysis and intra - arterial or intravenous abciximab , or heparin infusion during or after the procedure . immediately after the procedure furthermore , all patients underwent a non - enhanced brain computed tomography ( ct ) scan to evaluate possible hemorrhagic complications . unless the thromboembolic complication occurred or a stent was used , further antiplatelet or anticoagulant medications were not administered . the indications for long - term antiplatelet therapy of acetylsalicylic acid and/or clopidogrel were ; the formation of a thrombus at the end of the coil or thromboembolic events , stent - assisted coiling , and coil protrusion into the parent artery . occlusion was classified as ; complete when no contrast filling of the aneurysmal sac and neck was observed , near - complete when contrast filling of the neck of the aneurysm was slow or partial when any degree of contrast filling was observed within the aneurysm sac . clinical results were assessed upon discharge from hospital or at last visit using the modified rankin scale ( mrs ) , as follows : 0 , no symptoms at all ; 1 , no significant disability ; 2 , slight disability ; 3 , moderate disability ; 4 , moderately severe disability ; 5 , severe disability ; and 6 , death . complications were defined as all adverse events related to the procedure , and these were studied retrospectively using medical and operative reports . early rebleeding was defined as expanding hemorrhage with worsening of the patient 's condition within 30 days after coiling , and diagnostic confirmation was made by ct when it showed an increased amount of hemorrhage compared with immediate postprocedural ct . univariate and multivariate logistic regression analyses were used to identify risk factors associated with periprocedural rebleeding or a poor clinical outcome . results are presented as odds ratios ( ors ) and 95% confidence intervals ( cis ) . variables showing a univariate association with a dependent risk factor ( p<0.10 ) were included in the multivariate logistic analysis . chicago , il , usa ) . statistical significance was accepted for p values of < 0.05 . the microcatheter was navigated into the lumen of the pa aneurysm and coils were delivered in all 30 patient . the treatment of 19 aneurysms was straightforward , but the other 11 required an adjunctive technique , that is , double catheter , balloon - assisted , or a stent - assisted technique . immediate post - procedural angiograms showed complete aneurysm occlusion in 21 ( 21/30 , 70.0% ) and near - complete occlusion in 9 ( 9/30 , 30.0% ) . procedure - related thromboembolic complications developed in two patients ( 2/30 , 6.7% ) . in one patient , occlusion of a callosomarginal artery occurred due to thrombus formation at the artery origin , which resulted in right leg monoparesis . the other thromboembolism occurred in the inferior m2 segment of the middle cerebral artery during the procedure , a relevant infarction was observed 2 days after coiling . diagnosis of intraprocedural rupture was made based on the angiographic visualization of contrast material extravasation in two cases . the coil was observed partially outside the aneurysm with a sudden rise in arterial blood pressure but without angiographic demonstration of extravasation of contrast medium in one case . the mean delay between coiling and early rebleeding was 31 hours ( range , 2 - 83 hours ) . in these three patients , intravenous bolus of 3000 iu of heparin was injected during the procedure only in one case needed a stent . besides drip infusion of heparin during the procedure , further antiplatelet or anticoagulant medications were not administered in the other two cases . of these six patients with periprocedural rebleeding , five had small ( 4 mm ) aneurysms , but one had large ( 9.9 mm ) and multi - lobulated aneurysm . furthermore , the five patients ( 5/6 , 83.3% ) concerned had an adjacent hematoma on initial ct scans , and three ( 3/6 , 50.0% ) showed contrast retention and delayed washout by the aneurysm by preprocedural digital subtraction angiography . in these six patients with periprocedural rebleeding , no new neurologic deficit developed in three patients ( 3/6 , 50.0% ) , but deterioration of consciousness , resulting in death , occurred in two patients ( 2/6 , 33.3% ) . it could be questioned as to whether the early rebleeding had worsened clinical status in one patient , because the patient had initial severe sah and died within 5 days after sah onset . thus , the periprocedural mortality in our cohort was 6.7% ( 2/30 ) and morbidity was 3.3% ( 1/30 ) . table 3 provides a summary of the risk factors found to be associated with periprocedural rebleeding . the associations of all variables with periprocedural rebleeding were examined by univariate logistic regression analysis , and contrast retention was found to be associated with an increased risk of periprocedural rebleeding ( or : 23.000 ; 95% ci : 1.773 to 298.446 ; p=0.016 ) . furthermore , multivariate logistic - regression analysis adjusted for adjacent hematoma confirmed the validity of this relationship ( or : 21.352 ; 95% ci : 1.310 to 347.917 ; p=0.032 ) . only one patient ( 1/30 , 3.3% ) suffered from symptomatic vasospasm and , fortunately , no negative long - term effects were observed . clinical results were assessed upon discharge from hospital or at last follow - up visit using the mrs . at the end of the observational period , 18 patients were independent with a mrs score of 0 - 2 ( 18/30 , 60.0% ) , and the other 12 ( 12/30 , 40% ) were dependent ( mrs score , 3 - 6 ) . seven of the 12 had a mrs score of 6 ( due to a poor preoperative status in five and procedure - related rebleeding in two ) , three had a mrs score of 5 ( due to poor preoperative status or old age ) , one had a mrs score of 4 ( due to old age ) , and one had a mrs score of 3 ( due to a large ich ) . the 23 surviving patients were followed for 6 - 68 months ( mean , 32.7 months ) . no neurologic deterioration or hemorrhagic complication occurred during follow - up period in any of these patients . univariate logistic - regression analysis showed that age ( or : 1.080 ; 95% ci : 1.003 to 1.163 ; p=0.043 ) , adjacent hematoma ( or : 13.000 ; 95% ci : 2.074 to 81.479 ; p=0.006 ) , and hunt and hess grade 3 - 5 ( or : 13.750 ; 95% ci : 1.452 to 130.239 ; p=0.022 ) were associated with a poor clinical outcome . however , multivariate logistic - regression analysis adjusted for age and hunt and hess grade 3 - 5 showed that only adjacent hematoma was associated with a poor clinical outcome ( or : 47.734 ; 95% ci : 1.596 to 1427.240 ; p=0.026 ) . seventeen of the 23 surviving patients underwent follow - up conventional angiography between 1 and 65 months postoperatively ( mean , 16.5 months ) . according to the follow - up images obtained , 9 of the 10 initially occluded aneurysms remained completely occluded and the remaining one showed partial recanalization due to coil compaction . repeat follow - up angiography at 20 months showed in this patient that the condition was stable , and the patient concerned declined additional therapy . five of the 7 aneurysms initially showing near complete occlusion remained stable at follow - up . recoiling was undertaken in the patient showing major recanalization at 13 months postoperatively , and complete occlusion was achieved . thus , the overall stable occlusion rate was 82.4% ( 14/17 ) and the recanalization rate was 17.6% ( 3/17 ) . parent arteries of the aneurysm treated by y - stent - assisted coiling remained patent with no evidence of intimal hyperplasia or in - stent stenosis . the microcatheter was navigated into the lumen of the pa aneurysm and coils were delivered in all 30 patient . the treatment of 19 aneurysms was straightforward , but the other 11 required an adjunctive technique , that is , double catheter , balloon - assisted , or a stent - assisted technique . immediate post - procedural angiograms showed complete aneurysm occlusion in 21 ( 21/30 , 70.0% ) and near - complete occlusion in 9 ( 9/30 , 30.0% ) . procedure - related thromboembolic complications developed in two patients ( 2/30 , 6.7% ) . in one patient , occlusion of a callosomarginal artery occurred due to thrombus formation at the artery origin , which resulted in right leg monoparesis . the other thromboembolism occurred in the inferior m2 segment of the middle cerebral artery during the procedure , a relevant infarction was observed 2 days after coiling . however , the patient concerned did not experience any neurological symptom . diagnosis of intraprocedural rupture was made based on the angiographic visualization of contrast material extravasation in two cases . the coil was observed partially outside the aneurysm with a sudden rise in arterial blood pressure but without angiographic demonstration of extravasation of contrast medium in one case . the mean delay between coiling and early rebleeding was 31 hours ( range , 2 - 83 hours ) . in these three patients , intravenous bolus of 3000 iu of heparin was injected during the procedure only in one case needed a stent . besides drip infusion of heparin during the procedure , further antiplatelet or anticoagulant medications were not administered in the other two cases . of these six patients with periprocedural rebleeding , five had small ( 4 mm ) aneurysms , but one had large ( 9.9 mm ) and multi - lobulated aneurysm . furthermore , the five patients ( 5/6 , 83.3% ) concerned had an adjacent hematoma on initial ct scans , and three ( 3/6 , 50.0% ) showed contrast retention and delayed washout by the aneurysm by preprocedural digital subtraction angiography . in these six patients with periprocedural rebleeding , no new neurologic deficit developed in three patients ( 3/6 , 50.0% ) , but deterioration of consciousness , resulting in death , occurred in two patients ( 2/6 , 33.3% ) . it could be questioned as to whether the early rebleeding had worsened clinical status in one patient , because the patient had initial severe sah and died within 5 days after sah onset . thus , the periprocedural mortality in our cohort was 6.7% ( 2/30 ) and morbidity was 3.3% ( 1/30 ) . table 3 provides a summary of the risk factors found to be associated with periprocedural rebleeding . the associations of all variables with periprocedural rebleeding were examined by univariate logistic regression analysis , and contrast retention was found to be associated with an increased risk of periprocedural rebleeding ( or : 23.000 ; 95% ci : 1.773 to 298.446 ; p=0.016 ) . furthermore , multivariate logistic - regression analysis adjusted for adjacent hematoma confirmed the validity of this relationship ( or : 21.352 ; 95% ci : 1.310 to 347.917 ; p=0.032 ) . only one patient ( 1/30 , 3.3% ) suffered from symptomatic vasospasm and , fortunately , no negative long - term effects were observed . clinical results were assessed upon discharge from hospital or at last follow - up visit using the mrs . at the end of the observational period , 18 patients were independent with a mrs score of 0 - 2 ( 18/30 , 60.0% ) , and the other 12 ( 12/30 , 40% ) were dependent ( mrs score , 3 - 6 ) . seven of the 12 had a mrs score of 6 ( due to a poor preoperative status in five and procedure - related rebleeding in two ) , three had a mrs score of 5 ( due to poor preoperative status or old age ) , one had a mrs score of 4 ( due to old age ) , and one had a mrs score of 3 ( due to a large ich ) . the 23 surviving patients were followed for 6 - 68 months ( mean , 32.7 months ) . no neurologic deterioration or hemorrhagic complication occurred during follow - up period in any of these patients . univariate logistic - regression analysis showed that age ( or : 1.080 ; 95% ci : 1.003 to 1.163 ; p=0.043 ) , adjacent hematoma ( or : 13.000 ; 95% ci : 2.074 to 81.479 ; p=0.006 ) , and hunt and hess grade 3 - 5 ( or : 13.750 ; 95% ci : 1.452 to 130.239 ; p=0.022 ) were associated with a poor clinical outcome . however , multivariate logistic - regression analysis adjusted for age and hunt and hess grade 3 - 5 showed that only adjacent hematoma was associated with a poor clinical outcome ( or : 47.734 ; 95% ci : 1.596 to 1427.240 ; p=0.026 ) . seventeen of the 23 surviving patients underwent follow - up conventional angiography between 1 and 65 months postoperatively ( mean , 16.5 months ) . according to the follow - up images obtained , 9 of the 10 initially occluded aneurysms remained completely occluded and the remaining one showed partial recanalization due to coil compaction . repeat follow - up angiography at 20 months showed in this patient that the condition was stable , and the patient concerned declined additional therapy . five of the 7 aneurysms initially showing near complete occlusion remained stable at follow - up . recoiling was undertaken in the patient showing major recanalization at 13 months postoperatively , and complete occlusion was achieved . thus , the overall stable occlusion rate was 82.4% ( 14/17 ) and the recanalization rate was 17.6% ( 3/17 ) . parent arteries of the aneurysm treated by y - stent - assisted coiling remained patent with no evidence of intimal hyperplasia or in - stent stenosis . ruptured pa aneurysms may be technically difficult to clip because of the narrow exposure afforded by an interhemispheric approach , difficulty control of the parent artery , and sacrifice of a bridging vein to achieve adequate surgical exposure . surgical morbidity has been reported to be relatively high , with incidences ranging from 0 to 25%141221 ) . endovascular treatment of ruptured pa aneurysms has been reported to be technically difficult , primarily because of the long access route , which complicates microcatheter manipulation , and the small aneurysmal lumen . the introduction of new microcatheter and microguidewire with improved trackability , pushability , and torque and novel hydrophilic coatings have facilitated navigation into distant cerebral arteries , such as , the pa . furthermore , the availabilities of various soft coils may have contributed to the higher success rate of small aneurysm coiling without intraoperative rupture . we found that the pa could be reached and coiling of the aneurysm accomplished in all 30 patients . in fact , even a 2.5 mm sized aneurysm located at a distal junction was occluded successfully . in our series , an independent daily living activity level ( mrs score , 0 - 2 ) was achieved by 60% of our patients ( 18/30 ) . the poor clinical outcomes ( mrs score , 3 - 6 ) of the other 12 patients resulted from poor preoperative status , periprocedural rebleeding , old age , and large ich . waldenberger et al.25 ) reported that the most important negative contributor to the outcome of pa aneurysm coiling was the presence of ich , and this was followed by thromboembolic complications and poor preoperative status . ruptured pa aneurysms cause ich , in addition to sah , in more than one - half of cases , and are associated with poorer outcomes after rupture than aneurysms in other locations311 ) . in the present study , 15 of the 30 patients ( 50.0% ) had a concomitant ich that was visualized on initial ct scans , and 10 of the patients ( 10/15 , 66.7% ) had a poor clinical outcome ( mrs score , 3 - 6 ) . furthermore , multivariate logistic - regression analysis showed that adjacent hematoma was the only independent predictor of a poor clinical outcome ( or : 47.734 ; 95% ci : 1.596 to 1427.240 ; p=0.026 ) . other variables , such as , age and hunt and hess grade were of borderline significance , possibly due to the small number of patients enrolled ( table 4 ) . we have summarized the reported studies in which more than 10 patients with ruptured pericallosal artery aneurysms were treated with endovascular treatment in table 5 . in a recently reported meta - analysis of endovascular treatment of pa aneurysms including 16 studies , the authors concluded that endovascular treatment of pa aneurysms is associated with high angiographic occlusion rates , but the complication rates are higher compared with other aneurysms in the circle of willis . procedure - related ischemia and iatrogenic rupture occurred in 5% and 7% of patients , respectively . procedure - related permanent morbidity rate was 8%23 ) . in the present study , the overall procedure - related complication rate was 26.7% ( 8/30 cases ) . two patients experienced thromboembolic events and acute infarctions and developed corresponding neurologic deficits , though fortunately showed no neurological deficit at follow - up . intraprocedural rupture occurred from three aneurysms ( 10.0% ) , which was markedly greater than anticipated . a review of the literature revealed pa aneurysms have a higher procedure - related rupture rate than non - pa aneurysms . keston et al.9 ) reported an intraprocedural rupture rate of 18% ( 3/17 patients ) for pa aneurysms , which compared with an overall departmental five year ( 1999 - 2003 inclusive ) intraprocedural rupture rate of only 1.1% ( 7/640 patients ) for non - pa aneurysms . nguyen et al.15 ) reported a 12% ( 3/25 patients ) procedure - related perforation rate in their endovascular series of pa aneurysms . based on these reports and the fact that previous reports included unruptured cases , whereas we did not , we believe that our intraprocedural rupture rate is not excessive . it is well known pa aneurysms are smaller than non - pa aneurysms tend and that they tend to rupture at a smaller size101327 ) . in a study of a large number of aneurysm cases , the average diameter of ruptured aneurysms was reported to be 8.2 mm8 ) , whereas the average aneurysm diameter in our patients was 5.0 mm . we believe this difference could explain the higher intraprocedural rupture rate in the endovascular treatment of ruptured pa aneurysms . in addition , to aneurysm size , the risk of procedure - induced perforation may also be related to anatomical peculiarities . a more distal location leads to catheter positioning via a longer segment in a small - caliber vessel , which reduces the degree of catheter deflection when a coil encounters resistance15 ) . prior studies on early rebleeding after coiling have reported rupture rates of 1.4% to 2.6%6722 ) . incomplete embolization , adjacent hematoma , small aneurysms , and post - procedural anticoagulation have been reported to be associated with early rebleeding after coiling . the cerebral aneurysm rerupture after treatment study reported that the risk of posttreatment rebleeding was closely associated with the degree of aneurysm occlusion7 ) . however , we experienced three cases with early rebleeding after coiling ( 10.0% ) , despite all three aneurysms being complete embolized during the procedure . the presence of adjacent hematoma is a strong , independent risk factor of early rebleeding and a thrombosed pseudoaneurysm and hematoma could cause early reopening and rehemorrhage22 ) . in our series , all three cases with early rebleeding accompanied by adjacent hematoma . at coiling , a pseudoaneurysm may or may not be visible by angiography . if a pseudoaneurysm is apparent by angiography , coiling of the true aneurysm and the pseudoaneurysm can be considered , but the fragile wall of the pseudoaneurysm is prone to perforation , and this has been prompted to advise against catheterization into false lumen16 ) . on the other hand , cho et al.2 ) , who reported on early recurrent hemorrhage after coil embolization in ruptured intracranial aneurysms , hypothesized another possibility that rebleeding with ich may be caused by delayed hemorrhage or propagation of the initial ich due to vulnerability of the parenchyma adjacent to the ich , rather than by rebleeding of the coiled aneurysm . early rebleeding after coiling of a ruptured aneurysm and intra - procedural rupture are major concerns , because associated mortality rates are high . when a ruptured pa aneurysm accompanied by adjacent hematoma , decisions regarding endovascular treatment should be taken only after carefully considering the possibility of periprocedural rebleeding . therefore , it may be better to consider the surgical clipping of a ruptured pa aneurysm with adjacent hematoma as a first option to prevent periprocedural rebleeding . with regard to treatment durability , recurrence requiring recoiling due to the risk of re - rupture , developed in one of our 17 cases , who developed major recanalization . in the present study , the overall recanalization rate was 17.6% ( 3/17 ) but the major recanalization rate was only 5.9% ( 1/17 ) . recurrence after coiling has been reported to be associated primarily with aneurysm size7162226 ) . in one study , follow - up angiographic studies after complete occlusion showed recurrence rates of 14% for aneurysms of < 5 mm , 27% for aneurysms of 5 - 10 mm , and 57% for aneurysms of 10 mm23 ) . therefore , the lower rate of recurrence in our series could have been due to the lower proportion of cases with aneurysms 5 mm ( 9/30 , 30.0% ) . however , in a recent series of 16 cases of pa aneurysm coiling , there was a comparatively high recurrence rate ( one major and five minor recurrences , 37.5% ) , attributed to the loose coil packing rate19 ) . this study is limited by its retrospective design , patient - selection bias , small cohort , relatively short angiographic follow - up , by the fact that it was conducted at a single institution . nonetheless , our findings suggest that the endovascular treatment of ruptured pa aneurysms appears to be a valid treatment strategy given suitable levels of institutional and operator expertise . our experiences show that the endovascular treatment of ruptured pa aneurysms is both feasible and effective . however , they show periprocedural rebleeding occur far more frequently ( 20.0% ) than is generally considered and that these rebleeding are associated with preprocedural contrast retention . accordingly , our findings caution that decisions regarding endovascular treatment in cases showing preprocedural contrast retention should approached by carefully considering the possibility of periprocedural rebleeding . furthermore , an existing adjacent hematoma was found to have a severe negative influence on clinical outcome . cardiac implantable electronic device ( cied ) implantation continues to grow with permanent pacemaker implantation in the usa increasing by 56% between 1993 and 2009 . in europe , there has been a 75 and 115% increase in implantable cardiac defibrillator ( icd ) and cardiac resynchronization therapy pacemaker / defibrillator ( crt - p / crt - d ) implants between 2004 and 2008 . as a result of increasing indications for complex pacing , there is an expanding need for patients with pre - existing devices to undergo system revision due to lead failure or upgrade to allow icd and/or crt implant . in a recent european survey , 28% of crt implants were performed in patients with pre - existing devices , and recent lead advisories have necessitated an increase in lead extraction cases . the combination of such factors means that the need for system revision and upgrade is likely to increase in the future . a major obstacle to device revision and/or upgrade is the presence of asymptomatic ipsilateral central venous obstruction . older reports suggested obstruction occurred in up to 50% of cases with symptoms affecting only 13% , but more contemporary reports have demonstrated a lower incidence closer to 30% . this perhaps reflects improvements in lead design over time , but it remains clear that asymptomatic venous obstruction is not infrequently encountered . various strategies to overcome venous occlusion exist including contralateral lead or device implantation , venoplasty , and surgical epicardial lead implantation . the addition of extra pacemaker and/or icd leads is not without its drawbacks . in a recent prospective us registry of pacemaker / icd generator replacements , the need for an additional lead increased the rate of major complications from 4.0 to 15.3% . lead extraction may be an alternative option to overcome this problem , but it is not without risk and this should be weighed against the benefits of removing any leads and also the likelihood of symptom recurrence in cases where there are symptoms of venous occlusion . the practice of laser lead extraction to re - canalize venous obstruction has previously been described in a limited number of patients and the suitability of this technique on a larger scale has not been reported . we describe our experience using laser lead extraction to overcome venous occlusion to enable ipsilateral device revision and/or upgrade . guy 's and st thomas ' hospital is a quaternary referral centre for cied extraction . all patients undergoing device extractions are prospectively entered into a computer database recording patient demographics , comorbidities , device and lead type , reason for extraction , procedural success , and complications . complications are classified according to those recommended in the heart rhythm society consensus report on transvenous lead extraction . deaths were adjudicated by senior cardiologists within our department , none of whom had any input in the current study . patients from this database were included in the present study if the indication for lead extraction was to upgrade or revise an existing device in the presence of ipsilateral venous obstruction . in all cases , venous obstruction was identified on the basis of venography performed ( either in the radiology department at least 1 day prior to the procedure or in the cardiac catheter laboratory on the day of the procedure ) prior to each procedure to ascertain the patency of the venous system ( figure 1 ) . all patients provided written informed consent and all procedures were performed in our cardiac catheter laboratory under general anaesthesia . in patients with ipsilateral venous occlusion , or stenoses severe enough such that a hydrophilic guide wire would not cross the obstruction , laser lead extraction with retention of the outer sheath in the vascular tree was performed . in cases of device upgrade , any non - functional leads were removed ; if there was no non - functional lead ( for example , upgrade from dual - chamber pacing to crt - p ) , then the atrial lead was extracted with an attempt to preserve the existing right ventricular ( rv ) pacing lead . any redundant leads were also extracted . in cases of device upgrade , where there was also a failed lead in need of extraction , the failed lead was extracted and two hydrophilic wires passed through the outer sheath of the laser . after opening the existing generator pocket and disconnecting the leads from the generator , the suture sleeve of the lead being extracted was released . the proximal end of the lead was cut and a locking stylet ( liberator beacon , cook medical inc . or lld ez lead locking device ; spectranetics ) was advanced as distally as possible and locked in place . a silk suture was then tied to the lead to aid traction and this was fed through the laser sheath ( sls ii excimer laser sheaths ; spectranetics ) with the outer sheath also in position . both sheaths were advanced over the lead and the inner sheath advanced until resistance was met at which point laser energy was applied in short pulses to free the lead body from the surrounding vessel wall or cardiac musculature ( cvx-300 excimer laser system ; spectranetics ) . lasing was performed as necessary , up to the final 1 cm proximal to the distal electrode , and the lead was freed with counter - traction using the outer sheath . the lead and inner sheath were removed in their entirety leaving the outer sheath in place thereby maintaining vascular access . a venogram was performed to ensure that the sheath remained in the vascular / cardiac space and a hydrophilic wire ( terumo ) passed through the outer sheath when intravascular position was confirmed . a long haemostatic sheath(s ) was then placed to allow lead implantation in a standard fashion ( see figure 2 ) . ( a ) suggestive of collateral formation ( white arrow ) , which is confirmed with more medial panning during the venogram ( white arrows ; b ) . figure 2sequential images of the steps taken to successfully extract an atrial pace / sense ( patient in atrial fibrillation and in need of upgrade to a biventricular pacemaker ) , with maintenance of venous access across the level of occlusion . ( a ) the inner sheath is advanced with the outer sheath trailing . as demonstrated in this case , the laser is often required to overcome fibrosis at the clavicular level before the outer sheath can be advanced ( b ) . ( c ) further lasing upto and beyond the point of occlusion with passage of the outer sheath beyond the occlusion aided by rotational torque ( d ) . ( e ) the atrial lead is successfully extracted in its entirety using a combination of forward pressure on the outer sheath and manual traction on a locking stylet . ( f ) the inner sheath and lead are removed leaving the outer sheath in the vascular space just beyond the level of occlusion . ( g ) a hydrophilic wire is then passed through the outer sheath allowing passage of an introducer sheath and subsequent lv lead placement ( h ) . ( a ) suggestive of collateral formation ( white arrow ) , which is confirmed with more medial panning during the venogram ( white arrows ; b ) . sequential images of the steps taken to successfully extract an atrial pace / sense ( patient in atrial fibrillation and in need of upgrade to a biventricular pacemaker ) , with maintenance of venous access across the level of occlusion . ( a ) the inner sheath is advanced with the outer sheath trailing . as demonstrated in this case , the laser is often required to overcome fibrosis at the clavicular level before the outer sheath can be advanced ( b ) . ( c ) further lasing upto and beyond the point of occlusion with passage of the outer sheath beyond the occlusion aided by rotational torque ( d ) . ( e ) the atrial lead is successfully extracted in its entirety using a combination of forward pressure on the outer sheath and manual traction on a locking stylet . ( f ) the inner sheath and lead are removed leaving the outer sheath in the vascular space just beyond the level of occlusion . ( g ) a hydrophilic wire is then passed through the outer sheath allowing passage of an introducer sheath and subsequent lv lead placement ( h ) . between july 2004 and april 2012 , 242 upgrade / revision procedures were performed and of these 71 ( 29% ) were performed in patients with occluded or severely stenosed venous anatomy . complete ipsilateral occlusion was present in 52 of 71 patients ( 73% ) in this series . the remainder had severe stensoses that did not allow passage of hydrophilic guide wires and/or introducer sheaths and this was taken as indicating functional obstruction . the vast majority of obstructions were identified in the subclavian vein ( 67 of 71 ) with the remainder being at the junction of the subclavian vein with the superior vena cava ( svc ) ( see figure 1 ) . twenty - nine ( 41% ) patients had a history of ischaemic heart disease and 19 ( 27% ) had prior cardiac surgery ( coronary artery bypass surgery or valve surgery ) . the mean left ventricular ( lv ) ejection fraction was 38% ( derived from 2d echocardiography using simpson 's biplane method ) . three patients ( 4% ) had symptoms of venous occlusion ( arm swelling and pain on the side of device implant ) . table 1patient characteristicscharacteristicage ( years)62 15gender , n ( % ) male 55 ( 77)female 16 ( 23)ejection fraction ( % ) 38 15comorbiditiesihd , n ( % ) 29 ( 41)cardiac surgery , n ( % ) 19 ( 27)diabetes , n ( % ) 5 ( 7)hypertension , n ( % ) 16 ( 23)pvd , n ( % ) 3 ( 4)stroke , n ( % ) 5 ( 7)copd , n ( % ) 14 ( 20)ckd , n ( % ) 12 ( 17)ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . patient characteristics ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . most extractions were performed in patients with existing dual - chamber pacemakers , icds , or crt - ds ( 24 , 37 , and 27% , respectively ) . in total , 129 leads were extracted from 71 patients ( see table 2 ) . of these , 40 ( 31% ) were passive fixation right atrial ( ra ) or rv pacemaker leads , 33 ( 26% ) were active fixation ra or rv pacing leads , 41 ( 31% ) were single- or dual - coil defibrillator leads , and 15 ( 12% ) were coronary sinus lv pacing leads , which were extracted due to sub - optimal function ( phrenic nerve capture , or sub - optimal lead positioning ) . the commonest indications for extraction were lead malfunction ( 56% ) and need for device upgrade ( 40% ) . table 2device characteristicscharacteristicdevice , n ( % ) vvi2 ( 3 ) ddd17 ( 24 ) icd26 ( 37 ) crt - p7 ( 9 ) crt - d19 ( 27)indication for extraction , n ( % ) lead failure40 ( 56 ) upgrade28 ( 40 ) symptoms3 ( 4)nature of upgrade , n ( % ) ppm to icd8 ( 29 ) ppm to crt - p9 ( 32 ) icd to crt - d11 ( 39)number of leads extracted , n ( % ) total129 passive a / v40 ( 31 ) active a / v33 ( 26 ) icd41 ( 31 ) cs15 ( 12)mean duration of lead implant ( months)80 62vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . device characteristics vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . it was necessary to snare leads from the femoral venous approach in two cases following unsuccessful laser extraction when the lead fragmented despite the obstruction being crossed . new leads were successfully re - implanted via the laser sheath across the venous obstruction / stenosis in 67 ( 94% ) cases . in four cases , the laser sheath was unable to pass the obstruction due to intense fibrosis / calcification , and in three of these cases a subclavian vein puncture medial to the venous occlusion was performed to obtain venous access and successfully place the lead . in one case , a transvenous lead could not be placed and an epicardial lv pacing lead was surgically implanted during a later procedure . mean fluoroscopic screening time was 16 13 min and mean radiation dose was 837 1269 cgycm . there were two major complications ( 3% ) that were both cases of infection of a previously sterile site , and four ( 6% ) minor complications ( ipsilateral pneumothorax , phrenic nerve palsy , acute renal failure , and pocket haematoma ) . table 3procedural characteristics and outcomecharacteristicobstruction crossed , n ( % ) 67 ( 94)lead successfully extracted via laser sheath , n ( % ) 69 ( 97)transvenous lead successfully sited70 ( 99)procedural time ( min)116 32fluoroscopy time ( min)16 13radiation dose ( cgycm)837 1269complications major , n ( % ) 2 ( 3 ) minor , n ( % ) 4 ( 5 ) 30-day mortality , n ( % ) 2 ( 3 ) procedural characteristics and outcome medium- to long - term device follow - up was available in 65 of 71 ( 92% ) cases with a mean follow - up of 26 19 months . one patient developed non - procedure - related sepsis ( urinary sepsis following urethral instrumentation for urinary retention ) while recuperating from their procedure . the second death occurred in a patient with severe heart failure ( new york heart association iv ) with a pre - existing crt - d that had a non - functioning lv pacing lead . the patient had refractory hypotension and hyponatraemia limiting the use of medical therapy and a decision was made to attempt revision of the crt - d device as a procedure of last resort . the patient died in hospital from end - stage heart failure despite successful lead extraction . both deaths were adjudicated by senior physicians within our department , who were blinded to the interventions in the current study . it was necessary to further revise cied implants following the index procedure in four ( 6% ) cases . two cases required re - intervention for defibrillator leads with diminished sensing , one patient developed phrenic nerve capture from their lv pacing lead , and one patient deteriorated with a sub - optimal lv lead position that required revision . twenty - nine ( 41% ) patients had a history of ischaemic heart disease and 19 ( 27% ) had prior cardiac surgery ( coronary artery bypass surgery or valve surgery ) . the mean left ventricular ( lv ) ejection fraction was 38% ( derived from 2d echocardiography using simpson 's biplane method ) . three patients ( 4% ) had symptoms of venous occlusion ( arm swelling and pain on the side of device implant ) . table 1patient characteristicscharacteristicage ( years)62 15gender , n ( % ) male 55 ( 77)female 16 ( 23)ejection fraction ( % ) 38 15comorbiditiesihd , n ( % ) 29 ( 41)cardiac surgery , n ( % ) 19 ( 27)diabetes , n ( % ) 5 ( 7)hypertension , n ( % ) 16 ( 23)pvd , n ( % ) 3 ( 4)stroke , n ( % ) 5 ( 7)copd , n ( % ) 14 ( 20)ckd , n ( % ) 12 ( 17)ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . patient characteristics ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . most extractions were performed in patients with existing dual - chamber pacemakers , icds , or crt - ds ( 24 , 37 , and 27% , respectively ) . in total , 129 leads were extracted from 71 patients ( see table 2 ) . of these , 40 ( 31% ) were passive fixation right atrial ( ra ) or rv pacemaker leads , 33 ( 26% ) were active fixation ra or rv pacing leads , 41 ( 31% ) were single- or dual - coil defibrillator leads , and 15 ( 12% ) were coronary sinus lv pacing leads , which were extracted due to sub - optimal function ( phrenic nerve capture , or sub - optimal lead positioning ) . the commonest indications for extraction were lead malfunction ( 56% ) and need for device upgrade ( 40% ) . table 2device characteristicscharacteristicdevice , n ( % ) vvi2 ( 3 ) ddd17 ( 24 ) icd26 ( 37 ) crt - p7 ( 9 ) crt - d19 ( 27)indication for extraction , n ( % ) lead failure40 ( 56 ) upgrade28 ( 40 ) symptoms3 ( 4)nature of upgrade , n ( % ) ppm to icd8 ( 29 ) ppm to crt - p9 ( 32 ) icd to crt - d11 ( 39)number of leads extracted , n ( % ) total129 passive a / v40 ( 31 ) active a / v33 ( 26 ) icd41 ( 31 ) cs15 ( 12)mean duration of lead implant ( months)80 62vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . device characteristics vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . all 129 leads were successfully extracted in their entirety . it was necessary to snare leads from the femoral venous approach in two cases following unsuccessful laser extraction when the lead fragmented despite the obstruction being crossed . new leads were successfully re - implanted via the laser sheath across the venous obstruction / stenosis in 67 ( 94% ) cases . in four cases , the laser sheath was unable to pass the obstruction due to intense fibrosis / calcification , and in three of these cases a subclavian vein puncture medial to the venous occlusion was performed to obtain venous access and successfully place the lead . in one case , a transvenous lead could not be placed and an epicardial lv pacing lead was surgically implanted during a later procedure . mean fluoroscopic screening time was 16 13 min and mean radiation dose was 837 1269 cgycm . there were two major complications ( 3% ) that were both cases of infection of a previously sterile site , and four ( 6% ) minor complications ( ipsilateral pneumothorax , phrenic nerve palsy , acute renal failure , and pocket haematoma ) . table 3procedural characteristics and outcomecharacteristicobstruction crossed , n ( % ) 67 ( 94)lead successfully extracted via laser sheath , n ( % ) 69 ( 97)transvenous lead successfully sited70 ( 99)procedural time ( min)116 32fluoroscopy time ( min)16 13radiation dose ( cgycm)837 1269complications major , n ( % ) 2 ( 3 ) minor , n ( % ) 4 ( 5 ) 30-day mortality , n ( % ) 2 ( 3 ) procedural characteristics and outcome medium- to long - term device follow - up was available in 65 of 71 ( 92% ) cases with a mean follow - up of 26 19 months . one patient developed non - procedure - related sepsis ( urinary sepsis following urethral instrumentation for urinary retention ) while recuperating from their procedure . the second death occurred in a patient with severe heart failure ( new york heart association iv ) with a pre - existing crt - d that had a non - functioning lv pacing lead . the patient had refractory hypotension and hyponatraemia limiting the use of medical therapy and a decision was made to attempt revision of the crt - d device as a procedure of last resort . the patient died in hospital from end - stage heart failure despite successful lead extraction . both deaths were adjudicated by senior physicians within our department , who were blinded to the interventions in the current study . it was necessary to further revise cied implants following the index procedure in four ( 6% ) cases . two cases required re - intervention for defibrillator leads with diminished sensing , one patient developed phrenic nerve capture from their lv pacing lead , and one patient deteriorated with a sub - optimal lv lead position that required revision . we have described our experience using laser lead extraction to overcome ipsilateral venous obstruction in patients undergoing device revision and/or upgrade . to date , this is the largest series of cases , where laser lead extraction has been used to overcome venous obstruction thereby allowing ipsilateral lead revision or device upgrade . we have shown that the technique is feasible in the vast majority of cases . in 100% of cases , the targeted lead(s ) were completely extracted , and in 94% re - canalization of the obstructed vein allowed successful lead implantation via the laser sheath . in the remaining patients transvenous lead implantation was successful on the ipsilateral side with a medial puncture and in only one case did the patient need a further procedure ( surgical lv lead implant ) to achieve implant success . our complication rate is low even in a cohort of patients with reduced lv systolic function and significant comorbidities . the heart rhythm society ( hrs ) consensus statement on lead extraction in relation to venous obstruction states that lead removal is a class i recommendation in patients with ipsilateral venous occlusion preventing additional lead placement when there is a contraindication for using the contralateral side ( e.g. contralateral atrioventricular fistula , shunt or vascular access port , and mastectomy ) . lead removal in patients with ipsilateral venous occlusion and no contraindication to using the contralateral side is a class iia indication . obstruction and thrombosis of the access vein after implantation of permanent pacing and defibrillator leads are well described . the reported incidence of asymptomatic cases is up to 50% in older series and up to 30% in more contemporary series . symptomatic occlusion occurs in 13% , thus highlighting the importance of developing strategies for overcoming such obstacles at the time of device upgrade or lead revision . in our study , the finding of venous obstruction precluding device revision or upgrade was 29% in keeping with recent reports . in such cases , it may be possible to obtain venous access with a de novo puncture , but it is often not possible to advance introducer sheaths across a very tight stenosis and there is the added risk of increased lead lead interaction . these issues are avoided by using the technique described in this report and we have demonstrated that the procedure can be performed safely . laser technology is increasingly being used to facilitate lead extraction . in the plexes trial , use of the laser sheath resulted in complete removal of 94% of leads compared with only 64% where non - laser tools were used ( predominantly telescoping sheaths ) . more recently , bordachar et al . showed that laser extraction results in shorter procedures with lower radiation exposure to operators when compared with femoral snare techniques . the lexicon study was an observational retrospective study of 1449 consecutive laser lead extractions in north america and confirmed high success rates and low complication rates , particularly in high volume centres . the use of the laser sheath to overcome venous obstruction was first described by bracke et al . in three patients , where the laser was only used up to the point of obstruction and the lead left in situ . the largest previous series by gula et al . included 18 patients , where laser lead extraction was performed to facilitate system upgrade in the presence of central venous occlusion . in both earlier reports , our current study provides an expanded assessment of the technique in a larger number of patients . procedures were performed by three experienced operators in a single centre , each with extensive experience in laser lead extraction . our patients tended to have a longer duration of lead implant ( 80 62 vs. 70.8 43.5 months in the series of gula et al . ) . in addition , our cohort of patients is typical of those in whom these procedures are performed , namely depressed ejection fraction and attendant comorbidities . no outer sheath was used . in a minority of cases , tissue build - up at the tip of the laser sheath prevented the withdrawal of the lead tip through the laser sheath . this necessitated extraction of a functional atrial lead to ensure maintenance of venous patency . also , our study included patients requiring non - functional lead extraction rather than just device upgrade . in such cases , extraction may be preferable to adding extra leads . in our study , all leads were removed in their entirety , but it was necessary to snare two leads from the femoral venous route . therefore , 98% of leads were removed using the laser sheath alone and in total , venous access was maintained with the outer sheath in 94% of cases . no leads were unintentionally damaged and it was not necessary to extract any extra leads . this is important as it may have implications in reducing the risk of the procedure . there are inherent risks in extracting leads , particularly from the thin - walled ra , and so if extraneous extraction can be avoided this is preferable . in the current study , leads were successfully extracted in all cases after the laser sheath had successfully overcome fibrosis at the subclavicular level . there were two major procedural complications , both of which were cases of infection of a previously sterile site and this possibly reflects the added risk in performing upgrade or revision procedures . of the four minor complications , none were specifically related to the use of the laser sheath to overcome the obstruction . the alternative options available if ipsilateral venous occlusion is present include : insertion of new leads via the contralateral subclavian vein with either tunnelling of the leads across the sternum or abandonment of the pre - existing leads;venoplasty of the occluded vessels;alternative venous access;surgical epicardial lead implant.each of the techniques listed have their own specific drawbacks . the practice of adding leads is not without risk ; in the replace registry , there was a 15.3% major complication rate and a 1.1% 6-month mortality rate in patients undergoing generator change with a planned lead addition or revision . the hrs lead extraction consensus statement states that lead removal is reasonable in patients if cied implantation would require more than four leads on one side or more than five leads through the svc ( class iia recommendation ) . the presence of redundant leads is also associated with increased risks of infection and erosion . tunnelling across the sternum to the contralateral position of the existing generator potentially avoids the risk of svc syndrome , but there is a heightened risk of lead erosion , patient discomfort , and bilateral occlusion . venoplasty has been previously described , where a wire is passed through the occlusion , and balloon venoplasty performed to open a patent channel . the results of this procedure are encouraging with lead implantation possible in 96% of patients in a recent report . the technique may not be ideal in cases , where there is an indication for lead revision ( such as in defibrillator lead failure ) , where extraction of the malfunctioning lead may be the preferred option . lead malfunction was the primary indication for revision in 56% of our cases and the use of the laser sheath to overcome obstruction and remove the malfunctioning lead represents an attractive option in such cases . use of other venous access sites , such as the internal jugular vein , with subsequent tunnelling of leads has been described ; however lead erosion remains an issue . surgical implantation of epicardial leads negates the need for a transvenous approach but requires a thoracotomy and lead failure is not uncommon . insertion of new leads via the contralateral subclavian vein with either tunnelling of the leads across the sternum or abandonment of the pre - existing leads ; venoplasty of the occluded vessels ; alternative venous access ; surgical epicardial lead implant . this study is a single - centre experience with experienced operators and therefore our results may not be widely applicable in less experienced centres . lead extraction is not without risk and should only be performed in centres with experienced staff and necessary equipment / tools and with access to onsite emergency surgery . a prospective study of laser extraction to treat venous obstruction would be necessary to extrapolate our results . our results suggest that laser lead extraction to overcome ipsilateral venous obstruction is effective and safe , and therefore represents an attractive approach to deal with device upgrade / lead revision in patients with obstructed or severely stenosed venous anatomy . m.o.n . received research funding from st jude medical and biosense webster ; j.g . received research funding from biosense webster and st jude medical ; c.a.r . is a consultant to spectranetics and received research funding from st jude medical , boston scientific , and medtronic ; m.s .

objectiveaneurysms arising from the pericallosal artery ( pa ) are uncommon and challenging to treat . the aim of this study was to report our experiences of the endovascular treatment of ruptured pa aneurysms.methodsfrom september 2003 to december 2013 , 30 ruptured pa aneurysms in 30 patients were treated at our institution via an endovascular approach . procedural data , clinical and angiographic results were retrospectively reviewed.resultsregarding immediate angiographic control , complete occlusion was achieved in 21 ( 70.0% ) patients and near - complete occlusion in 9 ( 30.0% ) . eight procedure - related complications occurred , including intraprocedural rupture and early rebleeding in three each , and thromboembolic event in two . at last follow - up , 18 patients were independent with a modified rankin scale ( mrs ) score of 0 - 2 , and the other 12 were either dependent or had expired ( mrs score , 3 - 6 ) . adjacent hematoma was found to be associated with an increased risk of poor clinical outcome . seventeen of 23 surviving patients underwent follow - up conventional angiography ( mean , 16.5 months ) . results showed stable occlusion in 14 ( 82.4% ) , minor recanalization in two ( 11.8% ) , and major recanalization , which required recoiling , in one ( 5.9%).conclusionour experiences demonstrate that endovascular treatment for a ruptured pa aneurysms is both feasible and effective . however , periprocedural rebleedings were found to occur far more often ( 20.0% ) than is generally suspected and to be associated with preoperative contrast retention . analysis showed existing adjacent hematoma is predictive of a poor clinical outcome . aimsthe number of procedures involving upgrade or revision of cardiac implantable electronic devices ( cieds ) is increasing and the risks of adding additional leads are significant . central venous occlusion in patients with pre - existing devices is often asymptomatic and optimal management of such patients in need of device revision / upgrade is not clear . we sought to assess our use of laser lead extraction in overcoming venous obstruction.methods and resultspatients in need of device upgrade / revision underwent pre - procedure venography to assess venous patency . in patients with venous occlusion or stenosis severe enough to preclude passage of a hydrophilic guide wire , laser lead extraction with retention of the outer sheath in the vasculature was performed with the aim of maintaining a patent channel through which new leads could be implanted . data were recorded on a dedicated database and patient outcomes were assessed . between july 2004 and april 2012 , laser lead extractions were performed in 71 patients scheduled for device upgrade / revision who had occluded or functionally obstructed venous anatomy . new leads were successfully implanted across the obstruction in 67 ( 94% ) cases . there were two major complications ( infection ) and four minor complications with no peri - procedural mortality . device follow - up was satisfactory in 65 ( 92% ) cases with mean follow - up up to 26 19 months.conclusionlaser lead extraction is a safe and effective option when managing patients with central venous obstruction in need of cied revision or upgrade .

the incidence of age - related diseases is rising , seriously affecting the health of millions of people around the world . according to the united nations ( un ) and the world health organization ( who ) musculoskeletal , rheumatic , and arthritic conditions are leading causes of morbidity and disability throughout the world , giving rise to enormous healthcare expenditures and loss of work ( woolf and pfleger , 2003 ; source : http://www.arthritis.org/ ) , . many types of rheumatic diseases and arthritic conditions are essentially age - related inflammatory disorders where the inflammation facilitates disease progression . the term arthritis characterizes a group of conditions involving inflammatory damage to synovial joints ( di paola and cuzzocrea , 2008 ) . it involves pain , redness , heat , swelling , and other harmful effects of inflammation within the joint . however , the most common and important form of arthritis is osteoarthritis ( oa ) , also known as osteoarthrosis or degenerative joint disease ( djd ) . oa is the most prevalent of the chronic diseases affecting the elderly ( aigner et al . , 2004 ) . the majority of the population over 65 years of age demonstrate radiographic evidence of oa in at least one joint . although oa is rare in people under 40 , it becomes much more common with age . a 2005 study in the usa estimated that oa is one of the top five causes of disability amongst non - hospitalized adults [ source : center for disease control ( cdc ) , usa ] . in 2006 it was estimated that around 35 million to 40 million europeans suffer from oa and nearly 25% of people aged 60 and above suffer from oa induced disability . it is also anticipated that by the year 2030 , 20% of adults will have developed oa in western europe and north america . therefore , oa is expected to place a heavy economic burden on healthcare systems and community services throughout the world . the risk factors for oa are well known and include age , overweight / obesity , underlying metabolic or endocrine disease , genetics , and joint trauma ( lotz and kraus , 2010 ) . with increasing life expectancy , growth in the elderly population and an alarming escalation of chronic , inflammatory , and age - related conditions ( such as oa ) , it is a mechanically unique and resilient connective tissue responsible for load - bearing and low - friction movement in the synovial joints of all vertebrates ( buckwalter et al . , cartilage is avascular and as a consequence it has a very limited capacity for intrinsic repair ( brittberg , 1999 ; tew et al . , 2001 ) . it highly prone to structural degradation making it particularly difficult to restore once it is damaged or lost . the ecm consists of three classes of molecules : collagens , aggregating proteoglycans , and non - collagenous proteins . type ii , ix , and xi collagens form a fibrillar framework of macromolecules that give the tissue form , tensile stiffness , and mechanical strength ( buckwalter and mankin , 1998b ; eyre , 2004 ) . large aggregating proteoglycans ( predominantly aggrecan ) allow cartilage to swell and resist compressive forces ( hardingham and fosang , 1992 ; kuettner , 1992 ) . small proteoglycans including decorin , biglycan , and fibromodulin , bind to other matrix macromolecules and help to stabilize the ecm . other collagenous and non - collagenous macromolecules present within the ecm perform a variety of structural and informational roles , facilitate cell cell and cell - matrix interactions , and bind growth factors ( hardingham and fosang , 1992 ; feng et al . , 2006 ) . the chondrocyte is the only cell type present in articular cartilage ( archer and francis - west , 2003 ) . during embryonic development chondrocytes synthesize a cartilaginous template for endochondral ossification and skeletal development and in postnatal life they maintain the ecm by regulating the turnover of matrix components in response to biomechanical , biochemical , and endocrine signals ( goldring and marcu , 2009 ) . chondrocytes actively synthesize new ecm components as well as the proteolytic enzymes such as matrix metalloproteinases ( mmps ) , a disintegrin , and metalloproteinase ( adams ) and a disintegrin and metalloproteinase with thrombospondin motifs ( adamtss ) are responsible for tissue remodeling during development . these enzymes are also involved in the catabolic breakdown of cartilage in oa ( aigner et al . , osteoarthritis is a degenerative disease that involves joint inflammation , bone remodeling , and catabolic destruction of the articular cartilage component ( goldring and goldring , 2007 ; samuels et al . , 2008 ) . in oa there is an imbalance between the synthesis and degradation of ecm macromolecules ( felson , 2004 ) . this can be due to increased enzymatic activity of mmps ( okada et al . , 1992 ) , and pro - inflammatory mediators such as cytokines ( goldring and goldring , 2004 ) , prostaglandins , and nitric oxide ( goldring and berenbaum , 2004 ) , coupled with the reduced anabolic capacity of chondrocytes ( aigner et al . , 1997 ) and the tissue s inherently poor reparative capacity due to its avascular nature ( archer and francis - west , 2003 ) . the degradation and release of proteins and glycoproteins from cartilage in oa can vary according to the stage of the disease process . for example , elevated serum cartilage oligomeric matrix protein ( comp ) is correlated with the presence of oa and disease severity ( clark et al . , 1999 ) . aging is a major contributor to musculoskeletal degeneration and the development of oa ( hamerman , 1998 ; lotz and carames , 2011 ) . age - related changes in articular cartilage contribute to the development and progression of oa . although the degeneration of articular cartilage is not simply the result of aging and mechanical wear , aging nevertheless modifies the articular joint including cartilage , subchondral bone , muscle , soft tissues , synovial membrane , and synovial fluid ( buckwalter and mankin , 1998a ; hamerman , 1998 ) . although older age is the greatest risk factor for oa , oa is not an inevitable consequence of growing old ( shane anderson and loeser , 2010 ) . cell stress and oxidative damage contribute to chronic inflammation that promotes age - related diseases . in oa this results in senescence - associated secretory phenotype , which has many of the characteristics of an osteoarthritic chondrocyte in terms of the cytokines , chemokines , and proteases produced ( loeser , 2011 ) . a major focus of clinical research in recent years has been the identification of new disease markers that can facilitate early diagnosis and optimize individualized treatments . such markers can also facilitate the drug discovery process by reducing the high levels of attrition in clinical trials . a biomarker is classically defined as a biochemical entity that is used to measure the progress of a disease or the effects of treatment on clinical outcome . biochemical markers can be measured in blood , serum , and urine or a variety of other body fluids and tissues . the national cancer institute ( nci ) defines a biomarker as a biological molecule found in blood , other body fluids , or tissues that is a sign of a normal or abnormal process , or of a condition or disease , and the terms molecular markers or signature molecules have also been used to describe such markers . the term biomarker is all encompassing and can include proteins , protein fragments , metabolites , carbohydrates , nucleic acids ( rna and dna ) , cellular features , and images . osteoarthritis is unambiguously diagnosed when it is detected by the best available test . thus far the best test for this purpose has been radiography , the so - called gold - standard . this process also requires clinical signs in the patient , which often occur well into the progression of the disease . however , there is often early , pre - clinical evidence of disease provided by various biomarkers , which if detected , may facilitate earlier diagnosis and treatment . such an approach is particularly pertinent in the case of oa , a disease often characterized by a prolonged pre - clinical molecular phase , a pre - radiographic phase , and a recalcitrant radiographic phase by which time there are structural changes to joints along with pain and loss of function . biomarkers have the potential to provide an early warning of joint degeneration which could prompt earlier , more targeted treatment to prevent the tissue destruction that results in the characteristic chronic disability associated with oa . in this context , biomarkers could make a significant contribution to the early diagnosis of oa , as well as informing key aspects of disease prognosis , monitoring , and therapy . the national institute of arthritis and musculoskeletal and skin diseases ( niams ) established the osteoarthritis biomarkers network to develop and validate standardized , sensitive biomarker assays in blood and urine to facilitate the diagnosis of the pre - radiologic stage of oa in humans and in animal models . such markers can help us understand the biological processes involved in disease progression and allow us to monitor the effects of surgical or pharmacological treatment , thus accelerating the pace of drug discovery . such biomarkers could also potentially be used to identify patients at increased risk of developing oa . existing biomarkers of oa have major limitations : they do not flag the pre - radiographic phase of the disease ; they are not specific for the various stages of oa , and in some cases , may not even be specific for oa . considering these challenges , the osteoarthritis research society international ( oarsi ) and the us food and drug administration ( fda ) have recently established a new oa biomarkers working group , which has proposed the division of potential markers into two major groups : the so - called soluble or biomarkers , which typically reflect a modulation in an endogenous substance in body fluids such blood , serum , plasma , urine , or synovial fluid ) ; and the dry biomarkers , which usually consist of visual analog scales , performed tasks , or images of joints ( kraus et al . , 2011 ) . therefore , the ability to detect biomarkers of cartilage degradation and/or inflammation in biological samples , such as serum , urine , or synovial fluid , may enable clinicians to diagnose sub - clinical oa as well as determining the disease stage in both human and companion animals . identifying these biomarkers will also aid drug discovery and drug safety / efficacy monitoring in patients and in animal models . using combinations of biomarkers may be more effective in achieving these goals , thus having a panel of biomarkers will help researchers and the pharmaceutical industry to monitor disease progression as well as to assess responses to treatment in experimental models of oa ( rousseau and delmas , 2007 ; williams , 2009 ) . systems biology is increasingly applied in orthopedics and rheumatology to cartilage and synovium in arthritis . these techniques include genomics , transcriptomics , proteomics , metabolomics , glycomics , and bioinformatics and can be applied to the study of cartilage , synovium , synovial fluid , and even blood ( serum ) or urine from oa patients . proteomics involves the application of specialized analytical techniques that allow the evaluation of the protein composition of tissues , cells , and culture supernatants . proteomics is being increasingly applied in basic cartilage biology ( polacek et al . , 2010 ) and oa research ( ruiz - romero et al . , 2010 ) . characterization of cell lysates from isolated chondrocytes has yielded valuable information regarding the intracellular proteins of the chondrocyte proteome , and paved the way for future studies on cartilage pathologies such as oa ( ruiz - romero et al . , 2005 ; ruiz - romero and blanco , 2010 ) . studies of soluble proteins in cartilage tissue from oa patients has increased the knowledge of the proteins contained within the ecm of diseased versus normal tissue ( wu et al . , 2007 ) . a number of papers have reported on proteins secreted from the cartilage ecm in response to pathological insults such as interleukin ( il)-1 and all - trans - retinoic acid ( wilson et al . , 2008a , b ; ruiz - romero and blanco , 2010 ) , il-1 and tnf- ( cillero - pastor et al . , 2010 ) and mechanical compression ( stevens et al . , 2008 ; identifying proteins released from cartilage has the potential to give an indication of disease biomarkers likely to be present in the synovial fluid or blood of patients in the early stages of oa . understanding healthy aging is a key research priority , along with a better understanding of the pathophysiology of aging that occurs in a number of age - related diseases , such as arthritis . by gaining a better understanding of healthy musculoskeletal aging we can provide better care and new therapies for common musculoskeletal problems . physiology and pathophysiology of musculoskeletal aging is a research topic that is intended to bring together basic researchers and clinicians working in the broad area of musculoskeletal aging . the topic includes mechanisms of healthy aging in tissues of the musculoskeletal system ( i.e. , skeletal muscle , articular cartilage , subchondral bone , tendon , and ligament ) . the discovery and validation for biomarkers of oa has accelerated significantly as our understanding of joint tissue molecules and their complex interactions have increased ( kraus , 2005 ) . one of the main drivers in this context has been the urgent need for improved oa outcome measures in clinical trials ( kraus , 2005 ; hunter et al . , in particular there is a pressing need for new biomarkers that indicate early responses of the joint cartilage to degeneration that will be useful in detecting early , pre - radiographic changes . novel markers that characterize the status and prognosis of oa , and that can be used to monitor response to therapy are also required ( mobasheri and henrotin , 2010 ) . research aims to develop an analytical toolbox which is hoped will contribute to the clinical development process ( bay - jensen et al . , 2010 ; combinations of existing biomarkers may improve their prognostic accuracy and help identify at - risk patients ( williams , 2009 ) . omics - based technologies in order to identify sensitive and reliable pre - radiographic biomarkers that can be accurately and reproducibly measured in body fluids . biomarkers that flag early stage oa will be particularly useful in curbing disease progression by identifying patients that would benefit from early therapeutic intervention . in this research topic gharbi and co - workers ( gharbi et al . , 2011 ) their aim is to improve our understanding of the physiopathology of the disease its underlying mechanisms and to discover disease - specific biomarkers and identify new therapeutic targets . this timely and focused review summarizes the currently available data regarding proteomic techniques and their applications to oa research . the authors discuss technical limitations and solutions to real and practical problems including sample preparation . although proteomics has many potential applications in this area , there are technical challenges that still remain . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . a patient 's journey may begin with transfer from an ambulance to the emergency room and then sometimes continues to surgery and intensive care followed by a general ward . intensive care is designed and meant for the sickest patients with potential life threats and vital organs dysfunction ; it requires advanced monitoring , technique , diagnosis , and treatments . organizing and performing patient transfers in the continuum of care is part of the work of nurses and other staff of the multiprofessional healthcare team . whittaker and ball ( 2000 ) argue that it is important to perform the preparations for a transfer to the general ward accurately and correctly . if this is not done , the patient must be readmitted to the intensive care unit ( icu ) and be exposed to further stress . this depending on multiple reasons and common causes could be their present sedation or altered mental status . discharge planning in general is described as a process which should provide continuity of care for patients . in this study , icu transitional care is defined as care provided before , during , and after the transfer of an icu patient to another care unit with the aim of ensuring minimal disruption and optimal care for the patient . this care may be provided by icu nurses , acute care nurses , physicians , and other healthcare professionals . hence , in this study , care is defined as a mix of nursing care and medical care . it is important that patients ' transfers from the icu are done properly and at the right time when there is no longer a need for intensive care [ 4 , 5 ] . patients want to feel safe and secure both before and after the transfer , and they can easily become dependent on the staff . there are studies that describe how patients perceive their safety during transfer and how they feel about their recovery , but it has also been reported how perceived physical illness can affect experiences of displacement . patients sometimes also struggle with feelings of abandonment , vulnerability , helplessness , and unimportance . ambivalent feelings about the upcoming transfers are also shown to be common ; both positive and negative emotions have been reported [ 911 ] . different factors have impact on patients ' recovery from intensive care , premorbid state , social , family , psychological , physical status , and employment . the struggle for hospital bed placement is becoming more and more frequent , and nowadays hospitals often are overcrowded , which also implies that the organization of transfers is especially important for patient safety . discharge guidelines and policies are seen as important in the management of transfers [ 14 , 15 ] and are also considered an effective management tool to reduce length - of - stay in the icu and improve the utilization of icu resources [ 16 , 17 ] . however , the results of previous studies indicate that discharge planning often lacks guidelines and tends to be ad hoc and influenced by patient acuity [ 18 , 19 ] . priorities in icu may be necessary to enable admission for the most ill patients , leading to unplanned discharges even during night which are related to higher risks . a study by goldfrad and rowan ( 2000 ) found that the overall icu mortality is 25 times higher if the patient is discharged at night . in their study , the staff estimated that only 44% of these patients were fully ready for the transfer , compared with over 80% of patients who were transferred during the day . discharging patients recently discharged from the icu may be particularly at risk for adverse events and readmissions to icu . to complete a patient 's transfer from a high technological icu to a general ward , many studies demonstrate the experiences of the transition but there is no clear description of the process and how it could be organized in order to be safe [ 511 ] . based on the experiences described by those involved in the process , it can be argued that it is important to learn more about how to enhance and organize icu transitional care . therefore , the aim of this study was to describe , as experienced by intensive care and general ward staff , what strategies could be used when organizing patient 's care before , during , and after transfer from intensive care . before , during , and after transfer from the icu to a general ward , patients experience a transition process . the patients are transferred from the context of high technology to the culture of the general ward . the specific process of transition from the icu to the general ward has become a topic of interest because difficulties that arise during the process have been increasingly frequent . transitions can be initiated by such events as acute illness or injury , which also explains why the concept is a nursing concern . the process requires a beginning , middle , and end and how the person feels and perceives the situation is critical as the process continues . transition could result in a feeling of displacement and lack of control over their lives . the situation and time span vary and may consist of short periods or months and years ; an example of transition is hospitalization for an acute injury or illness . the study has been approved by the northern ethical committee in sweden ( d - number 07 - 159 ) . the first author informed and asked the nurses about participation in the study in accordance with verbal and written criteria . they were informed about confidentiality and their rights to withdraw their participation without giving reason . as the aim of the study was to describe and illuminate the transition process between icu and general ward , qualitative content analyses were considered [ 25 , 26 ] . the data were also used in a larger study that aimed to generate theories about main concerns in icu transitional care . data were collected between 2008 and 2010 in two hospitals located in sweden with different sizes . the participants were recruited in three icus and five general wards specializing in surgical , medical , or general fields . one of the hospitals had a step - down unit , and the second had no step - down unit . all interviews were conducted by the first author . there were totally 15 individual interviews : seven icu nurses from three icus in sweden , one anesthesiologist , one en ( enrolled nurse ) from an icu , and six rns working in different surgical or medical wards in two hospitals . the participants had different genders and lengths of experience ( 125 years ) ; 3 were males and 12 were women . their ages varied between 25 and 62 years . the staff in general wards was registered nurses and the nurses who worked in the icu had a one - year specialist certification in intensive care . the interviews were performed in a quiet place at the unit in the hospital and lasted between 30 and 50 minutes . the focus in this study was on how to organize patient transfer to the ward for further care and rehabilitation and not on patients transferred for palliative end - of - life care . could you please tell me about how the transfer process for icu patients is organized at your unit and your feelings about it ? clarifying questions such as what do you mean , when , why , and can you tell me more about that were asked when necessary to encourage interviewees to narrate their experiences . the data analyses were inspired by elo and kyngs ' ( 2008 ) description of content analysis . , the interview text was read several times to get a sense of the whole . the manifest analyses started with sorting the data in content areas : before , during , and after transfer from the icu to a general ward . this was followed by an open coding with handwritten notes and headings in the text , using as many headings as possible , close to the text , in order to describe all aspects of the content . with further abstraction , the codes were divided into subcategories and categories with similar incidents named with content - characteristic words that were all relevant to the aim and the research topic . the results show that the categories secure , encourage , and collaborate are strategies used in the three phases of the icu transitional care process before , during , and after transfer from the icu ( table 1 ) . the main category , a safe , interactive rehabilitation process , illustrates how all strategies were characterized by an intention to create and maintain safety during the process . it also included attempts to help the patient reach the best possible condition in the different phases so that their recovery and the rehabilitation proceeded as planned . one nurse stated : the goal for our care in this process is that the patients are going to be better in all ways , both physical and physiological . the goal for our care in this process is that the patients are going to be better in all ways , both physical and physiological . the main category and the strategies also illustrated a three - way interaction in the process : between staff and patient / families , between team members and involved units , and between patient / family and environment . the first category , secure , included activities that aimed at preserving patient safety during the transfer and preventing adverse events immediately after transfer or later . the second category , encourage , included activities focused on strengthening , inspiring , and giving hope and courage to patients and their families . the third category , collaborate , included activities aimed at intertwining the process : coordination , cooperation , and communication between the icu and the general wards . the first phase in icu transitional care included secure , optimizing vital signs and reducing and adjusting the intensive care , encourage , promoting self - ability and customizing information , and collaborate , communicating and coordinating with the ward and arranging a pretransfer meeting . optimize vital functions . securing the patients ' care and preserving patient safety during icu transitional care one essential issue was to optimize the patient 's vital functions prior to transfer , a key component to minimize risk for adverse and readmissions , which were commonly referred to in the interviews . the icu staff interacted with technology and other senses to assess and perform clinical judgments . the patient 's pain relief and vital signs ( especially respiration and hemodynamic ) were the focus and a point of decision in how and when the patient could be transferred out of intensive care.the most important in the transfer process is that the patients ' vital signs are stable enough ; we observe this extremely carefully . the most important in the transfer process is that the patients ' vital signs are stable enough ; we observe this extremely carefully . the decision for transfer mostly by anesthesiologists in consultation with the responsible physician from the ward . however , the icu nurses felt that they were involved and interacted with the physicians in the decision by either confirming that the patients were stable enough or postponing the transfer if the patient had respiratory issues , fever , or some other problematic clinical symptoms . if the patient was fragile or weak , it was seen as important to have a couple of extra days in the icu or , if possible , in an intermediary unit reassuring that intensive care was not needed anymore . this was expressed not only as one important strategy to prevent readmission but also as something that often could not be done . timing for transfer was crucial , and the interviews revealed that patients should not remain either too long or too short time in the icu . the nurses expressed that both compromised patient safety ; if they were transferred too early , they were not stable enough , and if they were transferred later than needed , it made them more immobilized and susceptible to infections and other symptoms . reduce and adjust . to secure patient care and to prevent adverse events later in the icu transitional care process , it was necessary to reduce technology and adjust how medication was administrated . weaning and timing of extubation was one important part , which was expressed as often time - consuming and difficult for less experienced nurses . the staff stated that patients should have time to recover from the state of full respiratory support , proceed to minor support ( noninvasive respiratory support ) , and finally manage to breathe at least 24 hours without respiratory support . it was also expressed as important to reduce and adjust the care for patient safety , both for the prevention for central line infections and for suiting the upcoming level of care and competence at the ward . not all ward nurses , for example , managed multilumen central lines and the like . in the interview , it was also apparent that technology should be reduced and only used when needed in the icu since the environment in the icu could cause trouble for the individual patient . instead , the use of technology should follow the need for hemodynamic assessment so that the patients and their families became used to fewer observations and monitoring once the patients were transferred . adjustments to care were often done in an interaction between ens , rns , and physicians and needed to be documented in the patient record to maintain consistency . it was also considered essential that administration of medications was altered to prepare the patients for the upcoming level of care , for example , shifting to sleeping pills instead of infusions prior to transfer.i think that patients that we try to wean that have been here a long time also should be used to not have full monitoring often , there are enough with a pulse oximetry part of the day . i think that patients that we try to wean that have been here a long time also should be used to not have full monitoring often , there are enough with a pulse oximetry part of the day . this included supporting the patient 's own initiative and promoting self - ability , since the icu period often had a negative impact on the patient 's muscle mass and initiative ability . it was expressed that the patient 's self - ability in this phase influenced the following phases in the transfer process . the staff meant that the patient 's self - ability should be strengthened prior to transfer as a part of their rehabilitation process and as a step toward managing the altered level of care . promoting the patient 's own ability required a lot of persuasion and sensitive interaction with the patient . the icu staff tried to support the patients to manage small things , for example , to use the bed alarm so they would be familiar with it later on to receive help at the ward . the staff described how they tried on a daily basis to encourage patients to sit up at the bedside and in chairs so that they would not be immobilized in bed at all times . it was also considered essential and helpful if the patients got early and frequent physiotherapy at the icu.we help the patient to drink water which includes helping them grip the glass and set it to the mouth we have to tell them that everything is better than doing nothing ; you can do it in your own way . we help the patient to drink water which includes helping them grip the glass and set it to the mouth we have to tell them that everything is better than doing nothing ; you can do it in your own way . encouragement based on providing repeated and customized information to patients and their families was considered by the staff to be an important tool in organizing icu transitional care . it was important to adjust and customize the information to the specific patient needs and inform the families about time for the upcoming transfer . it was expressed as essential to interact with the families and inform them several times about the progress , care plans , and goals . the icu and general ward staff expressed that anxiety could be minimized if patients and their families were encouraged and informed repeatedly that their condition was stabilized and that a transfer would soon occur . some patients and families needed to hear the information often and asked repeatedly about things related to their medical status and upcoming transfer while others asked nothing . it was expressed as important to give them consistent responses that did not differ , which could include visual information about the environment and routines at the ward preferably in form of a brochure or something similar . the staff experienced that the patients ' and/or families ' experience of encouragement and participation in their care depended on how well and how clearly the information was given.i think we can do even better , especially to work more with individualized information for the families . i think we can do even better , especially to work more with individualized information for the families . the staff described the coordination between the icu and the department to which the patient would be transferred as a foundation for collaboration , especially when the patient had been cared for in the icu for a long time . collaborating involved communicating and coordinating between the care units for transferred patients . the communication involved information about the patient needs and preparing the unit about what they should consider when the patient arrived at the ward . in some cases , it also involved care - planning meetings with families and different physicians . this made a common view for the patients ' planning , and it was expressed as a wish for mutuality between the icu and the ward , allotting responsibility and planning for the patients ' transfer . coordination was described as being better if it was done as early as possible , especially if the patient had a long length - of - stay in the icu . one unit used a liaison nurse a person with a specific responsibility for collaboration which was experienced in facilitating communication and planning for specific needs . the nurses expressed that if the patient had specific needs , they must be planned for and taken care of a strategy to manage care must be prepared . collaboration could also include planning for what room the patient should be placed in at the ward , considering individual needs and , if possible , what nurse should be responsible . one icu nurse said : it is important to communicate early and tell them ( the staff at the ward ) about every need that the patient may have and not cover or exclude anything . especially if the patient has been a long time in icu it is important to communicate early and tell them ( the staff at the ward ) about every need that the patient may have and not cover or exclude anything . especially if the patient has been a long time in icu arrange a pretransfer meeting . collaborating and arranging a pretransfer meeting between ward staff and the patients and their families before transfer was expressed as one appreciated strategy . this was described as extra important when the patient or the family was anxious or if the patient had been cared for in the icu for a long time and had extended needs . the ward nurses meant that this pretransfer meeting was valuable for them and for the patient , since it was an opportunity to create a contact with the patient and family before transfer . the meeting also provided a possibility to ask questions and get a picture about of the patient 's care needs . the meeting was described in two ways : at the icu or in the general ward , depending on the patient 's status . the nurses expressed that when a pretransfer meeting was organized , this helped to intertwine the healthcare chain.right now , for example , we have a gentleman at our ward that was 4 - 5 months in icu ; he has probably a length - of - stay of 8 months in the hospital ! and before he came up , we were down there and introduced us , told him that we would be responsible at the ward ! right now , for example , we have a gentleman at our ward that was 4 - 5 months in icu ; he has probably a length - of - stay of 8 months in the hospital ! and before he came up , we were down there and introduced us , told him that we would be responsible at the ward ! the strategies in this phase included secure , assess and summarize , hand over responsibility , and arrange a safe transfer , encourage , give confidence , and collaborate , negotiate and facilitate . the staff expressed that it was important prior to transfer to take time to assess and summarize the patient 's discharge status and the different nursing phenomena and actions that had been or should be taken for the patient . if the transfer was not planned in advance , this phase also included examining all the equipment that the patient still had and considering removing measurement and technology specific to intensive care and that were not wanted or needed at the general ward.i think it 's easier when we have someone who is responsible and keeps up the documentation and prepares for discharge we really want the patient 's record to be clear and that it is obvious what is planned . i think it 's easier when we have someone who is responsible and keeps up the documentation and prepares for discharge we really want the patient 's record to be clear and that it is obvious what is planned . the report was mentioned by the staff to be an essential tool to maintain continuity of care and was experienced as the way to hand over the patient without any loose ends . the staff from the general ward tried to prepare themselves by taking part of patient documents before they got to the icu , but they did not always have this opportunity.i try to keep up reading about the patient before the transfer so i do n't have to start from zero knowledge . for example , what surgery they 've done , drains , and also quickly check the latest values i try to keep up reading about the patient before the transfer so i do n't have to start from zero knowledge . for example , what surgery they 've done , drains , and also quickly check the latest values the handover often included a nurse - to - nurse report intended to focus briefly on the history and more specifically on the actual status and the planned care . the icu nurses expressed that their handover should be adjusted with adequate information but they wanted to tell as much as possible so that no loose ends would be left . some ward nurses expressed that they wanted less history and more of the actual status with planned x - rays , medications , and iv - fluids as well as more of a description of the patient 's own ability and problems . sometimes , the nurses from the general ward felt insecure but did not dare to ask the icu nurses about the patient , since they did not want to seem incompetent . most icu patients are fragile , and a safe transfer that maintains patient safety was expressed as essential . the patient often received oxygen during the transfer to avoid desaturation , and , sometimes , portable monitors were used to control vital signs . in the interviews , one nurse described an example of adverse events related to a transfer : well , we do n't want something critical to happen during the transfer but i will always remember one time i got to get a patient from icu i directly saw that she was hugely fragile and wondered if we really should move her from icu . but both our physician and icus physician insisted , and we took the patient and went away immediately when we came out from the elevator , the patient 's vital signs were deteriorating . we hurried as soon we could into the ward to get help and had to alarm for assistance . well , we do n't want something critical to happen during the transfer but i will always remember one time i got to get a patient from icu i directly saw that she was hugely fragile and wondered if we really should move her from icu . but both our physician and icus physician insisted , and we took the patient and went away immediately when we came out from the elevator , the patient 's vital signs were deteriorating . we hurried as soon we could into the ward to get help and had to alarm for assistance . the staff experienced that it was essential to give confidence prior to and during transfer and to talk in a positive way with patients and their families . the nurses expressed that some patients were anxious and showed feelings of disconnectedness when leaving the environment where they felt safe and staff they knew . the staff felt that the patients ' families also needed to feel that the transfer was a step forward and that it was a positive sign.when the day of the transfer comes , it may not be too ridden with anxiety for the patient either - but you have to play down the whole and try to make it easier it might be a grief to leave the place where you ( have ) been so long and become better . that you try to focus on that this is an important positive sign that the patient has to be transferred at a ward , emphasize that now is the worst of the crisis over ! when the day of the transfer comes , it may not be too ridden with anxiety for the patient either - but you have to play down the whole and try to make it easier it might be a grief to leave the place where you ( have ) been so long and become better . that you try to focus on that this is an important positive sign that the patient has to be transferred at a ward , emphasize that now is the worst of the crisis over ! collaboration was described by the staff as essential on the day of transfer and included negotiating a suitable time for transfer and facilitating the transfer for all involved staff members . it was important to seek resolutions for problems and to see each other as equal . the staff expressed that barriers could occur for collaboration that instead complicate the transfer , for example , blaming each other and not trying to understand each other 's work situation . hence , interprofessional respect between the staff at the units was essential and facilitated the process . the actual day of the transfer was expressed as more or less planned , depending on the acute situation at the unit . the nurses on general wards said that it was obvious that the icu , with rapid decision - making , led to rapid transfers , sometimes unplanned . they wished to be involved in deciding the time of transfer if possible , a time that suited staff from both the general ward and the icu . the staff at the general ward considered it extremely important to have the opportunity to prepare when they were about to get a patient from the icu , since it was often time - consuming . if they knew in time , they could plan their work , which made the process feel easier and safer . when the staff communicated about a suitable time for transfer , they also were informed about specific equipment needed , such as oxygen.most of the times , we can not influence the time for transfer , but we think that it more often should be possible to communicate about it , make it suit us all . most of the times , we can not influence the time for transfer , but we think that it more often should be possible to communicate about it , make it suit us all . the third phase included the strategies secure , take charge , encourage , create a good encounter and instill hope and courage , and collaborate , prepare discharge and follow up . when the patient arrived at the general ward , the staff stated that it was important to get a grip of the whole situation and to take charge of the patient . it involved an overall view from clinical judgments to take control over the paperwork and plan future care actions that were described as time - consuming . depending on the patient 's status , the ward nurses spent more or less time on continuing care , for example , supplying oxygen ; controlling wounds , drains , and drain holes ; monitoring feeding tubes and intravenous lines ; calculating fluid balance ; and checking vital signs . the nurses expressed that it also was vital that the physician at the ward checked out the ordinations and wrote an updated status in the patient 's record so that they had a tool for managing their care.it's not just ( that ) the patient must be in good condition when he or she is discharged from the icu ; the care must also continue with high quality at the general ward . it 's not just ( that ) the patient must be in good condition when he or she is discharged from the icu ; the care must also continue with high quality at the general ward . sometimes , it was necessary to change equipment and time for medication to suit the care at the ward . some of the ward nurses expressed that they felt that it was more often patients with a shorter length - of - stay at the icu who were hemodynamically unstable and readmitted . according to their experience , patients with longer length - of - stay more often were better optimized in their vital functions . the ward nurses mentioned that their control of vital signs sometimes required them to alarm a specific outreach team . this team included staff from the icu ( nurse / physician ) who could initiate treatment for the patient at the ward or decide to readmit the patient to the icu again . they expressed that the outreach team was a helpful tool and made them feel safer in their care but also meant that it was important to have personal clinical judgment.there are always some warning clocks when you are checking vital signs and something is wrong . sometimes , you can feel that something is breaking out , something will be wrong with the patient i think it depends of a combination of ( your ) own intuition and the patient 's history . there are always some warning clocks when you are checking vital signs and something is wrong . sometimes , you can feel that something is breaking out , something will be wrong with the patient i think it depends of a combination of ( your ) own intuition and the patient 's history . instill hope and courage . creating a good encounter with the patients and their families at the first meeting ( either in the icu or when arriving at the general ward ) a good encounter included a personal meeting , introducing the responsible staff , and interacting and supporting individual needs . the ward nurses wanted to have time to communicate and sense needs at the first encounter so that they could calm the patients and their families . they expressed that families often displayed anxiety and had questions about the differences in the environments . it was also important to inform the patient and other family members that staff members were available at the ward even though they were not physically present at the patient 's room all the time . it took some time to establish hope and courage , and some were more difficult than others.the patients and their families can be in a shock reaction when they arrive , and they still try to process and understand what happened according to my experience , this is more often seen if the intensive period were short . the patients and their families can be in a shock reaction when they arrive , and they still try to process and understand what happened according to my experience , this is more often seen if the intensive period were short . the ward nurses expressed that early mobilization in the icu was vital for how the patient 's rehabilitation process proceeded . the nurses expressed that the patient 's own ability to take initiative needed to be supported . depending on diagnosis , this ability differed , but the ward nurses expressed that the care in the icu often made patients immobilized and used to others taking care of their hygiene . nurses in the ward felt that the patients had become apathetic and then stepped aside , as others still were responsible for their body.some can stand on their legs immediately , but some must first be mobilized in the bed but you can really tell that they 've been working with early mobilization in icu . some can stand on their legs immediately , but some must first be mobilized in the bed but you can really tell that they 've been working with early mobilization in icu . collaboration in this phase included planning for further discharge for the patient , which depended on the individual patient 's status and home situation . sometimes , patients needed extensive planning and interaction with community nurses while other patients could be directly transferred at home without any extended help . the nurses explained that this planning often took time and was an important part of the patient 's healthcare chain . the icu and general ward staff often continued their collaboration around the patient following transfer a strategy that intertwined the icu transitional care process . sometimes , a ward visit was made by the icu staff primarily not only to meet the patient again but also to give the staff an opportunity to ask questions and to give medical advice if needed . in one of the hospitals , patients were further followed up within postintensive care meetings after they had been discharged and left the hospital.we try to go up and visit the patient at the ward a few days after the transfer , if we have time ; it 's appreciated by both the patient and the staff since they may ask if there is something missed . we try to go up and visit the patient at the ward a few days after the transfer , if we have time ; it 's appreciated by both the patient and the staff since they may ask if there is something missed . the main category of this study showed that strategies used by the staff before , during , and after transfer aimed to contribute to a safe , interactive rehabilitation process . the results showed how the strategies secure , encourage , and collaborate could be used to enhance and organize icu transitional care . patient safety and interaction with patient , family , environment , and other team members are essential for icu transitional care . the main category also illustrates that there is a three - way interaction in the process : between staff and patients / families , between different team members and involved units , and between patient / family and environment . the interaction between environment and patients has also been illuminated by theorists such as rogers , king , and nightingale , all of whom claimed that the patient is in constant interaction with his or her environment . the icu staff interacted with technology as a tool to determine if patients were stable enough for weaning and , further on , for transfer . the staff felt that the environmental differences between the icu and general wards are often a cause of concern for patients and their families . according to meleis therefore , we assumed that the care must be individualized even if there are routines and procedures to follow . transition stands for a change in health status , role relationships , expectations , or abilities . the transition event is dependent on , for instance , suddenness , personal meaning , and level of well - being . hence , it can be argued as logical to strive for a strengthening process that considers these needs and involves an active plan for a smoother transition . it is important to clarify and explain to the patient and family that observations and monitoring are reduced and adapted to the patient 's current health status , which may reduce the perceived stress during the transitional phase . overall , the results indicate that the transition experience is dependent on preparation of both patient and families for the first contact with the team at the ward and the time of transfer ; therefore , the planning must be taken seriously . timing seemed to be a critical point , and this is confirmed in a study by garland and connors ( 2013 ) . their study indicated that 30-day mortality increased not only if the patients had to leave earlier than planned from the icu but also if the transfer was delayed so they had to leave later than was optimal . this is in accordance with other researches and confirms that weaning is vital for patient safety . ( 2003 ) showed that readmission was more often seen when the time between extubation and discharge from the icu was short . a recent study including patients with traumatic brain injuries showed that reintubations within 48 hours ( i.e. , extubation failure ) were significantly associated with lengthened hospital stays , increased frequency of tracheotomy and of pulmonary complications , worsened functional outcomes , and increased mortality . intermediary units can be used as a way to reduce care and to prepare patients for the altered level [ 32 , 33 ] , but not all hospitals have this kind of unit . however , icus can identify beds at the unit aimed at intermediary care where patient care can proceed but with less technology and staff . the necessity of avoiding miscommunication during clinical handovers is also described in several other studies . miscommunication can lead to risk of prolonged stay , lack of continuity of care , suboptimal patient flow , readmissions , and patient dissatisfaction reports [ 34 , 35 ] . in a systematic review of foster and manser ( 2012 ) , handovers and transfer of patients in acute care were studied . the results showed that the handover process is multifacetted and can therefore be difficult to compare and evaluate , but they also show that standard report pages are one way to facilitate reporting . boutilier ( 2007 ) concluded that the most important thing in the icu transitional care process was to systematically communicate the necessary information to the receiving device so that patient safety was not threatened and to ensure the necessary continuity of care . the second strategy found in our study , encourage , is often mentioned in nursing . to encourage meant to instill hope , identity , and confidence for the patients and their families . the results of this study illustrate the importance of pep talk and supportive strategies to encourage the patient to manage the transition and recover from critical illness . according to the nurses , patients need to feel safe , a result also described by hupcey ( 2000 ) as a psychosocial need for icu patients . one characteristic of transition is disconnectedness associated with disruption of the links on which the person 's feelings of security depend . the nurses in this study wanted to be able to offer customized information prior to transfer since they thought that anxiety could be reduced with information . this can be compared with the result from strahan and brown ( 2004 ) , who found that patients often are anxious over how the transfer process will be done and wish to receive detailed information on what will happen . the nurses expressed that the patients and their families should be a part of the transfer . presence from relatives can affect the patient 's sense of participation and contribute to feelings of being cared for and of safety . it is important to minimize experiences of a gap between the icu and general wards . health and illness transitions include the sudden role change that can result from moving from a well state to an acute illness . the results of this study indicate that the patient 's role changes when arriving at the general ward from being more or less passive to being seen as active . furthermore , patients have a desire for normality and independence and wish to be able to have personal contacts also seen in a study by mckinney and deeny ( 2002 ) . the results also illustrate the importance of the team at the receiving ward having the opportunity to devote time to the patient and family when they first arrive . the ward nurses in our study expressed that patients showed mixed feelings about the transfer to the general ward and that they needed to instill hope and courage . our study indicated that both an immediate and later follow - up after leaving the icu could be useful , helping patients identify problems and find routes for rehabilitation and support . a study by schandl ( 2011 ) actually showed that multidisciplinary icu follow - up and the first six months after discharge are most important for follow - up . the results in this study showed that icu transitional care is a complex , multidisciplinary process that involves collaboration both within the icu and within other units involved . the findings of our study show that collaboration includes respectful , functional communication between units and different actions aimed to intertwine the healthcare chain . effective teamwork and coordination among staff can improve the icu patient discharge process and also reduce the gap separating icu and the general ward [ 14 , 21 ] . our study indicates that team members in their own unit and in other units need to treat each other with respect so everyone ask questions without fear or ridicule contempt . the results of their study showed , as did this study , that respect and equality are important . previous studies have focused on how a specific liaison nurse can be used to facilitate collaboration . a liaison nurse coordinates the transfer and is helpful for the patients and their families [ 43 , 44].the results of this study indicate that there are benefits to a function like that . many of the strategies during the process focus not only on identifying and minimizing risks for complications , such as pneumonia and central line infections , but also on strengthening mind and body . this process is described as an active , unique , nonlinear process with stages and phases . the results of this study follow a model called chime : connectedness , hope and optimism , identity , meaning in life , and empowerment . one important part of icu transitional care is to promote patients ' self - care capability and encourage patients ' autonomy . this is confirmed by chick and meleis ( 1986 ) , who also claim that transitions are linked to shifts in self - care capability . orem ( 1980 ) writes that independence was recognized by early nursing theorists as important for patients well - being and closely connected to their ability to perform daily activities and meet their own care needs . however , the nature of intensive care and its environment makes independence and autonomy difficult . hughes ( 2004 ) emphasized that the degree of autonomy is connected not only to the ability to actually be independent but also to the healthcare staff 's perception and understanding of the need for this assessment . the nurses in this study expressed that early mobilization was felt important not least for the patients ' ability to do things for themselves and manage the upcoming care . this can be compared with the results of an intervention study by korupolu et al . they saw that early mobilization in the icu and a strategy for whole - body rehabilitation in the early stage of critical illness showed better functional outcomes at hospital discharge , a shorter duration of delirium , and more ventilator - free days compared with standard care . mcfetridge ( 2011 ) discussed the importance of a structured , patient - centered rehabilitation program that patients can follow on their journey from critical care to ward and , finally , through discharge from hospital . mcfetridge also claims that interaction in care is essential so as to prevent actions taken from becoming fragmented . hence , the process should be seamless and transparent for all persons that are involved in the patient 's care journey and include a multidiscipline approach and a family perspective . this study illustrates how the strategies secure , encourage , and collaborate summarize specific actions that can be used as a basis for a patient - centered guideline for icu transitional care . however , the fact that guidelines on their own are not a solution to minimize the gap between icu and general wards must be discussed . a cultural gap has been identified between icu and general wards , and a study by van sluisveld et al . ( 2013 ) implies that there are social and cultural barriers to the implementation of guidelines and effective icu discharge . to summarize , no guidelines in clinical practice are useful if there is low adherence . also important are resources ( time , staffing ) and knowledge essential components in order to manage the organization of a safe transfer process from the icu to a general ward . nurses ' intention to support patients in icu transitional care often is balanced against the organization 's demands , work stress , and time restraint . a recent study showed that nursing care hours per patient in the icu and skill that mix significantly contribute to prevention of bloodstream infections and a shorter length - of - stay in the icu . ( 2011 ) found that caring for an increasing number of patients with complex problems caused stress for nursing staff already facing work overload in wards . qualitative content analysis was used in this study and was well suited since the aim was to describe experiences of intensive care and general ward staff . the results were derived from data from three icus and six general wards and , therefore , other hospitals may have other strategies for icu transitional care that have not been described in this study . the first and second author have their own experiences as rns from both icu and general wards , and their preconceptions have been bridled by thorough data analysis and discussion , by being open as possible to new perspective . this study involved staff with different professions from different hospitals , in order to enhance the variety of experiences . both authors were familiar with chosen method . during the analysis they were both part of the process and discussed how to label the codes , subcategories , and categories until agreement . by presenting the process of the analysis and presenting the result with quotes from the text , so it is possible for the readers to create their own value , important for the study 's credibility . the results of this study conclude that secure , encourage , and collaborate , called the sec - model , illustrates essential strategies suggested to use when organizing the care before , during , and after transfer from the icu to a general ward . the result is in concordance with other researches on meeting the needs of patients and families . a recent review described patients ' and families ' experiences about transfers from icu ; the themes were physical responses , psychological responses , information and communication , safety and security , and the needs of relatives all of which have been addressed in the sec - model developed through this study . the significance of this study is also strengthened by a study by lin et al . ( 2009 ) , who claim that clarification of guidelines and standardization of discharge decision - making and handover are needed . research also indicates that the care of acute ill ward patients is suboptimal which implies that this crucial link needs to be safer . according to massey et al . ( 2008 ) , suboptimal care at the wards depends on failure to seek advice , failure to appreciate clinical urgency , lack of knowledge , failure of the organization , and lack of supervision . to ensure safe and effective care transitions , strategies are needed to improve shared situational awareness , teamwork , patient flow , and resource efficiency in icu transitional care [ 59 , 60 ] . the result indicates that a successful icu transitional process aims to create an interactive , safe , forward - thinking process influenced by actions that preserve patient safety and promote individualized care . to sum it up , a safe transition involves coordination , optimal timing , early mobilization , participation , and a multidiscipline approach . also relatives perceive safety and a continuing care as very important in the icu transitional care process and want to participate . a recommendation for future studies is to explore safe transitions out of the family perspective in a systemic way .

the incidence of age - related musculoskeletal impairment is steadily rising throughout the world . musculoskeletal conditions are closely linked with aging and inflammation . they are leading causes of morbidity and disability in man and beast . aging is a major contributor to musculoskeletal degeneration and the development of osteoarthritis ( oa ) . oa is a degenerative disease that involves structural changes to joint tissues including synovial inflammation , catabolic destruction of articular cartilage and alterations in subchondral bone . cartilage degradation and structural changes in subchondral bone result in the production of fragments of extracellular matrix molecules . some of these biochemical markers or biomarkers can be detected in blood , serum , synovial fluid , and urine and may be useful markers of disease progression . the ability to detect biomarkers of cartilage degradation in body fluids may enable clinicians to diagnose sub - clinical oa as well as determining the course of disease progression . new biomarkers that indicate early responses of the joint cartilage to degeneration will be useful in detecting early , pre - radiographic changes . systems biology is increasingly applied in basic cartilage biology and oa research . proteomic techniques have the potential to improve our understanding of oa physiopathology and its underlying mechanisms . proteomics can also facilitate the discovery of disease - specific biomarkers and help identify new therapeutic targets . proteomic studies of cartilage and other joint tissues may be particularly relevant in diagnostic orthopedics and therapeutic research . this perspective article discusses the relevance and potential of proteomics for studying age - related musculoskeletal diseases such as oa and reviews the contributions of key investigators in the field . background . organizing and performing patient transfers in the continuum of care is part of the work of nurses and other staff of a multiprofessional healthcare team . an understanding of discharge practices is needed in order to ultimate patients ' transfers from high technological intensive care units ( icu ) to general wards . aim . to describe , as experienced by intensive care and general ward staff , what strategies could be used when organizing patient 's care before , during , and after transfer from intensive care . method . interviews of 15 participants were conducted , audio - taped , transcribed verbatim , and analyzed using qualitative content analysis . results . the results showed that the categories secure , encourage , and collaborate are strategies used in the three phases of the icu transitional care process . the main category ; a safe , interactive rehabilitation process , illustrated how all strategies were characterized by an intention to create and maintain safety during the process . a three - way interaction was described : between staff and patient / families , between team members and involved units , and between patient / family and environment . discussion / conclusions . the findings highlight that icu transitional care implies critical care rehabilitation . discharge procedures need to be safe and structured and involve collaboration , encouraging support , optimal timing , early mobilization , and a multidiscipline approach .

microglia are macrophage - like resident immune cells in the central nervous system ( cns ) and possess both neurotoxic and neuroprotective function . microglia accumulate in the lesions of a variety of neurodegenerative disorders , such as alzheimer 's disease ( ad ) , parkinson 's disease , and multiple sclerosis , and are thought to play both toxic and protective functions for neuronal survival . microglia are considered to be a first line defense and respond quickly to various stimuli . when activated , microglia undergo morphological changes to ameboid , proliferate , migrate toward injured areas , and release many soluble factors and phagocytosis of foreign substances or unwanted self - debris . appropriate migration of microglia to damaged area is controlled by chemokines and nucleotide atp [ 2 , 3 ] . phagocytosis seems to be important to prevent the senile plaque expansion in ad by removing amyloid ( a ) deposit . microglia not only engulf the a protein but also phagocytose apoptotic cells and degenerated neuronal debris . phagocytosis of apoptotic or degenerated neuronal debris is crucial to reduce inflammation and maintain healthy neuronal networks . another type of phagocytosis , phagocytosis with inflammation , occurs in chronic inflammatory - related neurodegenerative disorders including alzheimer disease [ 57 ] . degenerated neurons releases several signaling molecules , including nucleotides , cytokines , and chemokines , to recruit microglia and enhance their activities [ 8 , 9 ] . the phenomenon are now termed as find - me , eat - me , and help - me signals . in this paper , we focused on find - me , and eat - me signals from degenerated neurons to microglia . most distinguished and examined eat - me signal is phosphatidylserine ( ps ) , which is a component of cellular membrane and is everted on apoptotic cell membrane [ 10 , 11 ] . nucleotides are also considered as the eat - me signal lately ; microglia expresse various p2x and p2y receptors , nucleotide receptors , which regulate not only chemotaxis but also phagocytosis [ 8 , 12 ] . microglia express many other surface receptors , which have direct interaction with the target to initiate phagocytosis , including ps receptor , lipopolysaccharide ( lps ) receptor cd14 , the scavenger receptor cd36 , the purine receptor p2y6 , and the toll - like receptors ( tlrs ) ( table 1 , figure 1 ) . another surface receptor , the cx3c chemokine fractalkine receptor cx3cr1 , is almost exclusively expressed in microglia throughout the cns , which is involved in progression of neurodegenerative disease by altering microglial activities [ 16 , 17 ] ( figure 2 ) . deletion of cx3cr1 expression in microglia results in progressive neuronal cell death in an animal model of neurodegenerative disease , by inducing microglial dysfunction . it has been identified recently that neurons themselves produce cytokine and chemokine , such as fractalkine . as shown in figure 2 , we previously reported that interleukin-34 ( il-34 ) , a newly discovered cytokine , is produced by neurons , and that its receptor , colony - stimulating factor 1 receptor , is primarily expressed on microglia . fractalkine and il-34 might be important mediator between neurons and microglia , and it is important to clarify this cellular crosstalk signaling pathways for seeking future therapeutic target of neurodegenerative diseases including ad . in the following sections , we will discuss about recent advances of microglial chemotaxis and phagocytosis and their implications for ad therapy . the expression levels were increased under pathological conditions , which seem to facilitate the recruitment and trafficking of glial cells to the damaged area . microglia constitutively express several chemokine receptors ( table 1 ) , which are implicated in the recruitment and accumulation microglia in ad lesions . when exposed to a , microglia are induced to produce several chemokines , such as ccl2 , ccl4 , and cxcl12 . ccr2 , the receptor of ccl2 , deficiency resulted in reduction of microglial accumulation and higher brain a levels in mouse model of ad , which might be mediated via suppression of anti - inflammatory molecule , transforming growth factor ( tgf- ) . however , there is a conflicting report showing increased tgf- signaling in microglia surrounding a plaques in ccr2 knockout in ad model mouse ( appswe / ps1/ccr2/ ) . this ad model has been shown to have accumulation of oligomeric a and memory impairment . ccl2 expression level is also related to another neurodegenerative disorder , multiple sclerosis ( ms ) ; ccl2 level is downregulated in cerebrospinal fluid from ms patients . ccl21 triggers chemotaxis of microglia through cxcr3 , but not ccr7 which implicated in peripheral lymphoid organs . neuronal ccl21 upregulates p2x4 receptor , the nucleotide receptor , expressed in microglia . this cascade is implicated in pathophysiology of tactile allodynia to cause chronic neuropathic pain . a-induced microglial activation is mediated through p2x7 receptor that is reported as proinflammatory response conductive receptor . atp predominantly induced microglial migration among nucleotides through p2y receptors , especially p2y12 [ 2 , 37 ] . following cns injury , expression of p2y12 in microglia drastically reduced after microglial activation , suggesting that p2y12 is a primary and temporary receptor to induce microglial chemotaxis at early stages of the local cns injury . the other nucleotide udp increases mainly microglial phagocytosis ( uptake of microspheres ) via p2y6 receptor . in the condition brain damage by kainic acid administration , the cx3c chemokine cx3cl1 ( fractalkine , also called as neurotactin ) , which has been identified as two forms , soluble or membrane - anchored forms , plays a pivotal role in signaling between degenerating neurons and microglia . cx3cl1 and its receptor cx3cr1 are highly expressed in brain tissue , particularly in neurons and microglia [ 4042 ] . cx3cl1 directly induces various microglial functions including migration , proliferation , inhibition of fas - ligand - induced cell death and glutamate - induced neurotoxicity [ 45 , 46 ] , and inhibition of proinflammatory cytokine production [ 42 , 47 ] . recently , we have shown that soluble form of cx3cl1 also directly enhances microglial clearance of degenerated neuronal debris , which is mediated through phosphatidylserine ( ps ) receptor and production of milk fat globule - egf factor 8 protein ( mfg - e8 ) ( figure 2 ) . the membrane - anchored cx3cl1 is cleaved by several proteases including a disintegrin and metalloprotease ( adam ) family , adam-10 and adam-17 [ 4850 ] , and cathepsin s . when neurons are injured or exposed to glutamate , shedding of cx3cl1 occurs immediately [ 41 , 52 ] . however , little is known about direct connection with a-induced neuronal toxicity and the cx3cl1-shedding . another important function of adam family enzyme , except for cx3cl1 shedding , is an -secretase . it cleaves app in the centre of the a domain , and generated -app is considered to have neurotrophic function [ 5356 ] . cathepsin s is expressed predominantly in microglia and implicated in microglial activation of neuropathic pain . therefore , these cx3cl1-shedding proteases may regulate the microglial phagocytosis directly or indirectly through cx3cl1 expression , and may also play a role on pathogenesis of ad . there are some reports showing that ccl2 activates cx3cr1 expression , and the induction of cx3cr1 expression by ccl2-ccr2 axis is mediated through p38 mapk activation . cx3cr1 deficiency increases susceptibility to neurotoxicity in mouse models of parkinson 's disease , amyotrophic lateral sclerosis , and systemic lps administration . cx3cl1-induced neuroprotection in a rat model of parkinson 's disease has also been reported recently . in addition , there are some reports showing that pathologic features of ad mouse model are worsened by knockout of cx3cr1 [ 17 , 61 ] . the receptor leading to inflammatory status includes cd14 , cd36 , the receptor for advanced glycation end products ( rages ) , and toll - like receptor ( tlr ) 1 , tlr2 , tlr4 , and tlr6 . the receptor leading to anti - inflammatory status includes triggering receptor expressed on myeloid cells 2 ( trem2 ) and ps receptor ( psr ) , mfg - e8 . microglia also express many other phagocytosis - related receptors which are not yet unclear to the correlation of inflammatory status . cr3microglia express classical phagocytosis - related receptor , the m 2 integrin complement - receptor-3 ( cr3 ; mac-1 ; cd11b / cd18 ) , and scavenger - receptor ( sr)-ai / ii ( sr - ai / ii ; cd204 ) . cr3 synergistically activated sr - ai / ii - mediated myelin phagocytosis by microglia [ 63 , 64 ] . cr3 is implicated in clearance of bacteria through induction of major histocompatibility complex class ii ( mhc ii ) antigens . cr3 is involved in endocytosis in normal conditions and mhc ii antigens in an inflammatory state . microglia express sr - ai / ii and sr - bi during neonatal period , while in adult they lack the expression . microglia express classical phagocytosis - related receptor , the m 2 integrin complement - receptor-3 ( cr3 ; mac-1 ; cd11b / cd18 ) , and scavenger - receptor ( sr)-ai / ii ( sr - ai / ii ; cd204 ) . cr3 synergistically activated sr - ai / ii - mediated myelin phagocytosis by microglia [ 63 , 64 ] . cr3 is implicated in clearance of bacteria through induction of major histocompatibility complex class ii ( mhc ii ) antigens . cr3 is involved in endocytosis in normal conditions and mhc ii antigens in an inflammatory state . microglia express sr - ai / ii and sr - bi during neonatal period , while in adult they lack the expression . cd14cd14 is the lps receptor which is also considered as classical phagocytosis - related receptor in macrophage and microglia . cd14-mediated phagocytosis does not require expression of ps receptor and possibly induces inflammatory conditions through activation of cd14 signaling . the authors suggested that it might be due to reduction of insoluble , but not soluble , a . cd14 is the lps receptor which is also considered as classical phagocytosis - related receptor in macrophage and microglia . cd14-mediated phagocytosis does not require expression of ps receptor and possibly induces inflammatory conditions through activation of cd14 signaling . the authors suggested that it might be due to reduction of insoluble , but not soluble , a . fcreceptorexposure of fibrillar a to microglia induces phagocytosis through the receptors distinct from those used by the classical phagocytosis : immunoglobulin receptors ( fcri and fcriii ) or complement receptors . exposure of fibrillar a to microglia induces phagocytosis through the receptors distinct from those used by the classical phagocytosis : immunoglobulin receptors ( fcri and fcriii ) or complement receptors . cd36 and cd47a directly interacted with microglial cell surface receptor complex comprising the class b scavenger receptor cd36 , 6 1 integrin , and integrin - associated protein cd47 , all of them involved in migration and phagocytosis of microglia [ 7072 ] . cd36 is required for fibrillar a-induced chemotaxis and proinflammatory molecules including reactive oxygen species ( ros ) , tnf , il-1 , and several chemokines in microglia . a activates microglial recruitment to amyloid deposition site through cd36-dependent signaling cascade involving the src kinase family members , lyn and fyn , and the erk1/2 . cd47 is a membrane glycoprotein , broadly expressed in the various cell types in the cns , including neurons and microglia . signal regulatory protein- ( sirp ; cd172a ) is a receptor binding to cd47 , which is also expressed on neurons and myeloid cells . sirp is an inhibitory molecule of cd47 that downregulates mapk phosphorylation which is a downstream pathway of cd47 . sirp interacts with the protein tyrosine phosphatases shp-1 and shp-2 , which are predominantly expressed in neurons , dendritic cells , and macrophages . therefore , cd47 functions normally as a marker of self to protect intact body component [ 76 , 77 ] . a directly interacted with microglial cell surface receptor complex comprising the class b scavenger receptor cd36 , 6 1 integrin , and integrin - associated protein cd47 , all of them involved in migration and phagocytosis of microglia [ 7072 ] . cd36 is required for fibrillar a-induced chemotaxis and proinflammatory molecules including reactive oxygen species ( ros ) , tnf , il-1 , and several chemokines in microglia . a activates microglial recruitment to amyloid deposition site through cd36-dependent signaling cascade involving the src kinase family members , lyn and fyn , and the erk1/2 . cd47 is a membrane glycoprotein , broadly expressed in the various cell types in the cns , including neurons and microglia . signal regulatory protein- ( sirp ; cd172a ) is a receptor binding to cd47 , which is also expressed on neurons and myeloid cells . sirp is an inhibitory molecule of cd47 that downregulates mapk phosphorylation which is a downstream pathway of cd47 . sirp interacts with the protein tyrosine phosphatases shp-1 and shp-2 , which are predominantly expressed in neurons , dendritic cells , and macrophages . therefore , cd47 functions normally as a marker of self to protect intact body component [ 76 , 77 ] . ragein has been reported that the direct interaction of a peptide with the receptor for rage is important in ad pathophysiology . in ad brains , rage expression is increased , and rage directly induces neurotoxicity by production of oxidative stressors and indirectly by activating microglia . rage increases macrophage - colony stimulating factor ( m - csf ) from neurons via nuclear - factor-b- ( nf-b- ) dependent pathway and released m - csf induced interaction of cognate receptor c - fms on microglia which prolongs survival of microglia . rage recognizes multiple ligands other than a peptide , such as advanced glycation end products ( ages ) , ps , and high - mobility group box 1 protein ( hmgb1 ) [ 80 , 81 ] . these molecules act as an agonist of rage on microglia , by inducing proinflammatory molecules , such as no , tnf- , il-1 , and il-6 , via map - kinase - kinase ( mek ) and phosphatidylinositol 3-kinase ( pi3k ) pathways . activation of rage leaded to nf-b and mapk - mediated signaling to propagate and perpetuate inflammation status . rage also mediates the transport of peripheral a into the brain across the blood - brain barrier ( bbb ) . in has been reported that the direct interaction of a peptide with the receptor for rage is important in ad pathophysiology . in ad brains , rage expression is increased , and rage directly induces neurotoxicity by production of oxidative stressors and indirectly by activating microglia . rage increases macrophage - colony stimulating factor ( m - csf ) from neurons via nuclear - factor-b- ( nf-b- ) dependent pathway and released m - csf induced interaction of cognate receptor c - fms on microglia which prolongs survival of microglia . rage recognizes multiple ligands other than a peptide , such as advanced glycation end products ( ages ) , ps , and high - mobility group box 1 protein ( hmgb1 ) [ 80 , 81 ] . these molecules act as an agonist of rage on microglia , by inducing proinflammatory molecules , such as no , tnf- , il-1 , and il-6 , via map - kinase - kinase ( mek ) and phosphatidylinositol 3-kinase ( pi3k ) pathways . activation of rage leaded to nf-b and mapk - mediated signaling to propagate and perpetuate inflammation status . rage also mediates the transport of peripheral a into the brain across the blood - brain barrier ( bbb ) . cd200rcd200 is a transmembrane glycoprotein and is expressed on many different cell types including neurons , endothelial cells , lymphocytes , and dendritic cells . the receptor of cd200 , cd200r expression , is predominant in myeloid cells , macrophages , and microglia [ 84 , 85 ] . as in the case of sirp-cd47 , cd200 exerts inhibitory effect on cd200r , so that cd200-cd200r interaction can downregulate activity of microglia . in retina , it has been shown that activation of cd200r in microglia does not show direct effect on migration , but cd200-cd200r signaling restores lps / ifn-induced loss of migration . cd200 knockout leads to an expansion of the myeloid population in several tissues and increased expression of the activation markers in microglia , including the signaling adaptor protein dnax - activating protein of 12 kda ( dap12 ) , cd11b , cd45 , cd68 , and inducible no synthase ( inos ) . these observations suggest that cd200-cd200r signaling leads to anti - inflammatory state and protection against neurotoxic stimuli . cd200 and cd200r expression levels ( neurons and microglia , resp . ) are decreased in ad hippocampus and inferior temporal gyrus , indicating that inhibition of cd200-cd200r axis contributes to ad pathology . cd200 is a transmembrane glycoprotein and is expressed on many different cell types including neurons , endothelial cells , lymphocytes , and dendritic cells . the receptor of cd200 , cd200r expression , is predominant in myeloid cells , macrophages , and microglia [ 84 , 85 ] . as in the case of sirp-cd47 , cd200 exerts inhibitory effect on cd200r , so that cd200-cd200r interaction can downregulate activity of microglia . in retina , it has been shown that activation of cd200r in microglia does not show direct effect on migration , but cd200-cd200r signaling restores lps / ifn-induced loss of migration . cd200 knockout leads to an expansion of the myeloid population in several tissues and increased expression of the activation markers in microglia , including the signaling adaptor protein dnax - activating protein of 12 kda ( dap12 ) , cd11b , cd45 , cd68 , and inducible no synthase ( inos ) . these observations suggest that cd200-cd200r signaling leads to anti - inflammatory state and protection against neurotoxic stimuli . cd200 and cd200r expression levels ( neurons and microglia , resp . ) are decreased in ad hippocampus and inferior temporal gyrus , indicating that inhibition of cd200-cd200r axis contributes to ad pathology . trem2trem2 , a recently identified innate immune receptor , and its adaptor protein dap12 are expressed on microglia and cortical neurons , but not on hippocampal neurons , astrocytes , and oligodendrocytes . their expression correlates with clearance of apoptotic neurons by microglia without inflammation [ 9092 ] . however , endogenous ligand or specific agonist of trem2 had not been identified until recently . heat shock protein 60 ( hsp60 ) is a mitochondrial chaperone , which interacts with trem2 directly . hsp60-induced phagocytosis is only found in microglia which have robust expression of trem2 . in ad model mouse , trem2 expression was highest in the outer zone in a plaques , and the expression level correlated with the size of a plaque . forced expression of trem2 positively regulated microglial phagocytosis , the ability of microglia to stimulate cd4 t - cell proliferation , tnf- and ccl2 production , but not ifn production . trem2 , a recently identified innate immune receptor , and its adaptor protein dap12 are expressed on microglia and cortical neurons , but not on hippocampal neurons , astrocytes , and oligodendrocytes . their expression correlates with clearance of apoptotic neurons by microglia without inflammation [ 9092 ] . however , endogenous ligand or specific agonist of trem2 had not been identified until recently . heat shock protein 60 ( hsp60 ) is a mitochondrial chaperone , which interacts with trem2 directly . hsp60-induced phagocytosis is only found in microglia which have robust expression of trem2 . in ad model mouse , trem2 expression was highest in the outer zone in a plaques , and the expression level correlated with the size of a plaque . forced expression of trem2 positively regulated microglial phagocytosis , the ability of microglia to stimulate cd4 t - cell proliferation , tnf- and ccl2 production , but not ifn production . tlrs are class of pattern - recognition receptors in the innate immune system to induce inflammatory responses . 13 tlrs have been identified in human and mouse to date , except for tlr10 which is expressed only in human [ 95 , 96 ] . cd11b , a marker of macrophages and microglia , has been shown to interact with tlr signaling . cd11b knockout mouse exhibited enhanced tlr - mediated responses and subsequent more susceptibility to endotoxin shock . among the tlrs , lps receptor tlr4 potently activates microglia in various aspects , such as a phagocytosis and proinflammatory molecules production [ 98 , 99 ] . activation of tlr1 , tlr2 , tlr3 , and tlr9 by each selective agonist also increased phagocytosis and several cytokines and chemokine production [ 98 , 100 ] . the response of microglia to fibrillar a is mediated via cd14 , which act together with tlr4 and tlr2 to bind fibrillar a and induced microglial activation through p38 mapk . deficiency of cd200 induces the expression of tlr2 and tlr4 in glial cells and proliferation of cd11b / mhcii / cd40 activated microglia . these mice show memory impairment , suggesting that dual activation of tlr2 and tlr4 may induce an inflammatory phenotype of microglia which negatively regulate synaptic plasticity in the ad model . a triggers the inflammatory status in microglia via heterodimer of tlr4 and tlr6 , which is regulated by cd36 . therefore , cd14-tlr2-tlr4 and cd36-tlr4-tlr6 signaling are crucial to a-induced inflammatory response , and also in microglial phagocytosis . bacterial dna containing motifs of unmethylated cpg dinucleotides ( cpg - dna ) is a ligand of tlr9 , which is initially identified to activate microglia and strongly induces tnf- and il-12 production . however , we have shown that cpg activated microglia to produce neuroprotective molecule , such as hemeoxygenase-1 ( ho-1 ) and matrix metalloproteinase 9 ( mmp-9 ) without producing neurotoxic molecules , such as tnf- , glutamate and nitric oxide ( no ) , and enhanced a clearance to protect memory disturbance in vivo . there are the discrepancies about cpg effect between aforementioned two reports despite using the same origin cells ( mouse primary microglia ) . it may be due to concentrations of this tlr9 ligand used : higher concentration ( 10 m ) in former report , whereas lower concentrations ( 1 to 100 nm ) in latter report . moreover , the latter report revealed the difference on neuroprotective effect of cpg among synthetic oligodeoxynucleotides ( odns ) classes ( a to c ) . class a cpg did not activate microglia , but classes b and c cpgs increased microglial neuroprotective effect through induction of clearance of a and production of neuroprotectant . cpg increased chemokine ccl9 and its receptor ccr1 expression in macrophages and microglia via tlr9/myd88 signaling involving erk , p38 mapk , and pi3k pathways . phagocytotic cells recognize apoptotic cells by several mechanisms , including recognition of ps expressed on the cells . the receptors of ps ( psrs ) had not been clarified for a long time but have uncovered in recent years . these include mfg - e8 ( the lactadherin homolog in humans ) and t - cell immunoglobulin mucin domain 4 ( tim4 ) [ 107 , 108 ] . these act as a bridge between ps - expressing apoptotic cells and psr expressing phagocytes and trigger engulfment of cellular debris . mfg - e8 is expressed on various macrophage subsets in the periphery and on microglia in the cns . recently , we have shown that cx3cl1 induces mfg - e8 expression in primary mouse microglia to lead to the microglial clearance of degenerated neuronal debris . others also reported the induction of mfg - e8 by cx3cl1 in macrophages and rat microglia [ 109 , 110 ] . mfg - e8 bridges ps and integrins v 3 or v 5 on the surface of phagocytes [ 107 , 111 ] . high - mobility group box 1 protein ( hmgb1 ) is an intracellular protein that activates transcriptional factors , including p53 and nf-b . hmgb1 reportedly suppresses the interaction between mfg - e8 and v 3 integrin in macrophage and inhibits the phagocytosis of apoptotic cells through erk - mediated signaling pathway . mfg - e8 may also be involved in a phagocytosis , since its expression is reduced in ad . therefore , mfg - e8 may possibly lead to targeted clearance of unwanted molecules , such as a , without inflammation . the other well - studied psr , tim4 , is expressed in mac-1 cells in various mouse tissues , including spleen , lymph nodes , and fetal liver . among the other tim family members , tim1 , but neither tim2 nor tim3 , also specifically binds to ps . it has been shown recently that rage also recognizes ps and induces apoptotic cell clearance . however as mentioned previously , rage - guided intracellular signaling pathway induces prolonged inflammatory status . -secretase is a protein complex of four essential membrane proteins : aph-1 , pen-2 , nicastrin , and presenilin . a recent study suggests that presenilin increases microglial phagocytosis of a , and this -secretase has dual role for ad pathogenesis : one is cleavage of amyloid precursor protein ( app ) to produce pathologic a , and the other is reduction of microglial phagocytosis of a by -secretase inhibitor . vertebrates have two presenilin genes , psen1 ( located on chromosome 14 in humans ; encoded presenilin 1 ) and psen2 ( located on chromosome 1 ; encoded presenilin 2 ) . there is a report that presenilin 2 is identified as the predominant -secretase in mouse microglia ( but not presenilin 1 ) , which repressed microglial activation via its function as a -secretase , and its expression is increased by inflammatory stimuli ( ifn- ) . in order to explore the detailed mechanism how -secretase regulates microglial activity , further studies are needed since -secretase is a therapeutic target for ad . in traumatic injury of the brain , presenilin and nicastrin expressions -secretase mainly cleaves app to lead to accumulation of a 1 - 42 , which then results in aggregation of a protein to worsen ad pathology . microglia express a wide array of receptors characteristic to immune cell , such as cd molecules , integrins , chemokine receptors , and psr ( figure 1 ) . chemokine receptors not only induce migration of microglia but also contribute directly to ad pathogenesis through regulation of phagocytosis and neuroprotective activity . ps acts as eat - me signal , and psr - mediated phagocytosis so far is regarded as inducing anti - inflammatory responses . however , according to some recent reports , rage interacts with psr and facilitates phagocytosis with robust inflammation status [ 80 , 120 ] . therefore , if the other phagocytosis - related receptors including tlrs interact with psr , microglia would be activated to outbreak excessive phagocytosis with robust inflammation . microglia from old app / ps1 mouse , but not from younger ones , show the reduction of sra , cd36 , rage , and the a-degrading enzymes including insulysin , neprilysin , and mmp9 . thus , it is important to consider the microglial status depending on the disease stage , to treat ad effectively . as shown in figure 2 , fkn and il-34 may be an intrinsic neuroprotectant for damaged but still surviving neurons through activation of microglia . we conducted a qualitative study during september and november 2006 , in a peri - urban community on the outskirts of cape town , south africa . this township emerged during the last two decades because of rapid in - migration and is today a permanent fixture of formal and informal dwellings , where many of the younger generation are born in the township , but still maintain strong bonds to the rural areas of their family origin . generally , most people are living under very poor conditions with high unemployment rates , as well as high levels of crime , alcohol and drug use ( 23 , 24 ) . this qualitative study was undertaken in conjunction with a larger quantitative survey among men at high risk of hiv . the quantitative survey employed respondent - driven sampling ( rds ) to recruit participants ( 25 ) . rds is a chain - referral method that requires a pre - determined number of initial contacts and subsequent recruits to enlist a maximum of three new participants from their social network . the inclusion criteria for the quantitative survey were men older than 18 years , who had had more than one female sexual partner in the past 3 months , where at least one of these sexual partners was younger than 24 years , or 3 or more years younger than the participant . close to 56% had some high school education and 17.2% were unemployed . the majority ( 94.7% ) purposive sampling to identify individuals who were willing to participate in in - depth individual interviews was used among the participants in the quantitative study . twenty participants were selected and asked to participate in the qualitative component while they were waiting to complete the quantitative survey . interviews were conducted during weekends , as it was difficult to attract men to participate during the week . however , screening of the participants before the interviews was necessary to ensure that they were not under the influence of narcotics or alcohol , which is a common problem during weekends in the study setting . each interview took approximately one and a half hours and was conducted by the first author ( ar ) . a trained local interpreter was present during the interviews and translated the conversations into english and/or isixhosa when necessary . all interviews were audiotaped , transcribed verbatim and cross - checked with the initial recordings to ensure the quality of the transcription . participants were given a cellular telephone voucher worth 30 rand ( approximately us$4 ) as a token of appreciation . a thematic question guide ( tqg ) with open - ended questions was used during the interviews . the tqg provided a structure for the interviews while simultaneously allowing the interviewer to freely explore , probe and ask questions that would expand on or clarify particular topics . the tqg themes included sexual behaviours , social and sexual networks , masculinity and risk reduction strategies . the transcripts were analysed according to a latent content analysis suggested by graneheim and lundman ( 26 ) . descriptive , explicit areas of content with little attempt at interpretation were extracted first . these were examined for underlying meaning and situated in sub - themes that cut across categories . sub - themes were then grouped into overarching themes that expressed the latent content of the transcripts . data was discussed in detail by the research team to identify different themes for further analysis and to facilitate the uncovering of aspects of the underlying meaning , which contributed to the increased validity of the analysis . ethical clearance was obtained from the health sciences faculty research ethics committee at the university of cape town . this research was funded by the swedish research council , the swedish international development agency ( sida)/sarec and the national research foundation ( nrf ) , south africa . self - perceived social identities among hard - to - reach men in the study 's informal urban context suggested dominant masculine ideals and ways to legitimise specific behaviours that created a high - risk environment for sexually transmitted hiv . one common category among the study participants described reasons for having multiple , female sexual partners . themes that emerged and that promoted power imbalances in sexual relationships were lack of trust in women , disempowerment and biological determinism . the predominant ideal male identity was conceptualised in one word , the player , characterised by two symbols of status : wealth and women . thus , the player incorporated symbols believed to be important in the make - up of a hegemonic masculinity and clearly helped these men to position themselves in the community . money and material goods that could be visualised was crucial and seemed to override other status symbols such as education or societal position.it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king.you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king . you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . symbols such as cellular telephones , sunglasses and trendy clothes were important in overtly portraying economic status , and were used to position these men in relation to other men in the study context . in this manner , new and fashionable items became important in communication with other men and were key to forming hierarchical systems that incorporated specific forms of ideal masculinities . these symbols of material wealth also played an important role in men 's strategies to access sexual networks and sexual partners . besides displays of overt economic status , a player would be expected to show he could afford and handle several women at the same time . having multiple , often young female sexual partners further enhanced men 's social position and women served as a marker of both sexual and financial power.i can make an example about me because i have about eight girlfriends . it is my style and what i wear , my clothes and my money , because i have money.yes i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . it is my style and what i wear , my clothes and my money , because i have money . i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . seemingly , women responded to these strategies and aided in the socialisation of men 's sexual identities , with new gender power dynamics contrary to many of the previous norms in traditional initiation schools . furthermore , strong peer pressure to have many concurrent , young sexual partners played an important part in the creation of the masculine ideal within male social groups , manifesting in large sexual networks.i will take a lot of pressure from the boys . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all.other people will think that you do not have a game ( if not having multiple girlfriends ) . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all . other people will think that you do not have a game ( if not having multiple girlfriends ) . the pressure to live up to set norms further reinforced the meaning and status of the player . if a man adopted an alternative form of masculine ideal , he would risk being emasculated and thought not to have what it takes to be a real man according to prevalent norms in this specific context and group of men . it would seem that the masculinity norms that have evolved in the study context are making use of new symbols to express power that facilitate the redefinition of men in relation to other men thereby reinforcing risky sexual behaviours . the attributes suggested for a successful man also indicate that the representations of a man , like the player , exist and are integrated into a new urban form of masculinity . to explore forms of identities , underlying and associated reasons on different levels are needed to further explain why certain types of masculinity evolve . two themes closely associated with the player were biological determinism and frustration over a self - perceived disempowerment . the construction of the player not only involved the manifestation of material wealth and multiple girlfriends , but also offered a set of underlying reasons for gender - related power structures . irrespective of whether women had long - term concurrent or temporary sexual encounters , strong and negative views about them were revealed . a woman who crossed the strict boundaries that existed for gender relations was often labelled a bitch ; a term also sometimes used to describe women in general.they get a negative label , they are called bitches . they lose their respect and dignity.they look at them in a funny way . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . they get a negative label , they are called bitches . they lose their respect and dignity . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . by assessing women 's behaviour and what was perceived as unacceptable , men automatically felt that they had certain rights , which further legitimised a negative power play between men and women . applying restrictive social norms to certain female behaviours was not the only way to define men 's masculinity and to promote power imbalances . women were also considered physically vulnerable when engaging in sexual intercourse , especially with several sexual partners . biological determinism legitimised sexual risk taking among men , such as men having a greater need for several sexual partners , and being built for sexual encounters.the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished.it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it.because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished . it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it . because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . these biological explanations further reinforced strong , negative perceptions of women and female sexuality , which helped polarise men 's interpretation of gender constructions . the fact that women often challenged predominant gender stereotypes , by engaging in what was perceived to be normative male sexual behaviour , was used as an excuse for degrading attitudes and behaviours towards women . this could explain the rubbish can metaphor used for the vagina , and men 's association of women as being destroyed or thus , behaviours and the biology of women were themes that helped men to structure relations with women , which also promoted high - risk sexual behaviours in the form of multiple and concurrent sexual relations . another theme that occurred was that of an underlying frustration among these men that also reflected their position in society at large . bitches or women as passive victims , but rather as active agents who strategically played their cards . thus , a certain ambivalence was revealed in men 's views of women and the opposition between sexes.they are players like us because we think we play them and they are playing also their cards , they are playing us also.they ( women ) got so much power now . we got less power . [ ] because the women rights are too much . they are players like us because we think we play them and they are playing also their cards , they are playing us also . we got less power . [ ] because the women rights are too much . this underlying distress and insecurity among these men can be seen as a sign of a situation where the traditional hegemonic masculinity is contested , allowing new forms to evolve to maintain a certain power ( im)balance . the urban context characterised by lack of money and men 's self - perceived disempowerment in relation to women ( and society in general ) , created a situation where manhood was constantly questioned . many of the men believed that women actively engaged in concurrent transactional relationships for economic benefits . this interpretation of women as active agents in relationships created a deeply rooted insecurity among men that alienated them from women . the commonly expressed negative perceptions of women were thus multilayered and reflected an intricate power play that included lack of control and distrust towards women.according to me they can not keep one partner anymore because they do n't trust , that is why they are going around.they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . if i do not give you money they will go to the next person . according to me they can not keep one partner anymore because they do n't trust , that is why they are going around . they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . men 's distrust and a perceived disempowerment in relation to women supported the formation of large sexual networks characterised by unequal power dynamics . sexual relationships within these networks were often based on direct economic reciprocity , which is common in urban and peri - urban townships where people struggle to meet basic needs . the predominant ideal male identity was conceptualised in one word , the player , characterised by two symbols of status : wealth and women . thus , the player incorporated symbols believed to be important in the make - up of a hegemonic masculinity and clearly helped these men to position themselves in the community . money and material goods that could be visualised was crucial and seemed to override other status symbols such as education or societal position.it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king.you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king . you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . symbols such as cellular telephones , sunglasses and trendy clothes were important in overtly portraying economic status , and were used to position these men in relation to other men in the study context . in this manner , new and fashionable items became important in communication with other men and were key to forming hierarchical systems that incorporated specific forms of ideal masculinities . these symbols of material wealth also played an important role in men 's strategies to access sexual networks and sexual partners . besides displays of overt economic status , a player would be expected to show he could afford and handle several women at the same time . having multiple , often young female sexual partners further enhanced men 's social position and women served as a marker of both sexual and financial power.i can make an example about me because i have about eight girlfriends . it is my style and what i wear , my clothes and my money , because i have money.yes i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . it is my style and what i wear , my clothes and my money , because i have money . i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . seemingly , women responded to these strategies and aided in the socialisation of men 's sexual identities , with new gender power dynamics contrary to many of the previous norms in traditional initiation schools . furthermore , strong peer pressure to have many concurrent , young sexual partners played an important part in the creation of the masculine ideal within male social groups , manifesting in large sexual networks.i will take a lot of pressure from the boys . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all.other people will think that you do not have a game ( if not having multiple girlfriends ) . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all . other people will think that you do not have a game ( if not having multiple girlfriends ) . the pressure to live up to set norms further reinforced the meaning and status of the player . if a man adopted an alternative form of masculine ideal , he would risk being emasculated and thought not to have what it takes to be a real man according to prevalent norms in this specific context and group of men . it would seem that the masculinity norms that have evolved in the study context are making use of new symbols to express power that facilitate the redefinition of men in relation to other men thereby reinforcing risky sexual behaviours . the attributes suggested for a successful man also indicate that the representations of a man , like the player , exist and are integrated into a new urban form of masculinity . to explore forms of identities , underlying and associated reasons on different levels are needed to further explain why certain types of masculinity evolve . two themes closely associated with the player were biological determinism and frustration over a self - perceived disempowerment . the construction of the player not only involved the manifestation of material wealth and multiple girlfriends , but also offered a set of underlying reasons for gender - related power structures . irrespective of whether women had long - term concurrent or temporary sexual encounters , strong and negative views about them were revealed . a woman who crossed the strict boundaries that existed for gender relations was often labelled a bitch ; a term also sometimes used to describe women in general.they get a negative label , they are called bitches . they lose their respect and dignity.they look at them in a funny way . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . they get a negative label , they are called bitches . they lose their respect and dignity . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . by assessing women 's behaviour and what was perceived as unacceptable , men automatically felt that they had certain rights , which further legitimised a negative power play between men and women . applying restrictive social norms to certain female behaviours was not the only way to define men 's masculinity and to promote power imbalances . women were also considered physically vulnerable when engaging in sexual intercourse , especially with several sexual partners . biological determinism legitimised sexual risk taking among men , such as men having a greater need for several sexual partners , and being built for sexual encounters.the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished.it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it.because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished . it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it . because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . these biological explanations further reinforced strong , negative perceptions of women and female sexuality , which helped polarise men 's interpretation of gender constructions . the fact that women often challenged predominant gender stereotypes , by engaging in what was perceived to be normative male sexual behaviour , was used as an excuse for degrading attitudes and behaviours towards women . this could explain the rubbish can metaphor used for the vagina , and men 's association of women as being destroyed or thus , behaviours and the biology of women were themes that helped men to structure relations with women , which also promoted high - risk sexual behaviours in the form of multiple and concurrent sexual relations . another theme that occurred was that of an underlying frustration among these men that also reflected their position in society at large . the informants did not view bitches or women as passive victims , but rather as active agents who strategically played their cards . thus , a certain ambivalence was revealed in men 's views of women and the opposition between sexes.they are players like us because we think we play them and they are playing also their cards , they are playing us also.they ( women ) got so much power now . we got less power . [ ] because the women rights are too much . they are players like us because we think we play them and they are playing also their cards , they are playing us also . we got less power . [ ] because the women rights are too much . this underlying distress and insecurity among these men can be seen as a sign of a situation where the traditional hegemonic masculinity is contested , allowing new forms to evolve to maintain a certain power ( im)balance . the urban context characterised by lack of money and men 's self - perceived disempowerment in relation to women ( and society in general ) , created a situation where manhood was constantly questioned . many of the men believed that women actively engaged in concurrent transactional relationships for economic benefits . this interpretation of women as active agents in relationships created a deeply rooted insecurity among men that alienated them from women . the commonly expressed negative perceptions of women were thus multilayered and reflected an intricate power play that included lack of control and distrust towards women.according to me they can not keep one partner anymore because they do n't trust , that is why they are going around.they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . if i do not give you money they will go to the next person . according to me they can not keep one partner anymore because they do n't trust , that is why they are going around . they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . if i do not give you money they will go to the next person . men 's distrust and a perceived disempowerment in relation to women supported the formation of large sexual networks characterised by unequal power dynamics . sexual relationships within these networks were often based on direct economic reciprocity , which is common in urban and peri - urban townships where people struggle to meet basic needs . we found challenges to hiv prevention among men living in an urban south african township that urgently need to be addressed . the dominant masculine ideal , the player , thrived on money , multiple concurrent sexual relations and casual sex . strong social pressure within male core groups to pursue and maintain these concurrent sexual relationships and temporary sexual encounters existed , and helped legitimise specific behaviours that the player represented . the common use of derogatory words attributed to women or their genitals , such as bitch and rubbish can , dehumanised women and restricted female sexuality in order to retain , and in some instances , reclaim male superiority . women were perceived as too empowered and could not be trusted , making men feel alienated and lacking control in ( sexual ) relationships . the lack of trust in women 's fidelity was stated as an important reason for engaging in concurrent sexual relationships as well as casual sexual encounters , which is known to be a key driver of hiv transmission ( 10 ) . our findings thus support previous research showing that dominant masculinities can be characterised by large sexual networks as a means to express manhood and as a response to societal changes , unemployment and poverty , low self - esteem and perceived disempowerment ( 27 ) . the representation of a man and his associated attributes has evolved and been re - shaped into new sets of meanings , where traditional social expectations of conservative , restrained sexuality have largely changed . in this particular context , the ways in which manhood is defined has clearly put men and women at increased risk for sexually transmitted hiv as these gender structures profoundly influence men 's sexual identity , and how sexuality is used to manifest power , not least between men themselves . an urban and modern way of sexual socialisation that incorporates an ideal masculinity as the player , poses a clear risk that needs to be addressed . one potential way in which this risk could be addressed is during traditional initiation rituals that are key to the sexual socialisation of boys into men . the practice continues to be an important part of many young men 's transition to adulthood and should thus be revisited for its potential in integrating gender - related hiv prevention . previous research and designed interventions show weak support for the scaling up of traditional male circumcision as a biomedical intervention ( 28 ) , but that medical circumcision together with traditional initiation could be promising ( 29 ) . in the global debate , the potential benefits of circumcision in relation to hiv infection has largely had a biomedical focus , thereby ignoring the very important core of traditions and the context in which it is stipulated . by taking into account the traditional importance of rites of passage , the power of a successful intervention might not be solely in the removal of the foreskin , but rather in the development of structures in which boys can be sexually and gender socialised into responsible men . the potential in bridging traditional systems with medical interventions has shown promising results and is currently recommended ( 30 ) . however , further research is required to examine the effectiveness of bridging medical and traditional interventions as well as to assess potential harm reduction associated with , for example , circumcision ( 28 ) . traditional structures could be one such entry point as they are important and give meaning to people . this research was based on a selected group of men at high risk of sexually transmitted hiv in one specific urban environment in south africa . self - reported data does not fully explore underlying structures of social norms . however , these in - depth interviews gave the interviewees an opportunity to describe the quality of their social and sexual relationships and shed light on the normative systems that legitimise their behaviours in this peri - urban settlement . these norms represent a masculine ideal supported by males and accepted in society at large . this was a unique study in terms of the high - risk context in which it was conducted , and we believe we managed to reach males who are normally difficult to research , but whose behaviours are key to explaining the extremely high hiv prevalence in south african townships . although findings might only be representative of this group of men , we believe that this unique opportunity to reach men in this harsh urban setting provides important new knowledge on how contemporary masculine ideals affect gender dynamics that need to be addressed in hiv prevention . our results highlight the need to more firmly address sexuality and gender dynamics among men in the growing informal urban areas with strikingly high hiv prevalence in south africa . an understanding of a dominant urban masculinity and how key characteristics of that masculinity affect hiv transmission , as well as more innovative interventions that can help endorse alternative norms and behaviours is urgently needed . traditional and new structures might potentially serve as focal entry points for future preventive actions , where prevention efforts should focus on changing underlying masculine ideals and gender relations that promote and maintain concurrent and temporary sexual relationships as well as other high - risk behaviours . future research should try to explore the potential for using traditional structures in intervention strategies by testing innovative intervention models . the authors have not received any funding or benefits from industry to conduct this study .

microglia are multifunctional immune cells in the central nervous system ( cns ) . in the neurodegenerative diseases such as alzheimer 's disease ( ad ) , accumulation of glial cells , gliosis , occurs in the lesions . the role of accumulated microglia in the pathophysiology of ad is still controversial . when neuronal damage occurs , microglia exert diversified functions , including migration , phagocytosis , and production of various cytokines and chemokines . among these , microglial phagocytosis of unwanted neuronal debris is critical to maintain the healthy neuronal networks . microglia express many surface receptors implicated in phagocytosis . it has been suggested that the lack of microglial phagocytosis worsens pathology of ad and induces memory impairment . the present paper summarizes recent evidences on implication of microglial chemotaxis and phagocytosis in ad pathology and discusses the mechanisms related to chemotaxis toward injured neurons and phagocytosis of unnecessary debris . backgroundthe perspectives of heterosexual males who have large sexual networks comprising concurrent sexual partners and who engage in high - risk sexual behaviours are scarcely documented . yet these perspectives are crucial to understanding the high hiv prevalence in south africa where domestic violence , sexual assault and rape are alarmingly high , suggesting problematic gender dynamics.objectiveto explore the construction of masculinities and men 's perceptions of women and their sexual relationships , among men with large sexual networks and concurrent partners.designthis qualitative study was conducted in conjunction with a larger quantitative survey among men at high risk of hiv , using respondent - driven sampling to recruit participants , where long referral chains allowed us to reach far into social networks . twenty in - depth , open - ended interviews with south african men who had multiple and concurrent sexual partners were conducted . a latent content analysis was used to explore the characteristics and dynamics of social and sexual relationships.resultswe found dominant masculine ideals characterised by overt economic power and multiple sexual partners . reasons for large concurrent sexual networks were the perception that women were too empowered , could not be trusted , and lack of control over women . existing masculine norms encourage concurrent sexual networks , ignoring the high risk of hiv transmission . biological explanations and determinism further reinforced strong and negative perceptions of women and female sexuality , which helped polarise men 's interpretation of gender constructions.conclusionsour results highlight the need to address sexuality and gender dynamics among men in growing , informal urban areas where hiv prevalence is strikingly high . traditional structures that could work as focal entry points should be explored for effective hiv prevention aimed at normative change among hard - to - reach men in high - risk urban and largely informal contexts .

in the previous issue of critical care , chase and coworkers reported on their implementation into clinical practice and evaluation of the specialized relative insulin nutrition table ( sprint ) . this is an improved protocol in the form of a wheel - based system to control blood glucose levels and nutritional intakes in intensive care patients , which was developed a few years ago . blood glucose has become a key biological parameter in critical care since publication of the study conducted by van den berghe and colleagues , who demonstrated decreased mortality in surgical intensive care patients in association with tight glycaemic control ( tgc ) , based on intensive insulin therapy . however , two negative studies were recently reported , which were interrupted early because of high rates of severe hypoglycaemia , namely the visep study and the as yet unpublished glucontrol trial . hence , there is currently much debate regarding the actual benefits of such a strategy in intensive care patients in terms of outcomes . it is also uncertain whether the results of the ongoing multicentre , open label , randomized controlled trial nice sugar of the effects of blood glucose management on 90-day all - cause mortality in a heterogeneous population of intensive care unit ( icu ) patients will resolve remaining concerns about tgc in the icu . included among these concerns is the key issue of what is the most appropriate algorithm to achieve the desired blood glucose range . the major focus of the study conducted by chase and colleagues was on the method to achieve a predetermined blood glucose range by modulating both insulin infusion rate and nutritional inputs . as with the other reported studies comparing protocols , efficacy was evaluated by comparison with historical control patients . however , although the study reported by chase and coworkers was conducted with great care and rigour , it is but another case - control retrospective comparative study . nevertheless , there is a clear need to introduce efficient tools that will help clinicians and nursing staff to control blood glucose levels in icu patients , because hyperglycaemia superior to 10 mmol studies are required to provide clinicians with recommendations on the evaluation and comparison of the various protocols currently in use or that are soon to become available . benchmarking of tgc protocols must take in account all the dimensions of efficiency : performance , risk for severe hypoglycaemia , practical aspects ( ease of use , training time and required materials prior to implementation , error rate ) , integrated continuous monitoring , nursing workload ( evaluated on the mean time between controls ) . furthermore the best way to compare performance is controversial : is it the time with glucose within a common target range , the hyperglycaemia index , the recently described glycaemic penalty index , or the variability that would be associated with outcome ? this raises the question of whether the efficacy results from instructions regarding nutritional intake , allowing insulin infusion rates to be limited to a level lower than usual , or from the intrinsic quality of the algorithm used , which is based on the glucose - insulin regulatory system model ( capturing insulin utilization rate , insulin losses and saturation dynamics ) . also , sprint is apparently associated with few severe hypoglycemia events , which contrasts with the high rate of severe hypoglycaemic episodes reported in the second leuven study . finally , sprint should be relatively simple to implement in numerous icus as a paper - based protocol , presented in an original form using a wheel , without need for computational resources . weaknesses of sprint rest in its inability to monitor parameters related to the quality of glucose control as sprint is a paper - based protocol . most importantly , despite the favourable subjective opinions of care givers , sprint may not reduce workload because it requires measurements every hour or 2 hours . ultimately , evaluation of any tgc protocol must also include an assessment of its ability to be implemented easily and safely in another icu that did not participate in its development . the monocentric study of chase and coworkers may not ensure the ' exportability ' of their tgc protocol . the debate continues about the real benefits of tgc , with numerous questions being asked . what is the optimal target range ? which patients will benefit the most ? when during the icu stay should tgc be applied and to derive which benefits ? which is the best method to control glucose level intensive insulin therapy , and/or limitation of nutritional intakes during acute phase , and/or antidiabetic drugs ? however , the competition to develop the ideal tool with which to control blood glucose levels in the icu and perhaps throughout the hospital stay has begun , involving multidisciplinary teams of physicians and engineers who have specialized in control systems ( feedback control or model predictive control ) . icu = intensive care unit ; sprint = specialized relative insulin nutrition table ; tgc = tight glycaemic control . pk declares that he holds shares of lk2 ( saint - avertin , france ) . foot and mouth disease ( fmd ) is one of the highly contagious diseases of domestic animals , caused by foot - and - mouth disease virus ( fmdv ) , a member of the family picornaviridae , which has a colossal global impact , due to the huge number of animals affected . the fmd endemic countries collectively contain three - quarters of the world 's population ( robinson et al . , 2011 ) , indicating the global economic significance of the disease . at the national level in india , annual total economic loss due to fmd ranges from rs 12,000 crore to rs 14,000 crore ( singh et al . , 2013 ) . in spite of all the control measures taken , fmd continues to be an economically important disease in india due to poor surveillance and inadequate control programs . understanding of the mechanism of foot - and - mouth disease virus ( fmdv ) infection and replication of this virus is important to the control of this worldwide menace ( alexandersen et al . , 2003 , longjam et al . , 2011 ) . a vital question that has yet to be addressed concerns the role of viral receptors in the pathogenesis of fmd . integrins are the biologically important set of proteins used by the cells to bind and respond to the extracellular matrix which belong to a large family of integral membrane receptors that is required for cell adhesion . functionally active integrins consist of two noncovalently bound transmembrane glycoprotein subunits viz , alpha ( ) and beta ( ) ( springer , 2002 ) . evidence suggested that virus binding and infection of the integrin 6 ( itgb6 ) transfected cells are mediated through an rgd - dependent interaction ( baxt and becker , 1990 , jackson et al . , 2000 , mason et al . , 1994 ) . considering the role of host itgb6 receptor gene , the aim of the present study was to screen genetic variation within a snp ( rs136500299 ) at exon 14 region among different indigenous cattle . the polymorphism t / c is located at position 2145 of the reference itgb6 mrna and produces a missense change phe / ser in the position 667 of the polypeptide . previously , this polymorphism has been reported at least ten times : ss250661448 , ss263658018 , ss415630763 , ss420604742 , ss422472086 , ss679762826 , ss752601716 , ss828200981 , ss907724252 , and ss942564461 . in this study , we describe an accurate method on the basis of competitive polymerase chain reaction ( pcr ) so called tetraplex arms pcr , for identification of the snp among different animals . for analyzing the scenario of targeted snp among different zebu cattle , a total of 148 animals of different indigenous breeds of cattle { sahiwal ( 51 ) , kankrej ( 48 ) , ongole ( 38 ) and gir ( 11 ) } from different agricultural zones were used in the study . blood samples were collected from all the animals by jugular vein puncture using sodium heparin ( 10 iu / ml ) as an anticoagulant . immediately after collection , blood samples were stored in a portable refrigerator at 4 c , transported to the laboratory , and stored at 80 c until dna extraction . chloroform extraction method ( sambrook and russell , 2001 ) and the purity of genomic dna was assessed spectrophotometrically . the tetra - primer pcr procedure ( ye et al . , 2001 ) was used for genotyping the snp ( rs136500299 ) at exon 14 region of itgb6 receptor gene . the method employs four primers to amplify a fragment from dna containing the snp and amplicons representing each of the two allelic forms . primers can be designed to amplify fragments of differing sizes for each allele band in order for them to be easily resolved using agarose gel electrophoresis . details of the primer used for the present study and the amplicon size for different genotypes were shown in table 1 . pcr was performed in a total volume of 25 l containing approximately 50 ng dna , 2.5 l of 10x buffer , 2.0 mm mgcl2 , 0.2 mm dntps , 5 pmol of each outer primers , 10 pmol of each inner primers and 1 u of taq polymerase ( sigma aldrich , usa ) . the polymerase chain reaction ( pcr ) protocol was 94 c for 5 min , followed by 35 cycles of 94 c for 30 s , annealing at 55 c for 30 s and 72 c for 30 s , and a final extension at 72 c for 10 min . the pcr products were separated on 1.0% agarose gel ( sigma aldrich , usa ) including 0.5 g / ml of ethidium bromide and photographed under gel documentation system ( alpha imager ep ) . validation of the three different genotypes was analyzed by cloning and sequencing the 433 bp pcr products for each genotype . gene ( allele ) and genotype frequencies of itgb6 receptor gene were calculated by direct counting method . the four populations were tested for hardy weinberg equilibrium using proc allele ( sas inst . cary , nc ) . the chi - square ( ) test was used to find the difference in genotype frequencies in different breeds . the present study was aimed to develop a single tube tetraplex pcr based genotyping assay for snp ( rs13650029 ) at exonic region of bovine itgb6 receptor gene . snp genotyping techniques depend on amplification of the target dna using pcr technique , but differ in the means of discerning between the different alleles , which involves significant post - amplification manipulations . for instance , the restriction fragment length polymorphism typing method based on digestion of amplified pcr products with suitable restriction endonuclease . another extensively used snp typing technique allele specific oligonucleotide ( aso ) melting involves lengthy blotting and hybridization techniques . tetra - primer arms pcr method described in the present context circumvents the limitations of the earlier mentioned techniques . the technique used in the present study involves a single step pcr protocol with two sets of primers to detect the different banding pattern , without the downstream processing like re digestion or hybridization . the primers were designed in such a way to amplify fragments of differing sizes for each allele , in order to resolve differentially in agarose gel electrophoresis . the method described in the present study is a simple , swift and cost - effective method for snp genotyping in large number of individuals ( ye et al . , 2001 ) . correspondence between arms pcr and sequencing confirmed three genotype pattern of the targeted snp at exon 14 region of bovine itgb6 gene among different zebu cattle breeds ( fig . 1 ) . genotyping revealed that , the genotype tt is widely distributed among the targeted zebu cattle breeds . the study revealed that the frequency of t allele was higher in indigenous populations compared to the c allele . this trend was mostly seen in all the breeds except sahiwal where the heterozygotic frequency was comparatively higher , even though the difference is not statistically significant ( table 2 ) . the test for hw equilibrium among different populations showed that all the breeds were under equilibrium with respect to the itgb6 receptor gene indicating the absence of aggressive selection i.e. selection primarily based on this gene ( p > 0.05 ) . the frequency of animals having cc genotype was lowest in all the four breeds . in order to study the variation in the genotype frequencies among different indigenous breeds of cattle , the result showed in table 3 indicates that the genotype frequencies were not statistically different ( = 7.900 , p = 0.2455 ) among the breeds indicating that all the populations are having similar genetic constitution with regard to the itgb6 receptor gene . the snp ( rs136500299 ) t / c located at position 2145 of the reference itgb6 mrna produces a missense change phe / ser . serine is a polar amino acid with its smaller size , whereas phenylalanine is an aromatic amino acid with complex structure . thus it may be presumed that changes of amino acid may alter the conformational changes of the itgb6 receptor coding polypeptide , which however , needs to be confirmed through further studies . the results of the present study suggest that t allele is widely distributed among the indigenous breeds of cattle which can be associated with the resistance to fmd virus , as the susceptibility to fmd virus is lower than the tauras breeds . however , studies with larger sample sets and wide range of cattle breeds are still needed to confirm the exact genetic distribution pattern of the snp .

the report by chase and coworkers in the previous issue of critical care describes the implementation into clinical practice of the specialized relative insulin nutrition table ( sprint ) for tight glycaemic control in critically ill patients . sprint is a simple , wheel - based system that modulates both insulin rate and nutritional inputs . it achieved a better glycaemic control in a severely ill critical cohort than their previous method for glycaemic control in a matched historical cohort . reductions in mortality were also observed . the present study was aimed to screen genetic variation within a snp ( rs136500299 ) located at exon 14 region of bovine itgb6 gene among different zebu cattle breeds . the genotyping method describe in the present study is a tetraplex arms pcr , which offers extremely fast , economical , and simple detection tool . the distribution of the itgb6 genotypes among the different breeds studied suggested that the populations were under hardy weinberg equilibrium . our findings revealed that tt genotypes are widely distributed among different zebu cattle breeds , which can be associated with the resistance to fmd virus , as the bos indicus are more resistant to fmd virus in comparison to bos taurus .

huntington 's disease ( hd ) is a progressive , autosomal dominantly inherited neurodegenerative disorder , characterized by impaired motor control , cognitive decline , and occasional psychiatric illness . hd results from the expansion of a cag repeat in exon 1 of the huntingtin ( htt ) gene , yielding a protein with a polyglutamine ( polyq ) expansion tract near its amino terminus . this polyq - expanded huntingtin protein has a propensity to misfold , making it resistant to proteasomal and autophagy - mediated degradation . earlier age of disease onset and more rapid disease progression correlate with increasing cag repeat length , with hd manifesting in patients carrying 40 or more cag repeats . hd is the most common polyq repeat disease , with a prevalence of at least 1 in 10,000 in the usa and europe . in hd , polyq - expanded huntingtin protein is expressed throughout the brain , with the most severe degeneration occurring in striatal medium spiny neurons and cortical projection neurons that extend to the striatum . currently , hd research is conducted in rodent models , rodent primary neurons , and non - neural human cell lines , but there are questions as to whether such systems yield findings that are truly relevant to the mechanistic basis of human hd pathogenesis . about six years ago , takahashi and yamanaka developed a method for reprogramming human somatic cells so that they entered a pluripotent state , allowing for further differentiation into other cell types , including neurons . this induced pluripotent stem cell ( ipsc ) approach has ushered in a new era of human neurodegenerative disease modeling . two recent publications , one from the hd ipsc consortium and another by an et al . , respectively , reported the generation and characterization of ipsc - derived models for hd and the genetic correction of a disease - causing cag repeat expansion mutation in ipscs from individuals with hd . together , these two studies provide important insights into the utility and limitations of ipsc modeling of neurodegenerative disease . one of the greatest challenges facing the ipsc modeling field is the enormous variability of different ipsc lines and the neurons derived from them . as generation and characterization of ipsc lines can be laborious and costly , one obvious solution to assure the validity of ipsc disease models is to create research teams that work together on a particular disorder , sharing ipsc lines and derived neurons . this very strategy was applied in the hd ipsc consortium study , in which eight different groups produced ipscs from three individuals with hd and three control individuals and used them to assay a wide range of phenotypes . these included gene expression , cell adhesion , bioenergetics , glutamate toxicity , cell death , calcium flux , and trophic factor withdrawal . these phenotypes were assessed in neural stem cells ( nscs ) grown in spherical aggregates under defined growth factor conditions , as well as in differentiated striatal - like neurons obtained using both long and short differentiation protocols , which differed in the growth factors that were added . this comprehensive inventory of cellular and molecular assays revealed that certain phenotypes correlate with hd disease cag repeat length , whereas other phenotypes do not . the common results that were recapitulated across the various laboratories highlight the integrity of the developed ipsc lines , making the hd ipsc consortium study an impressive blueprint that is worthy of emulation . the study by an et al . had quite a different goal : to perform genetic correction in an hd - ipsc line , thereby creating an isogenic revertant ipsc line that would carry two normal - length htt alleles . these ' corrected ' controls had a genetic background that was identical to that of the ' uncorrected ' ipsc line , so any phenotypic or expression differences between the two could be solely attributed to the cag expansion . the ' corrected ' line was created successfully using a standard recombination approach , allowing the comparison of a number of cellular and molecular characteristics between the mutant progenitor line and the normalized derivative line . this study also demonstrated that the genetically corrected hd - ipsc line could be used to produce nscs using a protocol described previously by this group . furthermore , these nscs could be successfully transplanted into the striatum of hd mice , were able to populate this crucial brain region , and then underwent proper differentiation into neurons and glia . thus , the stage is set for the potential future application of this strategy as a therapeutic intervention in hd . in tandem , these papers catalogue a variety of phenotypes that were observed in hd - ipsc - derived neural progenitors and in medium spiny neurons . both groups performed gene expression analysis to compare hd - ipscs and their neuronal derivatives . the hd ipsc consortium findings implicated genes that are involved in cell signaling , cell cycling , axon guidance and neuronal development as dysregulated in nscs ( figure 1 ) . importantly , medium cag repeat expansions and longer cag repeat expansions expressed most of these genes differently . an and co - workers performed transcript expression analysis , although they did this in ipscs rather than in ipsc - derived nscs . they observed that , in comparison with corrected controls , hd - ipscs showed increased expression of genes that are involved in tgf- signaling and decreased expression of genes involved in cadherin signaling ( figure 1 ) . notably , the expression differences between the hd - ipscs and corrected hd - ipscs were of an order of magnitude less than those between non - related control ipscs and hd - ipscs . hd - ipscs , corrected ipscs , and ipsc - derived neurons reveal key disease - associated phenotypes . an et al . also generated corrected hd - ipscs by homologous recombination and showed that , when compared to corrected hd - ipscs , hd - ipscs have increased expression of genes that are induced by the tgf- signaling pathway and decreased expression of cadherin pathway genes . hd - ipsc lines developed by the hd ipsc consortium were further differentiated to form npcs and darpp-32-positive striatal - like neurons . these npcs yielded evidence for the altered expression in hd of genes that are involved in cell signaling , cell cycling , axon guidance , and neurodevelopmental pathways . as delineated here , future studies might compare these two datasets and thus could evaluate the expression of the target genes of those transcription factors known to interact with huntingtin , examine mitochondrial dysfunction in hd and investigate the exact identity of the dying cells in hd . npc , neural progenitor cell ; ros , reactive oxygen species ; tf , transcription factor . comparison of the two studies also highlights the shared relevance of certain cellular and molecular phenotypes , such as cell death and mitochondrial dysfunction ( figure 1 ) . initial studies had previously indicated that hd - ipsc - derived nscs display increased caspase activity , and that both hd - ipscs and derived neurons exhibit increased lysosomal activity . an important finding that is shared by the two groups relates to defects in energy metabolism . mitochondrial dysfunction , resulting in impaired cellular bioenergetics , is an emerging hallmark of hd . the hd ipsc consortium reported decreased intracellular atp levels in hd neural progenitor cells ( npcs ) , and an et al . both of these phenotypes revealed defects in energy metabolism in the hd models , but the importance of mitochondrial dysfunction requires further investigation . another hd - associated phenotype that was observed by both groups is increased cell death . the hd ipsc consortium used a variety of assays , including longitudinal survival tracking , nuclear condensation assays , and caspase 3/7 activation , to detect increased cell death in hd - nscs and striatal - like derivatives . an and colleagues also observed cag repeat - length - dependent cell death in similar assays . both groups demonstrated a more severe cell death phenotype upon withdrawal of brain - derived neurotrophic factor ( bdnf ) , adding to the mounting evidence to support a role for bdnf - associated toxicity in hd . bdnf is a secreted neurotrophin , which undergoes transcriptional repression in certain hd models , and there are indications that over - expression of bdnf can ameliorate the hd phenotype . cell death in the striatum and cortex are hallmarks of hd ; hence , this shared observation is significant , as it recapitulates in vitro what is seen in vivo . an et al . have taken an initial step toward the application of corrected hd - ipsc - derived neurons in cell replacement therapy . they showed that differentiated striatal - like neurons can survive transplantation into mouse brain , but they did not report whether transplanted r6/2 mice exhibited any recovery in phenotype . despite this uncertainty , it is quite clear that the newly derived hd - ipsc models hold great potential for use in drug screening . as illustrated in figure 1 , future studies should examine effects on htt interactor target genes , and compare the results so obtained with the data sets from these two studies . the observations from both studies regarding gene expression and mitochondrial dysfunction support findings generated in other models . the huntingtin protein is known to interact with a number of transcription factors and co - activators , including creb - binding protein ( cbp ) , sp1 , tafii130 , and ppar co - activator 1 ( pgc-1 ) . follow - up analysis of the expression of the target genes that encode these already - implicated transcription factors might be worthwhile ( figure 1 ) . diminished pgc-1 function appears to be a major contributor to hd pathogenesis , so the hd - ipsc model could be used to evaluate the mitochondrial phenotypes seen in hd mice , including decreased mitochondrial complex activities , decreased mitochondrial number , and increased oxidative stress . pgc-1 overexpression can rescue hd - associated phenotypes in mice by inducing transcription factor eb ( tfeb ) , a master regulator of autophagy . validation of the role of pgc-1 and tfeb dysfunction in hd - ipsc models would be worthwhile and could facilitate the development of new therapies . finally , a missing piece of the puzzle is the exact identity of the dying cells in hd . the hd ipsc consortium used a method for tracking live cells through longitudinal studies , which allowed them to identify the dying cells as being morphologically similar to neurons , although the specific neural type was unspecified . as hd patients with longer cag repeats suffer more widespread neuron cell death , it will be important to determine if the vulnerability of ipsc - derived neurons differs between neural cell types ( figure 1 ) . amelioration of cell death in a relevant ipsc - derived neural lineage could be a useful indicator of potential drug efficacy . once the hd field homes in on viable lead compounds , hd - ipsc models could serve as invaluable tools for validation and should accelerate therapeutic development . bdnf : brain - derived neurotrophic factor ; hd : huntington 's disease ; htt : huntingtin gene ; ipsc : induced pluripotent stem cell ; nsc : neural stem cell . the authors acknowledge funding support from the nih ( r01 ns065874 ) for their huntington 's disease research . in early december 2013 , during the zikv outbreak , a 44-year - old man in tahiti had symptoms of zikv infection : asthenia , low grade fever ( temperature from 37.5c to 38c ) and arthralgia . eight weeks later , he described a second episode of symptoms compatible with zikv infection : temperature from 37.5c to 38c , headache on days 13 , and wrist arthralgia on days 57 . the patient did not seek treatment , thus biological samples were not collected during the first 2 periods of illness . the patient fully recovered from the second episode , but 2 weeks later he noted signs of hematospermia and sought treatment . because the patient had experienced symptoms of zikv infection some weeks before , he was referred to our laboratory in the institut louis malard , papeete , tahiti for zikv infection diagnostic testing . the medical questionnaire revealed no signs of urinary tract infection , prostatitis , urethritis , or cystitis , and the patient stated that he did not had any recent physical contact with persons who had acute zikv infection . we extracted rna using the nuclisens easymag system ( biomrieux , marcy letoile , france ) from 200 l of blood and from 500 l of semen and urine ; both were eluted by 50 l of elution buffer . we tested blood and semen rna extracts using real - time reverse transcription pcr ( rrt - pcr ) as described using 2 primers / probe amplification sets specific for zikv ( 3 ) . the rrt - pcr results were positive for zikv in semen and negative in blood , and confirmed by sequencing of the genomic position 8581138 encompassing the prm / e protein coding regions of zikv . the generated sequence ( genbank accession no . km014700 ) was identical to those previously reported at the beginning of the zikv outbreak ( 3 ) . three days later , we collected a urine sample , then a second set of blood and semen samples . semen and urine from this second collection were not found to contain traces of blood by both direct and macroscopic examinations . rrt - pcr detected zikv rna in the semen and urine , but not in the blood sample . we quantified zikv rna loads using an rna synthetic transcript standard that covers the region targeted by the 2 primers / probe sets . rna loads were : 2.9 10 copies / ml and 1.1 10 copies / ml in the first and second semen samples , respectively , and 3.8 10 copies / ml in the urine sample . we cultured semen and urine as described for dengue virus cultured from urine ( 6 ) . briefly , 200 l of each sample diluted in 200 l of 1% fetal calf serum ( fcs ) minimum essential medium ( mem ) were inoculated onto vero cells and incubated for 1 h at 37c ; inoculum was then removed and replaced by 1 ml of culture medium . we also inoculated a negative control ( 200 l of 1% fcs - mem ) and a positive control ( 5 l of a zikv - positive serum diluted in 200 l of 1% fcs - mem ) . the presence of zikv in the culture fluids was detected by rrt - pcr as described . replicative zikv particles were found in the 2 semen samples but none were detected in the urine sample . ( 7 ) , but perinatal transmission ( 8) and potential risk for transfusion - transmitted zikv infections has also been demonstrated ( 9 ) . ( 10 ) , who described a patient who was infected with zikv in southeastern senegal in 2008 . after returning to his home in colorado , united states , he experienced common symptoms of zikv infection and symptoms of prostatitis . four days later , he observed signs of hematospermia , and on the same day , his wife had symptoms of zikv infection . because the wife of the patient had not traveled out of the united states during the previous year and had sexual intercourse with him 1 day after he returned home , transmission by semen was suggested . zikv infection of the patient and his wife was confirmed by serologic testing , but the presence of zikv in the semen of the patient was not investigated . infectious organisms , especially sexually transmitted microorganisms including viruses ( human papillomavirus or herpes simplex virus ) , are known to be etiologic agents of hematospermia ( 11 ) . to our knowledge , before the report of foy et al . ( 10 ) and this study , arbovirus infections in humans had not been reported to be associated with hematospermia , and no arboviruses had been isolated from human semen . we detected a high zikv rna load and replicative zikv in semen samples , but zikv remained undetectable by rrt - pcr in the blood sample collected at the same time . these results suggest that viral replication may have occurred in the genital tract , but we do not know when this replication started and how long it lasted . the fact that the patient had no common symptoms of zikv acute infection concomitantly to hematospermia suggests that the viremic phase occurred upstream , probably during the first or second episode of mild fever , headache , and arthralgia . the detection of zikv in both urine and semen is consistent with the results obtained in a study of effects of japanese encephalitis virus , another flavivirus , on boars . the virus was isolated from urine and semen of experimentally infected animals , and viremia developed in female boars that artificially inseminated with the infectious semen ( 12 ) . flaviviruses have also been detected in urine of persons infected with west nile virus ( wnv ) . wnv rna was detected in urine for a longer time and with higher rna load than in plasma ( 13 ) . wnv antigens were detected in renal tubular epithelial cells , vascular endothelial cells , and macrophages of kidneys from infected hamsters ( 14 ) , suggesting that persistent shedding of wnv in urine was caused by viral replication in renal tissue . dengue virus ( denv ) rna and denv nonstructural protein 1 antigen were also detected in urine samples for a longer time than in blood , but infectious denv has not been isolated in culture . concluded that the detection of denv by rrt - pcr was useful to confirm denv infections after the viremic phase ( 6 ) . also , yellow fever virus rna was isolated from the urine of vaccinated persons ( 15 ) , and saint louis encephalitis viral antigens , but not infective virus , have been detected in urine samples from infected patients ( 10 ) . furthermore , the observation that zikv rna was detectable in urine after viremia clearance in blood suggests that , as found for denv and wnv infections , urine samples can yield evidence ofzikv for late diagnosis , but more investigation is needed .

deconstructing the mechanistic basis of neurodegenerative disorders , such as huntington 's disease ( hd ) , has been a particularly challenging undertaking , relying mostly on post - mortem tissue samples , non - neural cell lines from affected individuals , and model organisms . two articles recently published in cell stem cell report first the generation and characterization of induced pluripotent stem cell ( ipsc)-derived models for hd , and second , the genetic correction of a disease - causing cag expansion mutation in ipscs from individuals with hd . taken together , these two studies provide a framework for the production and validation of ipsc materials for human neurodegenerative disease research and yield crucial tools for investigating future therapies . in december 2013 , during a zika virus ( zikv ) outbreak in french polynesia , a patient in tahiti sought treatment for hematospermia , and zikv was isolated from his semen . zikv transmission by sexual intercourse has been previously suspected . this observation supports the possibility that zikv could be transmitted sexually .

irritable bowel syndrome ( ibs ) is a common gastrointestinal disorder affecting approximately 1015% of the people in western world . the patient group is very heterogeneous and characterized by abdominal pain , discomfort and abnormal bowel habits . the pathophysiology of ibs is unknown , it would appear that the due to the heterogeneous patient material there are multiple explanations for the symptom development . currently , patients are subdivided according to their main symptom that have been proposed in the so called rome criteria ; ibs with diarrhea ( ibs - d ) , ibs with constipation ( ibs - c ) or ibs with mixed bowel habits ( ibs - m ) . no single unifying cause for ibs has been reported but several factors have been associated with the disease etiology , including genetic predisposition , anxiety and depression as well as female gender . moreover , there is strong evidence indicating that changes in the intestinal microbiota and their function could be one of the contributing factors in the development of ibs . several studies have reported differences in the intestinal microbiota composition of ibs patients and healthy controls , however the results show little overlap from study to study . this is likely caused by different methodological approaches in the analysis steps as well as the heterogeneity of the ibs patients . regardless of the lack of coherent changes in the microbial composition , there is clear evidence for the role of the microbiota in the pathogenesis of ibs . for one , the patients report improved symptoms after consuming agents targeted to modify the intestinal microbiota , including probiotics , antibiotics or changing diet , such as avoidance of fodmap carbohydrates . moreover , the strongest predictor for ibs is a prior gastroenteritic episode , which will increase the risk of developing ibs by 7-fold . it has been estimated that about 10% of the ibs cases begin after an episode of gastroenteritis , causing post - infectious ( pi ) ibs , indicating a cause and effect relationship . this is in line with a previously proposed model in which an event , such as an infection , affects the intestinal microbiota and generates an alternative stable state . recently , a number of bistable microbial groups , i.e. microbes with two stable homeostatic states , have been identified that could act as tipping points in this well known ecological process that is associated with resilience . in our recent study by jalanka and colleagues the differences in the microbial composition of pi - ibs patients were addressed by analyzing the faecal microbiota of the study subjects with a previously validated and benchmarked phylogenetic microarray , detecting the abundance of over 1000 bacterial phylotypes found in the human intestine . the studied cohort consisted of 57 british subjects divided into 5 study groups ; patients with a pi - ibs diagnosis ( group 1 , rome ii ) , patients whom 6 months after the gastroenteritic episode had a persisting bowel dysfunction , which however did not qualify for ibs ( pi - bd ) ( group 2 ) or did not experience any bowel dysfunction anymore ( pi - nonbd ) ( group 3 ) . the results were benchmarked against ibs - d patients ( group 4 , rome ii ) and healthy controls ( group 5 ) . our aim was to determine the microbial compositional differences and to address associations between the faecal microbiota and the clinical features of ibs from patients with a varying degree of symptoms . as the development of ibs is multifactorial , we hypothesized that the observed symptoms and development of pi - ibs may arise from the interplay between the faecal microbiota , the host immune response , and psychological factors . therefore , we analyzed in parallel the detailed records of the participants ' psychological well - being , intestinal symptoms , systemic immunological markers as well as the levels of host gene expression on rectal mucosa . this integrated approach , addressing the associations between clinical phenotype and the faecal microbiota allowed us to address the complex relationships between the host and microbiota in the pathogenesis of ibs and pi - ibs . since the intestinal microbiota is very heterogeneous and subject - specific , we hypothesized that instead of limiting the comparative microbiota analysis to individual taxa , it would be more relevant to search for a microbial signature that potentially differs between the patients and healthy controls . by using a novel bioinformatic approach ( bootstrap aggregated rda method ; baggedrda ) we identified a combination of 27 genus - like bacterial groups that significantly separated healthy controls and patients with ibs symptoms . moreover , we found that the microbial profile of the pi - ibs and pi - bd groups resembled those suffering from ibs - d . the major difference in the patients ' microbiota was the 12-fold increase of bacteroidetes phylum and the 35-fold decrease of uncultured clostridiales belonging to the firmicutes phylum . furthermore , when all participants were ranked according to the abundance of the discriminating bacterial taxa , a specific ordering was obtained , the so - called index of microbial dysbiosis ( imd ) . this rank ordering reflected the patient 's health status as it correlated with the increase of several immunological markers and intestinal complaints , where as no such associations were observed when subdividing the patients according to their clinical status . interestingly , there were no significant correlations between the imd and any of the psychological symptoms often associated with ibs . our results corroborate the previous reports that the microbial composition could be used to further stratify ibs patients and thus complement the clinical subgroupings . in a recent study it was estimated that circa 33% of the ibs patients exhibit anxiety and 13% suffer from depression . in addition , it was found that somatisation was significantly increased in the ibs patients compared to controls with gastrointestinal symptoms but without ibs . although the intestinal microbiota of these patients was not studied , in the light of the recent evidence it could be hypothesized that the etiology of the disease in the patients with psychological symptoms differs from those with microbial aberrations . taken together , these results suggest that the discriminant microbial profile could be used as an objective measure of disturbed bowel functions and therefore a potential novel way of stratifying this heterogeneous patient material in future studies and in clinical practise . to verify our findings in a larger patient material we combined the microbiota data form the british subjects with that of a swedish pi - ibs cohort reported recently by sundin and colleagues . both studies were conducted with an identical analysis pipeline , including the well established mechanical lysis for extraction of faecal dna , the hitchip phylogenetic microarray platform , and the normalization and analysis software suite . the combined data aimed to determine whether the pi - ibs patients from 2 countries share similarities in their microbiota compared to healthy controls . the swedish cohort introduced new subjects to both healthy controls ( n=16 ) and pi - ibs patients ( n=13 , rome iii ) therefore doubling the sample size to a total of 51 subjects . remarkably , the significant difference in the microbiota profiles between the healthy and pi - ibs patients was replicated ( p < 0.05 , fig . 1 ) . the strongest difference was observed with the significant increase in the bacteroides ssp . and decrease of members of the uncultured clostridiales in the pi - ibs patients . interestingly , both bacteroides fragilis and uncultured clostridiales have been identified as bistable microbial groups and predicted to act as tipping points of the gut ecosystem in opposite ways . however , not all of the imd taxa reported in the original study appeared as significantly different between the patients and controls , and there were new taxa that were significantly increased in the combined pi - ibs patient group . one of them was dialister , which was recently identified as bistable i.e. , being either abundant or nearly absent in contrast to most other bacteria that show gradual abundance distribution . this highlights the need of conducting the future research in large cohorts in order to verify the separating species between pi - ibs and healthy controls . figure 1.intestinal microbiota of pi - ibs patients is significantly different form that of healthy controls ( hc ) . the difference was measured with baggedrda that was used to identify a microbial signature that separates hc from pi - ibs patients from the two study cohorts . abundance of the discriminating taxa creates the so - called index of microbial dysbiosis ( imd ) . intestinal microbiota of pi - ibs patients is significantly different form that of healthy controls ( hc ) . the difference was measured with baggedrda that was used to identify a microbial signature that separates hc from pi - ibs patients from the two study cohorts . abundance of the discriminating taxa creates the so - called index of microbial dysbiosis ( imd ) . the role of bacteroides spp . in the pathophysiology of ibs is controversial , probably reflecting the heterogeneity of the patients as well as the genus bacteroides . previous studies have reported opposing results regarding the relevance of bacteroides genera in ibs , as there is evidence for both increased as well as decreased abundance in the patients . interestingly , elevated levels of bacteroides spp were among the microbiota changes that have been associated with susceptibility to enteric pathogens in mice . it has been suggested that susceptibility to infections could increase due to a imbalanced microbial composition , since complex interactions between the microbiota and host immune system might reduce the resistance to episodes of gastroenteritis . recently , similar observations were made in humans by dicksved and colleagues who followed abattoir workers who have a high risk of contracting c. jejuni infection . the participants donated faecal samples at the start of employment and were then regularly followed . the subjects who became c. jejuni infected had increased levels of bacteroides species prior the infection , as opposed to those individuals who did not contract the disease during the follow - up period . similar to these findings , it was recently shown that the subjects contracting a gastroenteritis had increased levels of bacteroides spp . and decreased levels of firmicutes immediately after the infection . although the infectious agents in this study were diverse , the differences in the microbiota profile were similar to what we found . altogether , these data from several studies allow to hypothesize that the subjects with a microbial profile resembling the imd could be more susceptible for contracting a gastrointestinal infection and later developing pi - ibs . more studies on patients before and after gastroenteritis are required to verify this hypothesis . moreover recent experiments in mice have indicated that innate and mucosal immune system play a deterministic role in shaping the gut microbiota . hence , the observed imd may be a reflection of the activity of the host 's immune system . while this alternative explanation may offer new insight in the etiology of ibs , it confirms the significance and application potential of imd in stratifying ibs subjects . to further analyze the crosstalk between the host and the microbiota , we concentrated on the 27 bacterial taxa of imd and separately studied the associations between bacterial abundance and the host gene expression in mucosal surface of rectum , the last part of large intestine . we mapped the microarray - derived gene expression data from rectal biopsies to known biological functions using gene set analysis ( gsa ) . there were numerous statistically significant associations between the abundance of the ibs - type microbiota and the level of expressed host genes that suggested an association between the discriminant microbiota and the physical barrier integrity of the host . the most prominent negative correlation was observed between 7 bacteroides ssp and the expression of glycine , serine and threonine metabolism pathway . in general , glycine , serine and threonine are known to be important in the maintenance of gut integrity and the structure of the mucin layer . for example , the majority of the dietary threonine is utilized for synthesising the secretory mucin and its dietary restriction results into impaired gut barrier . moreover , the mucus structure and the extent of its glycosylation shape the microbiota composition and similar compositional changes have also been previously associated with barrier function abnormalities . fucosyltransferase-2 is involved in the formation of the abo - blood groups and its activity also creates adhesion receptor for several microbes . therefore , it is interesting that fut2 deficient mice ( fut2 ) have been shown to display similar differences in their microbiota composition to what was found in our study , i.e. increased amounts of several bacteroides species and decreased levels of clostridiales . in addition to alterations in the mucosal barrier genes , we identified several other pathways , including those that regulate cell junctions and inflammatory responses , to be associated with the imd bacterial taxa . the results led us to hypothesize that both the physical and immunological gut barrier of subjects with an ibs - type microbiota has been compromised resulting in a low - grade inflammation , a feature of some ibs patients . changes in the inflammatory response , barrier function and microbiota composition could therefore lead into changes in the other host parameters such as gut - brain axis receiving signals from these systems . moreover , we observed that the imd correlated with the ibs symptoms and clinical features whereas the scores of anxiety and depression did not correlate with the imd , indicating that patients with a more psychological basis for the disease have a rather undisturbed microbiota . this is in line with previous findings and of great significance as it allows proposing a model in which some subjects with a relatively healthy microbiota report ibs symptoms while others with a microbiota resembling that of the ibs patients have no complaints as they have compensating factors that overcome the microbial component and hence are scored healthy ( fig . 2 ) . the pi - ibs patients can be stratified according to the imd ( or more generally the microbial composition ) to those with ibs - like microbiota ( red ) and those with a composition resembling healthy individuals ( turquoise ) . objective measurements such as low - grade inflammation and altered gene expression correlated with the imd . psychological aspects , such as depression or anxiety do not correlate with the imd and these patients , though diagnosed with ibs , show a healthy - like microbiota profile . the correlations between host response and microbiota aberration identified in our study are indicated in bold . proposed model on how microbial aberrations could explain the disease etiology in ibs . the pi - ibs patients can be stratified according to the imd ( or more generally the microbial composition ) to those with ibs - like microbiota ( red ) and those with a composition resembling healthy individuals ( turquoise ) . objective measurements such as low - grade inflammation and altered gene expression correlated with the imd . psychological aspects , such as depression or anxiety do not correlate with the imd and these patients , though diagnosed with ibs , show a healthy - like microbiota profile . the correlations between host response and microbiota aberration identified in our study are indicated in bold . ibs has a heavy impact on the health care system and currently the patient diagnosis is based on subjective symptoms and exclusion of other gastrointestinal diseases . results from our study as well as from others suggest that diagnosis based on symptoms alone is not optimal and should be accompanied with objective markers , such as the microbiota composition . we introduced an index of microbial dysbiosis ( imd ) that can potentially be used to objectively stratify patients into smaller groups where the patients with psychological basis of the syndrome could be separated from those with a peripheral gut abnormality . by using a relatively small but carefully phenotyped cohort we identified several associations between the imd taxa , hosts ' gene expression and clinical markers suggesting that impairment of intestinal barrier may underlie both immunological and microbiological deviations often associated with pi - ibs . here we show that the microbial signatures that were observed in our british pi - ibs cohort were reproduced in an independent swedish pi - ibs cohort ( fig . 1 ) . this testifies for the robustness of these signatures in different cohorts and countries as well of the used microbial analysis pipeline . the main observed microbial aberration included an increased level of bacteroides spp and decreased level of uncultured clostridiales , both recently identified as contrasting tipping point taxa that reflect alternative stable states of the gut ecosystem . recently , similar bacterial signatures have been also detected in subjects prior to gastroenteritis , suggesting that perhaps the microbial composition already at the time of the initial infection predisposes certain subjects for pi - ibs . this is of significance as it allows not only to diagnose these subjects but also may provide basis for treatment . dietary corrections represent the major approach in managing the ibs symptoms and it is expected that their efficacy depends on the individual 's gut microbiota as microbial activity on food metabolism directly or indirectly contributes to many ibs symptoms . this hypothesis is supported by a pilot study among pediatric ibs patients in us . of note , those children whose symptoms decreased during the test diet had lower abundance of bacteroides and dialister spp . that we found to typify the pi - ibs microbiota , further stressing the relevance of these organisms in ibs . we believe that the proposed model ( fig . 2 ) built on microbiota - based patient stratification will not only give insight into the observed microbiota heterogeneity among ibs patients as well as its partial overlap with healthy controls , but also represents a concrete step toward improved diagnostics and novel treatment options with improved efficacy . we are grateful to our collaborators , notably prof robin spiller md and prof robert jan brummer md , for helpful discussions . this work was partly supported by the finland academy of sciences ( grants 137389 , 141140 and 1272870 ) and the unrestricted spinoza of the netherlands organization for scientific research . intracranial dural arteriovenous fistula ( davf ) is abnormal arteriovenous shunts that occur within the leaflets of the dura mater and comprised 10 - 15% of intracranial arteriovenous malformation8 ) . in a meta - analysis of 377 patients with davf reported before 1989 , tentorial davf was only 8% ( 32/ 377 ) of all intracranial davf , but tentorial davf had the most aggressive neurological behavior , with 97% ( 31/32 ) causing hemorrhage or progressive focal neurological deficits110 ) . unlike the less aggressive davfs of the lateral and cavernous sinuses that drain into venous sinuses , tentorial davf drains into pial veins11 ) . ct and mr imaging revealed an acute hemorrhage at left tectum of mid brain and fourth ventricle ( fig . cerebral angiography showed a davf around torcular fed by meningeal branch of ascending pharyngeal artery , transosseus branch of the right occipital artery and drained into cortical vein of left occiput and basal vein of rosenthal ( fig . an intravenous bolus of 3000 iu of heparin was administered after placement of a 6-fr guiding sheath in the right femoral artery . a 6-fr guiding catheter ( envoy , cordis endovascular , miami lakes , fl , usa ) was placed at external carotid artery . we planned to superselective catheterization of meningeal branch of ascending pharyngeal artery but failed due to tortous curve at cranial base . we could access to fistula point through transosseus branch of occipital artery using microcatheter ( apollo , ev3 inc . , the volume of 0.7 ml onyx-18 ( ethyl vinyl alcohol , ev3 inc . , irvine , post - embolization angiogram demonstrated onyx occupying the retrograde drainage of cortical vein and obliteration of davf ( fig . the patient was discharged 7 days later and his postoperative course was uneventful . a 74-year - old man presented with progressive headache and dementia symptom for several months . seven years ago , he had spontaneous hemorrhage in 4th ventricular and been treated for davf by transarterial embolization using n - butyl cyanoacrylate ( nbca ) . both middle meningeal arteries ( using 33 and 50% glue ) and transosseus branch of left occipital artery ( using 20% glue ) were selected for embolization . although arteriovenous shunting flow was not completely abolished , retrograde cortical flow was remarkably decreased . he refused further treatment and did not seek hospital since then . by this time of visit , he has been well . ct and mr image showed a prominent , tortous vascular structure around medial tentorium and multiple fine vessels engorgements at both thalamic and putamen areas ( fig . angiogram demonstrated recanalization and recruitment from both middle meningeal arteries and posterior choroidal arterial supply from vertebral artery . the retrograde leptomeningeal drain of infratentorial vermian vein showed dilated and saccular change ( fig . we embolized davf only through the right middle meningeal artery and inject onyx-18 1.0 mg as described above . korea mini - mental state examination ( k - mmse ) score was improved from 14 to 28 . ct and mr imaging revealed an acute hemorrhage at left tectum of mid brain and fourth ventricle ( fig . cerebral angiography showed a davf around torcular fed by meningeal branch of ascending pharyngeal artery , transosseus branch of the right occipital artery and drained into cortical vein of left occiput and basal vein of rosenthal ( fig . an intravenous bolus of 3000 iu of heparin was administered after placement of a 6-fr guiding sheath in the right femoral artery . a 6-fr guiding catheter ( envoy , cordis endovascular , miami lakes , fl , usa ) was placed at external carotid artery . we planned to superselective catheterization of meningeal branch of ascending pharyngeal artery but failed due to tortous curve at cranial base . we could access to fistula point through transosseus branch of occipital artery using microcatheter ( apollo , ev3 inc . , the volume of 0.7 ml onyx-18 ( ethyl vinyl alcohol , ev3 inc . , irvine , post - embolization angiogram demonstrated onyx occupying the retrograde drainage of cortical vein and obliteration of davf ( fig . a 74-year - old man presented with progressive headache and dementia symptom for several months . seven years ago , he had spontaneous hemorrhage in 4th ventricular and been treated for davf by transarterial embolization using n - butyl cyanoacrylate ( nbca ) . both middle meningeal arteries ( using 33 and 50% glue ) and transosseus branch of left occipital artery ( using 20% glue ) were selected for embolization . although arteriovenous shunting flow was not completely abolished , retrograde cortical flow was remarkably decreased . he refused further treatment and did not seek hospital since then . by this time of visit , he has been well . ct and mr image showed a prominent , tortous vascular structure around medial tentorium and multiple fine vessels engorgements at both thalamic and putamen areas ( fig . angiogram demonstrated recanalization and recruitment from both middle meningeal arteries and posterior choroidal arterial supply from vertebral artery . the retrograde leptomeningeal drain of infratentorial vermian vein showed dilated and saccular change ( fig . we embolized davf only through the right middle meningeal artery and inject onyx-18 1.0 mg as described above . korea mini - mental state examination ( k - mmse ) score was improved from 14 to 28 . davf associated with retrograde leptomeningeal venous drainage exhibit a high incidence of aggressive behavior3912 ) and those with only leptomeningeal venous drainage was classified type iii by borden et al.2 ) . in tentorial davf patients , 22/22 cases ( 100%)14 ) , 26/31 cases ( 84%)10 ) and 13/14 cases ( 93%)6 ) were classified borden type iii and needed prompt treatment . the tentorial davf is a rare type of dvaf and should be treated even though without remarkable clinical symptoms because natural history is very disappointing . to our knowledge , tentorial davf is located in the tentorial dura mater and fed primarily from branches of the meningohypophyseal trunk , middle meningeal arteries , and occipital artery . the multitude of small dural arteries , a variety of venous pathways , and high flow may prevent spontaneous closure11 ) . davf usually has multiple fistula points by multiple feeding arteries but drains into single vein . the aim of the davf treatment with retrograde leptomeningeal venous drainage is complete and permanent obliteration of the all arterial supply or occlusion of proximal draining vein . therapeutic options for treating davf include transvenous and/or transarterial embolization and surgical excision of the davf nidus , ligation of the draining vein . usually the transvenous embolization was preferred when the venous anatomic features were open and not too tortuous for catheterization of the draining vein and fistula13 ) . these combinations of transvenous and transarterial embolization results in high obliteration rates for most davf , but tentorial davf are somewhat different . more importantly , tentorial davf often drain exclusively to subarachnoid veins rather than to their associated sinus ( borden type iii ) , which prevents transvenous access261011 ) . huang et al.6 ) , reported complete cure of tentorial davf in 78.6% ( 11/14 ) by transarterial embolization using onyx . in case of partial obliteration of the arterial supply , patients with borden type ii or iii lesions should considered surgery because they may experience temporary palliation of their symptoms but can not provide protection from the risk of hemorrhage or progression of the effects of venous hypertension414 ) . hwang et al.7 ) and lawton et al.10 ) recommended surgical option for incomplete embolization of tentorial davf by simple interruption of draining vein . surgical interruption of simple venous drainage is analogous to transarterial and/or tranvenous embolization of outlet from the nidus without effect of normal venous drainage . the surgical interruption of proximal venous drain could be considered according to types of tentorial davf by location and drain vein . the tentorial sinuses drain the supra- and infratentorial veins as well as the deep veins of the brain stem . lasjaunias et al.9 ) , divided tentorial davf into torcular , basal tentorium and marginal tentorium by their location and draining veins . lesions in marginal tentorium drain into tentorial vein , vein of rosenthal , and mesencephalic veins . picard et al.12 ) , divided tentorial davf to 3 groups by draining veins as medial , lateral and marginal tentorial sinus group . lesions in the medial tentorial sinus group are situated adjacent to the torcular and drain into it or the lateral or straight sinus . they primarily receive venous drainage from the cerebellar hemispheres and vermis ( infratentorial drainage ) . lesions in the lateral tentorial sinus group lie adjacent to the lateral sinus and receive supratentorial drainage from the lateral and inferior surfaces of the temporal and occipital lobes . lesions in the marginal tentorial sinus group lie along the free edge of the tentorium and receive venous drainage from the basilar and lateral mesencephalic veins . fistulae at this location may have infra- or supratentorial drainage or drain into spinal veins . case 1 was torcular type by lasjaunias et al.9 ) and lawton et al.10 ) , and then marginal tentorial sinus group by picard et al.12 ) which was located around tocular area and drained to supratentorial cotical vein into basal vein of rosenthal and causes brain stem hemorrhage . case 2 was medial tentorial sinus group by picard et al.12 ) and straight sinus type by lawton et al.10 ) which was located around midline torcular and drained to infratentorial vermian vein and causes venous hypertension . various embolic agents have been used including particles , platinum coils , nbca and onyx for arterial approach . in case of nbca , solidification time of the material should be matched to the local flow velocity and to the angioarchitectural features of the lesion . although nbca is usually considered as permanent embolic agent but may result in recanalization as our case in delayed fashion5 ) . furthemore posterior choroidal artery could not be selected for embolization of complete angiographic cure by nbca . recently due to the superior ability of penetration and controllability , onyx is used as the treatment of choice for transarterial embolic agent of davf and very effective for obliteration of multiple fistula point and proximal draining vein by single transarterial access . contrast to nbca , in case 2 , onyx could penetrate deeper venous sac and reach posterior choroidal arterial supply from middle meningeal artery and results in complete angiographic cure . tentorial davf should be treated aggressively and transarterial embolization with onyx can be prior option when transvenous access is difficult . transarterial approach using onyx also can be applied for delayed recanalization after embolization with nbca . if partially treated tentorial davf by embolization procedure , simple surgical interruption of proximal draining vein should be considered according to the location and drain veins .

irritable bowel syndrome ( ibs ) is a multifactorial and heterogeneous disorder estimated to affect over 10% of the western population . a subset of the patients reports the start of the disease after an episode of gastroenteritis . the alterations in the intestinal microbiota of the post - infectious ibs ( pi - ibs ) patients were recently investigated in a british cohort and shown to differentiate from the healthy controls and resemble that of diarrhea - predominant ibs ( ibs - d ) patients . the altered 27 genus - like groups created a microbial signature , which could be used to objectively stratify patients and healthy controls . in this addendum , we combine the microbiota data derived from the british cohort with that of a recently reported swedish pi - ibs cohort . remarkably , robust and reproducible microbiota signatures were observed in these pi - ibs patients . we discuss these results with attention on the emerging role of microbiota in the classification , development and treatment of pi - ibs . tentorial dural arteriovenous fistula ( davf ) is a rare vascular disease , which has high risk of intracranial hemorrhage . we present two cases of tentorial davf which were successfully treated with single trial of transarterial embolization using onyx . we briefly reviewed the types of the tentorial davf and strategies of treatment .

over the last 3 decades , population - based studies aiming to determine the true burden of neurological disorders have increasingly been acknowledged . nevertheless , in developing countries , the limited access of neurologists to rural areas and the logistic and economic limitations to perform neuroepidemiological studies have significantly complicated their implementation.1 however , a series of epidemiological studies was conducted in upper egypt in the last 2 decades . epidemiologic research has progressed in egypt , particularly over the last 6 years , through two major projects to evaluate the incidence and prevalence of different neurological disorders in al kharga district , new valley and al quseir city , red sea governorate , upper egypt . this project aimed to evaluate the incidence and prevalence of epilepsy and stroke ; the incidence of bell s palsy ; and the prevalence of dementia , chorea , athetosis , dystonia , parkinson s disease , cerebellar ataxia , multiple sclerosis , migraine , trigeminal neuralgia , cerebral palsy , and spinal cord disorders . the project was designed to assess major neurological disorders in al quseir city as a representative coastal city lying on the red sea . al quseir city has a long history , dating from the pharaonic era when it was named thagho . at the time of the batlamic and romanic eras it was named leukos limen and poptus albus , respectively . its population is representative of native inhabitants and immigrants from different governorates who reside there to participate in tourist activities , fishing , and mining.2 all eligible inhabitants who had been living in al quseir city for at least 6 months at the time of the interview were included in the survey . the study duration extended from july 1 , 2009 to june 31 , 2012 through different stages of data collection , a preparatory stage , screening , data entry , statistical manipulation , and tabulation . all households ( n = 33,285 ; 7497 families ) and both sexes , with 49.4% males ( n = 16,428 ) and 50.6% females ( n = 16,857 ) , were screened by three neurologists , in addition to 15 female social workers ( for sociodemographic data collection ) via a door - to - door survey . a standardized questionnaire , the sensitivity and specificity of which are 96% and 93.2% , respectively,3 was applied by three neurologists to every member of each family ( children and elderly were questioned through their caregivers ) . a total population of 31,754 ( 95.4% ) were free from any neurological disorder , versus 1531 ( 4.6% ) of both sexes ( 3.9% [ n = 647 ] males and 5.2% [ n = 884 ] females ) had different neurological disorders . full history data and examinations ( general , systemic , and neurological ) were carried out by the three neurologists in collaboration with nine other senior staff members from neurology departments in assiut , al - azhar , sohag , and qena universities . all neurological disorders were finally diagnosed after evaluation by the three neurologists , each separately , as well as specific investigatory tools . diagnoses of different neurological disorders were based on the accepted definition and diagnostic criteria given by the world health organization.4,5 the definition of incidence rate , prevalence , and lifetime prevalence in this study are according to those provided by abramson.6 ethical approval for the study was obtained from the research ethics committee of assiut university and from the ministry of health to carry out this project in al quseir city , red sea governorate . each participant provided written informed consent ( children , illiterate , and disabled individuals consented through the responsible person in the family or their caregivers ) . data management was carried out by two specialists in data entry and three medical statisticians using spss software ( v 16 ; ibm corporation , armonk , ny , usa ) , excel ( microsoft corporation , redmond , wa , usa ) , and epicalc 2000 ( microsoft corporation ) . the project was designed to assess major neurological disorders in al quseir city as a representative coastal city lying on the red sea . al quseir city has a long history , dating from the pharaonic era when it was named thagho . at the time of the batlamic and romanic eras it was named leukos limen and poptus albus , respectively . its population is representative of native inhabitants and immigrants from different governorates who reside there to participate in tourist activities , fishing , and mining.2 all eligible inhabitants who had been living in al quseir city for at least 6 months at the time of the interview were included in the survey . the study duration extended from july 1 , 2009 to june 31 , 2012 through different stages of data collection , a preparatory stage , screening , data entry , statistical manipulation , and tabulation . all households ( n = 33,285 ; 7497 families ) and both sexes , with 49.4% males ( n = 16,428 ) and 50.6% females ( n = 16,857 ) , were screened by three neurologists , in addition to 15 female social workers ( for sociodemographic data collection ) via a door - to - door survey . a standardized questionnaire , the sensitivity and specificity of which are 96% and 93.2% , respectively,3 was applied by three neurologists to every member of each family ( children and elderly were questioned through their caregivers ) . a total population of 31,754 ( 95.4% ) were free from any neurological disorder , versus 1531 ( 4.6% ) of both sexes ( 3.9% [ n = 647 ] males and 5.2% [ n = 884 ] females ) had different neurological disorders . full history data and examinations ( general , systemic , and neurological ) were carried out by the three neurologists in collaboration with nine other senior staff members from neurology departments in assiut , al - azhar , sohag , and qena universities . all neurological disorders were finally diagnosed after evaluation by the three neurologists , each separately , as well as specific investigatory tools . diagnoses of different neurological disorders were based on the accepted definition and diagnostic criteria given by the world health organization.4,5 the definition of incidence rate , prevalence , and lifetime prevalence in this study are according to those provided by abramson.6 ethical approval for the study was obtained from the research ethics committee of assiut university and from the ministry of health to carry out this project in al quseir city , red sea governorate . each participant provided written informed consent ( children , illiterate , and disabled individuals consented through the responsible person in the family or their caregivers ) . data management was carried out by two specialists in data entry and three medical statisticians using spss software ( v 16 ; ibm corporation , armonk , ny , usa ) , excel ( microsoft corporation , redmond , wa , usa ) , and epicalc 2000 ( microsoft corporation ) . the incidence rates of epilepsy , stroke , and bell s palsy compared to worldwide rates are illustrated in table 1 . the prevalence of the neurological disorders studied in al quseir city is illustrated in tables 24 . table 5 shows a record of the prevalence of different neurological disorders studied in al quseir city and worldwide . the increase in the number of different major projects to study neurological disorders worldwide during the last 3 decades will help all of us to construct a map of neurological disorders throughout the world and clarify the burden of neurological disorder among different countries . this study is a new one in the series of large epidemiological studies carried out in our country to assess the burden of neurological disorders at different localities in egypt . screening was carried out through a door - to - door survey , including every household , by using a standardized questionnaire,7 with subsequent clinical evaluation of all suspected cases by neurologists ( three specialists as well as nine senior staff members ) in al quseir general hospital and assiut university hospital . the limitations of this study include : the stigma of some neurological disorders ( eg , the view of epilepsy as an evil attack and dementia as a normal aging process ) necessitates extraneous effort from the project team to clarify the nature of these disorders and their suitability to treat . noncompliance by the patients with the prescribed treatments due to scarce financial resources and lack of health care systems suited for long term needs of chronic neurological disorders . these limitations required an extra effort on the project team to encourage participation of most inhabitants through : detailed explanations of the project goals by the members of the project team and responsible authorities in the defined area of the study . recruitment of well - known key persons , as well as female social workers , among native inhabitants , who accompanied the screening team to facilitate family interviews and encourage participation . a capture - recapture method for those who were not present at their homes during the first visit , the incidence rate of epilepsy ( 48/100,000 per year ) in al quseir city is within the international rate and lower than that recorded in sub - saharan africa10 and northern tanzania.11 meanwhile , the incidence rate of epilepsy ( 48/100,000 ) in this study was higher than that recorded in new york , ny , usa ( 16/100,000)12 and in the netherlands ( 29/100,000).13 although the incidence rate of stroke in al quseir city was lower than that recorded in france14 and other areas of egypt ( al kharga district , new valley),7 it is similar to that recorded in sohag , upper egypt.15 the incidence rate of bell s palsy in al quseir city was higher than that recorded worldwide.7,1618 the great variation in the incidence rate of epilepsy , stroke , and bell s palsy in al quseir city in comparison to incidence rates worldwide could be attributed to the different genetic characteristics of the population in al quseir city , where the population contains a mixture of native inhabitants and immigrants from different adjacent governorates , and thus a variable ( not distinct ) genetic predisposition for various neurological disorders . study of age - specific prevalence of some neurological disorders in al quseir city revealed that dementia ( 3.83% of those aged 60 years ) was the most frequent , followed by migraine ( 2.8% ) , stroke ( 6.2/1000 ) , epilepsy ( 5.5/1000 ) , parkinson s disease ( 452.1/100,000 ) , and cerebral palsy ( 3.6/1000 ) . however , less frequent neurological disorders were spinal cord injury ( 63.0/100,000 ) , dystonia ( 39.1/100,000 ) , cerebellar ataxia ( 30.0/100,000 ) , trigeminal neuralgia ( 28/100,000 ) , chorea ( 21.0/100,000 ) , athetosis ( 15.0/100,000 ) , and multiple sclerosis ( 13.7/100,000 ) . macdonald et al s uk study found that stroke was the most frequent neurological disorder , followed by epilepsy , then parkinson s disease , and , finally , multiple sclerosis.9 on the other hand , in a mexican rural community in 2011 , quet et al found that headache was the most frequent neurological disorder , followed by epilepsy , stroke , parkinson s disease , and cerebellar ataxia.19 in the al kharga district , the order of frequency of prevalence of the neurological disorders studied was as follows : dementia , epilepsy , stroke , parkinson s disease , cerebral palsy , cerebellar ataxia , chorea , dystonia , and athetosis.7 these variable results of different epidemiological studies might be attributed to differences in the methodology and types of neurological disorders studied ; the impact of different environmental factors in diverse geographic areas ; and different genetic compositions of the studied populations . the prevalence of multiple sclerosis ( 13.7/100,000 ) and spinal cord disorders ( 63/100,000 ) ( 18/100,000 traumatic and 45/100,000 nontraumatic causes ) in al quseir city are the first results of a community - based epidemiological study in egypt . while radhakrishnan et al recorded a lower prevalence of multiple sclerosis in libya ( 5.9/100,000)20 than that recorded in our study , macdonald et al recorded a much higher prevalence ( 200/100,000 ) in the uk.9 on the other hand , an intermediate figure ( 33.9/100,000 ) for the prevalence of multiple sclerosis was recorded by elik et al in the metropolitan area of edrine city , turkey.21 this wide variation in the prevalence of multiple sclerosis worldwide could be attributed to differences in climate , race , immigration , and emigration , as well as varied genetic predispositions of the studied sample frame . this study clarified important issues regarding the epidemiology of the studied neurological disorders , which can be summarized as follows : the high prevalence of parkinson s disease ( 452.1/100,000 ) in comparison with worldwide studies and those in other localities in egypt , which could be attributed to environmental pollution by heavy metals ( manganese , lead , and phosphorus ) in this area . the higher prevalence of cerebral palsy ( 3.6/1000 ) than in worldwide records and previously recorded data in al kharga district , egypt could be explained by a lack of antenatal , perinatal , and postnatal care in al - quseir city . the lower prevalence of chorea in al - quseir city ( 21.03/100,000 ) in comparison to the other two studies conducted in our country ( al kharga district : 31.96/100,000;7 assiut : 62.00/100,00022 ) for rheumatic chorea could be attributed to governmental efforts for the eradication of rheumatic activities in the last few years . door - to - door surveys , with direct personal interviews with both health - seeking and non - health - seeking individuals , gives a better chance for diagnosing rare neurological disorders . it remains the gold standard method of epidemiological studies , especially in the absence of existing coded data due to a lack of medical registry systems and health insurance coverage . this study had the aim of evaluating the prevalence of trigeminal neuralgia , multiple sclerosis , and spinal cord disorders for the first time in egypt . the study shows a high prevalence of parkinson s disease , cerebral palsy , and stroke in al quseir city compared with other cities worldwide . the study shows a marked decline in the prevalence of chorea in egypt , compared to a previous study.22 repair of anorectal malformation ( arm ) with rectourethral ( ru ) fistula involves the separation of two systems . this involves risk of injury to the urethra , ureters , seminal vesicles , bladder , and important nerves responsible for urinary control and sexual function . group a included patients of arm with ru fistula who had undergone posterior sagittal anorectoplasty ( psarp ) without closure of ru fistula , from february 2006 to january 2010 . the rest of the psarp procedure was the same as conventionally done , the only difference being that we did not close the ru fistula after separating it from the rectum . we just separated the rectum from the urethra and left the urethral fistula as it is without closing it . group b included 34 previous successive patients who had undergone psarp before january 2006 in whom the ru fistula was closed using interrupted sutures . all the patients in both the groups had undergone staged repair of arm and not primary psarp . micturating cystourethrogram ( mcu ) and distal colostogram was done in all these patients prior to psarp . all the patients were evaluated during follow - up both clinically and with investigations like mcu and cystoscopy . the patients were studied for parameters like urinary stream , urinary dribbling , urinary tract infections , and recurrent ru fistula . moreover , all patients had undergone urethrocystoscopy three months after psarp to check for the status of the urethra and bladder . patients who had sacral agenesis were excluded from the study group because such congenital sacral defects can lead to a neurogenic bladder . the following were the observations in group a ( a ) in the immediate postoperative period , there was no urinary leakage , urinary retention , or any other complication ; thus all these patients had an uneventful recovery . ( b ) urinary stream was normal ; there was no evidence of urinary dribbling or retention , urinary tract infection , and recurrent rectourinary fistula during follow - up . mcu showed normal urethra , without any evidence of stenosis or stricture , urethro - ejaculatory duct / vasal reflux , or diverticulum in any of the cases . however , in group b , complications like urethral stenosis , urethral diverticulum , and neurogenic dysfunction were seen . a comparative analysis of the two groups was done and overall complications were listed [ table 1 ] . during psarp , urological injuries in male patients are known complications.[13 ] excessive traction on the urethra during dissection leads to transection or injury to the urethra . it is extremely important for the surgeon to bear in mind that in arm with ru fistula , the rectum is intimately attached to the urethra and that meticulous dissection and separation are necessary . urethral stenosis can occur due to traction on the ru fistula , that is , indirect traction on the urethra during separation and closure of fistula . urethral stenosis can be avoided by applying less traction on the fistula during separation and avoiding the closure of fistula ; we have seen in our series that urethral stenosis was not seen in any of the cases of group a , where the ru fistula was not closed . closing another point to note is that if we separate the rectum from the urethra very near the urethral wall and use interrupted sutures for its closure , it also increases the chances for urethral stenosis . urethral stenosis due to ligation or closure of the ru fistula may result in recurrent epididymo - orchitis . urethral diverticulum is the result of a segment of the rectum left attached to the urethra and the separated end closed . such patients usually present with recurrent urinary tract infections , stone formations in diverticulum again leading to dysuria , urinary tract infections , and so on . this complication can be avoided by separating the rectum away from the urethra without leaving any segment of the rectum attached and leaving the fistula as it is without closing it , so that nothing like a pouch / diverticulum is formed . neurogenic dysfunction after psarp has been reported in the form of neurogenic bladder , impotence , or loss of ejaculation . postoperatively , a neurogenic bladder may reflect a poor surgical technique with denervation of bladder and bladder neck during repair . by avoiding the closure of the ru fistula , we can avoid excessive traction on the fistula and hence on the urethra , and also prevent the excessive dissection during fistula closure and minimize the chance of neurogenic dysfunction . damage to the external vesical sphincter has also been reported during ligation or closure of the fistula . thus by avoiding closure of the fistula , we avoid this complication also and , hence , neurovesical dysfunction . thus we have seen that by not doing something , that is , by not closing the ru fistula during psarp , we can avoid many complications ; so , not doing something is preferable here .

a door - to - door survey , including every household , was conducted for all inhabitants of al quseir city ( 33,283 ) , red sea governorate , egypt by three specialists of neurology as well as nine senior staff members of neurology and 15 female social workers to assess the epidemiology of major neurological disorders . over six phases , from july 1 , 2009 to january 31 , 2012 , screening of all eligible people in the population was carried out , by which case ascertainment of all major neurological disorders included in the study was done according to the accepted definitions and diagnostic criteria of the world health organization . the order of frequency of prevalence of the studied neurological disorders was dementia ( 3.83% for those aged > 60 years ) , migraine ( 2.8% for those aged > 8 years ) , stroke ( 6.2/1000 for those aged > 20 years ) , epilepsy ( 5.5/1000 ) , parkinson s disease ( 452.1/100,000 for those aged > 40 years ) , cerebral palsy ( 3.6/1000 among children < 18 years ) , spinal cord disorders ( 63/100,000 ) dystonia ( 39.11/100,000 ) , cerebellar ataxia ( 30.01/100,000 ) , trigeminal neuralgia ( 28/100,000 for those aged > 37 years ) , chorea ( 21.03/100,000 ) , athetosis ( 15/100,000 ) , and multiple sclerosis ( 13.74/100,000 ) . the incidence rates of stroke , epilepsy , and bell s palsy were 181/100,000 , 48/100,000 , and 98.9/100,000 per year , respectively . aim : to study the effect of nonclosure of rectourethral ( ru ) fistula and to do a comparative analysis of the complications with and without nonclosure of ru fistula during posterior sagittal anorectoplasty ( psarp ) in anorectal malformation cases ( arm).materials and methods : a total of 68 cases of arm were included in the study group , of which 34 cases were those in whom ru fistula was not closed ( group a ) during psarp . another 34 successive cases were included in study group b in whom the ru fistula was closed as is conventionally done by using interrupted sutures.results:comparatively , group a had none or minimum urological complications as compared to group b.conclusion:ru fistula closure is not mandatory during psarp and nonclosure avoids urological complications . it especially avoids urethral complications , which are 100% preventable .

middle ear cholesteatoma is characterized by keratinocytic hyperproliferation with bony destruction and granulation tissue by chronic inflammation . the pathogenesis of middle ear cholesteatoma has been debated for a century , but there are four basic theories : invagination , basal cell hyperplasia , epithelial ingrowth and squamous metaplasia . during last century , etiopathogenicity of middle ear cholesteatoma raised many controversies and still a unanimous concept does n't exist . from a histological point of view this pathological tissue the interaction between cellular components is essential for the major histopathological features of cholesteatoma - hyperproliferation and bone destruction . in 1964 , the american anatomist gray referred to middle ear cholesteatoma as " skin in the wrong place " ( 1 ) . this simplified misdefinition stuck into literature and has been cited profusely in many scientific papers . the metaplasia theory ( 2 ) and existence of so - called " congenital " cholesteatoma put into question the " skin origin " of middle ear cholesteatoma and gave etiopathogenetical evidences against the simplified definition " skin in the wrong place " . the list of histological differences includes the absence of cutaneous adnexa and specialized dermal structures . neither hypodermal structures , nor blood vessels , or nerve endings have been ever described in cholesteatoma . the only similarity that is left between them is the presence of multistratified squamous epithelium , but in cholesteatoma this epithelium is with hyperproliferative properties . experimental models are critical for studying pathogenesis of middle ear cholesteatoma . in vivo models were tried to establish by chemical injection into the middle ear ( 3 ) , ligation of external auditory canal ( 4 ) and transplantation of skin ( 5 ) . keratinocytes of middle ear cholesteatoma were cultured by tissue culture ( 6 ) , passaged subculture ( 7 ) and air - liquid interface system ( 8) . however , stable experimental models of middle ear cholesteatoma are not established until now . our study has aimed to create an " adequate " in vitro model , based on the cell - to - cell interaction between keratinocytes and fibroblasts and to assess the grade of homogeneity between cholesteatoma and skin keratinocytes by focusing on the cytokeratin profile . enzymatic disaggregation protocol for simultaneous isolation of primary keratinocytes and fibroblasts from cholesteatoma tissue has been developed . the biopsy samples were harvested from patients undergoing surgical treatment in the department of otolaryngology , ajou university hospital , korea . an informed consent form approved by the institutional review board of ajou university hospital cholesteatoma keratinocytes were cultured in defined keratinocytes serum free medium ( gibco , brl , gaithersburg , md , usa ) supplied with growth supplement and 5 mg / ml gentamicin ( gibco , brl ) according to the manufacture instructions . cholesteatoma primary fibroblasts were successfully cultured in dulbeco modified eagle medium ( gibco , brl ) supplemented with 10% fetal bovine serum ( fbs , clonetics , walkersville , md , usa ) and 1% penicillin - streptomycin mixture ( gibco , brl ) . primary keratinocytes ( passage 1 ) and fibroblasts ( between passages 1 - 5 ) were used for establishment of the co - cultured system . hacat cell line was kindly provided from the department of dermatology in ajou university hospital . feeder layer from cholesteatoma fibroblasts louis , mo , usa ) in concentration 0.25 g / ml . fibroblasts were incubated overnight at 37 , 5% co2 in humidified atmosphere . on the next day the feeder layer was subsequently trypsinized from the substrate and transferred on polyester membrane in transwell ( costar , cambridge , ma , usa ) system to develop a subconfluent feeder layer . cholesteatoma keratinocytes and hacat cells were seeded at density 210 cells / ml over the feeder layer , co - cultured for 3 - 5 days and air - exposed for a period of 14 days . transwell membranes were fixed in 4% formaldehyde overnight at 4 , dehydrated in graded alcohols , cleared in histoclear ( national diagnostics , atlanta , georgia ) and embedded in paraffin . six m - thick sections were prepared and subjected to routine hematoxylin - eosin staining . polyacrylamide gel electrophoresis has been used for protein separation and staining with coomassie blue was performed to compare the protein profile between cholesteatoma keratinocytes and hacat cells . blocking of nonspecific staining was accomplished with membrane incubation with 5% non - fat skim milk for 1 hr at room temperature ( rt ) . membranes were then incubated with primary mouse monoclonal anti - cytokeratin antibody clone k 8.12 ( sigma ) which is immunospecific for cytokeratin 13 ( ck 13 ) and cytokeratin 16 ( ck 16 ) , with dilution factor 1:100 , for 1 hr at rt . after washing membranes were incubated with goat anti - mouse horseradish peroxidase conjugate ( zymed , san francisco , ca , usa ) . the positive reaction was visualized with ecl western blotting detection system ( amersham , bucks , uk ) . the localization of cytokeratin 13 and 16 in middle ear cholesteatoma tissue was accomplished by ihc study as described before ( 9 ) . five middle ear cholesteatoma samples were compared with the corresponding retroauricular skin samples obtained during surgery . six m thick formalin - fixed paraffin - embedded tissue sections were processed with abc - method . primary mouse monoclonal anticytokeratin antibody ( sigma ) at concentration 1:20 was incubated overnight at rt . vectorlab elite abc kit ( vector laboratories , burlingame , ca , usa ) was used for detection of positive reaction using diaminobenzidine substrate . enzymatic disaggregation protocol for simultaneous isolation of primary keratinocytes and fibroblasts from cholesteatoma tissue has been developed . the biopsy samples were harvested from patients undergoing surgical treatment in the department of otolaryngology , ajou university hospital , korea . an informed consent form approved by the institutional review board of ajou university hospital cholesteatoma keratinocytes were cultured in defined keratinocytes serum free medium ( gibco , brl , gaithersburg , md , usa ) supplied with growth supplement and 5 mg / ml gentamicin ( gibco , brl ) according to the manufacture instructions . cholesteatoma primary fibroblasts were successfully cultured in dulbeco modified eagle medium ( gibco , brl ) supplemented with 10% fetal bovine serum ( fbs , clonetics , walkersville , md , usa ) and 1% penicillin - streptomycin mixture ( gibco , brl ) . primary keratinocytes ( passage 1 ) and fibroblasts ( between passages 1 - 5 ) were used for establishment of the co - cultured system . hacat cell line was kindly provided from the department of dermatology in ajou university hospital . feeder layer from cholesteatoma fibroblasts louis , mo , usa ) in concentration 0.25 g / ml . fibroblasts were incubated overnight at 37 , 5% co2 in humidified atmosphere . on the next day the feeder layer was subsequently trypsinized from the substrate and transferred on polyester membrane in transwell ( costar , cambridge , ma , usa ) system to develop a subconfluent feeder layer . cholesteatoma keratinocytes and hacat cells were seeded at density 210 cells / ml over the feeder layer , co - cultured for 3 - 5 days and air - exposed for a period of 14 days . transwell membranes were fixed in 4% formaldehyde overnight at 4 , dehydrated in graded alcohols , cleared in histoclear ( national diagnostics , atlanta , georgia ) and embedded in paraffin . six m - thick sections were prepared and subjected to routine hematoxylin - eosin staining . polyacrylamide gel electrophoresis has been used for protein separation and staining with coomassie blue was performed to compare the protein profile between cholesteatoma keratinocytes and hacat cells . blocking of nonspecific staining was accomplished with membrane incubation with 5% non - fat skim milk for 1 hr at room temperature ( rt ) . membranes were then incubated with primary mouse monoclonal anti - cytokeratin antibody clone k 8.12 ( sigma ) which is immunospecific for cytokeratin 13 ( ck 13 ) and cytokeratin 16 ( ck 16 ) , with dilution factor 1:100 , for 1 hr at rt . after washing membranes were incubated with goat anti - mouse horseradish peroxidase conjugate ( zymed , san francisco , ca , usa ) . the positive reaction was visualized with ecl western blotting detection system ( amersham , bucks , uk ) . the localization of cytokeratin 13 and 16 in middle ear cholesteatoma tissue was accomplished by ihc study as described before ( 9 ) . five middle ear cholesteatoma samples were compared with the corresponding retroauricular skin samples obtained during surgery . six m thick formalin - fixed paraffin - embedded tissue sections were processed with abc - method . primary mouse monoclonal anticytokeratin antibody ( sigma ) at concentration 1:20 was incubated overnight at rt . vectorlab elite abc kit ( vector laboratories , burlingame , ca , usa ) was used for detection of positive reaction using diaminobenzidine substrate . the low selectivity and potential damages of cells in case of prolonged incubation were major disadvantages of the procedure . as an inflammatory tissue , cholesteatoma posed some difficulties in isolation of primary keratinocytes , because of the attendant inflammation and possible contamination , which requires an adequate decontamination . the first primary keratinocytes appeared usually 7 - 10 days after trypsinization and generally within the next 10 days keratinocytes reached a confluence . the individual variations in proliferative activity of cholesteatoma keratinocytes were related to cholesteatoma localization and sex of the patient . for instance , attic cholesteatoma frequently resulted in isolation of primary keratinocytes with bigger proliferative ability . another interesting correlation was the fact that cholesteatoma samples obtained from young female patients were characterized by higher proliferative activity in vitro . primary cholesteatoma keratinocytes were uniform in shape and arranged in a typical monolayer pattern ( fig . in passage 2 an abrupt deceleration in proliferation was established , accompanied by morphological alterations such as enlargement and flattening of the cells , as well as vacuolization of the cytoplasm . in passage 3 primary cholesteatoma fibroblasts were steadily maintained without significant changes of doubling time until passage 10 . bigger stratification and proliferation have been observed in the proposed 3d in vitro system when primary cholesteatoma keratinocytes were co - cultured in the presence of fibroblasts ( fig . 3b ) in comparison with the control group without fibroblasts ( fig . 3a ) . 4a ) . disrupted differentiation in the suprabasal layers was revealed when cholesteatoma keratinocytes were air - exposed in the presence of fibroblasts mimicking the parakeratosis state , which is typical for this pathological tissue in vivo ( fig . coomassie blue staining of the protein extracts from primary cholesteatoma keratinocytes and hacat cells revealed significantly different protein profiles ( fig . stronger expression of ck 16 ( 48 kda ) and ck 13 ( 51 kda ) was established in cholesteatoma keratinocytes in comparison with hacat cells ( fig . 5b ) . using mouse monoclonal antibody clone k 8.12 , tissue localization of ck 13 and ck 16 the intensity of the ck 13 , 16-positive staining varied in different areas of the same cholesteatoma sample . the positive control revealed specific expression of ck 13 and 16 confined to the islands of neoplastic squamous cells in laryngeal carcinoma specimens . the low selectivity and potential damages of cells in case of prolonged incubation were major disadvantages of the procedure . as an inflammatory tissue , cholesteatoma posed some difficulties in isolation of primary keratinocytes , because of the attendant inflammation and possible contamination , which requires an adequate decontamination . the first primary keratinocytes appeared usually 7 - 10 days after trypsinization and generally within the next 10 days keratinocytes reached a confluence . the individual variations in proliferative activity of cholesteatoma keratinocytes were related to cholesteatoma localization and sex of the patient . for instance , attic cholesteatoma frequently resulted in isolation of primary keratinocytes with bigger proliferative ability . another interesting correlation was the fact that cholesteatoma samples obtained from young female patients were characterized by higher proliferative activity in vitro . primary cholesteatoma keratinocytes were uniform in shape and arranged in a typical monolayer pattern ( fig . in passage 2 an abrupt deceleration in proliferation was established , accompanied by morphological alterations such as enlargement and flattening of the cells , as well as vacuolization of the cytoplasm . in passage 3 primary cholesteatoma fibroblasts were steadily maintained without significant changes of doubling time until passage 10 . bigger stratification and proliferation have been observed in the proposed 3d in vitro system when primary cholesteatoma keratinocytes were co - cultured in the presence of fibroblasts ( fig . 3b ) in comparison with the control group without fibroblasts ( fig . 3a ) . 4a ) . disrupted differentiation in the suprabasal layers was revealed when cholesteatoma keratinocytes were air - exposed in the presence of fibroblasts mimicking the parakeratosis state , which is typical for this pathological tissue in vivo ( fig . coomassie blue staining of the protein extracts from primary cholesteatoma keratinocytes and hacat cells revealed significantly different protein profiles ( fig . ck 13 ( 51 kda ) was established in cholesteatoma keratinocytes in comparison with hacat cells ( fig . using mouse monoclonal antibody clone k 8.12 , tissue localization of ck 13 and ck 16 was verified both in the basal and suprabasal layers of cholesteatoma ( fig . the intensity of the ck 13 , 16-positive staining varied in different areas of the same cholesteatoma sample . the positive control revealed specific expression of ck 13 and 16 confined to the islands of neoplastic squamous cells in laryngeal carcinoma specimens . the putative interactions between major cellular components of cholesteatoma tissue have been extensively studied in the literatures . we demonstrate a summarized schematic model of possible intercellular " cross - talking " between epithelial - mesenchymal components of cholesteatoma in fig . pro - inflammatory cytokines such as interleukin-1 and interleukin-1 , ( il-1 , il-1 ) released from matrix keratinocytes stimulate keratinocytes growth factor ( kgf ) expression , which in turn induced proliferation of the cholesteatoma keratinocytes ( 10 ) . granulocyte - macrophage colony stimulating factor ( gm - csf ) is another fibroblasts - derived growth factor induced by il-1 , with acknowledged stimulating effects both on proliferation and terminal differentiation of keratinocytes ( 11 ) . keratinocytes - derived parathyroid hormone - related protein ( pthrp ) induces expression of kgf . kgf is then released by perimatrix fibroblasts and accelerates the proliferative response in matrix keratinocytes . a possible connection between pthrp and bone destructive lesions have also been reported ( 12 ) . transforming growth factor alpha ( tgf- ) is constitutively expressed in keratinocytes and regulates keratinocyte proliferation and differentiation in an autocrine manner ( 13 ) . on the other hand , a possible synergism between tgf- and epidermal growth factor ( egf ) was proposed with possible effect on keratinocytes proliferation through epidermal growth factor receptor ( egf - r ) , indicating existence of an alternative paracrine regulatory mechanism ( 14 ) . transforming growth factor - beta ( tgf- ) is expressed in hyperproliferative epithelium and granulation tissue ( 15 ) and influences the epithelial morphogenesis both in an autocrine and paracrine fashion . the above statements about cellular interactions were taken into consideration for creating an adequate in vitro middle ear cholesteatoma model . as it was mentioned before , the proliferative capacity of primary cholesteatoma keratinocytes in vitro is limited and expires within 2 passages . to improve their plating efficiency and survival rate at low density 3t3-mouse embryo fibroblasts that used routinely in cell culture practice display different morphological patterns ( cobble - stone morphology ) , which differs from in vivo state and complicates result interpretation . feeder layer secretes nutrients , growth factors and matrix constituents . as a positive control in our study we choose hacat cell line in order to avoid donor - to - donor and site - to - site variations in proliferative capacity of normal skin keratinocytes . hacat cells are spontaneously immortalized human adult skin keratinocytes raised in reduced calcium level and elevated temperature conditions ( 16 ) . hacat found an extensive application in tissue engineering for development of artificial skin equivalents ( 17 ) . the proposed organotypic in vitro model revealed stronger proliferation and bigger stratification , when hacat cells were co - cultured with fibroblasts , in comparison with the control group without fibroblasts . histopathological diagnosis of cholesteatoma includes : unrestrained proliferation , uncoordinated differentiation , migration , and invasion ( 18 ) . consistent with the above - mentioned observation , the proposed 3d - model disclosed disrupted differentiation and presence of nucleated cells in the upper layers of the cholesteatoma ali with fibroblasts . squamous metaplasia of middle ear epithelium is considered to be an adaptive tissue response towards persistent pathological stimuli such as chronic inflammation and possible etiopathogenetical mechanism for middle ear cholesteatoma . an experiment has been reported on the confirmation of a possible transformation of differentiated middle ear epithelium towards functionally inferior multilayered squamous epithelium ( 19 ) . epithelial cells are characterized by the presence of a specific set of intermediate protein filaments named cytokeratins , specific for different epithelial types . one of our primary objectives was to assess the grade of homogeneity between hacat cells and cholesteatoma keratinocyte . ck 16 was suggested in the literature as a proliferation marker , while ck 13 is considered as a marker of differentiation ( 20 ) . immunohistochemical investigation in our study disclosed non - uniform patterns of expression of ck 13 and ck 16 in middle ear cholesteatoma tissue . variation in intensity of the positively stained areas between different cholesteatoma samples as well as between different areas in the same sample was probably related to the different proliferative capacity of cholesteatoma . the expression of nonepidermal cytokeratins in middle ear cholesteatoma tissue gives arguments against the probable skin origin of mech keratinocytes . protein separation and coomassie blue staining revealed significantly different protein profile between cholesteatoma and hacat cells . precised western - blotting analysis demonstrated comparatively stronger expression of ck 13 and 16 in cholesteatoma keratinocytes with regard to hacat cell line . the different protein and cytokeratin profiles established between cholesteatoma keratinocytes and hacat cells favors metaplasia as a possible cause for development of mech . the proposed in vitro model underlines the importance of fibroblasts as a major component of 3d - in vitro system . our results emphasize the integrity of cholesteatoma as a separated pathological tissue , composed of two closely related functional and structural parts : the matrix and the perimatrix . dynamic biomolecules often undergo large - scale structural changes and visit distinct conformational states during their biological function . it is of great biological and pharmaceutical interest to characterize their structures and conformational transition pathways . ideally , detailed free energy landscapes are sought to understand the functional mechanisms of biomolecules in a quantitative manner . however , due to large energy barriers , conformational transitions of biomolecules usually take place on time scales of milliseconds or even longer . this has presented a grand challenge for computational molecular dynamics ( md ) simulations that are limited to typically hundreds - of - nanoseconds to tens - of - microseconds . to address this challenge , biasing simulation methods have been found useful in enhanced sampling and free energy calculation of the biomolecules . these methods include umbrella sampling , conformational flooding , metadynamics , adaptive biasing force ( abf ) calculations , and orthogonal space sampling , etc . during the simulations , a potential or force bias is applied along certain reaction coordinates ( or collective variables ) to facilitate the biomolecular conformational transitions across high - energy barriers . typical reaction coordinates include atom distances , torsional dihedrals , root - mean - square deviation ( rmsd ) relative to a reference configuration , eigenvectors generated from the principal component analysis , and so on . the definition of the reaction coordinates , however , often requires expert knowledge of the studied systems . furthermore , the predefined reaction coordinates largely place constraints on the pathway and conformational space to be sampled during the biasing simulations . it often leads to slow convergence of the simulations when important reaction coordinates are missed during the simulation setup . accelerated molecular dynamics ( amd ) is an enhanced sampling technique that works often by adding a non - negative boost potential to smoothen the biomolecular potential energy surface . the boost potential , v , decreases the energy barriers and thus accelerates transitions between the different low - energy states . with this , amd is able to sample distinct biomolecular conformations and rare barrier - crossing events that are not accessible to conventional md ( cmd ) simulations . unlike the previously mentioned biasing simulation methods , amd does not require predefined reaction coordinate(s ) , which can be advantageous for exploring the biomolecular conformational space without a priori knowledge or restraints . amd has been successfully applied to a number of biological systems , ( 10 ) and hundreds - of - nanosecond amd simulations have been shown to capture millisecond - time - scale events in both globular and membrane proteins . while amd has been demonstrated to greatly enhance conformational sampling of biomolecules , it suffers from large energetic noise during reweighting . the boost potential applied in amd simulations is typically on the order of tens - to - hundreds of kilocalories per mole , which is much greater in magnitude and wider in distribution than that of other biasing simulation methods that make use of predefined reaction coordinates ( e.g. , several kilocalories per mole ) . it has been a long - standing problem to accurately reweight amd simulations and recover the original free energy landscapes , especially for large proteins . our recent studies showed that when the boost potential follows near - gaussian distribution , cumulant expansion to the second order provides improved reweighting of amd simulations compared with the previously used exponential average and maclaurin series expansion reweighting methods . the reweighted free energy profiles are in good agreement with the long - time - scale cmd simulations as demonstrated on alanine dipeptide and fast - folding proteins . however , such improvement is limited to rather small systems ( e.g. , proteins with less than 35 amino acid residues ) . in simulations of larger systems , the boost potential exhibits significantly wider distribution and , a gaussian accelerated molecular dynamics ( gamd ) approach is presented to reduce the energetic noise for simultaneous unstrained enhanced sampling and free energy calculation of biomolecules , even for large proteins . gamd makes use of harmonic functions to construct the boost potential that is adaptively added to the biomolecular potential energy surface . a minimal set of simulation parameters is dynamically adjusted to control the magnitude and distribution width of the boost potential . as such , the resulting boost potential follows gaussian distribution and allows for accurate reweighting of the simulations using cumulant expansion to the second order . in this study , gamd is demonstrated on unconstrained simulations of the alanine dipeptide , chignolin folding , and ligand binding to the t4-lysozyme . gaussian accelerated molecular dynamics enhances the conformational sampling of biomolecules by adding a harmonic boost potential to smoothen the system potential energy surface ( figure 1 ) . consider a system with n atoms at positions = { 1, ... ,n } . when the system potential v ( ) is lower than a threshold energy , e , a boost potential is added as1where k is the harmonic force constant . the modified system potential , v * ( ) = v ( ) + v ( ) , is given by2otherwise , when the system potential is above the threshold energy , i.e. , v ( ) e , the boost potential is set to zero and v * ( ) = v ( ) . when the threshold energy is set to the maximum potential ( e = vmax ) , the system potential energy surface is smoothened by adding a harmonic boost potential that follows gaussian distribution . the coefficient k0 in the range of 01 determines the magnitude of the applied boost potential . with greater k0 , higher boost potential is added to the original energy surface in conventional molecular dynamics ( cmd ) , which provides enhanced sampling of biomolecules across decreased energy barriers . in order to smoothen the potential energy surface for enhanced sampling , first , for any two arbitrary potential values v1 ( ) and v2 ( ) found on the original energy surface , if v1 ( ) < v2 ( ) , v should be a monotonic function that does not change the relative order of the biased potential values ; i.e. , v1 * ( ) < v2 * ( ) . by replacing v * ( ) with eq 2 and isolating e , we then obtain3second , if v1 ( ) < v2 ( ) , the potential difference observed on the smoothened energy surface should be smaller than that of the original ; i.e. , v2 * ( ) v1 * ( ) < v2 ( ) v1 ( ) . similarly , by replacing v * ( ) with eq 2 , we can derive4with vmin v1 ( ) < v2 ( ) vmax , we need to set the threshold energy e in the following range by combining eqs 3 and 4:5where vmin and vmax are the system minimum and maximum potential energies . to ensure that eq 5 is valid , vmax vmin + ( 1/k ) and k has to satisfy the following:6we define k k0(1/(vmax vmin ) ) ; then 0 < k0 1 . as illustrated in figure 1 , k0 determines the magnitude of the applied boost potential . with greater k0 , higher boost potential is added to the potential energy surface , which provides enhanced sampling of biomolecules across decreased energy barriers . third , the standard deviation of v needs to be small enough ( i.e. , narrow distribution ) to ensure accurate reweighting using cumulant expansion to the second order:7where vav and v are the average and standard deviation of the system potential energies and v is the standard deviation of v with 0 as a user - specified upper limit ( e.g. , 10kbt ) for accurate reweighting . provided eq 5 gives the range of threshold energy e , when e is set to the lower bound e = vmax , we plug in e and k and obtain8we define the right - hand side in eq 8 as k0 = ( 0/v)((vmax vmin)/(vmax vav ) ) . for efficient enhanced sampling with the highest possible acceleration , k0 can then be set to its upper bound as9the greater v that is obtained from the original potential energy surface ( particularly for large biomolecules ) , the smaller k0 may be applicable to allow for accurate reweighting . alternatively , when the threshold energy e is set to its upper bound e = vmin + ( 1/k ) according to eq 5 , we plug in e and k in eq 7 and obtain10we define the right - hand side in eq 10 as k0 ( 1 ( 0/v ) ) ( ( vmax vmin)/(vav vmin ) ) . when k0 0 with v 0 , k0 can be theoretically set to any value between zero and 1 , although k0 = 1 is applied in this case for the current implementation of gamd ( see appendix a ) . note that a smaller k0 will give higher threshold energy e , but smaller force constant k. when 0 < k0 1 , k0 can be set to either k0 for the highest threshold energy e or its upper bound 1.0 for the greatest force constant k. in this case k0 = k0 is applied in the current gamd implementation . when k0 > 1 , we have to lower the threshold energy e to ensure that 0 < k0 1 ; e.g. , e = vmax for default k0 = 1 . given e and k0 , we can calculate the boost potential as11similar to amd , gamd provides options to add only the total potential boost vp , only dihedral potential boost vd , or the dual potential boost ( both vp and vd ) . the dual - boost simulation generally provides higher acceleration than the other two types of simulations for enhanced sampling . the simulation parameters comprise of the threshold energy values and the effective harmonic force constants , k0p and k0d for the total and dihedral potential boost , respectively . to characterize the extent to which v follows gaussian distribution , its distribution anharmonicity , , is calculated as was done previously:12where v is dimensionless as divided by kbt with kb and t being the boltzmann constant and system temperature , respectively , and smax = ( 1/2 ) ln(2ev2 ) is the maximum entropy of v . reweighting by approximating the exponential average term with cumulant expansion to the second order is able to accurately recover the original free energy landscape ( appendix b ) . as increases , the v distribution becomes less harmonic and the reweighted free energy profile obtained from cumulant expansion to the second order deviates from the original . as demonstrated on the alanine dipeptide , chignolin , and t4-lysozyme in this study , a gaussian distribution of the boost potential is normally achieved in the gamd simulations . the anharmonicity of v distribution serves as an indicator of the enhanced sampling convergence and accuracy of the reweighted free energy . gamd is currently implemented in the gpu version of amber 12 ( see appendix a for implementation details ) . simulations of the alanine dipeptide , chignolin , and t4-lysozyme were performed using the amber ff99sb force field on gpus . , the ligand benzene was removed from the x - ray crystal structure of the leu99ala mutant ( protein data bank ( pdb ) , 181l ) . another four benzene molecules were placed in the bulk solvent at least 40 away from the ligand - binding site in the starting configuration . by solvating the structures in a tip3p water box that extends 810 from the solute surface , the alanine dipeptide system contained 630 water molecules , 2,211 waters for chignolin , and 9,011 waters for t4-lysozyme . the total number of atoms in the three systems are 1,912 ; 6,773 ; and 29,692 for alanine dipeptide , chignolin , and t4-lysozyme , respectively ( table 1 ) . bonds containing hydrogen atoms were restrained with the shake algorithm , and a 2 fs time step was used . weak coupling to an external temperature and pressure bath was used to control both temperature and pressure . the electrostatic interactions were calculated using the pme ( particle mesh ewald summation ) with a cutoff of 8.0 for long - range interactions . the three systems were initially minimized for 2,000 steps using the conjugate gradient minimization algorithm , and then the solvent was equilibrated for 50 ps in an isothermal isobaric ( npt ) ensemble with the solute atoms fixed . another minimization was performed with all atoms free , and the systems were slowly heated to 300 k over 500 ps . final system equilibration was achieved by a 200 ps isothermal isovolumetric ( nvt ) and 400 ps npt run to ensure that the water box of the simulated systems had reached the appropriate density . in the present study , the system threshold energy is set as e = vmax for all gamd simulations . the maximum , minimum , average , and standard deviation values of the system potential ( vmax , vmin , vav , and v ) were obtained from an initial 2 ns nvt simulation with no boost potential . for optimal acceleration , the greatest 0 and k0 were determined through short testing simulations ( e.g. , 2 ns ) : with increasing 0 , either k0 calculated using eq 9 reaches 1.0 ( highest acceleration level ) or v reaches 10kbt ( the upper limit of the v distribution width for accurate reweighting ) . each gamd simulation proceeds with a 2 ns equilibration run , followed by production simulations . dihedral potential boost only to identify the optimal 0 and k0 acceleration parameters ( supporting information tables s1s3 ) . long - time dual - boost gamd simulations were then obtained for analysis , including three independent 30 ns simulations of alanine dipeptide and three independent 300 ns simulations of chignolin . complete binding of benzene to the target ligand - binding site was observed in one of the five simulations . even when the simulation was extended to 1,800 ns , time courses of dihedral angles , rmsd , radius of gyration ( rg ) , and residue distances in the amber simulation trajectories were analyzed using the cpptraj tool . particularly , the backbone dihedral angles and were calculated for alanine dipeptide ( figure 2a ) . for chignolin , the rg and rmsd of simulation frames relative to the pdb native structure ( figure 3a ) were calculated for the protein c atoms with the terminal residues gly1 and gly10 excluded . for ligand binding to the t4-lysozyme ( figure 5a ) , the symmetry - corrected rmsd of benzene was obtained by generating six symmetrically imaged reference benzene configurations from the 181l crystal structure , calculating the rmsds for the diffusing benzene molecules in each frame after aligning the protein c - terminal domain ( residues 80160 ) and then extracting the minimum value of the calculated rmsds . moreover , the number of protein atoms found within 5 of benzene ( ncontact , only heavy atoms are considered ) was calculated using pbwithin in vmd that accounts for the periodic boundary conditions . the pyreweighting toolkit was used to reweight the gamd simulations for calculating the pmf profiles and to examine the boost potential distributions . two - dimensional ( 2d ) pmf profiles were computed for backbone dihedrals ( , ) in alanine dipeptide . a bin size of 6 is selected to balance between reducing the anharmonicity and increasing the bin resolution as discussed earlier . two - dimensional pmf profiles were also constructed using ( rmsd , rg ) for chignolin with a bin size of ( 1.0 , 1.0 ) . for benzene binding to the t4-lysozyme , 2d pmf was constructed using ( ligand rmsd , ncontact ) with a bin size of ( 1.0 , 5 ) . when the number of simulation frames within a bin is lower than a certain limit ( i.e. , cutoff ) , the bin is not sufficiently sampled and thus is excluded for reweighting . the cutoff can be determined by iteratively increasing it until the minimum position of the pmf profile does not change . the final cutoff was set as 10 , 50 , and 1000 for reweighting of gamd simulations on alanine dipeptide , chignolin , and t4-lysozyme , respectively . gamd provides enhanced sampling of conformational transitions in alanine dipeptide , chignolin folding , and ligand binding to the t4-lysozyme . furthermore , the boost potential applied in the present gamd simulations follows gaussian distribution , which allows accurate reweighting using cumulant expansion to the second order and recovery of the original biomolecular free energy landscapes , even for proteins as large as the t4-lysozyme . notably , hundreds - of - nanoseconds gamd simulations are able to capture complete folding of chignolin and ligand binding of benzene to the t4-lysozyme that take place on significantly longer time scales . in order to balance between achieving the highest acceleration ( large v ) and ensuring accurate reweighting ( small enough standard deviation , v ) , short gamd simulations ( 2 ns ) were tested on alanine dipeptide to search for optimal acceleration parameters . for the total potential boost , when 0p was adjusted from 1.0 to 2.0 , the resulting k0p calculated using eq 9 was increased from 0.21 to 1.0 , accompanied by increases of the vp standard deviation from 1.03 to 1.75 kcal / mol and the vp average from 2.36 to 3.85 kcal / mol ( see supporting information table s1a ) . after k0p reached its maximum 1.0 , slight changes were observed when 0p was further increased from 2.0 to 3.0 . for the dihedral potential boost , the calculated k0d reached 1.0 when 0d was increased to 3.0 ( supporting information table s1b ) . therefore , ( 0p,0d ) were set to ( 3.0 , 3.0 ) for the production dual - boost gamd simulations of alanine dipeptide , which enables the highest acceleration as well as accurate reweighting with both k0p and k0d equal to 1.0 ( supporting information table s1c ) . as shown in figure 2b , the boost potential v applied in gamd simulation of the alanine dipeptide follows gaussian distribution . the average and standard deviation of v are 10.9 and 2.9 kcal / mol , respectively ( table 1 ) . figure 2c plots the 2d pmf of backbone dihedrals ( , ) that are obtained by reweighting the three 30 ns gamd simulations combined using cumulant expansion to the second order . the reweighted pmf is able to recover five energy minimum wells that are centered around ( 162 , 12 ) and ( 72 , 12 ) for the right - handed helix ( r ) , ( 48 , 18 ) for the left - handed helix ( l ) , ( 156 , 162 ) for the -sheet , and ( 66 , 156 ) for the polyproline ii ( pii ) conformation . the corresponding minimum free energies are estimated as 0 , 1.40 , 1.07 , 2.94 , and 4.27 kcal / mol , respectively . this is in good agreement with the pmf results of an exceptionally long 1000 ns cmd simulation as presented earlier . additionally , the distribution anharmonicity of vof frames clustered in each bin of the 2d pmf exhibits values smaller than 0.10 in the low - energy regions ( figure 2d ) , which suggests sufficient sampling for reweighting using cumulant expansion to the second order . greater anharmonicity is found in high - energy regions ( especially the energy barriers ) , for which low boost potential is applied and less sampling is normally achieved . therefore , the anharmonicity of v distribution appears to be a good indicator for sufficiency of the enhanced sampling and accuracy of the reweighted free energy . ( a ) scheme representation of backbone dihedrals and in alanine dipeptide . ( c ) 2d potential of mean force ( pmf ) of backbone dihedrals ( , ) calculated from three 30 ns gamd simulations combined using cumulant expansion to the second order . the low - energy wells are labeled corresponding to the right - handed helix ( r ) , left - handed helix ( l ) , -sheet ( ) , and polyproline ii ( pii ) conformations . ( d ) distribution anharmonicity of v of frames found in each bin of the pmf profile . for chignolin , short 2 ns testing gamd simulations showed that k0p reached the 1.0 maximum for the highest total potential boost when 0p was increased to 3.0 ( supporting information table s2a ) . for the dihedral potential boost , the simulation crashed when 0d was increased to 0.9 and stable simulation was achieved only when 0d was increased to 0.6 ( supporting information table s2b ) . thus , ( 0p,0d ) were set to ( 3.0 , 0.6 ) for the production dual - boost gamd simulations of chignolin ( supporting information table s2c ) . the resulting boost potential follows gaussian distribution and the calculated = 9.22 10 ( figure 3b ) . the average and standard deviation of v are 9.8 and 2.4 kcal / mol , respectively ( table 1 ) . folding of chignolin captured in gamd simulations : ( a ) comparison of simulation - folded chignolin ( blue ) with the pdb ( 1uao ) native structure ( red ) that exhibits 0.2 rmsd , ( b ) distribution of the boost potential v , ( c ) 2d ( rmsd , rg ) pmf calculated by reweighting the three 300 ns gamd simulations combined , and ( d ) the distribution anharmonicity of v of frames found in each bin of the pmf profile . started from an extended conformation , gamd simulations were able to capture complete folding of chignolin into its native structure ( supporting information movie s1 ) . the rmsd obtained between the simulation - folded chignolin and nmr experimental native structure ( pdb , 1uao ) reaches a minimum of 0.2 ( figure 3a ) . using the protein rmsd relative to the pdb native structure and rg , a 2d pmf profile was calculated by reweighting the three 300 ns gamd simulations combined ( figure 3c ) . the reweighted pmf allows us to identify three distinct low - energy conformational states : the folded ( f ) , unfolded ( u ) , and an intermediate ( i ) . the folded state corresponds to the global energy minimum at ( 1.0 , 4.0 ) , the unfolded state is 3.68 kcal / mol higher in a local - energy well centered at ( 6.0 , 7.0 ) , and the intermediate in a 3.06 kcal / mol free energy well centered at ( 4.0 , 5.5 ) . the energy barrier for chignolin folding between the unfolded and folded states is 4.0 kcal / mol , and 3.5 kcal / mol for transitions between the intermediate and folded states ( figure 3c ) . in comparison , when the three 300 ns gamd simulations were analyzed separately , the reweighted pmf profiles exhibit significant differences ( supporting information figure s2 ) . whereas the three folded , intermediate , and unfolded low - energy states were captured in sim2 , the unfolded and intermediate states were not sufficiently sampled ( no converged low - energy wells ) in sim1 and sim3 , respectively . figure 3d plots the distribution anharmonicity of v for frames found in each bin of the 2d pmf as shown in figure 3c . the anharmonicity exhibits values smaller than 0.05 in the simulation sampled conformational space , suggesting that v achieves sufficient sampling for reweighting using cumulant expansion to the second order . compared with the native structure , the unfolded chignolin exhibits an extended conformation without proper secondary structure formed in the protein backbone as shown in figure 4a . the intermediate conformation observed during folding of chignolin is characterized by hydrophobic interactions between the pro4 and trp9 residue side chains and a turn at thr8 . such conformation was also observed in previous microsecond - time - scale cmd simulations . in the simulation - derived folded state , the protein residue side chains exhibit closely similar conformations as in the nmr native structure ( figure 4c ) . residues tyr2 and pro4 form hydrophobic interactions between their side chains . hydrophilic residues asp3 , glu5 , thr6 , and thr8 expose side chains to the solvent . structures of chignolin observed during gamd simulations corresponding to the ( a ) unfolded ( u ) , ( b ) intermediate ( i ) , and ( c ) folded ( f ) states ( blue ) aligned to the pdb native structure ( red ) . residues including tyr2 , asp3 , pro4 , glu5 , thr6 , thr8 , and trp9 are represented by sticks . finally , a 2d free energy profile of chignolin was calculated using the rmsd of the protein c atoms relative to the native pdb structure and the backbone dihedral of residue gly7 ( supporting information figure s3 ) . this allows for identification of a misfolded ( m ) low - energy state , in addition to the f , i , and u states as observed in figure 3c . the m conformation of chignolin shows 180 rotation of the c - terminal strand about its long axis relative to the native pdb structure . the free energy of the misfolded chignolin is found to be 1.89 kcal / mol greater than that of the folded state . this is consistent with previous findings that the misfolded chignolin was observed with higher probability in simulations with the amber ff99sb force field , which is used in the present study . short testing gamd simulations of the t4-lysozyme showed that k0p reached the 1.0 maximum for the highest total potential boost when 0p was increased to 3.0 ( supporting information table s3a ) . for the dihedral potential boost , the simulation crashed when 0d was increased to 5.0 ( supporting information table s3b ) . stable simulation was achieved when 0d was increased to 4.0 and the calculated k0d was 0.35 . with further testing for stable dual - boost gamd simulation , ( 0p,0d ) were set to ( 3.0 , 4.0 ) for the final production simulations of the t4-lysozyme ( supporting information table s3c ) . gamd captured complete binding of benzene to the deeply buried ligand - binding cavity in the leu99ala t4-lysozyme within 100 ns in one of the five independent 800 ns simulations ( supporting information movie s2 ) . benzene remained bound in the ligand - binding site even when the simulation was extended to 1,800 ns . by aligning the c - terminal domain ( residues 80160 ) of the t4-lysozyme , the rmsd of the diffusing benzene molecules relative to the bound pose in the 181l x - ray crystal structure reaches a minimum of 0.1 ( figure 5a ) . the boost potential applied during the 1,800 ns gamd simulation follows gaussian distribution , and its = 1.39 10 ( figure 5b ) . the average and standard deviation of v are 36.5 and 4.7 kcal / mol , respectively ( table 1 ) . although the v average values exhibit variations between five independent simulations , the v standard deviations are closely similar to each other provided that ( 0p,0d ) were set to ( 3.0 , 4.0 ) ( supporting information table s3c ) . using the rmsd of benzene relative to the bound pose and the number of protein heavy atoms that are within 5 of benzene ( ncontact ) , a 2d pmf profile was calculated by reweighting the 1,800 ns gamd simulation ( figure 5c ) . the reweighted pmf allows us to identify three distinct low - energy states : the unbound ( u ) , intermediate ( i ) , and bound ( b ) states . the bound state corresponds to the global energy minimum located at (0 , 30 ) , the unbound state in a local - energy well centered at (33 , 0 ) , and the intermediate centered at (11 , 20 ) . it is important to note that since the complete binding of benzene to the target ligand - binding site was observed only once , the calculated binding free energy between the bound and unbound states is subject to the error of limited sampling . nevertheless , benzene visits the intermediate site many times during the 1800 ns gamd simulation with the ligand rmsd decreased to 11 ( supporting information figure s2a ) . repeated sampling of the intermediate state was observed in the other four 800 ns gamd simulations as well ( supporting information figure s2 ) , for which a local - energy well appears around ( 11.0 , 20 ) in the 2d pmf profiles ( supporting information figure s3 ) . the relative free energy between the intermediate and unbound states is estimated to be 0.53 0.46 kcal / mol from pmf profiles of the five gamd simulations . furthermore , benzene was observed to bind another intermediate 2 ( i2 ) site that is located in the pocket formed by the hinge c helix and the b helix from the n - terminal domain ( supporting information figure s2 ) . a corresponding local energy well of the i2 state appears in the calculated 2d pmf profiles . figure 5d plots the v distribution anharmonicity , , for frames found in each bin of the 2d pmf . it exhibits relatively large values in the high - energy regions ( less sampling ) , notably the energy barrier between the intermediate and bound states . the ligand entry from the intermediate to the bound state is thus suggested to be the rate - limiting step for benzene binding . in comparison , exhibits values smaller than 0.01 in the energy well regions , suggesting that v achieves sufficient sampling for reweighting using cumulant expansion to the second order . binding of benzene to the leu99ala t4-lysozyme simulated via gamd : ( a ) comparison of simulation - derived complex structure that captures benzene binding ( blue ) with 0.1 ligand rmsd relative to the 181l pdb structure ( red ) , ( b ) distribution of the boost potential v , ( c ) 2d ( ligand rmsd , ncontact ) pmf calculated by reweighting the 1,800 ns gamd simulation , and ( d ) distribution anharmonicity of v of frames found in each bin of the free energy profile . a complete binding pathway of benzene that was observed in the gamd simulation is shown in figure 6a . benzene diffuses from the bulk solvent to the protein surface formed by the d and g helices and then to the target ligand - binding site in the protein c - terminal domain . figure 6b depicts the unbound pose of a benzene molecule that is located far away from the ligand - binding site in the 181l x - ray crystal structure . in the intermediate state , benzene interacts with residues lys83 , pro86 , and val87 from the d helix and the thr115 , thr119 , and gln122 residues from the g helix ( figure 6c ) . in the bound pose , benzene is superimposable with the ligand cocrystallized in the 181l crystal structure . it forms hydrophobic interactions with residues ile78 , leu84 , tyr88 , val87 , leu91 , val111 , leu118 , and leu121 in the deeply buried protein cavity ( figure 6d ) . supporting information figure s5 shows a transient snapshot observed during benzene binding between the intermediate and bound poses . benzene appears to slide into the ligand - binding cavity , interacting with residues gln81 , lys83 , leu84 , val111 , phe114 , thr115 , and leu118 from the d , f , and g helices . ( a ) pathway of benzene binding to the t4-lysozyme observed during the gamd simulation . ( b d ) the ( b ) unbound ( u ) , ( c ) intermediate ( i ) , and ( d ) bound ( b ) poses of the protein ligand complex ( blue ) with the protein c - terminal domain ( residues 80160 ) aligned to the pdb native structure ( red ) . the protein and benzene are represented by ribbons and spheres , respectively , and they are colored by blue for the simulation structure while red for the pdb native structure , except that in a the simulated benzene is represented by lines and colored by simulation time in a bwr color scale . residues with heavy atoms found within 3 of benzene are represented by sticks . by adaptively adding a harmonic boost potential to smoothen the system energy surface , gamd provides both unconstrained enhanced sampling and free energy calculation of biomolecules . important statistical properties of the system potential , such as the average , maximum , minimum , and standard deviation values , are used to calculate the simulation acceleration parameters , particularly the threshold energy , e , and force constant , k0 . for alanine dipeptide , both k0p and k0d can be increased to the 1.0 maximum for the greatest possible boost of the total and dihedral potential energies . the resulting standard deviation of v that follows gaussian distribution is 2.9 kcal / mol , which allows for accurate reweighting using cumulant expansion to the second order . notably , high - energy regions of the gamd sampled free energy surface ( especially the energy barriers ) exhibit increased anharmonicity compared with the low - energy wells ( figure 2d ) . thus , the free energy barriers appear unconverged and still suffer from insufficient sampling during the three short 30 ns gamd simulations . nevertheless , the gamd reweighted pmf profile is able to recover low - energy states of the system that are in good agreement with those observed in a 1000 ns cmd simulation . furthermore , gamd is able to fold chignolin from an extended conformation to the nmr native structure in three independent 300 ns simulations . while k0p can be increased to 1.0 for the greatest total potential boost , k0d can reach only 0.150.17 for stable simulation . nevertheless , this seems to provide sufficient sampling of the chignolin - folding process . among the three independent simulations , although the folded , intermediate , and unfolded low - energy states are captured in sim2 , the unfolded and intermediate states are not sufficiently sampled in sim1 and sim3 , respectively ( supporting information figure s2 ) . all three folded , intermediate , and unfolded states are identified from the resulting reweighted free energy profiles . particularly , the intermediate conformation was also observed in earlier microsecond - time - scale cmd simulations . gamd appears to achieve better convergence than the previous amd simulations that were not able to distinguish between the intermediate and unfolded states within the same simulation time . finally , gamd captured complete binding of benzene to the ligand - binding site of the t4-lysozyme . in one of the five independent simulations , benzene diffused into the deeply buried ligand - binding cavity within 100 ns and remained bound even when similar to chignolin , while k0p can be increased to the 1.0 maximum for the greatest total potential boost in the t4-lysozyme simulations , k0d can reach only 0.330.35 for the greatest dihedral boost . the resulting v standard deviation is 4.7 kcal / mol in the final dual - boost gamd simulations . such narrow distribution of v ensures accurate reweighting using cumulant expansion to the second order . distinct low - energy unbound , intermediate , and bound states were identified from the reweighted pmf profiles . the atomistic gamd simulation also elucidates a highly detailed binding pathway of benzene that diffuses from the bulk solvent to an intermediate site located on the protein surface formed by the d and g helices and then slides into the target ligand - binding cavity through a channel formed by the d , f , and g helices . the free energy difference between the intermediate and unbound states was found to be small at 0.53 0.46 kcal / mol as estimated from the five independent gamd simulations . benzene repeatedly visits the intermediate site on the protein surface . in comparison , the ligand entry from protein surface to the deeply buried protein cavity appears to be the rate - limiting step for complete benzene binding . it is important to note that the complete ligand binding was not observed in the four 800 ns gamd simulations , suggesting that the present gamd simulations still suffer from insufficient sampling of the ligand entry process and the reweighted free energy profiles remain unconverged ( figure 5c and supporting information figure s5 ) . this is also indicated by the increased anharmonicity corresponding to the free energy barrier between the intermediate and bound states as shown in figure 5d . nevertheless , our gamd simulation captured a binding pathway of benzene to the t4-lysozyme . the ligand entry site is indeed adjacent to the mobile f helix ( residues 108113 ) , which has been suggested earlier based on the finding that the f helix exhibits increased b - factors in the leu99ala complex structures compared to the apo structures . here , gamd is compared with the original amd , particularly on the performance of smoothening the potential energy surface and energetic reweighting . supporting information table s4 summarizes the statistical properties of the boost potential and the original and modified potential energies as obtained from amd and gamd simulations of the alanine dipeptide , chignolin , and t4-lysozyme , with reference to the cmd simulations . specifically , dual - boost amd simulations of the alanine dipeptide and chignolin obtained from our previous study were used for comparison . for the t4-lysozyme , restarting from the initial configuration of gamd simulations , a 200 ns dual - boost amd simulation was performed using the following acceleration parameters : edihed = vdihed_av + 4nres , dihed = 4nres/5 , etotal = vtotal_av + 0.2natoms , and total = 0.2natoms , where vdihed_av and vtotal_av are the average dihedral and total potential energies calculated from a short 10 ns cmd simulation . as shown in supporting information table s4 , although higher average boost potentials are applied in gamd simulations of the three systems than in the amd simulations , the boost potentials exhibit smaller standard deviations ( i.e. , narrower distribution ) in the gamd simulations , except for the alanine dipeptide . furthermore , anharmonicity of the boost potential distribution is significantly reduced in the gamd simulations . relative to cmd , both amd and gamd simulations mostly exhibit smaller standard deviations in the modified dihedral and total potential energies . notably for the t4-lysozyme , standard deviations of the modified potential energies are significantly smaller in the gamd simulations than in amd . thus , the potential energy surfaces appear more smoothened in gamd enhanced sampling of the t4-lysozyme . provided the narrower distribution and lower anharmonicity of the boost potential ( supporting information table s4 ) , gamd allows for more accurate approximation of exponential average reweighting factor using cumulant expansion to the second order and thus improved free energy calculation , especially for the t4-lysozyme . in the present simulations of ligand binding to the t4-lysozyme , complete binding of benzene was observed only once , largely because the target ligand - binding site is deeply buried in the protein . however , benzene binding to the intermediate site on the protein surface formed by the d and g helices was captured many times , which provides better statistics for free energy calculation . it has been suggested that a pmf - based approach is appropriate to calculate binding free energy for ligands ( especially the charged ) that bind to the protein surface . systems of this type for future applications of gamd might include binding of benzamidine to trypsin and allosteric modulators to the protein surface of the g - protein coupled receptors . in comparison with many enhanced sampling methods such as umbrella sampling , conformational flooding , metadynamics , abf calculations , and orthogonal space sampling , gamd has the advantage of no need to set predefined reaction coordinates . metadynamics , in particular , is another potential biasing technique that has been widely used to map the free energy landscapes of biomolecules such as protein conformational changes and protein ligand binding . by monitoring the energy surface of biomolecules during the simulation , metadynamics keeps adding small gaussians of potential energies to the low - energy regions . this will eventually fill the low - energy wells and achieve uniform sampling of the free energy surface along selected reaction coordinates . the usage of predefined coordinates greatly reduces the complexity of biomolecular simulation problems and facilitates the free energy calculations ( e.g. , significantly lower energetic noise compared with amd simulations ) . however , it is key to select proper reaction coordinates , which often requires expert knowledge of the studied systems . construction of biomolecular reaction coordinates or collective variables has thus been one of the main objectives in metadynamics studies . when important reaction coordinates are missed during the simulation setup , metadynamics simulations may suffer from slow convergence problems as discussed earlier . furthermore , the predefined reaction coordinates tend to place constraints on the sampled space and pathways . it seems difficult to identify certain intermediate states during the protein - folding and ligand - binding pathways , e.g. , intermediate 2 as observed during the binding of benzene to the t4-lysozyme . in comparison , amd simulations are not constrained by reaction coordinates , but this also leads to much higher energetic noise and presents grand challenge for accurate reweighting to recover the original free energy landscapes of biomolecules . although cumulant expansion to the second order was shown to improve amd reweighting when the boost potential follows near gaussian distribution , such improved reweighting is still limited to small systems such as protein with 35 residues . here , by constructing boost potential using a harmonic function that follows gaussian distribution , gamd enables rigorous energetic reweighting through cumulant expansion to the second order , even for simulations of larger proteins ( e.g. , t4-lysozyme ) . with this first , while the present gamd simulations seem to provide sufficient sampling of the low - energy regions , they appear to remain unconverged in sampling of the high - energy barriers . this is particularly true for the ligand entry step in the gamd simulation of benzene binding to the t4-lysozyme . it is worth recalling that the threshold energy for adding the boost potential is set to its lower bound in the present gamd simulations . it is subject to future investigation into whether using the upper bound of the threshold energy will facilitate sampling of the high - energy barriers in gamd simulations . second , based on a potential biasing approach , gamd mainly accelerates transitions across enthalpic energy barriers . improvement for its application to systems with high entropic barriers is still in need . on this regard , gamd can be potentially combined with the parallel tempering and replica exchange algorithms for further enhanced sampling . particularly , the combination of parallel tempering and metadynamics ( pt - metad ) has been shown to facilitate enhanced sampling of biomolecules over entropic barriers . in summary , without the need to set predefined reaction coordinates , gamd is generally applicable to a wide range of biomolecular systems as demonstrated on protein folding and ligand binding in this study . for systems of increasing size , the upper limit of the v standard deviation , 0 can be adjusted dynamically to ensure that the distribution width of the applied boost potential is narrow enough for accurate energetic reweighting using cumulant expansion to the second order . therefore , gamd provides both unconstrained enhanced sampling and free energy calculation for biomolecular simulations .

objectivesexperimental models are of importance to study the pathogenesis of middle ear cholesteatoma , however , they were not established until now . we aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue . hacat cell line was used as a " skin equivalent " and to compare the grade of homogeneity between cholesteatoma keratinocytes and hacat cells.methodsprimary keratinocytes were isolated from cholesteatoma tissue , co - cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air - exposed . the protein profile of cholesteatoma keratinocytes and hacat cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin ( ck ) 13 and 16 . tissue localization of ck 13 and 16 was accomplished with immunohistochemistry.resultsdifferent protein profile and stronger expression of ck 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with hacat cells . bigger stratification was observed in the 3d - in vitro systems when both cholesteatoma keratinocytes and hacat cells were respectively co - cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts.conclusion3d-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue . a gaussian accelerated molecular dynamics ( gamd ) approach for simultaneous enhanced sampling and free energy calculation of biomolecules is presented . by constructing a boost potential that follows gaussian distribution , accurate reweighting of the gamd simulations is achieved using cumulant expansion to the second order . here , gamd is demonstrated on three biomolecular model systems : alanine dipeptide , chignolin folding , and ligand binding to the t4-lysozyme . without the need to set predefined reaction coordinates , gamd enables unconstrained enhanced sampling of these biomolecules . furthermore , the free energy profiles obtained from reweighting of the gamd simulations allow us to identify distinct low - energy states of the biomolecules and characterize the protein - folding and ligand - binding pathways quantitatively .

a 40-year - old , previously healthy man presented with left hemiparesis and left facial weakness which had developed four hours before admission after being struck on the right side of the neck by the edge of a door . ct and mri of the brain revealed acute infarction in the vascular territory of the right middle cerebral artery . intravenous heparinization was initiated , thus maintaining a twice - normal activated partial thromboplastin time ( aptt ) . angiography performed on admission revealed near - complete occlusion of the suprabulbar portion of the right internal carotid artery and complete occlusion of the ipsilateral proximal middle cerebral artery , suggesting dissection of the vessel and subsequent arterial thromboembolism ( figs . following admission the patient 's mental status deteriorated suddenly , and ct revealed a large intracerebral hemorrhage within the territory of the infarcted right middle cerebral artery . follow - up angiography at day 20 revealed partial restoration of luminal patency ; the lumen , however , was largely compromised by the presence of a thick intimal flap ( fig . because of the risk of continuing anticoagulation therapy , endovascular treatment of the dissection was planned . two days before angioplasty , performed at day 35 , daily doses of aspirin ( 100 mg ) and ticlopidine ( 250 mg ) were initiated and continued thereafter . a heparin bolus of 5000 iu was injected intravenously , and in order to maintain aptt at 1.5 to 2 times normal , was continued for one day . atropine ( 1 mg ) was injected intravenously just before stent placement ; for primary stenting of the dissected segment , a self - expandable uncovered metallic stent 30 mm in length and 8 mm in diameter ( easy wallstent ; boston scientific corporation , watertown , mass . , u.s.a . ) was used ( fig . 1d ) , and for postdilatation , a 6-mm diameter angioplasty catheter ( ultra - thin ; boston scientific corporation , watertown , mass . , u.s.a . ) at a low inflation pressure was employed . intermittent neurological examinations were conducted throughout the procedure , and after stenting , focal residual dissected false lumen at the posterior portion of the artery was observed ( fig . interestingly , the symptom of left side weakness showed slight improvement two days after stent placement , motor power having increased from grade 2 to 3 . thirty - six days after the procedure , follow - up axial ct scanning and 2d reconstruction images showed complete reconstitution of the arterial lumen and disappearance of the false lumen . ticlopidine was discontinued . a 39-year - old man presented with multiple fractures of the lower extremities and a fractured left zygoma and skull base after a motor vehicle accident . brain ct performed immediately after arrival in the emergency room showed no significant abnormality and orthopedic management was initiated . on the fourth day following admission , however , the patient showed mild weakness of the right extremities , and brain ct revealed cortical areas of low density at the left frontal and occipital lobes . angiography was performed the following day , and focal but significant stenosis associated with a dissecting aneurysm was observed at the subpetrosal portion of the left internal carotid artery ( fig . because the patient was not in a fit condition for sustained anticoagulation therapy , percutaneous angioplasty aimed at reestablishing the vessel lumen was performed during the same session . a 7-fr guiding catheter was introduced into the proximal portion of the internal carotid artery , and to prevent thromboembolic complications a heparin bolus of 5000 iu was administered intravenously . for primary stenting of the affected portion of the vessel , a premounted balloon - expandable uncovered stent ( nir primo ; boston scientific scimed , inc . , maple grove , minn . this was 16 mm in length and was dilated to 4.2 mm with a balloon at a pressure of 10 atm ( fig . a postangioplastic angiogram showed improved luminal patency , with reduction of the aneurysmal sac ( fig . 2c ) . aspirin ( 100 mg / day ) and ticlopidine ( 250 mg / day ) were subsequently administered , and to maintain aptt at 1.5 to 2 times normal , [ jw2]heparinization was continued for five days after stenting . although the filled aneurysmal sac was significantly smaller , focal filling of a part of it was noted ( fig . the patient 's symptom was improved gradually after the procedure , and the motor power of the affected extremities returned to normal at the time of follow - up angiography . a 40-year - old , previously healthy man presented with left hemiparesis and left facial weakness which had developed four hours before admission after being struck on the right side of the neck by the edge of a door . ct and mri of the brain revealed acute infarction in the vascular territory of the right middle cerebral artery . intravenous heparinization was initiated , thus maintaining a twice - normal activated partial thromboplastin time ( aptt ) . angiography performed on admission revealed near - complete occlusion of the suprabulbar portion of the right internal carotid artery and complete occlusion of the ipsilateral proximal middle cerebral artery , suggesting dissection of the vessel and subsequent arterial thromboembolism ( figs . following admission the patient 's mental status deteriorated suddenly , and ct revealed a large intracerebral hemorrhage within the territory of the infarcted right middle cerebral artery . follow - up angiography at day 20 revealed partial restoration of luminal patency ; the lumen , however , was largely compromised by the presence of a thick intimal flap ( fig . because of the risk of continuing anticoagulation therapy , endovascular treatment of the dissection was planned . two days before angioplasty , performed at day 35 , daily doses of aspirin ( 100 mg ) and ticlopidine ( 250 mg ) were initiated and continued thereafter . a heparin bolus of 5000 iu was injected intravenously , and in order to maintain aptt at 1.5 to 2 times normal , was continued for one day . atropine ( 1 mg ) was injected intravenously just before stent placement ; for primary stenting of the dissected segment , a self - expandable uncovered metallic stent 30 mm in length and 8 mm in diameter ( easy wallstent ; boston scientific corporation , watertown , mass . , u.s.a . ) was used ( fig . 1d ) , and for postdilatation , a 6-mm diameter angioplasty catheter ( ultra - thin ; boston scientific corporation , watertown , mass . , u.s.a . ) at a low inflation pressure was employed . intermittent neurological examinations were conducted throughout the procedure , and after stenting , focal residual dissected false lumen at the posterior portion of the artery was observed ( fig . interestingly , the symptom of left side weakness showed slight improvement two days after stent placement , motor power having increased from grade 2 to 3 . thirty - six days after the procedure , follow - up axial ct scanning and 2d reconstruction images showed complete reconstitution of the arterial lumen and disappearance of the false lumen . a 39-year - old man presented with multiple fractures of the lower extremities and a fractured left zygoma and skull base after a motor vehicle accident . brain ct performed immediately after arrival in the emergency room showed no significant abnormality and orthopedic management was initiated . on the fourth day following admission , however , the patient showed mild weakness of the right extremities , and brain ct revealed cortical areas of low density at the left frontal and occipital lobes . angiography was performed the following day , and focal but significant stenosis associated with a dissecting aneurysm was observed at the subpetrosal portion of the left internal carotid artery ( fig . because the patient was not in a fit condition for sustained anticoagulation therapy , percutaneous angioplasty aimed at reestablishing the vessel lumen was performed during the same session . a 7-fr guiding catheter was introduced into the proximal portion of the internal carotid artery , and to prevent thromboembolic complications a heparin bolus of 5000 iu was administered intravenously . for primary stenting of the affected portion of the vessel , a premounted balloon - expandable uncovered stent ( nir primo ; boston scientific scimed , inc . , maple grove , minn . this was 16 mm in length and was dilated to 4.2 mm with a balloon at a pressure of 10 atm ( fig . a postangioplastic angiogram showed improved luminal patency , with reduction of the aneurysmal sac ( fig . 2c ) . aspirin ( 100 mg / day ) and ticlopidine ( 250 mg / day ) were subsequently administered , and to maintain aptt at 1.5 to 2 times normal , [ jw2]heparinization was continued for five days after stenting . follow - up angiography performed 65 days after stent placement showed good luminal patency . although the filled aneurysmal sac was significantly smaller , focal filling of a part of it was noted ( fig . the patient 's symptom was improved gradually after the procedure , and the motor power of the affected extremities returned to normal at the time of follow - up angiography . carotid dissection is initiated by hemorrhage into the medial layer of the artery due to various causes . where the dissection primarily involves the subintima , stenosis or occlusion of the artery usually results , and angiography reveals the so - called ' string sign ' . if the subadventitia is principally involved , an out - pouching sac , a dissecting aneurysm in the form of an outponihing sae usually arises . luminal stenosis can cause distal ischemia leading to stroke or transient ischemic attacks . in both subintimal and subadventitial dissections the resulting exposure of the basement membrane leads to platelet aggregation , and this serves as an embolic source ( 1 ) . it has been suggested that ischemic symptoms are more often secondary to embolic phenomena than to hypoperfusion caused by stenosis ( 2 ) . because many cases escape diagnosis or patients receive some form of therapy , the natural history of carotid dissection remains somewhat unclear . although its clinical course may be benign ( 1 ) , overall mortality rates have been consistently reported at 20 - 40% ( 3 ) . in addition , in a recent study of the time course of symptoms in extracranial carotid artery dissection , the authors observed ischemic events in 82.5% of afflicted patients and complete stroke in two - thirds ( 12 ) . these results imply that the time course of carotid dissection is not at all benign , and the same authors emphasized the need for early preventive measures to avoid ischemic complications ( 12 ) . the management goal in cases of extracranial carotid dissection is to prevent the development or worsening of neurologic deficits by preventing thrombosis and embolization ( 3 ) : options include observation , anticoagulation therapy , surgery , and endovascular treatment . fabian et al . suggested that anticoagulative measures should be used in all non - occlusive arterial dissections without contraindications , regardless of the degree of luminal narrowing ( 13 ) . anticoagulation is often initially achieved with heparin and then , after 1 - 3 weeks , maintained with warfarin , which is continued for up to 6 months . surgical repair is reserved for the management of symptomatic patients with lesions in an accessible location ( 3 ) , but the technique for the preservation of the artery is technically demanding and time consuming . for many years , endovascular occlusion of the affected artery has involved the use of detachable balloons or coils as an alternative to surgery . the advent of stenting techniques in carotid artery angioplasty has , however , led to their exploitation in the preservation of arterial patency . the techniques are much easier and safer than surgical repair , and published case reports have described their successful application ( 4 - 11 ) . various stents can be used for the restoration of luminal patency and occlusion of the sac of a dissecting aneurysm : some authors have insisted that a covered stent is especially useful ( 11 ) , though stents without fabric covering have also been used for the same purpose with successful results , as in our cases . to obliterate dissecting aneurysms , guglielmi detachable coils along with stents have also been used ( 10 ) . for suprabulbar internal carotid artery dissection we applied a self - expandable stent , and for subpetrosal internal carotid artery dissection , the balloon - expandable type : in both cases the results were successful . emphasized the superiority of self - expandable stents over the balloon - expandable type in terms of their radial expansile force and longitudinal flexibility ( 5 ) . they surmised that the first of these two factors was helpful for effectively sealing off the dissecting aneurysm , and in a case involving the use of a balloon - expandable stent the aneurysm sac also effectively disappeared . the balloon - expandable stent we used was also flexible , a feature which allowed us to place it in an angled portion of the vessel . follow - up showed that the softness of the stent did not compromise luminal patency of the vessel . selection of a stent must be individualized on the basis of factors which include the characteristics and location of the lesion and the diameter and geometry of the vessel . during the management of case 1 , we delayed stent placement because of an infarction in the patient 's right middle cerebral artery territory due to embolic occlusion . where there is overt parenchymal infarction , as in that patient for the stabilization of already - infarcted parenchyma to which anticoagulative measures have been applied , a wait of three to four weeks is recommended , though in case 1 there was unexpected hemorrhagic transformation of the infarction . because flow through the patent anterior communication artery was good , management could have involved occlusion of the dissected ica : because of the relative ease and safety of the endovascular recanalization methods now available , however , a destructive method of this kind was not considered . in case 2 , the cause of the left cerebral cortical infarction was not clear . because a dissecting aneurysm is already known to be a potential embolic source , the dissected segment was recanalized after initial angiography without evaluating the significance of the stenosis . angiography appeared to indicate that this was narrow enough to cause a watershed infarction due to hemodynamic insufficiency , and that stenting of the stenotic segment could recanalize the stenosis as well as seal the aneurysmal sac . follow - up angiography , however , revealed a focal residual sac . suggesting that the dissection had opened : in such cases endovascular stent placement is a safe and effective method for the management of patients with extracranial carotid artery dissections with or without associated aneurysm .

extracranial carotid artery dissection may manifest as arterial stenosis or occlusion , or as dissecting aneurysm formation . anticoagulation and/or antiplatelet therapy is the first - line treatment , but because it is effective and less invasive than other procedures , endovascular treatment of carotid artery dissection has recently attracted interest . we encountered two consecutive cases of trauma - related extracranial internal carotid artery dissection , one in the suprabulbar portion and one in the subpetrosal portion . we managed the patient with suprabulbar dissection using a self - expandable metallic stent and managed the patient with subpetrosal dissection using a balloon - expandable metallic stent . in both patients the dissecting aneurysm disappeared , and at follow - up improved luminal patency was observed . techniques for isolation and culture of fetal type ii alveolar epithelial cells , as well as the morphologic and biochemical characteristics of these histotypic cultures , are described . type ii alveolar epithelial cells can be isolated from fetal rat lungs and grown in an organotypic culture system as described in this review . the fetal type ii cells resemble differentiated rat type ii cells in morphology , biochemistry , and karyotype as they grow in culture for up to 5 weeks . the cells of the mature organotypic cultures form alveolarlike structures while growing on a gelatin sponge matrix . the type ii cells also synthesize and secrete pulmonary surfactant similar in biochemical composition to that produced in vivo . this system has been used to study the effects of hormones on surfactant production and composition . the organotypic model has many potential applications to the study of pulmonary toxicology.imagesfigure 1.figure 2 .

allogeneic bone marrow transplantation ( bmt ) is widely used in the treatment of various hematologic disorders , such as leukemia and aplastic anemia , and the major complication of bmt is graft - versus - host disease ( gvhd ) . we discuss whether autologous melanocyte transplantation is an appropriate way as a treatment of the vitiligo after allogeneic bmt . in this report we describe a patient with hodgkin 's lymphoma who developed universal vitiligo after allogeneic bmt from his sister . he underwent four times melanocyte - keratinocyte transplantation ( mkt ) for the treatment of vitiligo ; all treated depigmented patches had a significant repigmentation . an 18-year - old patient was diagnosed with epstein - barr virus - related hodgkin - like lymphoma in 2003 , and antivirotic had been used to control his lymphoma , but the treatment was ineffective . then in march 2009 , he received the histocompatibility leukocyte antigen ( hla)-matched allogeneic bmt from his sister . cyclosporine and methotrexate were used as a prophylaxis strategy to prevent gvhd for 6 months after the allogeneic bmt . during the 6-month recovering period , the patient developed a small erythematous rash , which was consistent with the clinical features of graft - versus - host disease ( gvhd ) of the skin . no external agents were used and the rash dissipated in a short period of time . small depigmented macules started to appear on his cheek 11 months after allogeneic bmt in february 2010 , and followed by rapid progression to the whole body from the face to hands within 1 month . we indicated it to be a typical universal vitiligo by the reason that the depigmentation macules had a chalky white appearance under wood 's light . moreover , leukotrichia appeared at the same time associated with his vitiligo , affecting back and arms [ figure 1 ] . the patient with extensive vitiligo of the back and arms the depigmentation macules did not expanded any more after he received whole - body nb - uvb therapy twice a week for 2 months . simultaneously , following the doctor 's advice , 0.1% tacrolimus was used and traditional chinese medicine was orally taken . the patient underwent the therapies mentioned above for 8 months in total , but with poor repigmentation . then he discontinued medical therapies and performed non - cultured mkt on january 17 , 2011 , including left forehead and temple [ figure 2 ] . almost complete repigmentation was shown after 8 months since the first transplantation [ figure 3a ] . then he received mkt on his right face on september 29 , 2011 . and 8 months after the second mkt , 28 months after the first - time transplantation , all of the transplanted areas had almost completely repigmented and the color of the repigmented area matched with the normal surrounding skin excellently [ figure 3c ] . then the patient underwent the third and the fourth mkt procedure on the right and the left side of his neck respectively on may 28 , 2012 and january 22 , 2013 [ figure 4a ] . significant improvement was shown in the area around the left side of his neck while there 's also a majority repigmentation on the right side [ figure 4b and c ] . no hyperpigmentation , scar , infection and any other adverse effects on both recipient and donor sites by now were noticed . leukasmus involving the face symmetrical , around the nose , eyes and mouth repigmentation of vitiligo by mkt on his face . ( c ) 28 months after the first time transplantation , significant repigmentation obtained and normal skin achieved mkt performed on his neck . ( b ) 12 months after the surgery of right neck showing nearly 100% repigmentation . vitiligo is a most common depigmentation disorder and affects 0.5 - 2.0% of the world population . the etiology of vitiligo remains obscure , the most prevalent hypothesis today considers vitiligo as an autoimmune disease and focuses on a melanocyte - specific cytotoxic t - cell immune reaction in the destruction of melanocytes . supported that there perhaps be four explanations for the development of vitiligo after bmt , such as the destruction of melanocytes stimulated by pretransplantation chemotherapy and radiotherapy , chronic gvhd , infusion of a larger number of lymphocytes and adoptive passive transfer from the donor to the recipient . in the case of our patient , vitiligo might be a result of an immune response directed against the melanocyte destruction initiated by gvhd . there are reported cases about allogeneic posted - bmt vitiligo . but most of the pre - bmt diagnosis were chronic myelogenous leukemia , none of them were diagnosed with hodgkin 's lymphoma as our report . in terms of treatment during those cases , just partial of them accepted therapies , but none of them performed with transplantation . in the bmt context , the extensive and relentless vitiligo progression indicated an aggressive alloimmune process , so the destruction of melanocyte is alloimmune in nature . there was no report to make discriminate of melanocyte destruction between alloimmune and autoimmune , but au et al . proposed the success of melanocyte autografts for autoimmune vitiligo could be used in an alloimmune setting . therefore , we came up with the idea of vitiligo melanocyte transplantation while other treatment was invalid for the patient . melanocyte grafting technique include cultured autologous melanocyte suspension transplantation and autologous noncultured melanocyte - keratinocyte . in our report , the patient obtained universal vitiligo after allogeneic bone marrow transplantation for hodgkin 's lymphoma . in the condition of vitiligo failed to respond to non - surgical treatment , the patient underwent four times of mkt during 2 years . the largest area of treated lesions was 50 cm ; repigmentation rate achieved more than 95% after each transplantation without any adverse effects or koebner 's phenomenon . the pattern of the repigmentation was uniform in the first three times mkt but diffuse in the last one ; whatever , more follow - up time is needed in the future . after 2 years of follow - up , graft sites still repigmented obviously , and the color was similar to the surrounding normal skin which illustrated mkt can treat allogeneic vitiligo successfully . this is the first time to report about treating vitiligo after allogeneic bone marrow transplantation by mkt . we suggest that autotransplantation especially autologous grafting with noncultured melanocyte is simple , safe , and inexpensive for remedying the loss of melanocyte after bmt in the case of depigmented macules is stable . but whether the melanocyte transplantation used in alloimmune vitiligo is effective as in autoimmune , we need more follow - ups . this is the first time to report about treating vitiligo after allogeneic bone marrow transplantation for hodgkin 's lymphoma by non - cultured melanocyte - keratinocyte transplantation without koebner 's phenomenon and adverse effects but all successful . it probably suggests that therapy of transplantation can be used in the treatment of vitiligo after allogeneic bone marrow transplantation .

defining alterations in signalling pathways in normal and malignant cells is becoming a major field in proteomics . a number of different approaches have been established to isolate , identify and quantify phosphorylated proteins and peptides . in the current report , a comparison between scx prefractionation versus an antibody based approach , both coupled to tio2 enrichment and applied to tmt labelled cellular lysates , is described . the antibody strategy was more complete for enriching phosphopeptides and allowed the identification of a large set of proteins known to be phosphorylated ( 715 protein groups ) with a minimum number of not previously known phosphorylated proteins ( 2 ) . the treatment of vitiligo is derisory since the pathogenesis of vitiligo is not clear at present . most conservative treatments are difficult to approach satisfactory therapy . so transplantation is the only way left when the disease becomes insensitive to those conservative treatments . here we describe an 18-year - old patient who developed vitiligo , which was triggered by graft - versus - host disease after a allogeneic bone marrow transplantation for the treatment of hodgkin 's lymphoma from his sister . in the following treatment to vitiligo , the patient successfully performed the transplantation of autologous uncultured melanocyte on the premise of poor reaction to other conservative methods . we infer that transplantation can be a treatment of the vitiligo after allogeneic bone marrow transplantation .

spondylolisthesis is defined as an anterior migration of a vertebral body in relation to the vertebra located immediately caudal . in 1930 junghanns was the first to describe anterior translation of a lumbar vertebra without any defect in the neural arch following this the term degenerative spondylolisthesis ( ds ) was introduced by newman in 1955 . five types of spondylolisthesis have been described including dysplastic , isthmic , traumatic , pathologic , and degenerative . there are many predisposing factors like sagittally - placed facet joint , a high iliac crest [ 4 - 6 ] , etc . ds which is characterized by an intact vertebral ring is presumed to result from degeneration of facet joints and intervertebral discs with aging and thus has traditionally been considered to represent instability of the vertebral segment . a 66-year - old gentleman , farmer by occupation , came with complaints of lower backache for 2 years which was insidious in onset , gradually progressive and non - radiating . he also complained of acute retention of urine since 15 days for which he was catheterized . he also had a history of neurogenic claudication at a distance of 100 m. there was no history of any trauma . a detailed systemic and neurological examination revealed power of flexor hallucis longus(fhl ) and flexor digitalis longus(fdl ) as 4/5 , ankle jerks were absent , sensory deficits in s1-s2 dermatome and per - rectal examination revealed decreased perianal sensations and anal tone with absent anal wink . plain radiographs of lumbo - sacral spine revealed spondylolisthesis of s1-s2 ( meyerding s grade 1 ) ( fig.1 ) . magnetic resonance imaging and computed tomography scan of the spine revealed lumbarization of s1 with spondylolisthesis of s1 over s2 , facetal hypertrophy at l5-s1 and canal stenosis at s1-s2 ( figs . 2 , and 3 ) . anteroposterior ( a ) and lateral ( b ) radiographs showing grade 1 spondylolisthesis ats1-s2b . the patient underwent posterior spine surgery where decompression was done with laminotomy of s1 bilaterally and then pedicular screw fixation was done bilaterally at l5 , s1 , and s2 ( fig.4 ) . s1 and post - operative radiographs - anteroposterior ( a ) and lateral ( b ) view . the bladder symptoms disappeared after 3 weeks and the power of fhl / fdl improved from 4/5 to 5/5 . the patient underwent posterior spine surgery where decompression was done with laminotomy of s1 bilaterally and then pedicular screw fixation was done bilaterally at l5 , s1 , and s2 ( fig.4 ) . s1 and post - operative radiographs - anteroposterior ( a ) and lateral ( b ) view . the bladder symptoms disappeared after 3 weeks and the power of fhl / fdl improved from 4/5 to 5/5 . the patient underwent posterior spine surgery where decompression was done with laminotomy of s1 bilaterally and then pedicular screw fixation was done bilaterally at l5 , s1 , and s2 ( fig.4 ) . s1 and post - operative radiographs - anteroposterior ( a ) and lateral ( b ) view . the bladder symptoms disappeared after 3 weeks and the power of fhl / fdl improved from 4/5 to 5/5 . the deformity occurs at l4 - 5 6 times more often than at other lumbar levels and four times more often above a sacralized l5 . the lumbosacral junction and middle lumbar spine are most often involved , but the lesion is also found in cervical or rarely the thoracic vertebra . to the best of our knowledge , ds of sacral vertebrae has not been reported in the available english literature till now . the prevalence of complete lumbarization is 1.8% and to get a spondylolisthesis is even rarer . there have been many publications in the literature mentioning incidence of spondylolisthesis with sacralization but hardly any on spondylolisthesis with lumbarization . further case series or longitudinal studies of such cases may help understand better the pathomechanics related to spondylolisthesis at this level . ds of s1-s2 is a very rare entity and further case reports will help us to explore the biomechanics at this level . the research protocol was approved by the institutional review board at the oregon health & science university ( ohsu ) and performed in accordance with the tenets of the declaration of helsinki . written informed consent was obtained from each participant after explanation of the nature and possible consequences of the study . participants were recruited at the casey eye institute / ohsu according to the aig study protocol . the ohsu ancillary site followed the same eligibility and endpoint protocol as the aig study , but used an advanced swept - source oct prototype system instead of commercially available oct instruments . briefly , normal control participants met the following criteria in both eyes : iop of less than 21 mm hg in both eyes , and a normal humphrey visual field ( hvf ) on achromatic standard automated perimetry by swedish interactive threshold algorithm 24 - 2 testing ( hfa ii ; carl zeiss meditec , inc . , dublin , ca , usa ) with mean deviation ( md ) , glaucoma hemifield test ( ght ) , and pattern standard deviation ( psd ) within normal limits . in addition , normal subjects had a normal - appearing optic nerve head ( onh ) and nfl on ophthalmoscopic examination , and an open angle by gonioscopy . glaucoma participants were required to have a glaucomatous onh rim or nfl thinning on ophthalmoscopic examination . glaucomatous eyes were classified in the pg subgroup if they have visual field ( vf ) psd or ght outside normal limits ( p < 0.05 and p < 1% , respectively ) in a consistent pattern on two qualifying vf exams . exclusion criteria for all groups included visual acuity less than 20/40 , age < 40 or > 79 years at enrollment , any ocular surgery other than uncomplicated cataract extraction , other diseases that might cause vf or onh abnormality , and factors that might preclude the participant from performing study procedures or completing the study . a prototype high speed swept - source fourier - domain oct system was used in this study . the device operated at an axial scan speed of 100 khz using a swept - source cavity laser operating at 1050 nm with a tuning range of 100 nm . a resolution of 5.3 m axially and 18 m laterally at an imaging depth of 2.9 mm in tissue was achieved . the ocular light power exposure was 1.9 mw , within the american national standards institute ( ansi ) safety limit . participants were scanned using a high density 8 8 mm raster scan pattern , centered at the onh . in the fast transverse scan direction , the time to acquire each 3d volumetric scan was 4.3 seconds . in the scan protocol , one eye of each participant was scanned with 4 scans consisting of 2 horizontal and 2 vertical scans . an orthogonal registration algorithm was applied to register and merge all 4 scans to reduce eye motion . concentric , cylindrical , cross - sectional oct images of varying radii were resampled from the volume scan ( fig . we selected rings with radii of 1.5 , 1.7 , 2 , 2.5 , 3 , and 3.5 mm for analysis . the disc center was decided manually by one of us ( ll ) by matching the disc boundary with an ellipse on the en face view ( fig . ( a ) en face view ( average projection of log reflectance intensity ) of the 8 8 mm volumetric cube scan of the onh region in a glaucomatous eye . the 3-mm radius circle centered on the onh ( b ) the 3-mm radius cylindrical cross section is resampled from the cube scan . the ilm contour ( white dashed line ) is measured from this circular cross section . ( c ) en face oct showing resampling circles at radii of 1.5 , 1.7 , 2.0 , 2.5 , 3.0 , and 3.5 mm . the ilm was detected as the most inner boundary of retina in the cylindrical cross - section images . an automated algorithm was developed to follow the vessel bumps and avoid artifacts , such as points of vitreous macular adhesion . the ilm elevation profile ( contour ) one of us ( ll ) inspected the cross - sectional images with ilm contour overlay and performed manual correction of the contour if necessary . the ilm elevation profile was transformed to spatial frequency domain using the fast fourier transform ( fig . the spatial frequency components at lowest frequencies were removed because they simply relate to the average axial position and the tilt of the oct scan beam relative to the onh plane . the highest frequency components also were nondiagnostic because of high noise and little anatomic information . the middle frequency band that optimized the detection of retinal vessel relief and nfl bundle defects was determined empirically based on the difference between the average normal and glaucoma ilm spatial frequency spectrum ( fig . the rscv is defined as the average log value within a middle spatial frequency band of the fourier transform to the elevation profile of the inner retinal surface . ( a ) spatial frequency spectrum of ilm elevation showing the averages of all glaucoma eyes and all normal eyes derived from the 3.0-mm oct cylindrical sections . ( b ) shows the difference spectrum ( glaucoma normal ) and the optimize frequency band ( between dotted lines ) used to compute the rscv . optimized passbands for rscv calculation at different radii . on the cylindrical cross - sectional oct image with radius of 1.7 mm the wilcoxon rank sum test was performed to determine statistical significance between the study groups . the area under receiver operating characteristic curve ( aroc ) was calculated for the diagnostic accuracy . pearson correlation was used to determine correlations between rscv , axial length , nflt , and visual field test md . a coefficient of variance ( cv ) the first one compares rscv value at r = 2.8 mm and r = 3.0 mm . the second test compares results graded independently by two of the authors ( ot and ll ) . all image processes and statistical analyses were done using matlab ( mathworks , natick , ma , usa ) . the research protocol was approved by the institutional review board at the oregon health & science university ( ohsu ) and performed in accordance with the tenets of the declaration of helsinki . written informed consent was obtained from each participant after explanation of the nature and possible consequences of the study . participants were recruited at the casey eye institute / ohsu according to the aig study protocol . the ohsu ancillary site followed the same eligibility and endpoint protocol as the aig study , but used an advanced swept - source oct prototype system instead of commercially available oct instruments . briefly , normal control participants met the following criteria in both eyes : iop of less than 21 mm hg in both eyes , and a normal humphrey visual field ( hvf ) on achromatic standard automated perimetry by swedish interactive threshold algorithm 24 - 2 testing ( hfa ii ; carl zeiss meditec , inc . , dublin , ca , usa ) with mean deviation ( md ) , glaucoma hemifield test ( ght ) , and pattern standard deviation ( psd ) within normal limits . in addition , normal subjects had a normal - appearing optic nerve head ( onh ) and nfl on ophthalmoscopic examination , and an open angle by gonioscopy . glaucoma participants were required to have a glaucomatous onh rim or nfl thinning on ophthalmoscopic examination . glaucomatous eyes were classified in the pg subgroup if they have visual field ( vf ) psd or ght outside normal limits ( p < 0.05 and p < 1% , respectively ) in a consistent pattern on two qualifying vf exams . exclusion criteria for all groups included visual acuity less than 20/40 , age < 40 or > 79 years at enrollment , any ocular surgery other than uncomplicated cataract extraction , other diseases that might cause vf or onh abnormality , and factors that might preclude the participant from performing study procedures or completing the study . a prototype high speed swept - source fourier - domain oct system was used in this study . the device operated at an axial scan speed of 100 khz using a swept - source cavity laser operating at 1050 nm with a tuning range of 100 nm . a resolution of 5.3 m axially and 18 m laterally at an imaging depth of 2.9 mm in tissue was achieved . the ocular light power exposure was 1.9 mw , within the american national standards institute ( ansi ) safety limit . participants were scanned using a high density 8 8 mm raster scan pattern , centered at the onh . in the fast transverse scan direction , the time to acquire each 3d volumetric scan was 4.3 seconds . in the scan protocol , one eye of each participant was scanned with 4 scans consisting of 2 horizontal and 2 vertical scans . an orthogonal registration algorithm was applied to register and merge all 4 scans to reduce eye motion . concentric , cylindrical , cross - sectional oct images of varying radii were resampled from the volume scan ( fig . we selected rings with radii of 1.5 , 1.7 , 2 , 2.5 , 3 , and 3.5 mm for analysis . the disc center was decided manually by one of us ( ll ) by matching the disc boundary with an ellipse on the en face view ( fig . ( a ) en face view ( average projection of log reflectance intensity ) of the 8 8 mm volumetric cube scan of the onh region in a glaucomatous eye . the 3-mm radius circle centered on the onh ( b ) the 3-mm radius cylindrical cross section is resampled from the cube scan . the ilm contour ( white dashed line ) is measured from this circular cross section . ( c ) en face oct showing resampling circles at radii of 1.5 , 1.7 , 2.0 , 2.5 , 3.0 , and 3.5 mm . the ilm was detected as the most inner boundary of retina in the cylindrical cross - section images . an automated algorithm was developed to follow the vessel bumps and avoid artifacts , such as points of vitreous macular adhesion . the ilm elevation profile ( contour ) one of us ( ll ) inspected the cross - sectional images with ilm contour overlay and performed manual correction of the contour if necessary . the ilm elevation profile was transformed to spatial frequency domain using the fast fourier transform ( fig . 3a ) . the spatial frequency components at lowest frequencies were removed because they simply relate to the average axial position and the tilt of the oct scan beam relative to the onh plane . the highest frequency components also were nondiagnostic because of high noise and little anatomic information . the middle frequency band that optimized the detection of retinal vessel relief and nfl bundle defects was determined empirically based on the difference between the average normal and glaucoma ilm spatial frequency spectrum ( fig . the rscv is defined as the average log value within a middle spatial frequency band of the fourier transform to the elevation profile of the inner retinal surface . ( a ) spatial frequency spectrum of ilm elevation showing the averages of all glaucoma eyes and all normal eyes derived from the 3.0-mm oct cylindrical sections . ( b ) shows the difference spectrum ( glaucoma normal ) and the optimize frequency band ( between dotted lines ) used to compute the rscv . on the cylindrical cross - sectional oct image with radius of 1.7 mm , the lower nfl boundary also was detected using a method described previously . the wilcoxon rank sum test was performed to determine statistical significance between the study groups . the area under receiver operating characteristic curve ( aroc ) was calculated for the diagnostic accuracy . pearson correlation was used to determine correlations between rscv , axial length , nflt , and visual field test md . a coefficient of variance ( cv ) the first one compares rscv value at r = 2.8 mm and r = 3.0 mm . the second test compares results graded independently by two of the authors ( ot and ll ) . all image processes and statistical analyses were done using matlab ( mathworks , natick , ma , usa ) . of 17 participants in the glaucoma group ( 8 in the pg and 9 in the ppg subgroups ) , 15 had early vf damage ( md > 6 db ) and 2 had more severe vf damage . we enrolled 25 participants in the normal control group , but due to their younger average age , only the 17 oldest control participants were used in the present analysis . the age matching was not exact , as the control participants still averaged 9 years younger ( table 1 ) . as expected , the glaucoma group had significantly worse vf md and thinner nfl in the overall area , superior and inferior quadrants . the image quality of the normal group is significantly higher than that of the glaucoma group based on signal strength index . participant characteristics the spatial frequency transforms of the ilm profile were averaged for the normal and glaucoma groups ( fig . optimized bands were detected at frequency components where the glaucoma group was significantly larger than the normal group ( fig . we averaged rscvs in the optimized bands for each circle and the rscv distribution for normal and glaucoma groups are illustrated in figure 5 . the rscv was significantly higher at all sampling radii in the glaucoma group compared to the normal control group ( table 2 ) . distribution of rscv values for normal ( n ) and glaucoma ( g ) groups at sampling radii between 1.5 and 3.5 mm . retinal surface contour variability the diagnostic accuracy of nfl and rscv were assessed using aroc values at each sampling radius ( table 3 ) . the diagnostic accuracy of rscv improved with larger radii and the highest aroc value ( 0.90 ) was found at a radius of 3.5 mm . this was higher than that for nflt parameters , but only significantly in the temporal and nasal quadrants ( < 0.01 ) . we examined averages of rscv over various ranges of radii and found that the average over 2.5 to 3.5 mm had the highest aroc ( 0.91 ) . the combination had marginally higher aroc ( 0.91 ) than nfl only ( p = 0.08 ) . diagnostic accuracy comparison venn diagram analysis ( fig . 6 ) showed that rscv had higher sensitivity for glaucoma diagnosis ( 53% ) than nflt ( 29% ) . it also was notable that all abnormal eyes detected by nflt also were detected by rscv . however , the difference in sensitivity was not statistically significant ( p = 0.13 ) . the threshold for rscv was set 2.33 sd above the mean of the normal group ( 99 percentile cutoff assuming normal distribution ) and the threshold of nfl was 2.33 sd below the mean of normal group . in the normal group , all eyes were within the cutoff for both parameters , indicating 100% specificity . in the glaucoma groups , rscv detected abnormality in nine participants , including the five detected by nfl . the correlations between averaged rscv and nflt , or between averaged rscv and vf md , were estimated for glaucomatous participants . significant correlations were found between rscv and nfl ( r = 0.54 , p = 0.03 ) but not md ( r = 0.32 , p = 0.21 ) . a significant correlation also was found between nfl and md ( r = 0.68 , p = 0.002 ) . we also evaluated the correlation between rscv and nfl for normal participants , which was not significant ( r = 0.10 , p = 0.70 ) . we tested whether the rscv was sensitive to transverse magnification changes ( i.e. , axial eye length variation ) by comparing the values evaluated at the sampling radii of r = 2.8 and 3 mm . no significant difference was found between the two radii in either the glaucoma or normal group ( p > 0.26 ) . 1.3% for glaucoma eyes . we also tested the between - grader ( ot and ll ) reproducibility of rscv because the onh boundary and center were defined manually . the reproducibility ( cv ) was 1.6% for normal eyes and 1.1% for glaucoma eyes . we did not find a significant correlation between axial length and rscv parameter ( r = 3.5 mm ) for the normal ( r = 0.06 , p = 0.83 ) and glaucoma ( r = 0.42 , p = 0.09 ) groups . we also did not find a significant correlation between axial length and rscv parameters at any radius for the normal group ( p > 0.32 ) . in this pilot study , we reported , to our knowledge , the first use of spatial frequency analysis to detect small scale retinal surface contour change in glaucoma . the rscv , measured in the peripapillary area , was significantly larger in glaucoma eyes . the diagnostic accuracy of rscv was at least as good as nflt , although a larger study would be needed to see if there is a significant advantage . in early glaucoma , nflt has limited diagnostic sensitivity , with literature values of 33.3% to 67.4% at a fixed specificity of 99% . this is not surprising as there is a relatively wide range of population variation in the average nflt , which has been reported as between 9.0% and 10.0% sd . because the rscv measures small scale , focal changes that are unlikely to be due to the inborn variation of nflt , it may be able to better detect glaucoma in the earlier stages , when nfl damage is small and variable in the location . in this study , nflt was abnormally ( 99% specificity cutoff ) thin in only 5 of 17 participants in the glaucoma group , while rscv was abnormally elevated in 9 of 17 glaucoma participants . while this advantage is not statistically significant , it shows a promising trend that deserves further study . because the nflt and the rscv measure different aspects of nfl damage while we previously have developed another parameter to measure focal peripapillary nfl and macular ganglion cell complex ( gcc ) thinning called focal loss volume ( flv ) , that algorithm was optimized to detect relatively larger areas ( 500 m superpixels ) of focal damage . by contrast the rscv spatial frequency analysis detects changes in the 2 to 100 radians / mm , which corresponds to spatial features as small as 60 m ( the diameter of a small arteriole ) . thus , we believe all three parameters will be synergistic based on the fact they measure glaucoma damage on different spatial scales . we found that the rscv appeared to have higher diagnostic accuracy in circles with large radii ( 2.53.5 mm ) than small radii ( 1.52 mm ) . firstly , we observed that , in the normal eye , larger retinal vessels submerge into the retina further from the disc margin . thus , the inner retinal surface is smoother and rscv is lower further from the disc . this observation also might be due to the fact that glaucoma damages axon bundles nonuniformly within the onh ( laminar pores , and so forth ) and these bundle losses may be more detectable within the nfl where all bundles are more spread out . another assumption is that the thinner nfl away from the disc is going to be less likely to mask blood vessels and loss of axon bundles may reveal blood vessels more readily in these regions . the second observation is that the population variances were tighter with larger radii ( table 2 ) . we suspected this was simply due to the larger number of a - scans in sampling circles of larger radii . thus , maximizing the oct scan density in the 2.5- to 3.5-mm radius annulus around the optic disc may improve rscv evaluation . an additional advantage of rscv over nflt is that the rscv value is relatively insensitive to magnification variation or decentration relative to the optic disc center . between the scan radii of 2.5 to 3.5 mm , the mean rscv values varied less than 2% in the normal and glaucoma groups . intergrader variation in disc boundary identification also affected rscv by less than 2% . by comparison furthermore , nfl quadrant thickness averages are known to be exquisitely sensitive to decentration of the sampling circle relative to the optic disc . therefore , rscv may be a more robust diagnostic parameter that is relatively resistant to the introduction of bias due to patients , oct operators , or graders . in previous studies , contour variability was accessed by heidelberg retinal tomography ( hrt ) but was considerably inferior in its ability to diagnose glaucoma or progression compared to nfl measured by oct . we believe there are two reasons why contour variability by hrt was considerably inferior in diagnostic power . thus , it is difficult for hrt to catch small changes in the contour variability caused by protrusions of retinal vessels . , we found that the contour variability is less sensitive in location near the disc edge . one of the limitations of this study is the relatively small number of participants , especially for advanced glaucoma ( vf < 6db ) . a power calculation shows that our sample size of 17 normal and 17 glaucoma subjects should be able to detect a difference in aroc of 0.67 compared to the aroc of rscv of 0.90 with a probability of 0.80 at a significance threshold of p < 0.05 . however , rscv was not significantly affected by sex in our control group ( p = 0.62 ) . second , the glaucoma group also is 9 years older than the normal group . as a consequence , the oct image quality in normal however , the ssi is good for the normal ( 60 9 ) and glaucoma ( 50 12 ) groups . the rscv parameters are not sensitive to ssi when the image quality is good enough for accurate ilm boundary detection . we also tested the correlation between ssi and rscv but did not find any significance for the normal and glaucoma groups ( p > 0.38 ) . furthermore , rscv was not correlated with age in the normal group ( p = 0.43 ) . retinal surface contour variability also may be sensitive to focal nfl loss caused by other optic neuropathies , such as anterior ischemic optic neuropathy ( aion ) and optic neuritis . since rscv is a nonspecific detector of nfl loss on a small spatial scale , specific diagnosis of particular conditions would require the physician to consider the clinical presentation as well as patterns of nfl , gcc , optic disc topography , and vf changes . in summary , since the rscv detects small - scale focal damage and the average nflt measures global damage , they provide different diagnostic information that may be synergistic .

introduction : degenerative spondylolisthesis ( ds ) is usually seen at l4-l5 level and less frequently at l5-s1 level . this is a rare case report of spondylolisthesis of s1 over s2 with lumbarization of s1 . lumbarization of s1 is seen in just 1 - 2% of the population and to have spondylolisthesis in this segment is even rarer . the purpose is to report a rare case of ds at s1-s2 level.case report : this is a single case report of a 66-year - old gentleman who presented with complains of lower backache for 2 years and acute retention of urine to the emergency department . detailed clinical and radiological evaluation of the spine was done which revealed lumbarization of s1 with spondylolisthesis at s1-s2 and facetal hypertrophy at l5 , s1 , and s2 . he underwent decompression and stabilization at l5 , s1 , and s2 along with placement of autologous bone graft . the bladder symptoms disappeared after 3 weeks . at 1-year follow - up , patient s clinical symptoms were relieved , and he improved clinically.conclusion:to the best of our knowledge , this is probably the first case of ds of sacral vertebrae to be reported in english literature . the prevalence of complete lumbarization is around 1.8% and to get spondylolisthesis in this segment is even rarer , hence the lack of literature in this regard . since this is the first of its kind of case , further case series or longitudinal studies of such cases may help understand better the pathomechanics related to spondylolisthesis at this level . purposewe investigated the feasibility of glaucoma detection by measuring retinal surface contour variability ( rscv ) using optical coherence tomography ( oct).methodsthe peripapillary region in one eye of each participant was scanned over an 8 8 mm area with a swept source oct prototype . the retinal surface contour was sampled at approximately 1.5- to 3.5-mm radius circles centered on the optic nerve head . the rscv is defined as the average log value within a middle spatial frequency band of the fourier transform to the elevation profile of the inner retinal surface . the spatial frequency band was optimized to distinguish glaucoma from normal . nerve fiber layer thickness ( nflt ) was sampled around a 1.7-mm radius circle . glaucoma severity was assessed by automated static perimetry.resultswe enrolled 17 glaucomatous eyes and 17 healthy eyes . a great majority of the glaucoma group were in the early stage ( visual field mean deviation average 2.48 3.73 db ) . significant differences were found for rscv between glaucoma and control eyes ( p < 0.003 ) at all radii . the area under the receiver operating characteristic curve ( aroc = 0.90 ) of rscv was best at the 3.5-mm radius . this was not significantly better than nflt ( aroc = 0.84 ) . with the 99% specificity , the glaucoma detection sensitivity was 53% for rscv and 29% for nflt ( p = 0.13).conclusionsretinal surface contour variability was significantly increased in glaucoma patients . the diagnostic accuracy of rscv was equal to nflt in early glaucoma . since the rscv detects small - scale focal damage and the average nflt measures global damage , they provide different diagnostic information that may be synergistic .

organic foreign bodies are generally associated with severe inflammatory reaction and infection , while the nature of reaction elicited by inorganic foreign bodies depends on the material of the foreign body . graphite , which is the major constituent of pencil lead , has been reported to remain inert in the eye for a long time . however , it has also been reported to cause severe endophthalmitis - like reaction in the eye . we report a rare case of retained graphite pencil tip in the anterior chamber of a six - year - old girl . a six - year - old girl presented to us with history of mild pain in the left eye of two days duration . the child s mother gave a history of trauma with a graphite lead pencil about four months ago at school when she was accidentally poked in the left eye by another child . the child had not been examined by an ophthalmologist after the incident as she was apparently asymptomatic at the time . on examination , the best corrected visual acuity was 20/20 in the right eye and 20/40 in the left eye . there was a full thickness corneal scar ( figure 1 ) in the left eye . a small area of iris atrophy with a sphincter tear was noted at the edge of the pupil at the 6 o clock position . there was a black foreign body resembling a graphite pencil lead tip , measuring about 1.5 mm in size , on the iris at the 7 o clock position ( figure 1 ) . ocular ultrasonography of the left eye did not reveal any abnormality in the posterior segment . figure 1slit lamp photograph of the left eye showing the corneal scar and the graphite pencil lead tip on the iris . slit lamp photograph of the left eye showing the corneal scar and the graphite pencil lead tip on the iris . a corneal incision was made at 5 o clock position with a 2.8 mm keratome . the anterior chamber was filled with 2% methyl cellulose and the foreign body ( figure 2 ) was removed in toto with a bechert - mcpherson forceps . the remaining graphite particles were aspirated out with a simcoe cortex aspiration cannula using an anterior chamber maintainer . post - operatively , the patient was put on tapering doses of topical steroids and cycloplegics . on follow up , one month later , the best corrected visual acuity in the left eye was 20/40 . there was no inflammation ; the lens was clear and the fundus was normal . the reaction of the eye to a retained intraocular foreign body varies depending on its composition . foreign bodies comprised of materials like gold , silver and platinum have been reported to remain inert in the intraocular environment . there are only a few reports of ocular trauma with retention of graphite pencil lead in the eye . retained graphite has been described in the conjunctiva , cornea , angle of the anterior chamber and the posterior segment . a case of pencil - tip injury to the orbit with retained graphite foreign body associated with delayed orbital infection has also been described . however , the potential toxicity of the other constituents of pencil lead like animal fats and clay is not clearly known . there has been a report of severe endophthalmitis - like reaction incited by retained graphite foreign bodies in the vitreous . in this case , it is unclear whether the reaction was induced by the other constituents of pencil lead like the aluminium in the kaolinite or if there was an associated infection . in our case , we decided to surgically remove the intraocular foreign body in spite of the fact that it had obviously remained in the eye for some time ( as evidenced by the healed corneal scar and presence of iris atrophy ) without inciting an inflammatory response or causing much damage to the intraocular structures . there was a distinct possibility of causing damage to the lens and inciting an inflammatory reaction during surgical removal of the foreign body . this risk had to be weighed against that of the damage the foreign body might cause if it was left in the eye . there was a chance that the foreign body would get dislodged into the angle at a later stage and cause progressive damage to the angle structures and the cornea as in the case reported by han et al . honda et al . have reported a case of a five - year - old child with a graphite foreign body lodged in the peripheral retina , whom they followed up for six years with serial electroretinograms , fundus photographs and fundus fluorescein angiography . there was no evidence of any damage to the eye caused by the foreign body at the end of their follow up . however , in our case , such meticulous follow - up would have been quite impossible as the child s family belonged to a poor socio - economic background and the parents would not have been able to afford the cost of repeated hospital visits and investigations . our case also differed from honda et al.s case in that the foreign body was in the anterior segment and therefore , was at a more accessible site . there was a higher chance of removal of the foreign body without causing damage to the intraocular structures in our case . in conclusion , graphite foreign bodies may be retained in the eye without causing any inflammation or damage to the intraocular structures . on the other hand , there is also the possibility of progressive damage to intraocular structures by these foreign bodies due to various mechanisms . therefore , the decision about surgical removal of the foreign body has to be made on an individual basis after taking multiple factors into consideration and estimating the risk - benefit ratio in each patient . polyacrylonitrile ( pan ) are commercially important and used for the precursor for high - performance carbon fiber . in the fiber applications , pan is dissolved in a suitable solvent and spin to fibers by a wet or dry spinning process . the extensively used solvents are aqueous sodium sulfocyanate ( nascn ) , n , ndimethylformamide ( dmf ) , n , n - dimethylacetamide ( dmac ) , and n , n - dimethyl sulfone ( dmso ) . unfortunately , all the solvents mentioned above are unfriendly to the environment . to meet the requirements of green chemistry , scientists have been searching for a green solvent to be an alternative to these solvents . in recent years , room temperature ionic liquids ( rtils ) have received a lot of attention as the potential green and designable solvents . although the ionic liquids have been studied since the 1950s , but they can not to be handled under an inert atmosphere . these are not part of our studies until some kinds of water - stable and air - stable ionic liquids are found such as [ bmim]cl . because of its extremely low volatility , some of the rtils are promising environment - friendly solvents instead of the volatile organic solvents in a range of science and technology applications such as media for organic and inorganic reactions , materials processing electrochemistry and chemical separation . during the copolymerization of an and mma in rtils our research group find that rtils are good solvents for pan copolymer , and pan copolymer could be precipitated from the rtils solutions by the addition of water . therefore rtils are promising to be green solvents for pan . understanding the rheological properties of a solution is a convenient and effective way to gain a fundamental knowledge of the spinnability and structure - property relationships for the spinning solution . thus , this paper investigates the rheological properties of pan copolymer / bmimcl concentrated solutions in some detail . the pan copolymer , chlorobutane , 1-methylimidazole , ethyl acetate and acetone were used as supplied . firstly , pan powder was swollen by [ bmim]cl at room temperature to give a white , little viscous pulp . secondly the pulp was heated to 90c until a homogeneous , transparent solution was formed . the pan/[bmim]cl solution of 5 wt% to 22 wt% concentration was obtained within 5 hours at 90c . figure 1 shows the process of the dissolution of pan with ionic liquids [ bmim]cl at 90c at the concentration of 10 wt% . steady state rheological measurements were performed on a haake rs150l rotational rheometer using a 35 mm , 1 cone and plate . the apparent viscosity a is shown as a function of shear rate for the pan in two different solvents , [ bmim]cl and dmf ( see figure 2 ) . over the experimental range , the pan / dmf solution is newtonian fluid , while the pan/[bmim]cl solution is pseudoplastic , that is , the apparent viscosity decreases with increasing shear rate . according to the so - called power law equation , where k and n are constants , for pseudoplastic liquids the viscosity should decrease nearly linearly with shear rate on the log - log plot and the value of n is less than one . however , it is surprising to find that the data of pan/[bmim]cl solution should be considered having two linear stages ( see figure 2 ) . the value of n of the first stage is less than 1 , according with the shear thinning rheological behavior . while at the second stage the apparent viscosity decreases so dramatically that even the value of n is negative . it is known that n is used to indicate the non - newtonian property of fluids . the more the value of n deviates from 1 , the more non - newtonian the fluids would be . usually , the value of n of pan / traditional organic solvents solutions is between 0 and 1 . therefore , it could be postulated that there would be some much stronger interaction between pan and the new solvent [ bmim]cl which will be discussed in the following text . the data in figure 3 shows that the viscosity - shear rate curves for the pan/[bmim]cl solutions of different concentrations . these curves show that solutions of lower concentration remain newtonian in behavior at high shear rates than concentrated solutions . at high concentration , the rheological behavior of pan/[bmim]cl solutions acts like that of liquid crystalline polymers ( lcp ) . the solvent [ bmim]cl reduces the number of entanglements . reducing the number of entanglements at a given shear rate since the orientation of macromolecular is the major cause of non - newtonian behavior , increasing the shear rate would make the non - newtonian behavior more noticeable . besides , it could be found in figure 3 that when at high shear rate , the viscosity of high concentration solutions is lower than that of low concentration , for example , the viscosity of 22wt% solution is the lowest among all the solutions when the shear rate is close to 1000 1/s . as we known , liquid crystalline polymers usually have rigid chain segment such as aromatic polyamide and aromatic polyester . the flexibility of the c - c bond of the pan main chain is smaller than the c - o bond , c - n bond and decrease because of the strong polarity of the cn , but comparing to the liquid crystalline polymers , the pan chain segment can not be oriented because of lack of strong rigid chain segment . in this case , high concentration means more entanglements , which indicates that only low shear rates would be needed to orient the macromolecules . and the amount of entanglements is so large that there is no enough time and much more difficulties for most of them to slip and disengage . with the increasing of the shear rate the number of the oriented segments increases , so that the viscosity of the high concentration pan/[bmim]cl solution decreases greatly . commonly , the viscosity follows the andrade or arrhenius equation to a good approximation : in this equation , a is a constant , e is the activation energy . the slope of the straight line plotted by ln versus 1/t is e / r . the value of the energy of activation depends strongly on whether the viscosities at various temperatures are evaluated at constant shear stress or at constant shear rate . here since low molecular weight polymers has fewer entanglements than high molecular weight ones , it is not surprising that deviations from newtonian behavior start at higher shear rates for the low molecular weight solutions . however , in figure 5 , when the shear rate up to 210 1/s the viscosity of the high molecular weight pan/[bmim]cl solution is lower than the other one . the viscoelasticity of polymer is usually characterized by dynamic rheological experiment in which the sample is subjected to a sinusoidal strain of infinitesimal amplitude and fixed angular frequency . the term g , called the storage modulus , is the in - phase component of the modulus and represents the energy stored and recovered per cycle . correspondingly , the term g , called the loss modulus , is the out - of - phase component of the modulus and represents the energy dissipated as heat per cycle of deformation . figure 6 shows the modulus - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . the data shows that the loss modulus g is much higher than the storage modulus g. either module increases when the concentration increases or the temperature decreases . the storage modulus g increases faster than the loss modulus g because the elasticity of the solution increases . the data in fig . 7 shows that the complex viscosity - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . . the complex viscosity of the solution increases when the concentration increases or the temperature decreases . this non - newtonian behavior is of tremendous practical importance in the processing and fabrication of polymer material . first , the decreased viscosity makes the polymer solution easier to process or squirt through small channels , such as spinning . second , the decrease in viscosity is associated with the development of elasticity in the polymer solution . this elasticity produces such phenomena as die swell , or the puff - up of extruded strands . the possible dissolution mechanism of pan in ionic liquid [ bmim]cl is shown in figure 8 . it has been proved that the miscibility of ionic liquid with polymer is the function of their structural characteristic . we here focus on the polar group of pan which has strong interaction with the ionic liquid . because the electronegativity of the nitrogen atom on the nitrile grouping is very strong when the electron density is very large , the nitrogen atom on the nitrile grouping donate electrons and the carbon atom on the nitrile grouping attract electrons . as a result , there is strong interaction between the cation and the nitrogen atom on the nitrile grouping . similarly , there is strong interaction between the anion and the carbon atom on the nitrile grouping . the pan copolymer , chlorobutane , 1-methylimidazole , ethyl acetate and acetone were used as supplied . firstly , pan powder was swollen by [ bmim]cl at room temperature to give a white , little viscous pulp . secondly the pulp was heated to 90c until a homogeneous , transparent solution was formed . the pan/[bmim]cl solution of 5 wt% to 22 wt% concentration was obtained within 5 hours at 90c . figure 1 shows the process of the dissolution of pan with ionic liquids [ bmim]cl at 90c at the concentration of 10 wt% . steady state rheological measurements were performed on a haake rs150l rotational rheometer using a 35 mm , 1 cone and plate . the apparent viscosity a is shown as a function of shear rate for the pan in two different solvents , [ bmim]cl and dmf ( see figure 2 ) . over the experimental range , the pan / dmf solution is newtonian fluid , while the pan/[bmim]cl solution is pseudoplastic , that is , the apparent viscosity decreases with increasing shear rate . according to the so - called power law equation , where k and n are constants , for pseudoplastic liquids the viscosity should decrease nearly linearly with shear rate on the log - log plot and the value of n is less than one . however , it is surprising to find that the data of pan/[bmim]cl solution should be considered having two linear stages ( see figure 2 ) . the value of n of the first stage is less than 1 , according with the shear thinning rheological behavior . while at the second stage the apparent viscosity decreases so dramatically that even the value of n is negative . it is known that n is used to indicate the non - newtonian property of fluids . the more the value of n deviates from 1 , the more non - newtonian the fluids would be . usually , the value of n of pan / traditional organic solvents solutions is between 0 and 1 . therefore , it could be postulated that there would be some much stronger interaction between pan and the new solvent [ bmim]cl which will be discussed in the following text . the data in figure 3 shows that the viscosity - shear rate curves for the pan/[bmim]cl solutions of different concentrations . these curves show that solutions of lower concentration remain newtonian in behavior at high shear rates than concentrated solutions . at high concentration , the rheological behavior of pan/[bmim]cl solutions acts like that of liquid crystalline polymers ( lcp ) . the solvent [ bmim]cl reduces the number of entanglements . reducing the number of entanglements at a given shear rate reduces the amount of orientation of macromolecular . since the orientation of macromolecular is the major cause of non - newtonian behavior , increasing the shear rate would make the non - newtonian behavior more noticeable . besides , it could be found in figure 3 that when at high shear rate , the viscosity of high concentration solutions is lower than that of low concentration , for example , the viscosity of 22wt% solution is the lowest among all the solutions when the shear rate is close to 1000 1/s . as we known , liquid crystalline polymers usually have rigid chain segment such as aromatic polyamide and aromatic polyester . the flexibility of the c - c bond of the pan main chain is smaller than the c - o bond , c - n bond and decrease because of the strong polarity of the cn , but comparing to the liquid crystalline polymers , the pan chain segment can not be oriented because of lack of strong rigid chain segment . in this case , high concentration means more entanglements , which indicates that only low shear rates would be needed to orient the macromolecules . and the amount of entanglements is so large that there is no enough time and much more difficulties for most of them to slip and disengage . with the increasing of the shear rate the number of the oriented segments increases , so that the viscosity of the high concentration pan/[bmim]cl solution decreases greatly . commonly , the viscosity follows the andrade or arrhenius equation to a good approximation : in this equation , a is a constant , e is the activation energy . the slope of the straight line plotted by ln versus 1/t is e / r . the value of the energy of activation depends strongly on whether the viscosities at various temperatures are evaluated at constant shear stress or at constant shear rate . here the two molecular weights are high enough to become entangled . since low molecular weight polymers has fewer entanglements than high molecular weight ones , it is not surprising that deviations from newtonian behavior start at higher shear rates for the low molecular weight solutions . however , in figure 5 , when the shear rate up to 210 1/s the viscosity of the high molecular weight pan/[bmim]cl solution is lower than the other one . the viscoelasticity of polymer is usually characterized by dynamic rheological experiment in which the sample is subjected to a sinusoidal strain of infinitesimal amplitude and fixed angular frequency . the term g , called the storage modulus , is the in - phase component of the modulus and represents the energy stored and recovered per cycle . correspondingly , the term g , called the loss modulus , is the out - of - phase component of the modulus and represents the energy dissipated as heat per cycle of deformation . figure 6 shows the modulus - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . the data shows that the loss modulus g is much higher than the storage modulus g. either module increases when the concentration increases or the temperature decreases . the storage modulus g increases faster than the loss modulus g because the elasticity of the solution increases . the data in fig . 7 shows that the complex viscosity - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . the complex viscosity of the solution increases when the concentration increases or the temperature decreases . this non - newtonian behavior is of tremendous practical importance in the processing and fabrication of polymer material . first , the decreased viscosity makes the polymer solution easier to process or squirt through small channels , such as spinning . second , the decrease in viscosity is associated with the development of elasticity in the polymer solution . this elasticity produces such phenomena as die swell , or the puff - up of extruded strands . the possible dissolution mechanism of pan in ionic liquid [ bmim]cl is shown in figure 8 . it has been proved that the miscibility of ionic liquid with polymer is the function of their structural characteristic . we here focus on the polar group of pan which has strong interaction with the ionic liquid . because the electronegativity of the nitrogen atom on the nitrile grouping is very strong when the electron density is very large , the nitrogen atom on the nitrile grouping donate electrons and the carbon atom on the nitrile grouping attract electrons . as a result , there is strong interaction between the cation and the nitrogen atom on the nitrile grouping . similarly , there is strong interaction between the anion and the carbon atom on the nitrile grouping . within the limits of the experimental techniques accessible to us , the results obtained for pan/[bmim]cl solutions show that they are pseudoplastic , similar to the pan / traditional organic solvents solutions . the dependence on concentration and molecular weight shows that entanglements play an important role in the rheological behaviors of pan/[bmim]cl concentrated solutions . the dynamic rheological measurement indicates the elasticity of the solution increases when the concentration is increasing or the temperature decreasing . the rheological behaviors of the concentrated solution at high share rate are directive to the spinning of pan fiber .

retained intraocular graphite foreign bodies are uncommon . although they are generally inert , they have been reported to cause severe inflammatory reaction and progressive damage to intraocular structures . we report a case of a six - year - old girl with a retained intraocular graphite pencil lead foreign body in the anterior chamber of the eye and discuss the various considerations in the management of such cases . one of the room temperature ionic liquids ( rtils ) , 1-butyl-3-methylimidazolium chloride ( [ bmim]cl ) was chosen to prepare the concentrated solutions of polyacrylonitrile ( pan ) . the rheological behaviors of the solutions were measured with rotational rheometry under different conditions , including temperatures , concentration , and molecular weight of pan . the solutions exhibited shear - thinning behaviors , similar to that of pan / dmf solutions . the viscosities decreased with the increasing of shear rates . however , the viscosity decreased sharply at high shear rates when the concentration was up to 16wt% . the dependence of the viscosity on temperature was analyzed through the determination of the apparent activation energy . unusually , the viscosity of solutions of higher concentration is lower than that of lower concentration . similarly , the viscosity of low molecular weight pan was higher than high molecular weight pan at high shear rates . the dynamic rheological measurement indicates the loss modulus is much higher than storage modulus . the trend of complex viscosity is similar with the result of static rheological measurement . the interaction between pan and ionic liquid [ bmim]cl was discussed .

fistulae between the upper respiratory and gastrointestinal tracts are uncommon in adults . whereas developmental anomaly is the commonest cause in infancy and childhood , the etiology in adults is most frequently secondary to an esophageal malignancy . we report two cases of esophagobronchial fistulae one secondary to chronic chest tuberculosis and the other secondary to a squamous cell carcinoma of the upper esophagus diagnosed by multi - detector computed tomography ( mdct ) . traditionally , fluoroscopy with oral contrast swallow examination has been the mainstay radiological investigation for the diagnosis of these fistulae . however , it can be an inconvenient study with need for multiple projections to adequately demonstrate the fistula and its course . also , being a luminal study , the cause of the fistula is not adequately evaluated . use of the various post - processing features like thick maximum intensity projections ( mips ) and volume rendering techniques ( vrts ) enables better detection and depiction of these fistulae . use of virtual endoscopy also guides the clinician for the conventional endoscopy which would be needed for biopsy and treatment . a 15-year - old male presented with cough , mucopurulent expectoration , and dyspnea since 3 months . this finding of swallow cough sequence has been referred to as ono 's sign . a chest radiograph revealed complete opacification of the right hemithorax with volume loss suggesting complete collapse of the right lung with mediastinal shift to the right [ figure 1 ] . a plain and intravenous contrast - enhanced ct study with oral contrast swallow was performed on a 128-slice mdct scanner ( siemens somatom definition as , erlangen , germany ) . the study revealed complete collapse of the right lung with irregular dilated ectatic bronchi in the right lower lobe [ figure 2 ] . there was stenosis and diffuse narrowing of the right mainstem bronchus with nodularity of the mucosa seen best on the virtual bronchoscopy [ figure 3 ] . an air - filled tract was noted extending from one of the right lower lobar bronchi toward the posterior mediastinum with ill - defined soft tissue around it . the possibility of an esophagobronchial fistula was suspected , and we did a ct oral contrast swallow study to detect it . oral contrast swallow study performed with the patient in right decubitus position using diluted non - ionic iodinated contrast medium ( 1:20 dilution of iohexol with normal saline ) . it depicted the site and the fistula tract between the right lateral wall of the esophagus and one of the ectactic bronchi in the right lower lobe of the lung . thick mip images and vrt processing demonstrated the fistula site and tract [ figures 4 and 5 ] . these imaging findings led us to conclude that these changes were most likely the sequelae of chronic tuberculosis . histology of the lung specimen revealed distorted bronchioles with diffuse and focal dense infiltration by mononuclear cells and giant cells . frontal chest radiograph reveals severe volume loss and complete opacifi cation of the right hemithorax with ipsilateral tracheal and mediastinal shift . branching tubular radiolucencies are noted in the right lower zone representing bronchiectasis ( encircled area ) axial ( a ) and coronal ( b ) ct of the chest in lung window shows complete collapse of the right lung with shift of of the heart and the mediastinum to the right . dilated irregular fluid bronchi are seen in the right lower lobe ( thin black arrows ) with few having air - fluid levels within ( thick black arrow ) coronal ct of the chest in lung window ( a ) demonstrates narrowing of the right mainstem bronchus ( thick black arrow ) . a virtual bronchoscopy images ( b ) revealing stenotic orifice of the right mainstem bronchus with mucosal nodularity near the carina thick maximum intensity projection ( mip ) image of the ct contrast swallow study demonstrating the esophagus pulled to the right and opacification by oral contrast of the fistula tract between the esophagus and the dilated right lower lobe bronchi volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination demonstrating the fistula ( arrowheads ) between the right lateral aspect of the pulled esophagus and the right lower lobar bronchus with opacifi cation of the bronchial tree a 65-year - old male presented with progressive dysphagia to solids over the past 2 months . an ultrasound of the neck revealed few enlarged lymph nodes which on fine needle aspiration ( fna ) revealed neoplastic squamous cells . a plain and intravenous contrast - enhanced ct study with ct oral contrast swallow was performed . there was a loss of fat plane between the esophagus and the carina , left mainstem bronchus , and aorta . a defect was seen in the posterior wall of the proximal left mainstem bronchus suggesting formation of a fistula with the esophagus [ figure 6 ] . a ct oral contrast swallow study performed in prone position with diluted non - ionic contrast ( 1:20 dilution of iohexol in normal saline ) revealed a small fistula tract between the esophagus and the proximal left mainstem bronchus just distal to the carina [ figure 7 ] . post - processing of this contrast swallow study with volume rendering depicted the fistulous communication in 3d [ figure 8 ] . axial contrast enhanced ct ( a ) at a level just beyond the carina demonstrates defect in the posterior wall of the proximal left mainstem bronchus ( thick white arrow ) . a virtual bronchoscopy image ( b ) shows the site of the fistula opening ( thin white arrow ) oral contrast ct swallow- axial ct performed with patient in prone position shows the fistula tract ( white arrow ) opacifi ed by the diluted oral contrast volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination show the fistula tract between the esophagus and the proximal left mainstem bronchus a 15-year - old male presented with cough , mucopurulent expectoration , and dyspnea since 3 months . this finding of swallow cough sequence has been referred to as ono 's sign . a chest radiograph revealed complete opacification of the right hemithorax with volume loss suggesting complete collapse of the right lung with mediastinal shift to the right [ figure 1 ] . a plain and intravenous contrast - enhanced ct study with oral contrast swallow was performed on a 128-slice mdct scanner ( siemens somatom definition as , erlangen , germany ) . the study revealed complete collapse of the right lung with irregular dilated ectatic bronchi in the right lower lobe [ figure 2 ] . there was stenosis and diffuse narrowing of the right mainstem bronchus with nodularity of the mucosa seen best on the virtual bronchoscopy [ figure 3 ] . an air - filled tract was noted extending from one of the right lower lobar bronchi toward the posterior mediastinum with ill - defined soft tissue around it . the possibility of an esophagobronchial fistula was suspected , and we did a ct oral contrast swallow study to detect it . oral contrast swallow study performed with the patient in right decubitus position using diluted non - ionic iodinated contrast medium ( 1:20 dilution of iohexol with normal saline ) . it depicted the site and the fistula tract between the right lateral wall of the esophagus and one of the ectactic bronchi in the right lower lobe of the lung . thick mip images and vrt processing demonstrated the fistula site and tract [ figures 4 and 5 ] . these imaging findings led us to conclude that these changes were most likely the sequelae of chronic tuberculosis . histology of the lung specimen revealed distorted bronchioles with diffuse and focal dense infiltration by mononuclear cells and giant cells . frontal chest radiograph reveals severe volume loss and complete opacifi cation of the right hemithorax with ipsilateral tracheal and mediastinal shift . branching tubular radiolucencies are noted in the right lower zone representing bronchiectasis ( encircled area ) axial ( a ) and coronal ( b ) ct of the chest in lung window shows complete collapse of the right lung with shift of of the heart and the mediastinum to the right . dilated irregular fluid bronchi are seen in the right lower lobe ( thin black arrows ) with few having air - fluid levels within ( thick black arrow ) coronal ct of the chest in lung window ( a ) demonstrates narrowing of the right mainstem bronchus ( thick black arrow ) . a virtual bronchoscopy images ( b ) revealing stenotic orifice of the right mainstem bronchus with mucosal nodularity near the carina thick maximum intensity projection ( mip ) image of the ct contrast swallow study demonstrating the esophagus pulled to the right and opacification by oral contrast of the fistula tract between the esophagus and the dilated right lower lobe bronchi volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination demonstrating the fistula ( arrowheads ) between the right lateral aspect of the pulled esophagus and the right lower lobar bronchus with opacifi cation of the bronchial tree a 65-year - old male presented with progressive dysphagia to solids over the past 2 months . an ultrasound of the neck revealed few enlarged lymph nodes which on fine needle aspiration ( fna ) revealed neoplastic squamous cells . a plain and intravenous contrast - enhanced ct study with ct oral contrast swallow was performed . there was a loss of fat plane between the esophagus and the carina , left mainstem bronchus , and aorta . a defect was seen in the posterior wall of the proximal left mainstem bronchus suggesting formation of a fistula with the esophagus [ figure 6 ] . a ct oral contrast swallow study performed in prone position with diluted non - ionic contrast ( 1:20 dilution of iohexol in normal saline ) revealed a small fistula tract between the esophagus and the proximal left mainstem bronchus just distal to the carina [ figure 7 ] . post - processing of this contrast swallow study with volume rendering depicted the fistulous communication in 3d [ figure 8 ] . axial contrast enhanced ct ( a ) at a level just beyond the carina demonstrates defect in the posterior wall of the proximal left mainstem bronchus ( thick white arrow ) . a virtual bronchoscopy image ( b ) shows the site of the fistula opening ( thin white arrow ) oral contrast ct swallow- axial ct performed with patient in prone position shows the fistula tract ( white arrow ) opacifi ed by the diluted oral contrast volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination show the fistula tract between the esophagus and the proximal left mainstem bronchus esophagobronchial fistulae are uncommon and difficult to diagnose . in the elderly , they are most frequently seen with an intrathoracic malignancy and are most commonly associated with malignancy of the esophagus . in a large case series , subsequent ulceration and necrosis of the malignant tissue leads to tissue breakdown and fistula formation . non - malignant causes are infrequent and include trauma ( blunt , penetrating , or iatrogenic ) , chronic inflammation ( chronic infections like tuberculosis and histoplasmosis , crohn 's disease ) , late presentation of a congenital fistula , and rarely poisoning . over the years , iatrogenic trauma has become a commoner cause for such fistulae compared to infections . tuberculosis as a cause for these fistulae has been infrequently reported in the western literature . however , in the indian subcontinent where the incidence of tuberculosis is high , chest tuberculosis should be high in the list of differentials when a non - malignant fistula is encountered . in nonmalignant conditions , traumatic bronchial or tracheal wall necrosis or necrotizing inflammation is responsible for the fistulization . fluoroscopic oral contrast swallow examination with barium is the initial investigation of choice for evaluation of dysphagia and suspected fistulae , even though endoscopy is needed for definite evaluation . if esophageal perforation is suspected , an iodinated contrast medium should be used as barium extravasation can lead to mediastinitis . if there is no frank leak seen , then barium can be given as it produces better radiographic quality being of higher density than iodine . if respiratory fistula is suspected , barium still may be used as small quantity of barium in the tracheobronchial tree is harmless . however , ionic iodinated contrast medium should not be used as they can cause chemical pneumonitis and only non - ionic iodinated contrast medium should be used in such cases . the best initial agent to use in both these scenarios of perforation or fistula is a non - ionic iodinated contrast agent . fluoroscopy allows for dynamic evaluation of esophageal motility as well as evaluation of its lumen . even though barium swallow fluoroscopy examination has the advantage of being a real - time study , fistula tracts may not always be detected . if detected , depiction of the three - dimensional course of the fistula may be difficult in spite of use of multiple projections . also , being a luminal study , it would fail to show changes in the wall of the esophagus and in the mediastinum that are shown accurately by ct . no large case series has been published about its efficacy in adult fistulae , but few case reports have highlighted its role . the patient should be given a mouthful bolus of the preparation and asked to swallow it promptly on instruction to do so . since the oral and pharyngeal phases of deglutition take no more than 2 seconds , the acquisition can be triggered immediately after the instruction to the patient to swallow has been given . the patient position may be changed to better opacify the fistula tract as we used prone and right decubitus positions in our cases to better opacify the fistula tracts . a recent study demonstrated that ct contrast swallow was better tolerated and more sensitive than fluoroscopy at detecting post - esophagectomy anastomotic leaks . post - processing these studies with maximum - intensity projection and with volume rendering allows a three - dimensional evaluation of the fistula tract . supplementing oral contrast swallow to the chest ct protocol in cases where fistulae are suspected can improve the diagnostic ability of ct and also better demonstrate these fistulae . an individual , as a consumer researcher , wants to buy a refrigerator . with appliances of most sorts , people regard best practice decision making as consulting trustworthy comparison websites and magazines , ones that go beyond expressing opinions , or recording likes , to numerically rate the alternative products on a set of attributes or criteria . they want these decision support tools to give them ratings that can be trusted because they are produced free of any conflict of interest or other biases the consumer does not know , and does not want to know , why this refrigerator is given a 4*/80% rating on reliability , and a 3*/60% rating on environmental impact , and another one the opposite ratings . feeling justified in assuming a common sense , lay understanding of the terms reliability and environmental impact , they do not have neither the time nor motivation to find out more about what these concepts mean , in terms of the mechanical functioning of the refrigerator , the quality of its components , the emissions it produces or whatever else contributes to these ratings made by the expert assessors some may wish to establish whether consumers have made an informed decision by seeing how well they score on a test of refrigerator knowledge . giving considerable and fixed weight to knowledge in their measures of decision quality , consumers decisions might be regarded as poor quality , because their knowledge sub - score is low . in contrast , consumers may regard themselves as having made good decisions , indeed the best possible decisions they could make , given the time and cognitive effort they are willing to devote to research into the decision - making process including accessing and accumulating knowledge deemed important , even essential , by others . but surely health care decisions are different from buying refrigerators ? choosing between surgery and medical options for newly - diagnosed cancer , or pain management for chronic osteoarthritis , is not like buying a household appliance , is it ? in fact , nothing really changes for the individual , whom we now conceptualize as a researcher conducting a continuing , informal n - of-1 study into his / her health . the affective and emotional differences between the two situations may well produce differences in the decision - making process , but the patient accepts that this will not necessarily enhance the quality of the decision as he / she defines it . patients may become interested in finding out more about their medical condition than they would about refrigerators , and actually do so . but unless it leads to a change in a performance rating for an available option on one of their criteria especially the bean for a criterion they weigh heavily the additional information they now possess is decision - neutral . people - as - researchers may feel better informed in some sense , but they realize they will not necessarily be in a position to make a better decision and therefore have not ended up more decisionally empowered . they may even simply have become more anxious and regretful about the opportunity costs of acquiring the information , in the form of the foregone benefits from other activities in which they could have engaged . informed decision ? according to themselves and us , absolutely , since they have consulted a transparent set of option performance ratings on relevant criteria , originating from a source that they have decided is the most trustworthy . their decision quality score may well be low according to an instrument that weights highly the knowledge that they are assumed by others to need to make an the growing number of condition - specific decision quality instruments being developed , notably by karen sepucha and colleagues , all give very heavy weight to a knowledge subcomponent . there could be no clearer confirmation of the issue at stake here than the title of one of the background papers to these projects : how does feeling informed relate to being informed? trust is crucial here . in either shared or unshared decision making , trust relates to the inputs into decision making , since we have left behind the notion of an agency relationship , previously dominant in conceptualising medical practice . trust is always a matter of degree , rather than a binary all or nothing , whether it relates to the beans provided by the clinician , or by a decision support tool . even if there is only one , dubious , source , it will be the most trustworthy . unless , that is , the person rates his / her own estimates as more trustworthy than the best source , since so we envisage an individual regarding the respected consumer magazine s beans on refrigerators as the most trustworthy in relation to that purchase decision . people s task in health care decisions , given a restricted willingness to devote time and energy to processing information , is to assess the trustworthiness of the available sources of beans for the outcomes and other criteria important to them . they would expect a clinician , or a team developing the ratings for a decision aid , to be highly trustworthy and to be provided with evidence for this , especially in the case of an aid . the key information the person - as - researcher requires is labelling that ensures he / she will get what it says on the tin when they open an aid . with this meta - information , they can make an informed choice about which tins of what size to open . the other major problem with any imposed information requirement is that it condemns many on the continuum of health literacy , and especially health numeracy , to receiving little or no help . we fully support attempts to reduce health illiteracy and innumeracy , especially their decision - focused forms . however , it is too much to expect of a decision support tool or a clinician to overcome the limitations of previous education and socialization in these respects . moreover , it is important to accept that even if aid users are able to register and report the relative numbers of sad and smiley faces in frequency diagrams , or repeat back 1 in x statements about which there is considerable doubt this does not in any way ensure that they can meaningfully incorporate the numerical probabilities they have correctly registered ( say 10% and .05% , or 1 in 10 and 1 in 2000 ) , into their decisions . this is not to say that a decision aid should not contain help in this respect , including guidance on how the person can best avail themselves of what it offers , and information on the bases of that offering . it is to suggest that much of this should be provided on an opt - in basis . nothing in what we have said is intended to imply that the community is not entitled to apply community - level criteria and weights to what it provides , or allows to be provided , to whom , under what conditions , and at what cost , in the pursuit of goals such as efficiency , equity and justice . formal laws and regulations ( including those on informed consent and clinician liability ) and resource allocation policies ( including reimbursement decisions ) will be the context in which the individual decision is made , and they will frequently be in conflict with what an individual sees as best for him / herself , given personal criteria and weights . external consequences for others may trump individuals preferences , as in the case of infectious diseases . trickier are the issues of social responsibility or morality which are not dealt with formally . apart from issues of environmental and social impact ( such as those arising from hormonal treatments and opioids ) , there are all those that arise in resource - constrained and interdependent systems simply as a result of those constraints and interdependencies . in these cases , we say two things . first , it is not the function of individual decision support tools to mandate the inclusion or exclusion of social criteria in an individual s set , such as concern for others health , or insist that these be given specific weights . second , that in order to be regarded as having made a high - quality decision , the individual should not be required to be informed about the social criteria they do select , other than having the processed beans available to them from a trustworthy source . normative checklists for decision support tools , such as those constructed in accordance with the guidelines of the ipdasi collaboration , are clearly intended to promote person - as - patient empowerment . but most decision aids that comply with these guidelines are designed for use only within the context of shared decision making , in which the person is assigned the status of patient . in many cases , the support can be accessed only within the clinical encounter , or with provider permission . they all perpetuate the idea that only a decision informed in a particular way and to a particular extent can be a good decision . we do not need the concept of an informed decision , only that of a good , better or best possible decision . for none of these will there be a definition that is not multi - dimensional and therefore preference - sensitive . there can only be one answer : the patient s or the person s if they are not a patient .

we report two cases of esophagobronchial fistulae diagnosed by multi - detector computed tomography ( mdct ) oral contrast swallow examination . it is helpful to supplement the ct study with an oral contrast swallow as it aids in confirmation of a suspected fistula and also demonstrates the fistula tract better . we present the clinical details and the imaging findings on mdct of two cases of esophagobronchial fistulae one secondary to chronic chest tuberculosis and the other secondary to a squamous cell carcinoma of the upper esophagus followed by discussion of the etiology , pathogenesis , and imaging of these fistulae . most guidelines for clinical practice , and especially those for the construction of decision support tools , assume that the individual person ( the patient ) needs to be in possession of information of particular sorts and amount in order to qualify as having made an informed decision. this often implicitly segues into the patient having made a good decision. in person - centred health care , whether , in what form , and with what weight , information is included as a criterion of decision quality is a matter for the person involved , to decide in the light of their own values , preferences , and time and resource constraints .

pancreatic intraepithelial neoplasia ( panin ) is a nomenclature for microscopic proliferative epithelial lesions of the pancreas which was proposed in 2001 by hruban et al . . panin is assumed to be a precursor lesion of invasive ductal carcinoma ( idc ) . precursor lesions of idc also include intraductal papillary mucinous neoplasm ( ipmn ) and mucinous cystic neoplasm . panin is divided into three grades , panin-1 , -2 , and -3 , according to the cytological and architectural atypia . panin-1 is further subdivided into flat ( panin-1a ) and papillary types ( panin-1b ) . this hypothesis has arisen from the observation of the resected specimens , in which synchronous or metachronous occurrence of panin and idc were seen . panin had been reported to be more common in the pancreas with idc than without [ 2 , 3 ] . it has also been reported that idc occurred in the remnant pancreas several years after partial pancreatectomy for high - grade panin had been performed [ 4 , 5 ] . several studies have demonstrated higher frequencies of genetic alterations such as k - ras mutation or dpc4 inactivation in higher - grade panin lesions [ 6 7 8 9 ] . thus , it is hypothesized that pancreatic ductal lesions may progress from a histologically normal duct through flat lesion ( panin-1a ) , papillary lesion ( panin-1b ) , atypical lesion ( panin-2 ) to severely atypical lesion ( panin-3 ) . panin-3 is likely to develop eventually into idc because panin-3 and idc share critical genetic abnormalities . however , the histological evidence that panin-3 invades beyond the basement membrane of pancreatic ductal epithelium , that is , the moment panin-3 becomes idc , has not been captured yet . as defined by the consensus guideline , panin is a microscopic papillary or flat , non - invasive epithelial neoplasia that is usually < 5 mm in diameter . , panin-3 obstructed several branch pancreatic ducts and subsequently caused pancreatitis which developed clinical symptom and was detectable as a pancreatic mass on imaging studies . since pancreatic cancer was suspected , further examinations were performed . histological examination of the resected specimens showed panin-3 slightly invading beyond the basement membrane of the ductal epithelia accompanied by fibrotic changes caused by occlusion of branch ducts . a 65-year - old woman had been admitted to a former hospital for acute pancreatitis , which recurred 2 months thereafter . she was referred to our center for further examination of her pancreas after the second pancreatitis had been relieved . she was slightly thin , showing a body mass index of 20.2 , and had neither smoking nor drinking habits . laboratory data showed no abnormalities including serum levels of amylase and tumor markers related to pancreatic diseases . abdominal ultrasound revealed a low echoic mass of 13 mm in diameter in the pancreatic body without upstream dilatation of the main pancreatic duct ( mpd ) ( fig . endoscopic ultrasound showed a low echoic mass of 20 mm in diameter in the pancreatic body ( fig . since pancreatic cancer was suspected in the pancreatic body , she underwent endoscopic retrograde pancreatography , which showed a strictured segment of 2 mm in length in the mpd at the pancreatic body ( fig . cytological examination of the pancreatic juice obtained during endoscopic retrograde pancreatography revealed adenocarcinoma ( fig . 1 g ) . the patient was diagnosed with pancreatic body cancer without obvious vascular involvement ( t3 , n0 , m0 , stage iia , according to the uicc classification ) and underwent distal pancreatectomy . the resected specimens were fixed in formalin and cut at a slice thickness of 5 mm as shown in fig . histological examination revealed papillary growth of intraductal epithelia with significant nuclear atypia which was classified as panin-3 according to the definition of the consensus guideline mainly in the branch ducts ( fig . panin-3s were located separately in branch ducts with normal epithelia in the mpd between them ( fig . 2b , h , i ) , indicating that they originated from different sites of the branch ducts . histologically , this case was diagnosed as idc of the pancreatic body ( t1 , n0 , m0 , stage i ) . high - grade panin ( panin-3 ) is recognized as a best - defined precursor lesion of idc based on genetic as well as histological observations . panin-3 has been reported to share some genetic alterations with idc [ 6 7 8 9 ] and to be found usually in the pancreas with idc [ 2 3 4 5 ] . however , the direct evidence of the moment when panin-3 invades beyond the basement membrane to become idc has not been captured yet . this is the first case report in which microinvasion of panin-3 was histologically confirmed in the resected pancreatic specimens . considerable ambiguities had existed in the distinction between panin and ipmn , therefore the definitions of these two lesions were revised at the consensus meeting held at johns hopkins hospital in 2003 . according to the revised definitions , panin is a microscopic , papillary or flat epithelial neoplasm arising in the pancreatic duct and differs from ipmn , which is defined as a grossly visible , mucin - producing , predominantly papillary epithelial neoplasm . in this case , pancreatic epithelial neoplasm itself was not identified in any radiological examinations , and mucin production was not evident in pancreatic ductal epithelia by microscopic observation . a few reports had suggested that minimally invasive carcinoma should be distinguished from invasive ipmn because this subgroup of ipmn showed apparently better prognosis [ 11 , 12 13 ] . however , the precise definition of minimally invasive carcinoma has not been established [ 14 , 15 ] and has been recently proposed by some studies [ 16 , 17 ] . in this case , the patient has been alive without any signs of recurrence for more than 3 years . since there has been no concept of minimally invasive panin to date , this case is classified as idc in spite of excellent outcome . a new notion that minimally invasive panin should be distinguished from idc might be widely accepted in the future . in this case , panin-3 lesions were located separately in several branch ducts without involving the epithelia of the main duct between them . this fact indicates that panin does not arise from one small portion of pancreatic ductal epithelium and spread creeping along the epithelia , but arises multicentric . it is well known that ipmn , another precursor lesion of idc , occurs multicentric . as the whole pancreas may be involved in the genetic alteration which is related to carcinogenesis , it is not difficult to understand that panin occurs independently in different sites of the pancreas . several cases dealing with multiple occurrence of idc have been reported as rare clinical manifestation [ 18 , 19 ] . this observation could explain the rapid growth of pancreatic ductal carcinoma as follows : microinvasion initially occurs in several pancreatic ducts ; each invasion independently progresses and eventually makes a fusion mass . this observation could also explain the multiple occurrence of idc when microinvasions occur independently distinct from each other . unlike ipmn , panin-3 is hardly detected in clinical practice because panin-3 itself is not visible on imaging examination . in this case , panin-3 was successfully detected due to pancreatitis caused by the intraductal growth of panin-3 and subsequent occlusion of the pancreatic ducts . pancreatitis might be a candidate for the diagnostic clues to the early detection of pancreatic ductal carcinoma within the preinvasive stage . there are other known diagnostic clues to early detection , such as pancreatic cysts or dilatation of the mpd . in this case , however , neither pancreatic cyst nor dilatation of the mpd was observed . the detection of pancreatic ductal carcinoma in the preinvasive stage is the most promising way of improving patient survival . we hope that our report will contribute to the further understanding of early development of pancreatic ductal carcinoma . ductal adenocarcinoma of the prostate was first reported by melicow and pachter in 1967 as an endometrial carcinoma prostatic utricle . since then , ductal adenocarcinoma of the prostate has been found to account for 0.27.5% of all prostate carcinomas . a 73-year - old man was referred to our hospital due to an elevated prostate - specific antigen ( psa ) level of 23.4 ng / ml . he had no remarkable medical history . the hematological and biochemical data showed no abnormal findings aside from the elevated psa levels . in february 2016 , a prostate needle biopsy detected gleason score 4 + 4 adenocarcinoma in his left prostate . computed tomography ( ct ) and magnetic resonance imaging ( mri ) showed a higher density on his left peripheral zone ( fig 1a , b ) . in may 2016 , radical prostatectomy with lymph node resection histologically , there were many large , clear - edged cells and cancer cells with low differentiation forming a circular shape . based on these findings , ductal adenocarcinoma and gleason score 4 + 4 = 8 acinar adenocarcinoma with positive surgical margin were diagnosed . the patient has not experienced recurrence or biochemical recurrence in the 10 months since radical prostatectomy . histologically , there were many large , clear - edged cells and cancer cells with low differentiation forming a circular shape . based on these findings , ductal adenocarcinoma and gleason score 4 + 4 = 8 acinar adenocarcinoma with positive surgical margin were diagnosed . no adverse perioperative events were observed . the patient has not experienced recurrence or biochemical recurrence in the 10 months since radical prostatectomy . ductal adenocarcinoma of the prostate was first reported as endometrial carcinoma of the prostatic utricle in 1967 . recent studies have suggested that ductal adenocarcinoma of the prostate developed from the ductal epithelium , based on findings from immunohistochemical and electron microscope analyses . histologically , ductal adenocarcinoma of the prostate is characterized by high cylindrical epithelium collate papillary or etat cribriform . the histological differences between ductal adenocarcinoma and acinar adenocarcinoma are thought to be clear . in this case , although the prostate needle biopsy showed acinar adenocarcinoma , the surgical specimens showed ductal adenocarcinoma . the first is a mixed type with acinar adenocarcinoma and accounts for < 75% of ductal prostate specimens . mixed - type ductal prostate adenocarcinomas account for 5.06.6% of all prostate cancer cases , and pure - type ductal prostate adenocarcinomas account for 0.40.8% of all prostate cancer cases . because ductal carcinomas account for 90% of all prostatic carcinoma cases , our case was assumed to be pure type . because of its extension toward the urethra , the tumor was not palpable on a digital rectal examination and showed a low psa level . ductal adenocarcinoma of the prostate usually extends toward the urethra and shows macrohematuria and urinary symptoms at an early stage . reported that ductal adenocarcinoma of the prostate showed a significantly poorer prognosis than acinar prostate adenocarcinoma in nonmetastatic cases . however , in metastatic cases , there were no prognostic differences between these 2 groups . other reports have found no marked differences in the 5-year survival rate between ductal adenocarcinoma and gleason score 810 acinar adenocarcinoma . reported therapies of ductal adenocarcinoma of the prostate are also the same as for acinar adenocarcinoma , including radical prostatectomy , androgen deprivation therapy , and radiation therapy or a combination of these therapies . reported that pure ductal adenocarcinoma tended to extend into the submucosal urethra ; as such , pure ductal adenocarcinoma carries a higher risk of a positive surgical margin in the urethra . although we are not performing adjuvant therapy in this patient at present , careful observation including ct , mri , or positron emission tomography - ct should be performed , as psa does not always accurately represent cancer progression .

high - grade pancreatic intraepithelial neoplasia ( panin-3 ) is recognized as a precursor lesion of invasive ductal carcinoma ( idc ) . however , histological evidence that panin-3 invades beyond the basement membrane of pancreatic ductal epithelium , that is , the moment panin-3 becomes idc , has not been captured yet . this may be because panins which are microscopic papillary or flat lesion rarely develop clinical symptoms and are not detectable on imaging examination . on the other hand , most idcs were found in the advanced stage with massive invasion . in this report , panin-3 obstructed several branch pancreatic ducts and subsequently caused pancreatitis which developed clinical symptom and was detectable as a pancreatic mass in imaging studies . a 65-year - old woman was referred to our institution for further examination of her repeated pancreatitis . abdominal ultrasound revealed a low echoic mass of 13 mm in diameter in the pancreatic body without upstream dilatation of the main pancreatic duct ( mpd ) . endoscopic retrograde pancreatography showed a strictured segment of 2 mm in length in the mpd at the pancreatic body . cytological examination of pancreatic juice revealed adenocarcinoma and distal pancreatectomy was performed . a resected specimen revealed a whitish mass of 15 mm in diameter in the pancreatic body , which was identified as pancreatitis by histological examination . papillary growth of panin-3 was seen mainly in the branch ducts . each panin-3 was located separately in the branch ducts with normal epithelia in the mpd between them . in three adjacent branch ducts , panin-3 was observed to be invading microscopically beyond the basement membrane . ductal adenocarcinoma is an unusual variant of adenocarcinoma of the prostate . a 73-year - old male was referred to our hospital for the further examination of an elevated prostate - specific antigen level of 23.4 ng / ml . radical prostatectomy ( rp ) was performed based on the diagnosis obtained by a prostate needle biopsy . the rp specimen revealed ductal adenocarcinoma of the prostate with positive capsular penetration . we herein report a rare case of ductal adenocarcinoma of the prostate .

patients treated with salvage chemotherapy had a response rate of only 9% and a median time to tumor progression ( ttp ) of 9 weeks . although bevacizumab can offer a significantly higher response rate of 55% and a period of clinical stabilization with a median ttp of 26 weeks , the tumor remains in the brain and continues to proliferate despite clinical and radiological appearances of improvement . as a result , bevacizumab has a questionable impact on the overall survival of patients [ 3 , 4 ] . therefore , new and novel treatments are needed for patients with recurrent glioblastoma who failed initial treatment with radiotherapy and temozolomide . the novottf-100a device is a new treatment approved by the united states food and drug administration ( fda ) for recurrent glioblastoma . the device emits alternating tumor treating electric fields ( ttfields ) via 2 pairs of transducer arrays placed orthogonally on the scalp . the ttfields work by interrupting tumor cells during mitosis , resulting in violent blebbing during cytokinesis , asymmetric chromosome segregation , and aneuploidy [ 5 , 6 ] . these cell biology effects ultimately result in apoptosis or slippage into a g0 state of the tumor cell , while simultaneously making it susceptible to immunogenic cell death . in the pivotal phase iii clinical trial , the novottf-100a device had a similar efficacy when compared to salvage chemotherapy , but without the toxicities associated with systemic chemotherapies [ 7 , 8 ] . here , we report a patient who had failed bevacizumab therapy for a recurrent cystic glioblastoma and , at the time of bevacizumab continuation , received add - on ttfields therapy by using the novottf-100a device . this treatment combination eventually resulted in the disappearance of cystic enhancement together with a marked reduction of the cyst size and the cerebral edema in the surrounding brain . the patient is a 76-year - old right - handed woman who came to the brain tumor center for an evaluation of her recurrent glioblastoma after bevacizumab failure . her initial neurological problems occurred 9 months prior to presentation and consisted of mental confusion and comprehension problems manifesting as a fluent aphasia . a gadolinium - enhanced head mri at an outside hospital showed a cystic enhancing mass in the left temporal lobe of the brain . she then received 6 weeks of external beam fractionated radiotherapy to 6,000 cgy ( 200 cgy in 30 fractions ) with concomitant daily temozolomide at 75 mg / m , followed by adjuvant temozolomide 200 mg / m for 5 days on a monthly basis . after 5 cycles of adjuvant temozolomide , new cystic enhancement was discovered while performing a head mri ( fig . 1a , b ) , and she was placed on 4 mg of dexamethasone 4 times / day . bevacizumab was subsequently started at a dose of 10 mg / kg every 2 weeks . after 2 cycles of bevacizumab , there was only a partial decrease in the gadolinium enhancement and the size of the cystic tumor ( fig . additional pathology testing revealed a negative idh1 immunohistochemical labeling but a positive olig2 , egfr amplification and methylated mgmt promotor status . because ttfields interrupt tumor cells during mitosis and have no appreciable overlapping toxicity with bevacizumab , we proceeded to add to her bevacizumab treatment ttfields therapy using the novottf-100a device . the treatment compliance was recorded by sensors , embedded within the transducer arrays and can be downloaded by computer for review in clinic visits . after a total of 6 cycles of bevacizumab plus ttfields therapy , with a respective mean and median compliance of 17.6 hours ( 73% of the day ) and 18.4 hours ( 77% of the day ) ( range 3.6 to 22.8 h ) , there was a near complete resolution of gadolinium enhancement and a 65% reduction in the size of the cystic tumor ( fig . 1e , f ) . however , there were also recurrent tumors detected in the left internal capsule and the medial left frontal brain ( fig . 2a , b ) , which were located outside of the prior radiation treatment fields . therefore , the recurrent tumors were treated with fractionated cyberknife radiosurgery to 2,100 cgy ( 700 cgy in 3 fractions ) . despite this radiosurgery intervention , the patient continued to deteriorate neurologically and her deterioration was most likely caused by the microscopic invasive glioblastoma . she eventually died 15 months after her first recurrence and 22 months after initial diagnosis . the addition of novottf-100a to bevacizumab therapy in our patient appears to be safe and may provide added efficacy after initial incomplete response to bevacizumab alone . the rationales to combine ttfields therapy with bevacizumab are threefold . first , there is no overlapping side effect and , therefore , this combination does not appear to cause synergistic or additive toxicities . in patients with glioblastoma , we retrospectively analyzed 20 patients treated with this combination and found no instance of intracranial hemorrhage . other treatment side effects are minor in severity and they include the expected scalp rash , electric shock sensation from poorly applied transducer arrays and vivid dreams , all of which resolved upon the application of corticosteroid cream and adjustment of the arrays . second , ttfields therapy mimics the effects of chemotherapy by interference with tumor cell mitosis while not having the conventional side effects of chemotherapy [ 5 , 7 ] . when first approved by the fda , bevacizumab was combined with various cytotoxic agents , including carboplatin and irinotecan , carboplatin and etoposide as well as lomustine or carmustine , but the side effects do not warrant the routine clinical use of bevacizumab combined with cytotoxic chemotherapy . third , both bevacizumab and novottf-100a are listed in the national comprehensive cancer network practice guideline for recurrent glioblastoma . therefore , there appear to be strong rationales to combine novottf-100a with bevacizumab for recurrent glioblastoma . the resolution of our patient 's cystic tumor is a notable response to novottf-100a and bevacizumab after initial bevacizumab failure . although the response assessment in neuro - oncology criteria deem it as nonmeasurable , cystic tumors can not be measured in a traditional bidimensional fashion for response assessment , unless there is an associated solid nodule measuring 10 mm or greater in diameter . however , the disappearance of enhancement in the cystic tumor is still remarkable . in their retrospective series of 51 recurrent high - grade gliomas treated with bevacizumab and irinotecan , zuniga et al . reported only 8% of patients with a complete response while the majority had either a partial response ( 63% ) or no response ( 29% ) . similarly , when bevacizumab was used as a single agent , kreisl et al . and friedman et al . these data indicate that bevacizumab alone rarely results in a complete radiographic response , and the partial response seen in a majority of patients suggests that there are probably multiple proangiogenic pathways activated in the glioblastoma . a prior post hoc analysis of the response characteristics from the pivotal phase iii trial indicates that secondary glioblastomas as well as low or no dexamethasone usage are potentially important predictors of response in patients treated with the novottf-100a device alone [ 7 , 17 ] . first , secondary glioblastomas may have a different genetic composition that makes the tumor cells more susceptible to ttfields . indeed , verhaak et al . have shown that most of the secondary glioblastomas fall in the proneural genotype with amplification of pdgfra and olig2 as well as mutations in idh1 and tp53 . although our patient 's tumor appears to be a primary glioblastoma by the lack of idh1 immunohistochemical labeling , there could still be unidentified genetic mutations that make her tumor susceptible to ttfields therapy . indeed , 9 out of 14 responders in the phase iii trial did not have prior low - grade histologies , and they could have genetic mutations or epigenetic alterations that make them susceptible to novottf-100a monotherapy . second , the slower growth rate of our patient 's tumor may have helped to allow sufficient time for the ttfields to effect a treatment response . this is because the median time to response for ttfields therapy is longer than that for chemotherapies , 8.4 versus 5.8 months , respectively , as noted in the prior post hoc response analysis . last , our patient 's dexamethasone was completely discontinued 2 months after initiation of combination ttfields and bevacizumab therapy , but a near complete resolution of gadolinium enhancement and a significant reduction of the cystic tumor were not detectable until 6 months into treatment . consistent with this observation , the inverse relationship between response and dexamethasone dosage is probably a consequence of removing the immunosuppressive effect of dexamethasone , which would allow for better control of the glioblastoma by the patient 's immune system . therefore , allowing sufficient treatment time and removing dexamethasone are key parameters to maximize the probability of a response from ttfields . the pattern of relapse from combination ttfields and bevacizumab therapy is unknown . at the time of tumor recurrence , this type of distant recurrence of glioblastoma could be the consequence of progressive development of the invasive phenotype , intracranial inhomogeneity of the ttfields , or incomplete coverage of certain parts of the brain by ttfields . these hypothesis - generating observations would be important for future studies to correlate the location of the relapsed disease and the distribution of the electric fields within the brain . plaque - induced gingivitis is one of the most frequent periodontal diseases , affecting more than 90% of the population , regardless of age , sex or race20 . brazilian epidemiologic studies show a high prevalence of gingival inflammation , ranging from 74% to 100% , although the mean individual percentage of gingival bleeding varies from 28% to 35%5 . however , the inability of the normal adult population to perform adequate toothbrushing has led to the search for chemotherapeutic agents in order to improve plaque control12 . these chemicals , mainly triclosan and chlorhexidine , have been used as mouthrinses or added to dentifrices to avoid plaque formation and development of gingivitis10,12,14,22 . as some of these substances may have undesirable side effects , such as tooth staining and taste alteration , phytotherapic agents with antimicrobial and antiinflammatory properties have been investigated7,16,17 . the use of natural products in the prevention and treatment of oral conditions has increased recently and could be of benefit to low - socioeconomic level urban and rural communities4 . among the various currently available herbal agents , aloe vera , popularly known as " babosa " , is a plant commonly found the northeast of brazil . its foliage , extract and resin present antimicrobial , antiinflammatory and healing properties and are indicated to hepatic and stomach diseases9 . the antimicrobial effect of a dentifrice containing aloe vera has been demonstrated in an in vitro study , in which this phytotherapic agent inhibited the growth of diverse oral microorganisms , such as s. mutans , s. sanguis , a. viscosus and c. albicans 7 . the only study available evaluating the clinical effects of aloe vera showed a significant reduction of gingivitis and plaque accumulation after use of a mouthrinse containing this natural product20 . to the present date , there is no reported controlled trial evaluating the efficacy of a dentifrice containing aloe vera in the control of plaque and gingivitis . therefore , the purpose of the present study was to assess the antiplaque and antigingivitis effects of this phytotherapic agent compared to a fluoridated dentifrice . thirty adult subjects from the university of fortaleza , brazil ( 15 male and 15 female , aged 35 to 43 years ) were enrolled in this double - blind , parallel , controlled clinical trial . all randomly screened participants were informed about the nature of the study and signed an informed consent form in compliance with the guidelines of the brazilian national health council . the protocol was approved by the institutional ethics committee ( report cotica / unifor : 407/ 2006 ) . the subjects were entered in the study if they had gingival bleeding index ( gbi)1 > 40% , presence of at least 20 natural teeth and absence of supragingival calculus and other plaque retentive factors . subjects with medical disorders or probing depth > 3 mm , individuals under antimicrobial therapy at least 1 month prior to the study and using mouthrinses or dentifrices containing substances with antiinflammatory properties , as well as smokers and pregnant women were excluded from the trial . the participants were assigned to either the test group ( n=15 ) or the control group ( n=15 ) by random permutation of three . the test group used a herbal dentifrice containing aloe vera ( forever bright , forever living products , tempe , az , usa ) and the control group used a fluoridated dentifrice ( sorriso dentes brancos , kolynos do brasil , so paulo , sp , brazil ) , with no antiinflammatory properties , but with color and taste similar to those of the test dentifrice . a single , previously calibrated examiner3 scored the gingival bleeding index ( gbi)1 and the plaque index ( pi)19 , which were recorded on the buccal , mesial , distal and lingual surfaces of all teeth . the values of 4 sites of each tooth were averaged to determine the gbi and pi for each subject . in addition to this examination , the hard and soft oral tissues were visually inspected for the presence of any adverse reaction by the same examiner . after the initial examination , all teeth of each subject were polished with pumice and flossed to eliminate plaque remnants . a personal " kit " containing a new toothbrush ( leader ; facilit odontolgica e perfumaria ltda , rio de janeiro , rj , brazil ) and the test or control dentifrice was given to all participants . they were instructed to brush their teeth for 1 min , three times a day , using the bass technique , and to refrain from any other oral hygiene procedures throughout the period of the clinical trial . verbal and written instructions about the correct use of dentifrice were given to all subjects as well . the tubes containing the dentifrices were previously coded to warrant that neither the examiner nor the volunteers knew their content , which was revealed only after completion of the study . the subjects were asked to return their dentifrice tubes , that were weighted by a digital balance ( filizola , modelo bp6 , indstrias filizola s.a . , so paulo , sp , brazil ) , previously and after the trial , so that compliance could be indirectly evaluated . student 's t - test was used to evaluate statistical differences between the weights of dentifrice tubes on days 0 and 30 ( =0.01 ) . mann - whitney test was performed to evaluate statistical differences between control and test groups on days 0 and 30 ( =0.01 ) . in each group , the mean scores of gbi and pi were compared between baseline and the end of the trial by the wilcoxon test ( =0.01 ) . the test dentifrice had a good acceptance and did not show adverse effects , such as formation of abscess and ulcerations or allergic reactions . only one subject in the test group reported unpleasant taste , but he did not drop out the clinical trial . there was a significant reduction of dentifrice tube weights between days 0 and 30 in both groups ( p<0.001 ) , which indirectly indicated that the volunteers actually used the dentifrices ( table 1 ) . on day 0 , there was no statistically significant difference between the control and test groups with respect to gbi ( p=0.8774 ) and pi ( p=0.0477 ) means . these results indicated that both groups were well balanced at baseline ( tables 2 and 3 ) . at the 30th day , plaque ( p=0.2801 ) and gingival bleeding ( p=0.9346 ) were present in both groups , but the difference between them was not significant statistically ( tables 2 and 3 ) . comparing the means between baseline and day 30 in each group , there was statistically significant difference in both the gbi ( p=0.002 , control group and p=0.001 , test group ) and pi indexes ( p=0.002 , control group and p=0.001 , test group ) ( tables 2 and 3 ) . aloe vera is a natural product contained in herbal dentifrices with commercial appeal on the control of plaque and gingivitis . despite its free commercial use , this phytotherapic agent does not have sufficient data to support its antigingivitis and antiplaque claims21 . to the best of our knowledge , the present study is the first report about effect of a dentifrice containing aloe vera on gingivitis . the results showed that both toothpastes were efficient on plaque reduction ( 23% in test group and 19% in control group ) . this percent difference was not significant at the end of the trial . in spite of a reported in vitro inhibitory effect of aloe vera against microorganisms from supragingival biofilm7 , its in vivo antiplaque effect in the present study was not satisfactory . it is interesting to note that this previous study7 evaluated a dentifrice that also contained other antiplaque agents , which probably masked the effect of aloe vera . the test and control dentifrices reduced gingivitis significantly , although the fluoridated dentifrice presented a higher percent reduction on bleeding areas ( 56% versus 53% ) . the gbi is a generally used dichotomous index to evaluate gingivitis , but it does not access the severity of gingival inflammation . studies evaluating the reduction of gingivitis by a grading index could be interesting to complement these results . gingival index uses a scale in which color changes in the gingival tissues precede bleeding on probing ; however this parameter is not necessarily an accurate indicator of gingivitis18 . the findings of the present study are in agreement with those of villalobos , et al.20 ( 2001 ) , who observed a significant reduction on plaque and gingivitis after a 30-day use of mouthrinses containing aloe vera associated to toothbrushing . this study20 also showed an additional antiplaque and antigingivitis effect of this phytotherapic agent , which was not observed in the present clinical trial . villalobos , et al.20 ( 2001 ) used a higher concentration of aloe vera ( 50% ) , which could explain the better effect of this phytotherapic agent when compared to our findings . although the manufacturer does not inform the concentration of aloe vera in the product used in the present study , the percentage of therapeutic agent in a dentifrice usually range from 0.4% to 1.0% of the total formulation6 , which was probably the concentration used in our study and could explain those results . furthermore , villalobos , et al.20 ( 2001 ) used a mouthrinse containing only aloe vera as the active agent , favoring its action without interference of other components . the test dentifrice used on the present trial contains other agents that can promote a moderate antiplaque effect , such as menthol and sodium lauryl sulfate12,21 . since the last two components are also present in the control dentifrice and considering that no difference was found between groups the antagonism of diverse substances with similar effects , in the same product , should be considered as well . this fact has been highlighted by wua and savitt21 ( 2002 ) and confirmed by other clinical trials comparing fluoridated and herbal dentifrices11,15 . home - use dentifrice studies are often influenced by a number of factors which can mask the superiority of a test agent over the controls . participants in clinical trials may experience some improvement associated not specifically to the therapeutic properties of the test agent but rather related to a behavior change - hawthorne effect15 . subjects enrolled in oral hygiene studies usually improve their toothbrushing , irrespective of the product they receive11,13,15 . although the volunteers of the present study were not aware of which dentifrice they were using , another important factor is the novelty effect , which is the motivation of oral hygiene practice by the use of a new substance . on the other hand , lack of compliance in the correct use of dentifrice can occur as well15 . in order to minimize its occurrence , the participants were asked to bring the dentifrice tubes at the end of the trial to be weighed , so we could evaluate indirectly subject compliance . the significant difference between weights in both groups , before and after the study , indicates that the participants used the products , but does not confirm if they were used correctly . however , it may not be sufficient to show the superiority of the test dentifrice over the control toothpaste15 . further long - term studies must be performed to evaluate the antigingivitis effect of this herbal dentifrice . if its real benefit is confirmed , the use of aloe vera should be advantageous in cases where patients have little motor skills and toothbrushing is compromised . within the limits of this clinical study , it may be concluded that the dentifrice containing aloe vera did not show any additional effect on plaque and gingivitis control compared to the fluoridated dentifrice .

the novottf-100a device emits alternating tumor treating electric fields ( ttfields ) that interfere with cytokinesis and chromosome segregation during mitosis . because it has a similar efficacy to cytotoxic chemotherapy , the device has been approved by the united states food and drug administration for the treatment of recurrent glioblastoma . although bevacizumab has been in use for recurrent glioblastoma , patients who experience incomplete or no response to bevacizumab may be predisposed to early bevacizumab treatment failure . however , the addition of ttfields therapy may augment the efficacy from bevacizumab . we report a patient with recurrent cystic glioblastoma who received add - on ttfields therapy due to an incomplete response to single - agent bevacizumab . after 6 cycles of therapy , a resolution of cystic enhancement was noted , together with reduction of the tumor cyst and resolution of most of the cerebral edema in the surrounding brain . however , the patient also suffered from relapsed disease at locations distant from the original glioblastoma and the corresponding radiation fields received at initial diagnosis . we conclude that combination ttfields and bevacizumab therapy is safe and may be efficacious for patients with recurrent glioblastoma . a further study would be needed to determine the relapse pattern and the distribution of the electric fields in the brain . the effect of aloe vera on the reduction of plaque and gingivitis was evaluated in a randomized , parallel and double - blind clinical trial . subjects were randomly allocated to the test group ( n=15 ) dentifrice containing aloe vera - or the control group ( n=15 ) fluoridated dentifrice . plaque index ( pi ) and gingival bleeding index ( gbi ) were assessed at days 0 and 30 . subjects were asked to brush their teeth with the control or test dentifrice , three times a day , during a 30-day period . there was a significant reduction on plaque and gingivitis in both groups , but no statistically significant difference was observed among them ( p>0.01 ) . the dentifrice containing aloe vera did not show any additional effect on plaque and gingivitis control compared to the fluoridated dentifrice .

there have been sporadic cases reported since it was first described by lennox et al . , in 1952 . we report an additional case of pmc of the skin in a 70-year - old male presenting with a swelling in the lateral canthal region of his left eye , and review the pertinent literature . a 70-year - old male noticed a swelling near the the left canthus since one - and - a - half years . a raised , freely mobile , firm skin mass of approximately 3.0 2.5 cm was present on the lateral canthal region on the left eye . gross examination revealed a well - circumscribed , spherical soft tissue mass , partially skin covered , measuring 2 cm in diameter . the cut surface of the tumor had a gray - brown gelatinous appearance [ figure 1 ] . microscopically , the sections revealed a tumor in the dermis composed of tumor cells arranged in nests , glands , and cribriform patterns [ figure 2 ] . the cells were seen floating in large pools of mucin separated by thin fibrovascular septa [ figure 3 ] . [ figure 4 ] further investigations , including a colonoscopy , ultrasonographic examination of the abdomen , and computerized tomography ( ct ) scans of the chest , abdomen , and pelvis were found to be normal . thus , the lesion in the skin was reported to be pmc of the skin . gross specimen revealing a spherical , well - circumscribed tumor with a gelatinous cut surface . tumor in the dermis composed of cells arranged in nests , glands , and cribriform patterns , floating in mucin lakes ( h and e , 20 ) small nests of adenocarcinoma lying in pools of extracellular mucin that were separated by fibrocollagenous septae ( h and e , 40 ) photomicrograph showing pools of pas positive , diastase - resistant mucin between the tumor nests ( pas stain , 20 ) primary mucinous carcinoma of the skin ( pmcs ) is a rare adnexal neoplasm with sweat gland differentiation . this tumor was first described by lennox et al . , in 1952 , and later designated by mendoza and helwig , in 1971 . . recently studied approximately 200 cases of pmcs documented in the literature and found that the mean annual age standardized incidence of pmcs during the period 1978 to 2003 was 0.07 per million person - years . although some debate exists as to the apocrine or eccrine origin of this tumor , most authors favor the eccrine differentiation based on the evidence obtained from the immunohistochemical studies and electron microscopic ultra structural analysis . it has been suggested that pmcs may develop as a progression from abnormal apocrine or eccrine ducts , analogous to the progression seen in the mucinous carcinoma of the breast . a ductal in - situ component can occasionally be seen ranging from ductal hyperplasia to frank carcinoma in - situ . rosai originally suggested that the ductal proliferation continues until the overproduction of mucin results in islands of tumor cells breaking off and floating in the mucinous pools . primary mucinous carcinoma of the skin is slightly more common in men than women and typically affects people in the age range of 50 to 70 years . the large majority of specimens of pmcs arise on the face , especially on the eyelids and scalp . the primary lesion of mesenchymal chondrosarcoma ( mcs ) is often solitary , and the history is often of a small cutaneous lesion that grows slowly for many months or years and suddenly enlarges . by the time it is first brought to medical attention , the neoplasm is a nodule that varies in diameter from about 0.7 8.0 cm . mucinous carcinoma rarely originates in the skin ; the majority of examples in the skin are actually metastatic to it . common sites of origin of mucinous carcinomas are the breast , gastrointestinal tract , salivary glands , lacrimal glands , nose , paranasal sinuses , bronchi , renal pelvis , and ovary . metastatic lesions from the breast or colon are most likely to mimic mucinous carcinoma of the skin . differentiating primary mucinous carcinoma from metastatic tumors , particularly from these sites , the absence of expression of ck20 by immunohistochemical staining may exclude the diagnosis of metastatic colorectal mucinous carcinoma . it is important to recognize , however , that the likelihood of mucinous carcinoma from the breast , metastasizing to the skin ( much less on the face or eyelid ) is exceedingly low . cases of pmcs have been found to be estrogen receptor , progesterone receptor , and gcdfp-15 positive . qureshi et al . , suggest that finding an in - situ component of a tumor that stains for myoepithelial cells ( positive stains for p63 and ck5/6 among others ) can help to exclude metastatic mucinous breast carcinoma . the distinction of mcs from a mucinous carcinoma metastatic to the skin is important because the prognosis of mucinous carcinoma metastatic to the skin is poorer than that of mcs . primary lesions can be differentiated from metastatic lesions by their more organized epithelial cells , less hyperchromasia , and fewer mitoses in individual cells . in addition , in the case of metastatic carcinoma , tumor cells invade between the collagen bundles at the margin of the nodule . due to the difficulty in distinguishing these histologically , a thorough workup for metastatic lesions no primary tumor was detected in the breast , digestive tract , salivary glands , lacrimal glands , paranasal sinuses , lungs or kidneys . primary mucinous carcinoma of the skin is a slow - growing benign tumor with a high local recurrence rate . treatment of mucinous carcinoma entails local excision . on account of the high rate of recurrence , adequate excision with generous margins other treatments , such as chemotherapy and radiation , are generally not employed in the management of these tumors . patients should be counseled about the importance of frequent follow - ups for evaluation of local tumor recurrence or development of regional lymphadenopathy . incidental detection of small renal masses is increasing . this has led to an increase in biopsy of small renal masses , a proportion of which needle biopsy of small renal masses is controversial owing to the risk of seeding malignant cells along the needle tract . needle tract seeding is a rare event ; the incidence is estimated to be less than 1 in 10,000 cases of all biopsies . eight other cases of needle tract seeding in a renal mass biopsy have been described in the medical literature , two as recently as 2013 ( table 1 ) . we report our experience of a man with renal cell carcinoma ( rcc ) seeding along a biopsy tract and compare the circumstances and biopsy techniques with reported cases in the literature . a 66-year - old man was incidentally found to have a 32-mm right lower pole renal mass on a computed tomography ( ct ) scan ( fig . two samples were obtained by use of a 16-gauge temno core biopsy needle ( carefusion , san diego , ca , usa ) and a 22-gauge francine needle . histopathology revealed a well - circumscribed 30-mm clear cell rcc , predominantly fuhrman grade 2 with focal areas of grade 3 . there was an area where the capsule was interrupted that corresponded to a hemorrhagic area on the cortical surface ( fig . a tumor deposit was also noted in the perinephric fat . these features suggested that the tumor deposit in the fat was likely due to tumor seeding rather than a metastasis and that the tumor seeding could have resulted from the needle biopsy . his tnm staging was pt3a nx mx , at least stage 3 disease ( american joint committee on cancer , 7th edition , 2010 ) and his leibovich score was 5 ( intermediate risk ) . six months after the operation , there was no radiological evidence of tumour recurrence on a ct scan . aside from the potential for false - negative results , a key risk of renal mass biopsy is seeding of the biopsy tract with malignant cells . several factors in theory could affect the risk of biopsy tract seeding , such as needle size , the number of needle passes , and the use of a coaxial needle . biopsy tract seeding has been reported in renal mass biopsies using needles as fine as 23-gauge and as large as 14-gauge . theoretically , a larger - bore needle would increase the risk of seeding owing to an increased area of defect on the surface of the tumor and an increased circumference or surface area of the needle . however , because of the scarcity of cases , it is difficult at this stage to accurately determine a relationship between needle size and the risk of seeding . it is also difficult because of underreporting to associate the risk of needle tract seeding with the number of needle passes through a tumor . use of a coaxial needle allows multiple passes through the renal mass with only one pass through the surrounding normal tissue . this theoretically reduces the risk of needle tract seeding into normal tissue and potentially reduces patient discomfort as well . although it is interesting to note that a coaxial needle was not used in any of the currently reported cases of needle tract seeding after renal mass biopsy ( table 1 ) , there are just too few cases to establish a firm relationship between the risk of biopsy tract seeding and the use of a coaxial needle . visualization of larger coaxial needles on ultrasound or ct may be easier than with smaller biopsy needles , and this may improve accuracy . histological evidence of biopsy tract seeding may not always be found after definitive surgery to remove the renal mass . seeding into excised perinephric tissues can be found soon after surgery but seeding into surrounding muscle , fascia , and skin may only be apparent months , or even years , after surgery . as was seen with this case , the biopsy needle traversed skin , subcutaneous tissue , multiple muscle and fascia layers , and perinephric fat before reaching the renal lesion ( fig . thus , the tumor could theoretically seed into one or more of these tissues ; seeding as superficial as the subcutaneous tissue has been reported ( table 1 ) . this delayed presentation may increase the risk of adverse outcomes such as further metastasis and poorer prognosis . time to presentation or diagnosis of tumor seeding after renal mass biopsy has ranged from 24 days to 84 months in previously reported cases where tumor seeding was not found on the initial histopathological analysis ( table 1 ) . in conclusion , a common feature in all reported cases of needle tract seeding from a renal mass biopsy is that a coaxial needle was not used . however , because of the paucity of cases , there is currently no satisfactory association between the risk of needle tract seeding and needle size or the number of needle passes . it is important to consider that histopathological evidence of needle tract seeding may not be apparent in all cases , especially if seeding occurred beyond the excised tissues .

primary mucinous carcinoma of the skin is a rare adnexal tumor of sweat gland origin . a case report is presented of a 70-year - old male , who presented with a slow growing mass near the lateral canthus of his left eye . the case was clinically diagnosed as a fibroma . an excisional biopsy of the lesion revealed mucinous carcinoma of the skin . investigations excluded the possibility of metastatic mucinous carcinoma . thus , the lesion in the lateral canthus region was diagnosed as primary mucinous carcinoma of the skin , a rare site of occurrence . a 66-year - old man underwent computed tomography - guided needle biopsy of a suspicious renal mass . two months later he underwent partial nephrectomy . histology revealed a 30-mm clear cell renal cell carcinoma , up to fuhrman grade 3 . an area of the capsule was interrupted , which corresponded to a hemorrhagic area on the cortical surface . under microscopy , this area showed a tongue of tumor tissue protruding through the renal capsule . a tumor deposit was found in the perinephric fat . these features suggest that tumor seeding may have occurred during the needle biopsy .

despite its ubiquity in cell membranes , there is little quantitative information on the effects of small mole fractions of cholesterol on the phase diagrams , molecular organization , and surface viscosity of mixtures of phospholipids and cholesterol . understanding cholesterol s effects in altering the local molecular organization and rheology of lipid monolayers may give clues to the mechanisms that stabilize nanometer - scale rafts in complex lipid - cholesterol mixtures ; the raft hypothesis has emerged in recent years as a general organizing principle for the structure of eukaryotic cell membranes . rafts are hypothesized to result from nanometer - scale phase separation of ordered and viscous domains in which cholesterol , saturated lipids , and membrane proteins preferentially accumulate , surrounded by a sea of less viscous , unsaturated lipids with greater protein diffusivity . however , the fundamental physics underlying raft formation is still being developed , especially how interactions between saturated phospholipids and cholesterol determine membrane phase behavior , viscosity and diffusivity . the interactions of cholesterol and saturated phospholipids also play an important , but poorly understood , role in the properties of human lung surfactant ( ls ) , a lipid - protein monolayer necessary to reduce the surface tension in the lung alveoli . at present , even the existence of cholesterol in native ls is questioned , as the lung lavage required to harvest ls inevitably causes blood and cell debris to be coextracted , potentially contaminating ls with cholesterol . this lack of consensus is reflected in the composition of replacement lung surfactants , which are used to treat neonatal respiratory distress syndrome ( nrds ) , which occurs in 20 00030 000 premature infants each year in the u.s . survanta and curosurf , two clinically approved animal extract replacement surfactants for treatment of nrds , have all cholesterol removed after harvesting . infasurf , the third clinically approved surfactant , retains 4 - 5 wt % cholesterol . resolving this controversy is difficult , as there is little information on the effects of small cholesterol fractions on the organization and dynamics of phospholipid monolayers . our previous work has shown that the surface viscosity of dipalmitoylphosphatidylcholine ( dppc ) monolayers decreases by an order of magnitude per wt % cholesterol , up to about 3 wt % , suggesting that the cholesterol - containing infasurf would have significantly different monolayer dynamics than cholesterol - free survanta and curosurf . interfacial viscosity is believed to have a significant effect on monolayer collapse , surfactant spreading during breathing , and transport from the type ii epithelial cells , where surfactant is produced , to the alveolar air water interface . interfacial viscosity may also be important during the instillation of replacement surfactant and the reopening of bronchial airways . however , the origin of this dramatic effect of cholesterol on dppc viscosity is not yet known . cholesterol may also play a role in lung surfactant inactivation in acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) . ards occurs as a rapid onset of respiratory failure and affects about 150 000 people per year in the u.s . with a mortality rate of 3040% . the pathogenesis of ards is not fully understood , but in both ali and ards , surfactant is inactivated by some primary pathogenesis such as lung inflammation , trauma , pulmonary infection , near - drowning , etc . the cholesterol content of ards patients shows an increase in cholesterol content and in vitro , cholesterol levels greater than 10 mol % increase the minimum surface tension at monolayer collapse , which may exacerbate lung damage . grazing incidence x - ray diffraction ( gixd ) shows that up to 7 mol % added cholesterol does not change the basic alkane packing of dppc , but does decrease the extent of ordering , or coherence area , suggesting an increased number of lattice defects . we postulate that the free area available for diffusive transport in a two - dimensional analog of the classic cohen and turnbull free volume model of viscosity is inversely proportional to the number of molecules in a coherence area . using this relationship , the surface viscosity data for all surface pressures and cholesterol fractions collapses to a simple logarithmic relation with no adjustable parameters . this suggests that the decreased molecular ordering caused by the incompatibility of cholesterol with the alkane chain lattice is the origin of the orders of magnitude decrease in surface viscosity . 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( dppc , r - enantiomer ) and dihydrocholesterol ( avanti , alabaster , al ) with 0.1 wt % texas - red dhpe ( n-(texas red sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , invitrogen , grand island , ny ) were mixed in the appropriate ratios and diluted to 0.2 mg / ml in hplc - grade chloroform ( fisher scientific , st . dihydrocholesterol ( chol ) was used instead of cholesterol to minimize oxidation but has little impact on phase behavior . surface pressure area isotherms were recorded at 25 c using a teflon trough ( nima , coventry , england ) with custom designed stainless steel ribbons which reduce film leakage at high surface pressures . a filter paper wilhelmy plate ( riegler and kirstein , gmbh ; potsdam , germany ) was used to measure surface pressure . the open area of the trough used was 125 cm and each complete compression / expansion cycle took about 8 min ( 0.42 cm / s ) . for fluorescence imaging , the langmuir trough was mounted on a nikon optiphot optical microscope with a custom designed stage equipped with long working distance objectives designed for fluorescent light . a dichroic mirror / barrier filter assembly directed the excitation light onto the monolayer films at a normal angle of incidence and filtered the emitted light and the images were detected by a silicon intensified ccd camera . videos of the monolayer film were recorded during the compression - expansion cycle directly onto the computer using the pinnacle studio capture software . the continuous , fluid lipid phase appears bright due to the preferential segregation of the texas red dye , while the better ordered domains exclude the dye molecules and appear dark . two - dimensional gixd experiments were carried out at the chemmatcars station at beamline 15-id at the advanced photon source , argonne national laboratory . dppc / chol at the appropriate ratios were dissolved in chloroform solution at 1 mg / ml and spread dropwise onto the air / deionized water interface in a custom langmuir trough , which was temperature - controlled at 22 c. after waiting 30 min for solvent evaporation , the monolayers were compressed to the desired surface pressure ( 20 , 30 , or 40 mn / m ) and annealed for an additional 30 min . the trough was enclosed in a helium - filled chamber and the oxygen level was constantly monitored during exposure to the x - ray beam . the analysis of gixd data for two - dimensional films at the air - water interface follows that of kaganer et al . it is well established that the bragg peaks correspond to ordering within the alkane chains of the lipid tailgroups ; the organization of the headgroups is inferred from changes in the tailgroup lattice in response to changes in surface pressure and cholesterol fraction . circular ferromagnetic probes ( microbuttons ) of diameter 20 m , thickness 1 m , with button holes of diameter 3.5 m were fabricated by photolithography . . a uniform magnetic field of magnitude , b , and orientation , , was generated by the output of two independent pairs of electromagnets controlled by a custom labview code to exert a controlled torque , l , on a microbutton of moment m and orientation . to measure the frequency - dependent linear viscoelastic response , a sinusoidal magnetic field was applied to generate a time varying applied torque . the microbutton orientation was determined from bright field images of the holes in the microbuttons as a function of applied torque , to determine the rotational resistance . from the rotational resistance , the linear viscoelastic surface moduli were obtained from the solution of the hydrodynamic problem of a rotating cylinder within a viscoelastic monolayer atop a viscous subphase . in terms of measured experimental properties , the surface loss modulus , g is the out of phase component of the rotational resistance , the surface storage modulus , g , is in the in - phase component as in conventional 3-d rheology ( figure 6b ) . the surface viscosity for the newtonian response we observed over the frequency range of 0.110 hz is s = g/ , or for the 1 hz frequency used in figure 6 , s = g/2. the same exponential dependence of surface viscosity on surface pressure was obtained using a 100 m probe showing that continuum values of elasticity and viscosity were being measured . the 100 m probe is more than an order of magnitude larger than the domain sizes we have seen ( figures 5 and 6 ) . recent results using macroscopic wire rings ( 10 cm diameter ring , 0.7 mm diameter wires ) showed identical trends with surface pressure and good agreement for similar monolayers . the maximum surface viscosity that we could measure with our rheometer was 100 pams ; the surface viscosity of pure dppc at 40 mn / m was greater than this and was not measured . freshly - cleaved mica substrates ( s&j trading inc . ; glen oaks , ny ) connected to a computer - controlled dipping mechanism in a commercial circular nima l - b trough ( biolin scientific , inc . , linthicum heights , md ) were pulled through the monolayer at 5 mm / min at a constant surface pressure of 20 mn / m . transfer ratios were determined by recording the interfacial area change of the trough during transfer and comparing this to the surface area of the mica substrate . a transfer ratio of 1 means that these areas are equal ; only films with transfer ratios of 1 were examined . the mica substrates were glued to stainless steel discs and affixed to the magnetic holder of an mmafm-2 afm ( digital instruments ; santa barbara , ca ) with a cantilever tip ( asylum research , ac160ts ; santa barbara , ca ) designed for tapping mode operation . two - dimensional gixd was carried out at chemmatcars , sector 15-id at the advanced photon source , argonne national laboratory on dppc monolayers with various mole fractions of dihydrocholesterol ( chol ) . figure 1 shows the gixd intensity maps for ( a ) pure dppc at 20 mn / m and ( b ) 6.4 mol % chol / dppc monolayers at 40 mn / m . figure 2 shows the qz - integrated intensity profiles ( arbitrary units ) for dppc / chol monolayers at 20 , 30 , and 40 mn / m for 0 to 7 mol % chol ( each spectra offset by 1000 ) . two - dimensional x - ray maps of ( a ) pure dppc at = 20 mn / m and ( b ) 6.4 mol % chol in dppc at = 40 mn / m . two bragg reflections are visible , the degenerate reflection at positive qz and lower qxy and the at higher qxy and qz = 0 , indicating nearest neighbor tilt . the peak remains at the same location for all cholesterol or surface pressures ( dotted lines ) . the basic motif of the dppc lattice is unchanged by cholesterol , although the tilt is reduced with increasing cholesterol . cholesterol broadens both peaks consistent with a decrease in the size of the correlated areas in the monolayer . we assign the bragg peak in figure 1 at lower qxy and positive qz as due to the degenerate ( 11 ) and ( 11 ) reflections of distorted hexagonal packing ; the second peak at higher qxy and qz = 0 is due to the nondegenerate ( 02 ) reflection . as the ( 11 ) reflection is located at qz > 0 , and the ( 02 ) reflection is centered at qz = 0 , the alkane chains are tilted in the nearest neighbor ( nn ) direction . regardless of composition or surface pressure , all ordered areas in the monolayers had the same distorted hexagonal packing with nn tilt ( figure 3b , inset ) . ( a ) d11 and ( b ) d02 as a function of cholesterol and surface pressure . d11 decreases with increasing surface pressure and cholesterol fraction . this is consistent with a decrease in tilt , which decreases d11 , while the alkane chain packing normal to the chain axis , hence d02 remains the same . one gray and one white circle are the two alkane chains in a rectangular unit cell of dimensions a and b. translation of the pair of gray and white circles by a and/or b generates the lattice . the spacing between the dotted lines corresponds to the d - spacings in table 2 and a , b. the qz - integrated intensity profiles ( figure 2 ) show that the ( 11 ) bragg peak broadens and moves to higher qxy with increasing cholesterol content ( dotted lines ) for all surface pressures , while the ( 02 ) peak also broadens with increasing cholesterol content but remains at constant qxy . from the qij values in table 1 , the real space lattice dimensions are dij = 2/qij ( table 2 and figure 3 ) . the tilt angle , , for nearest neighbor tilt is given by tan = qz[q112 ( q02/2 ) ] . the coherence length is determined from the full width at half - maximum ( fwhmij ) of each peak after correction for the instrumental resolution , lij = ( 0.92)/(fwhmij ) ( table 2 ) . q02 and q11 are the lattice parameters in fourier space for the two strong reflections from the qz averaged data . all d - spacings are referenced to a two molecule rectangular unit cell with a = d10 = [ d112 ( 2d02 ) ] and b = 2d02= d01 ( see inset to figure 3b ) . the error in a is larger than b as the ( 11 ) reflection is much broader than the ( 02 ) reflection ( see figure 2 ) . is the tilt angle of the alkane chains in degrees with respect to the water surface , tan = qz[q112 (q02/2 ) ] from table 1 ( see inset to figure 3b ) . the chain area is the cross sectional area of the chains in the direction perpendicular to the chains , or ( ab cos )/2 . l02 and l11 are the coherence lengths in the ( 02 ) and ( 11 ) directions , lij ( 0.92)/(fwhmij ) . figure 3a shows that , at a fixed surface pressure , adding chol decreases d11 while figure 3b shows that d02 remains constant . for pure dppc , d11 decreases from 4.79 to 4.58 as the surface pressure increases from 20 to 40 mn / m ; d02 is constant at 4.30 ( table 1 ) . cholesterol and surface pressure influence the lattice in a similar way ; the same linear relationship between d11 and the tilt angle holds over the range of surface pressures and cholesterol fractions examined ( figure 4 ) . decreases with increasing surface pressure , and with some scatter , increasing cholesterol fraction from 35 for pure dppc at 20 mn / m to 18 for 7 mol % chol at 40 mn / m . this change in tilt causes a decrease in the area per dppc molecule at the air water interface from 49.9 1 to 43.9 1 . molecular tilt angle measured from the monolayer normal , , decreases in the same manner with increasing cholesterol fraction as with increasing surface pressure ( over this range of , sin tilt tilt ; to convert to degrees of tilt , = 180(tilt/ ) ) . black symbols 20 mn / m surface pressure , open symbols 30 mn / m and gray symbols 40 mn / m . with some scatter , the tilt decreases with increasing cholesterol fraction . this linear relationship between d11 and is the same for changes in cholesterol fraction and surface pressure , which suggests that the local alkane packing of dppc does not change with added cholesterol , but that both surface pressure and cholesterol act to decrease the mismatch between the lipid headgroup and tailgroup area in the same way . from the values in table 2 , we determine a rectangular two - molecule unit cell of dimensions , a = d10 = [ d112 ( 2d02 ) ] and b = 2d02 = d01 ( figure 3b inset ) , with a decreasing from 5.8 0.1 at 20 mn / m , to 5.4 0.1 at 40 mn / m ; b remains constant at 8.6 0.05 . these unit cell dimensions are consistent with a hexagonal packing of the alkane chains , which are tilted to accommodate the mismatch in projected area between the dppc headgroup and the close - packed chains . as is the case for other lipids , accommodation of the larger dppc headgroup area occurs by dilation of the alkane chain area via an increase in the tilt angle . tilt costs little favorable alkane chain contact energy , as tilt occurs without changing the distances between the alkane chains . tilt in the nn direction causes a , which is measured in the plane of the monolayer , to increase . however , b remains constant as this spacing does not change with tilt if the alkane chains retain their close - packed configuration . increasing the surface pressure provides a uniform compression of the dppc headgroup , which results in a decrease in the headgroup - chain incompatibility , and hence the tilt , without altering the alkane chain packing . the area per alkane chain perpendicular to the alkane chains , 20.5 0.3 = ( ab cos )/2 , is constant within the experimental error for all surface pressures and also for all cholesterol fractions . pure chol has an untilted hexagonal lattice with a d spacing of 5.7 , and an area per molecule of 35 , much larger than the 20.5 area per alkane chain we measure . the invariance of d02 ( or b ) , and the linear relationship between d11 and is consistent with the bulk of the chol not intercalating uniformly into the dppc lattice as it does at higher mole fractions , but separating primarily into a second phase . figure 5 shows afm images of dppc / chol monolayers transferred to mica substrates at 20 mn / m showing two distinct morphologies . at 0.8 mol % chol , dark gray , 10100 nm circular areas are dispersed in extended light gray domains . the dark gray nanodomains localize preferentially at the boundaries of the light gray domains ( arrows ) . increasing the chol fraction causes the circular nanodomains to percolate into linear structures ( 3.7 mol % ) , although the width of the linear structures remains 10100 nm . the linear structures eventually break up the light gray domains ( 5 mol % ) . afm force spectroscopy showed that the nanodomains were more compliant and easier to deform than the dppc domains , suggesting that the nanodomains are disordered and do not contribute to the gixd signature of the monolayer . the alkane chains of dppc prefer to be untilted to maximize the van der waals contact between the chains , but are frustrated by the conflicting cross - sectional area requirements of the phosphocholine headgroup . this mismatch requires the tailgroup lattice to dilate , which responds by the lower energy tilt deformation in order to fill space efficiently . however , chol has a complementary shape to dppc , with a relatively small alcohol headgroup and a relatively large sterol ring tailgroup . hence , this shape complementarity suggests that chol can relieve the packing frustration of dppc by making up some of the mismatch between the headgroups and alkane chains . palmitic acid ( pa ) and hexadecanol ( hd ) , which also have complementary shapes with dppc , also lead to a decreased tilt at a given surface pressure , but do not show nanodomains in afm images . hd is the same as the c16 alkane chain of dppc , which allows pa and hd to be incorporated into the dppc lattice . pa and hd increase the correlation length of the mixed crystal and the surface viscosity . tapping - mode afm images of dppc / cholesterol monolayers transferred by langmuir - blodgett deposition to mica substrates at 20 mn / m . for 0.8 mol % cholesterol , dispersed , circular 10100 nanodomains appear ( darker gray indicates more compliant relative to the lighter gray background phase ) , which preferentially locate at the boundaries of the light gray domains ( arrows ) . for 3.7 mol % chol , the circular nanodomains have condensed into linear features begin to break up the light gray domains , but the light gray domains remain continuous . for 5.0 mol % chol the nanodomains make up a cocontinuous network separating the light gray domains , while decreasing the size of the light gray domains to 100200 nm . fluorescence images of dppc monolayers with 0.0 , 0.2 , and 0.4 mol % cholesterol at coexistence between the disordered le ( light ) and ordered lc ( dark ) phases at 10 mn / m . contrast is due to doping the monolayer with 0.1 wt % of the fluorescent ( n-(texas red sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , ( invitrogen ) which segregates to the more fluid le phase . the domain width decreases with increasing cholesterol , indicative of a decrease in line tension . although the shape of cholesterol can relieve the frustration between the area of the headgroup and alkane chains of dppc , the sterol rings can not efficiently pack into the alkane chain lattice . this leads to a second type of frustration ; the decrease in tilt due to the accommodation of the area mismatch results in disrupting the alkane chain lattice , which is likely higher in energy than decreasing the tilt . the combination of the sterol rings of chol and more disordered dppc chains gives the nanodomain phase excess tailgroup area , which may relieve part of the packing frustration of the headgroups in the adjacent ordered dppc domains , albeit at longer range than if the cholesterol intercalated within the dppc lattice . the size of domains in typical phase - separated monolayers is governed by a competition between line tension , , which leads to larger domains , and entropy and electrostatic interactions , which favor smaller domains . fluorescence images ( figure 6 ) at the coexistence surface pressure ( 910 mn / m ) show that increasing chol leads to a dramatic decrease in the width of the domains , which means a large decrease in . this decrease may be sufficient to stabilize the nanodomains against coalescence . an additional factor stabilizing the nanodomains may be the energy of reducing the tilt in the adjacent dppc domains . the time for diffusive mixing of the nanodomains with the bulk is of order seconds over the 100 nm length scales of the nanodomains , so even with electrostatic interactions slowing bulk coalescence , molecular diffusion should eliminate the nanodomains in minutes if this structure were not stable . this stability may also be an indication that we are seeing an example of the recently proposed 2-d analogs of 3-d microemulsions , in which compositional variations within a single phase are due to coupling between monolayer curvature ( i.e. incompatible area requirements of headgroups and tails ) and composition . the evolution of the structure between discrete circular nanodomains ( analogous to spherical micelles in 3-d ) to extended linear structures ( rod - like micelles ) to an interconnected network ( bicontinuous microemulsion ) suggests this possibility . figure 7a shows that the coherence length , l02 , in the untilted direction for pure dppc is about 70 lattice repeats , or > 300 , more than five times that in the tilted direction , l11 60 or about 12 lattice repeats ( table 2 ) . for both 20 and 30 mn / m , l02 decreases monotonically with increasing cholesterol fraction to 20 lattice repeats , but l11 only decreases to 10 lattice repeats . at 40 mn / m , l02 does not monotonically decrease with cholesterol fraction , the scatter in l02 is much greater than at lower surface pressures , and l02 is always less than expected from the results for the lower surface pressures . this is likely due to a decrease in film stability caused by a combination of leakage under the trough barriers and slow monolayer collapse during the 3 - 5 h required for gixd . the afm images in figure 5 show that the average dppc ( light gray ) domain size decreases from microns to 100200 nm with cholesterol . even with the decreasing domain size , the positional ordering given by the coherence lengths are orders of magnitude smaller than the domain size for a given cholesterol fraction . however , the orientational order extends for tens of microns as shown by the spiral domain textures in figure 6 . dppc / chol monolayers have nanometer - range positional order and micron - range orientational order , similar to tilted smectic c liquid crystals and other langmuir films that are classified as hexatics . ( a ) coherence lengths , l11 , in the ( 11 ) or tilt direction , and l02 , in the ( 02 ) or untilted direction , normalized by their respective lattice constants , d11 and d02 . l02 decreases significantly with cholesterol fraction , but is less affected by surface pressure . l02 for 40 mn / m has more scatter and is nonmonotonic with cholesterol fraction , likely the result of film instabilities due to trough leakage and monolayer collapse at higher surface pressure . ( b ) the surface viscosity of dppc / chol monolayers measured with a microbutton magnetic rheometer decreases exponentially with cholesterol fraction for a given surface pressure for small mole fractions of cholesterol , then plateaus in the same fashion as l02 . ( surface viscosity for pure dppc at 40 mn / m was too high for the viscometer to measure . ) ( c ) free area model for dppc / chol monolayers ( eqs 6 - 8 ) provides an excellent correlation between the surface viscosity and the number of correlated molecules , ( l02l11)/ab , over the entire range of cholesterol fraction . the lines are linear regression fits of eq 7 to the data ( p < 0.01 ) . ( d ) normalizing to a reference state ( taken to be that of pure dppc for 20 and 30 mn / m and 0.4% chol for 40 mn / m surface pressures ) , collapses the data onto a single universal curve relating surface viscosity to the molecular organization . the line is a linear regression fit of eq 8 to the data with p < .001 showing that the data is well described by the free area model . figure 7b shows that the surface viscosity , s , of dppc / chol monolayers decreases exponentially with increasing cholesterol fraction at a given surface pressure . in previous work , we found that the exponential dependence of surface viscosity on surface pressure was well - correlated by a free area the free - volume model was developed to explain the divergence in the viscosity of a liquid at the glass transition . the premise underlying the model is that in order to diffuse , a molecule in a liquid or other condensed phase has to have sufficient free volume , that is , volume not occupied by other molecules , in order to escape the cage formed by its neighboring molecules . each molecule has a minimum van der waals volume , v0 , and moves randomly with thermal velocity u , within confining cages of diameter d0 defined by its nearest neighbors . cohen and turnbull calculated the probability for fluctuations in free volume relative to the average free volume , v. if the local fluctuation in free volume exceeds v0 , a hole is created in the confining cage sufficiently large to allow a diffusional jump of the solute molecule into the hole . diffusion occurs if another molecule fills the hole left by the solute molecule before the original molecule returns to its starting position . the probability p(v0 ) that the free volume , vf , rearranges to give a void volume , v0 , large enough for a molecule to diffuse ( at constant energy ) is given by1thus giving a diffusivity2 in which g is a geometric factor . the parameter b in eqs 1 and 2 is to take into account overlaps of free volume , and cohen and turnbull suggest a range from 1/2 b 1 . in three dimensions , the diffusivity can be related to the bulk viscosity , , via a generalized stokes - einstein relationship , d = kt / f . for spherical particles , the friction factor , f , is given by the stokes drag on a sphere of diameter a : f = 3a . this leads to the free volume model for the viscosity:3 in applications of the model , the free volume is the difference between the measured volume per molecule , v , and v0 : vf = v v0 . in applications of the model , v0 is a fitting parameter ; theoretically , v0 is related to the volume per molecule at the glass transition where the viscosity diverges . for a 2-dimensional film of constant thickness , l:4 in analogy to the free volume , af( ) a0 , in which a( ) is the area per molecule determined at a surface pressure , , from a surface pressure - area isotherm and a0 is taken to be a fitting parameter . in 2-dimensions , the diffusivity and free area can be related to the surface viscosity , s , via the saffmann - delbrck model for a cylinder diffusing within a viscous monolayer , surrounded by a viscous subphase , to give an equation analogous to eq 3 in terms of the free area:5from fitting dppc viscosity data , we found that a0 40 , which is roughly equal to ab cos , the molecular area of a dppc molecule in a close - packed , untilted lattice ( table 2 ) . however , cohen and turnbull did not speculate on the molecular origins of af(19 ) as they were modeling an unstructured liquid . however , for semicrystalline monolayers , we postulate that the free area is proportional to the number of defects in the lattice , which is inversely proportional to the number of correlated molecules at that composition and surface pressure:6 lattice defects , such as dislocations , vacancies , and grain boundaries decorrelate the lattice , which creates free area and pathways for diffusion . we define as the free area ( or number of defects times the area per defect ) per number of correlated molecules ; we take to be constant , independent of concentration . combining eqs 5 and 6:7 in which = ba0/. linear regression to eq 7 ( figure 7c ) shows that for 20 , 30 , and 40 mn / m , the slopes , = 0.0134 0.0007 , 0.0131 0.002 and 0.0196 0.007 are the same within the experimental error . s0( = 20 mn / m ) = 0.09 0.02 , s0( = 30 mn / m ) = 0.37 0.2 , and s0( = 40 mn / m ) = 0.6 0.5 pms , although the physical significance of the surface pressure variation of s0 is not given by our model . the pearson correlation coefficient , r , for the three lines are 0.99 , 0.95 and 0.81 , respectively , giving a statistically significant fit of eq 7 to the data with p < 0.001 , 0.001 , and 0.05 , respectively . to better compare the data at different surface pressures , the surface viscosity and correlated area are normalized relative to a reference composition , xref , at the same :8ref(xref, ) is the surface viscosity and ( ( l02l11)/ab)ref is the number of molecules in the coherence area at xref ( taken to be pure dppc for = 20 and 30 mn / m and 0.4% chol for = 40 mn / m ) . linear regression to eq 8 gives = 0.0133 0.007 , which is the same as the fits to eq 7 . the pearson correlation coefficient is 0.91 , giving p < 0.001 , showing that eq 8 gives a statistically significant representation of the surface viscosity over the almost three orders of magnitude change in surface viscosity ( figure 7b ) . for a0 40 , 1500 ( for b = 1/2 ) 3000 ( for b = 1 ) . for pure dppc , ( l02l11)/ab 450 , giving af 36 from eq 6 , which is consistent with the variation in area per molecule , a(x, ) = a0 + af(x, ) , measured from dppc isotherms . adding cholesterol decreases ( l02l11)/ab to 100 , thereby increasing the free area per molecule to 1530 , resulting in the dramatic decrease in surface viscosity . these values of af are in agreement with the assumptions behind the cohen and turnbull free volume theory , which postulates that vf v0 , hence af a0 . in summary , gixd shows that increasing the chol fraction at constant surface pressure , or increasing the surface pressure at constant chol fraction , decreases the tilt of the dppc lattice , while leaving the alkane chains close - packed with an invariant area per molecule normal to the alkane chain direction . this confirms afm images that show cholesterol does not intercalate homogeneously into the dppc lattice but is expelled to disordered nanodomains that break up the dppc domains . the nanodomains evolve from isolated , circular 10100 nm diameter domains to 1050 nm wide linear nanodomain aggregates to an interconnected network structure with increasing cholesterol . however , the monolayer is homogeneous at micron - scale optical images ; macroscopic phase separation does not occur as at lower surface pressure ( figure 6 ) . hence , the dppc / cholesterol monolayer is better described as a nanostructured , single - phase monolayer , rather than a mixture of two distinct phases , similar to recently proposed two - dimensional microemulsions . as such , this system is analogous to 3-d nanostructured surfactant - oil - water bicontinuous microemulsions , or copolymer systems that separate into nanometer - scale regions more enriched in one or the other monomer , which are considered single phase . adding cholesterol does not change the local hexagonal dppc chain packing , but does reduce the molecular tilt , suggesting that cholesterol relieves some of the packing frustration between the dppc headgroup and tailgroups . most important to the monolayer dynamics , the extent of order , or the number of correlated molecules , decreases with increasing cholesterol fraction at all surface pressures , suggesting an increased number of lattice defects that create free area for visco - diffusive transport . at all cholesterol fractions , the positional order is much shorter ranged than the orientational order , consistent with an overall hexatic organization . we show that a simple model that proposes that the free area available for visco - diffusive transport is inversely proportional to the number of correlated molecules , which collapses the surface viscosity data for all surface pressures and cholesterol fractions onto a single universal curve . molecular defects and the associated decrease in domain size caused by the incompatibility of cholesterol packing into the alkane chain lattice enhance visco - diffusive transport in monolayers . our model and data show that the extent of molecular correlations is an excellent predictor of the effects of the cholesterol on the surface viscosity of model lung surfactant monolayers . pollution is predicated to all tiny particles in the air which are produced due to human or natural activities ( 1 ) . since the centralization of human activities is associated with metabolic human interactions in urban areas , different pollutants enter into air easily and cause the urban environment more vulnerable ( 2 ) . it is such that , world health organization ( 1992 ) , has addressed the air pollution as a serious problem ( 3 ) . improper use of fuel and inappropriate topography of tehran caused air pollution due to the entrance of approximately 1.5 million tons of pollutants annually . hence , the combination of natural and artificial factorscauses tehran to be one of the most polluted cities inthe world , standing by mexico city , beijing , cairo , sao paulo , shanghai , jakarta , and bangkok ( 4 ) . air pollution has also caused painful events all around the world : the pollution event in meuse ( 1930 ) , in which 63 people experienced respiratory problems ; or the death of more than 400 people in london due to the contaminated thick smog in 1952 are two examples of such irrecoverable events ( 1 ) . accor - ding to an environmental program of united nations 48 % of premature deaths are due to exposure to particulate matter in both outdoor and indoor environments , with potentially 500000 excess deaths annually due to particulate matter in outdoor situations ( 5 ) . likewise , the air quality control agency s report indicates that more than 4500 people are dying every year in tehran due to air pollution ( 6 ) . the most important sources of air pollution can be divided into four key groups including overcrowding ( 7 ) , economic growth ( 8) , natural factors ( exp : geographical situation and topography , temperature inversion etc.)(9 ) , and mobile and stationary sources ( exp . non - standards and poor fuel consumption of motor vehicles , industries in and around the city , home heating and cooling systems ) ( 1 , 10 ) . depending on the type and sources of pollutants , metropolises around the world took different measures to control the air pollution . among developing countries , south africa has benefited from a collection of best legal solutions in order to reduce the air pollution ; among them are objective and standard setting , status quo assessment and priority area delineation , control strategy preparation and implementation ( 11 ) . china , as a developed country has also taken effective measures to control air pollution including integrated monitoring program on acidification of chinese terrestrial systems ( impacts ) , permanent control of vehicles emissions , increasing the quality of fuels , taking advantage of new technologies , developing transportation systems , providing five - year plans to understand causes and sources of pollution , current status , effects , and control of acid rain ( 1216 ) . in italy , special measures have been taken for energy management in industry , transportation systems and domestic systems ( 17 ) . economic and industry growth plus overcrowding in india , caused city planners to take serious measures including banning polluting vehicles , developing roads , escalating standards and regulations enforcement and etc . just some of them have been successful to decline air pollution , while the others failed to effectively and efficiently control air pollution . in south africa , despite numerous plans and policies for controlling air pollution , they failed due to some major challenges such as lack of relationship between district and local municipalities , lack of relationship between provincial and local authorities , plan s integration , public s roles , technical capabilities in programs , extending a partial focus on some polluter sources and lack of systemic approach in air pollution planning , plans and climate change management , shortage of funding and etc.(11 ) . improper prioritization of environmental intervention , lack of funding , unwillingness of the countries to plan with a systemic approach , lack of enforcement and poor communication between all public and private sectors are the most principal factors causing the failure to successfully control air pollution ( 1821 ) . from 1334 , lots of case and cross - sectional plans , programs and projects had been developed individually or as a part of a national document to control the air pollution of tehran . they are including the first five - year development plan ( 19881993 ) , clean air plan ( 1995 ) , transportation emission reduction project ( 1997 ) , comprehensive plan of tehran s combating air pollution ( 1997 ) , second five - year development plan ( 19962000 ) , third five - year development plan ( 20012005 ) , 20 year visionary plan ( 2005 ) , forth five - year development plan ( 20062010 ) , tehran s master plan ( 2007 ) , fifth five - year development plan ( 20112015 ) and master plan of metropolises combating air pollution ( 2011 ) ( 22 ) . despite the fact that all these plans had been precisely codified , the present evidence implies that expected results based on reducing the air pollution have not been met due to lack of an integrated stewardship responsible for regulating , coordinating and monitoring the process and the gained results of air pollution control plans ( 23 ) . hence it is necessary to analyze the air pollution controlling plans with a pathologic approach to identify a set of practical solutions for tehran . this paper bears a twofold purpose : first , pathologically analyze air pollution control plans of tehran from the viewpoint of experts ; and secondly offer appropriate and effective solutions for controlling the air pollution . a descriptive case study method was used in 2012 for pathologic analysis of air pollution control plans in order to offer solutions for tehran metropolis as the capital city of iran . the research team provided a semi - comprehensive review of the literature which contained more than 70 articles of different metropolises all around the world for developing an initial conceptual framework as a springboard for developing the interview questions . the scope of the review is limited to developed and developing metropolises ( china , italy , india and south africa ) which faced the same air pollution difficulties as in tehran . by developing the framework , a qualitative content analysis was drawn to identify the manifest and latent contents relating to different air pollutants and plans for their control . the key contents around which the interview questions were organized consists of 1 ) causes and sources of air pollution , 2 ) challenges & obstacles towards effective performance of air pollution control plans and 3 ) the most effective controlling solutions for air pollution in metropolitan areas . a semi - structured face - to - face interview was done to survey tehran s air pollution control plans , from the aspects mentioned above . the participants were chosen by snow - balling sampling and according to their context of work ( practicing professionals and tehran , tarbiyat modarres and shahid beheshti university professors ) . the interviews were continued until the saturation in data and no more information was gathered by interviewing and about 80% of data were repeated by experts , so about 14 experts , professors and managers interviews were practical and applied for data analysis . the context of the interview consists of the extent to which air pollution control plans of iran are appropriate and the ways to cope with the air pollutants in different sectors ( industry , transportation systems and domestic system ) with regard to other metropolises experience in the very same field . in addition , they were asked to provide more information about the relevant issues , if possible . as the interview sessions precede , the quality , relevancy and comprehensiveness of questions were developed simultaneously . all the opinions ( meaning units ) were reviewed , condensed and labeled as a code through a back and forth movement between the whole and part of the interview texts . then the codes with the same meaning grouped together under higher order heading to create categories ( 27 sub - categories and 8 categories ) in a way that each group of codes dealt with a specific issue or content area . next , the primitive title and content of all categories and sub - categories were discussed by the article research team . finally , the underlying meanings which were the latent contents of the categories were formulated into four main themes . the analysis results of all 14 viewpoints from practicing professionals were categorized into four main themes . the first theme is demonstrated in table 1 as causes and sources of air pollution of tehran metropolis . as it is depicted in the table , the most vital sources of air pollution corresponds to the process of policy making , lack of attention to environmental changes , topogherafic status of tehran , pollutant due to mobile and stationary sources and the matter of improper energy management . the second theme is demonstrated in table 2 as challenges & obstacles towards effective performance of air pollution control plans of tehran metropolis . causes and sources of air pollution of tehran metropolis challenges & obstacles towards effective performance of air pollution control plans of tehran metropolis as it is shown , the most important challenges of tehran s air pollution management are focused on nine areas which we abstracted them into two wider categories including firstly , air pollution stewardship challenges and secondly , environmental ( political , economic , social and technical ) challenges . some factors such as lack of information for evidence - base decision making , poor vertical and horizontal coordination among urban developmental goals and plans , improper funding , follo - wing unfit pattern and mores are categorized as stewardship challenges , on the other hand political intervention in plans provision and performance , sanctions against iran , unaffordability of some families to exchange their non - standard cars with new ones and some more are identified as environmental challenges , which are presented in the table 2 in detail . in order to demonstrate the challenges importance , the amount of their repetitions were accounted and shown in table 3 in percentage : the third theme is demonstrated in table 4 as the most effective controlling solutions for air pollution in the metropolitan areas . with regard to the fact that air pollution is one of national planning priorities in metropolitan areas , the most effective controlling solutions for air pollution in the world the forth theme is demonstrated in table 5 as the most effective controlling alternatives for air pollution in tehran metropolis which is broken into two main categories , first of all technical & infrastructural factors , then managerial & administrative factors . planning based on reliable evidences , involving all responsible key stakeholders in planning process , developing the parks & green sites , monitoring the amount & type of different pollutant in the air by the help of new technology , fair distribution of facilities and development , non - individual based planning , considering the implementation & monitoring capacities in contemporary with planning , improving the mutual understanding and communication among authorities and citizens , considering revision loops for plan , developing environmental standards and mores are categorized as technical and infrastructural factors . furthermore , improving the cooperative & mutual communications among the education deputy of tehran municipality and the other authorities forculturalization & informing , developing public participation capacities , are categorized as managerial & administrative factors . tables 5 , there are those alternatives which suites to tehran s status and were mentioned by the interviewees . in order to estimate the alternatives importance , the amount of their repetitions were accounted and shown in table 6 in percentage . the most effective controlling alternatives for air pollution in tehran metropolis the priorities of most effective controlling alternatives for air pollution in tehran metropolis our findings confirm that tehran s air pollution has been due to five major reasons including improper policy making , no attention to the pollutants changes , geographical situation , mobile and stationary sources and nonstandard energy production . paying inadequate and incoherent attention to the sources of pollution caused the authorities face with some important challenges including those which could be due to not having a unique and organized stewardship and of course those challenges could be due to the environmental changes which are not controllable by the air pollution management authorities . as all metropolises confront such challenges , their authorities implement different plans in order to manage air pollution . some of the best and effective controlling solutions are focused on culturalization , infrastructural development such as public transportation ( 16 ) and applying economical control levers in china ( 13 ) , utilizing participatory capacities and strategic planning in south africa ( 11 ) , applying solar battery to produce clean energy in italy ( 24 ) , identifying emission limits for automobiles and industry in india ( 3 ) . in iran , same as other metropolises , several plans are prepared and some are implemented , but , unfortunately , thanks to some challenges mentioned above , they failed to control tehran s air pollution as it was expected to be achieved in the plans . to solve the challenges and improve the quality and effectiveness of controlling air pollution plans , should name paying more attention to the matter of stewardship in the field of air pollution management , cultural and infrastructural development , focusing on effective management system doing systematic studies before planning in other words systematizing the plan preparation process , standardization of fuel and energy production and consumption , enhancing public participation capacity , developing appropriate coordination and controlling mechanism , realization of social justice through capacity evaluation in regional scope , standardizing and utilizing new technologies and professional experts , stopping manufacture of the low quality automobiles and reducing import tariff of the high quality ones , applying economical control levers , making transparency in deaths statistic due to air pollution , providing environment organization s box to centralize polluting industries penalties , and legitimizing air pollution control plans to enhance their implementation s guarantee ( 22 ) . our findings about the air pollution control plans challenges replicate the finding of a survey on controlling air pollution plans in south africa conducted by niaiker et al . ( 11 ) . in the viewpoint of niaiker poor standards and regulations to support plans implementation , poor cooperation among stakeholders ( key authorities , planners , policymakers , citizens etc . ) , poor resource generation and poor attention to the type and source of pollution are the most important challenges caused the failure of control plans . also , asadollah ( 23 ) mentions that poor and disorganized stewardship is a key factor which leads to the failure of tehran s air pollution control plan . furthermore , our finding about controlling alternatives for tehran s air pollution is similar to the findings of a survey on the same subject in this metropolis ( 19 ) . he believed that developing an appropriate evaluation and monitoring mechanism to enforce different responsible authorities through evaluate the performance of them , realization of social justice and improving the participation capacities are success factors of plan implementation . these finding can help tehran s authorities to look at plan preparation process with a new approach and would be practical for every developing large cities which are confronting such problems . in other words , authorities can positively benefit the viewpoints of the practicing professionals who consider the plan s deficiencies from unlike aspects in diverse levels of initials in advanced studies requirements , plan s preparation to plans implementation . we considered only the most important challenges of tehran s air pollution control plans in order to offer effective controlling solutions . further , more researches should be done to determine priority of the offered solutions in accordance with tehran s facilities and requirements . controlling air pollution of tehran needs a serious attention from policymakers to make an effectual enforcement through applying a systemic cycle of preparation and revising effective and comprehensive plans , implementing enforcement and evaluating the environmental impact of the plans through involving all stakeholders ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission redundancy , etc . ) have been completely observed by the authors .

adding small fractions of cholesterol decreases the interfacial viscosity of dipalmitoylphosphatidylcholine ( dppc ) monolayers by an order of magnitude per wt % . grazing incidence x - ray diffraction shows that cholesterol at these small fractions does not mix ideally with dppc but rather induces nanophase separated structures of an ordered , primarily dppc phase bordered by a line - active , disordered , mixed dppc - cholesterol phase . we propose that the free area in the classic cohen and turnbull model of viscosity is inversely proportional to the number of molecules in the coherence area , or product of the two coherence lengths . cholesterol significantly reduces the coherence area of the crystals as well as the interfacial viscosity . using this free area collapses the surface viscosity data for all surface pressures and cholesterol fractions to a universal logarithmic relation . the extent of molecular coherence appears to be a fundamental factor in determining surface viscosity in ordered monolayers . abstractbackgroundthe centralization of human activities is associated with different pollutants which enter into environment easily and cause the urban environment more vulnerable . regarding the importance of air pollution issue for tehran metropolis , many plans and regulations have been developed . however , most of them failed to decline the pollution . the purpose of this study was to pathologically analyze air - pollution control plans to offer effective solutions for tehran metropolis.methodsa qualitative content analysis in addition to a semi - structured interview with 14 practicing professional were used to identify 1 ) key sources of tehran s air pollution , 2 ) recognize challenges towards effective performance of pertinent plans and 3 ) , offer effective solutions.resultsrelated challenges to air - pollution control plans can be divided into two major categories including lack of integrated and organized stewardship and pest challenges.conclusionfor controlling the air pollution of tehran effectively , various controlling alternatives were identified as systematization of plan preparation process , standardization and utilization of new technologies & experts , infrastructural development , realization of social justice , developing coordination mechanisms , improving citizens participatory capacity and focusing on effective management of fuel and energy.controlling air pollution in tehran needs a serious attention of policymakers to make enforcements through applying a systemic cycle of preparation comprehensive plans . further , implement the enforcements and evaluate the environmental impact of the plans through involving all stakeholders .

the syndrome is named after french neurosurgeon octave crouzon , who described this rare genetic disorder first time in 1912 . although it is encountered rarely , crouzon syndrome constitutes almost 5% of all craniosynostoses with an approximate birth prevalance of 1/25,000 - 1/50,000 . the syndrome is characterized by abnormal head shape , midfacial hypoplasia , maxillary hypoplasia , mandibular prognathism , ocular hypertelorism , proptosis , and airway obstruction due to premature fusion of multiple calvarial and skull base sutures within the first year of life . however , the clinical picture may vary greatly from mild to severe midfacial and orbital anomalies . the relationship between craniosynostosis and chiari malformation type i ( cm - i ) has been well - documented . cm - i has a tendency to accompany syndromic craniosynostosis more commonly than sporadic synostosis . the incidence of cm - i in crouzon syndrome is about 70% . herein , we present a 16-year - old boy who admitted with symptoms related to cm - i and underwent suboccipital decompression . however , on physical examination his cruzonoid features drawed attention . a 16-year - old , formerly healthy boy admitted to the outpatient clinic with occasional headache and neck pain . on physical examination it was remarked that he had characteristic features of syndromic craniosynostoses : hypertelorism , proptosis , midfacial hypoplasia , and abnormal head shape [ figures 1a and b ] . however , the patient and his family did not admit to a hospital for this reason before , despite prominent cruzonoid features . the patients neurological examination revealed no abnormalities including pinprick , touch , pain , and temperature sensations in both upper extremities . anteroposterior ( ap ) and lateral plain radiographs of head and a cranial computed tomography ( ct ) demonstrated midfacial and orbital hypoplasia , the fusion of bilateral coronal and lambdoid sutures and sagittal suture , alongside with the increase of ap diameter of the head [ figures 2a and b ] . cranial and spinal magnetic resonance imaging ( mri ) studies were obtained and it showed a 18 mm cerebellar tonsil herniation into the foramen magnum with accompanying syringomyelia between th 4 and th 7 [ figure 3 ] . surgery was planned in order to decompress the posterior cranial fossa . using a median incision , the dura was opened in y - form and after arachnoid dissection duraplasty was performed . there were no significant events in the postoperative course and the patient was discharged from the hospital with no neurological deficits . the patient was then referred to the genetics department for the further evaluation of the craniosynostosis . phenotypical features like the typical dismorphic facies , ocular proptozis and hypertelorism , parrot - like nose , and frontal bossing alongside with the patients mothers history of four recurrent intrauterine fetal losses supported the diagnosis of crouzon syndrome in our patient . regular follow - up examinations were scheduled for the observation of orbital deformities and vision . control mri showed complete resolution of the tonsillar herniation and significant reducement of the syringomyelia both in length and thickness [ figures 4a and b ] . four years after the operation the patient is still doing well with no neurological or ocular deficits and with the relief of his symptoms at the admission . ( a and b ) photographs show characteristic features of syndromic craniosynostoses in our patient : hypertelorism , proptosis , midfacial hypoplasia , and abnormal head shape . ( published with permission and informed consent of the patient ) ( a ) anteroposterior ( ap ) and lateral plain radiographs of head shows increase of ap diameter of the head and midfacial - orbital hypoplasia . fusion of multiple calvarial sutures is also remerkable , ( b ) axial cranial computed tomography scan demonstrated the abnormal head shape and the fusion of bilateral coronal and lambdoid sutures alongside with sagittal suture sagittal t2-weighted magnetic resonance imaging shows a 18 mm cerebellar tonsil herniation into the foramen magnum with accompanying syringomyelia between th 4 and th 7 with the largest thickness of 11 mm ( a ) sagittal t2-weighted mri of the craniocervical junction showed complete resolution of 18 mm cerebellar tonsil herniation , ( b ) sagittal t2-weighted thoracal mri showed relative resolution of the accompanying syringomyelia between th 4 and th 7 with the largest thickness of 7 mm the premature fusion of cerebral sutures has been postulated as the mechanism leading to the development of cm - i in patients with syndromic craniosynostosis . especially premature fusion of the lambdoid suture has been accepted as a crucial developmental anomaly , which results in a relatively small posterior fossa . cinalli et al . , have reported that cm - i is present in 70% of patients with crouzon syndrome . on contrary , they have proposed , that this relationship is due to earlier closure of sagittal and lambdoid sutures in crouzon syndrome compared to apert syndrome . hydrocephalus , jugular venous stenosis leads to venous hypertension and associated brain malformations have been postulated as the other mechanisms leading to cm - i development . mutations in three of four fibroblast growth factor receptor ( fgfr ) genes have been demonstrated to be responsible for various types of syndromic craniosynostoses including crouzon syndrome . fujisawa et al . , have demonstrated that a missense mutation in fgfr2 gene ( tyr281cys ) is responsible for the development of cm - i in patients with crouzon syndrome . , the patient or his family was never attempted to seek for professional medical help . the syndrome was only diagnosed when the patient was admitted to our department with symptoms of cm - i . strahle et al . , presented a series of patients with cm - i associated with craniosynostosis . in their series of 29 patients , however , the mean age of the whole patient group was 1.8 years ( range 2 months-9 years ) . this data shows that both craniosynostosis or craniosynostosis related cm - i are expected to be diagnosed in early childhood . cm - i could be diagnosed before the craniosynostosis in some instances , but the age of our patient at diagnosis was 16 . to our knowledge , this is the only case report indicating a late adolescent diagnosis of crouzon syndrome through clinical symptoms of an associated cm - i . surgical approach to craniosynostosis related cm - i may include cranial vault remodeling with an adequate posterior fossa decompression . however , most neurosurgeons agree with the conservative follow - up of patients with cm - i unless it is not symptomatic or associated with spinal syringomyelia . strahle et al . , reported some of his patients cm - i was resolved or regressed with cranial vault remodeling only , without posterior fossa decompression . , we do not consider cranial vault remodeling as an option , instead we applied a classical posterior fossa decompression for cm - i . strahle et al . , underlined the risk of venous bleeding due to abnormal venous sinuses and increased venous hypertension , therefore suggested a posterior fossa decompression without dural opening and c1 arcusectomy . we did not encounter any venous bleeding during the surgery , where we opened dura and performed a duraplasty . pediatric patients with cm - i should be carefully examined for the clinical signs and features of crouzon syndrome or other syndromic craniosynostosis . in mild clinical forms or in case of a late diagnosis , posterior fossa decompression without cranial remodeling should be kept in mind as a treatment option . permanent postoperative hypoparathyroidism results from the unintentional removal or injury of parathyroid glands during thyroid and parathyroid surgery . permanent hypoparathyroidism is defined as persistent hypocalcemia requiring calcium and vitamin d supplementation 6 months after surgery . the risk is nominal during a minimally invasive parathyroidectomy for a single adenoma but is greatest after a subtotal or total parathyroidectomy , thyroid resection and nodal dissection for large and extensive thyroid cancers , and reoperative neck operations . even though the risk of transient hypocalcemia can be high during an extensive neck dissection , the permanent hypoparathyroidism rate is typically around 1% in the hands of experienced endocrine surgeons at high - volume centers . the accidental onset of permanent hypoparathyroidism can be agonizing for the patient and the clinician alike . for the patient , its negative impact includes a reduced quality of life , expensive lifelong medication supplementation , frequent laboratory testing , and the potential for frequent hospital admissions . in addition , the persistent absence of parathyroid hormone ( pth ) has long - term systemic effects on the body , such as the development of osteoporosis ( due to the decreased function of osteocytes ) , premature cataracts , cardiac dysfunction , and neurologic dysfunction [ 1 , 2 ] . in the late 1960s and 1970s , several new techniques were introduced in an attempt to prevent the detrimental health and social consequences of permanent hypoparathyroidism . for example , intraoperative autotransplantation of parathyroid tissue into the sternocleidomastoid muscle or the brachioradialis muscle was recommended . however , not all patients at risk of permanent hypoparathyroidism actually develop it , nor do all patients require an immediate autotransplant . in fact , an unnecessary autotransplant , if performed during parathyroid surgery , could result in persistent hyperparathyroidism . additionally , the autograft could become autonomously hyperfunctional , posing a diagnostic and treatment dilemma in the future ; wells et al . overcame that limitation and transformed our approach to prevent hypoparathyroidism by introducing autotransplantation of cryopreserved parathyroid tissue . cryopreservation permits parathyroid tissue to be stored for potential reimplantation , thereby avoiding the risk of needlessly implanting parathyroid tissue during initial surgery . the clinician is able to accurately determine whether any residual parathyroid tissue will recover function or whether a delayed autotransplant will be needed . the disadvantages of cryopreservation of parathyroid tissue include the potential of graft failure and the risk of graft - dependent hypercalcemia . this paper provides an up - to - date , comprehensive review of the cryopreservation of parathyroid tissue and its current role in thyroid and parathyroid surgery . the clear indication for an autotransplant of cryopreserved parathyroid tissue is permanent postoperative hypoparathyroidism . in the hands of experienced endocrine surgeons , the risk is particularly low in patients with sporadic primary hyperparathyroidism ( phpt ) , most of whom have a single parathyroid adenoma . nonetheless , certain patients have a higher risk of developing permanent hypoparathyroidism after their initial neck surgery ( table 1 ) . most commonly at risk are patients with multigland parathyroid hyperplasia , especially those with familial primary hyperparathyroidism . such patients may undergo either a subtotal ( 3.5-gland ) parathyroidectomy or a total parathyroidectomy with an immediate autotransplant . both a subtotal parathyroidectomy and a total parathyroidectomy can result in aparathyroidism , so cryopreservation of parathyroid tissue is recommended at the time of the initial surgery . in addition , it has been reported that the use of intraoperative parathyroid hormone ( iopth ) monitoring during parathyroid surgery can accurately predict patients at risk of developing postoperative hypocalcemia . a drop of > 80% of iopth at 10 minutes is a significant factor for postoperative hypocalcemia . therefore , cryopreservation of parathyroid tissue should be considered during parathyroid surgery when the iopth drop > 80% . patients with end - stage renal disease are at high risk of developing secondary ( shpt ) and tertiary ( thpt ) hyperparathyroidism . such patients have persistent stimulation of the parathyroid glands secondary to abnormalities in the metabolism of calcium , phosphorus , and vitamin d , resulting in multigland parathyroid hyperplasia . they have an inherently high risk of disease recurrence if a subtotal parathyroidectomy is performed but a high risk of permanent hypoparathyroidism if a total parathyroidectomy and an immediate autotransplant are performed . moreover , such patients may require multiple operations ; to prevent aparathyroidism as a consequence of the initial or subsequent operations , cryopreservation of parathyroid tissue is recommended . the risk of aparathyroidism is nominal with only a thyroidectomy but increases with more extensive surgical resections . routine central neck dissections for thyroid cancer have a complication rate of permanent hypoparathyroidism of up to 14% . given such risks , the role of prophylactic central neck dissections for papillary thyroid cancer continues to be debated . an immediate intraoperative autotransplant is preferred during a neck dissection for thyroid cancer , yet cryopreservation of parathyroid tissue fragments is also warranted given the multiple operations that may be required in the future . subsequent operations increase the risk of permanent aparathyroidism , an outcome that cryopreservation of parathyroid tissue may prevent . in contrast to initial neck operations , the risk of permanent hypocalcemia after a redo neck operation is not minuscule , but rather as high as 30% [ 6 , 10 ] . the risk of aparathyroidism is higher during a redo neck operation because the viability of the remaining parathyroid glands can not be adequately determined . during the initial surgery , parathyroid glands left in situ may have unknowingly been devascularized or damaged . during a redo neck operation , removal or inadvertent injury of any remaining functional gland this scenario can be particularly problematic in patients who develop hyperparathyroidism after previously undergoing a thyroidectomy ; during a redo neck operation , any parathyroid gland removed must be assumed to be the last viable parathyroid gland and should be autotransplanted immediately to optimize transplantation success . a fragment of the parathyroid tissue may be cryopreserved for a possible delayed autotransplantation if future neck reoperations result in aparathyroidism . other common reasons for parathyroid reoperations include persistent hyperparathyroidism ( hypercalcemia < 6 months after the initial surgery ) and recurrent hyperparathyroidism ( hypercalcemia > 6 months after the initial surgery ) . operative success for redo parathyroid surgery is < 90% and is fraught with an 18% rate of causing permanent hypoparathyroidism . immediate autotransplantation of parathyroid tissue to prevent hypoparathyroidism impedes determination of surgical outcome , which is unpredictable during redo parathyroid surgery . accurately assessing the number of parathyroid glands previously resected , or the viability of the remaining parathyroid glands , is difficult . cryopreservation of parathyroid tissue , rather than an immediate autotransplant , allows the surgeon to appropriately predict surgical outcome , the functionality of any remaining parathyroid tissue , and the need for a delayed parathyroid autotransplant . operative failure and disease recurrence are infrequent in patients with sporadic phpt ; reoperations are more commonly needed in those with shpt and thpt . up to 15% of patients with shpt and thpt tend to have more than four parathyroid glands , increasing the risk of operative failure . cryopreservation of their parathyroid tissue is especially important , because the long - term renal effect of prolonged shpt and thpt predisposes patients to transient hypocalcemia from bone hunger . before any parathyroid autotransplant , it is critical to differentiate transient from permanent hypoparathyroidism . similarly , patients with thyroid cancer who require repeating operations are at risk of hypoparathyroidism , because their remaining parathyroid gland function can not properly be assessed . furthermore , if parathyroid glands are immediately autotransplanted into the neck muscles during the initial thyroid surgery , the function of those glands can not be properly evaluated . during reoperations for thyroid cancer , a portion of any inadvertently removed or devascularized parathyroid glands should be sent for cryopreservation before any immediate autotransplant . patients who develop aparathyroidism during subsequent operations might later benefit from a delayed autotransplant of the cryopreserved parathyroid remnant . is dissected into 30 to 40 pieces of 1 1 1 mm . the supernatant is decanted ; about 10 tissue fragments are transferred into each sterile freezing vial to be resuspended in the freezing media . several media have been proposed in the literature since the initial report by wells et al . . the typical freezing medium contains roswell park memorial institute ( rpmi ) 1640 solution [ 2 , 4 , 6 , 10 , 1316 ] . most institutions use an 80% rpmi 1640 solution [ 2 , 4 , 10 , 13 ] , but some use either a 60% rpmi solution 1640 or no rpmi 1640 solution . in addition , some authors recommend supplementing the rpmi 1640 solution with 2 mm of glutamine [ 11 , 16 ] and 5 g / ml of penicillin streptomycin or 50 g / ml of gentamicin . dimethyl sulfoxide ( dmso ) in a 10% concentration is added as a cytoplasmic stabilizer [ 2 , 4 , 10 , 11 , 15 ] . other reported concentrations of dmso range from 7.5% to 20% [ 16 , 18 ] . the last component of the storage medium is either a 10% to 30% autologous serum [ 2 , 10 , 11 , 15 , 16 ] or a 10% to 20% fetal bovine serum [ 4 , 11 ] . when the rpmi 1640 solution is excluded , a 90% fetal bovine serum is used . the goal of the freezing process is to preserve cellular function by maintaining cellular integrity through the temperature change . to accomplish that goal , a cooling technique developed at the mayo clinic entails placing the vials in an ice chest filled with dry ice prechilled to 55c to 60c for 1 hour to allow cooling by 1c per minute . other authors recommend placing the vials in a 60c ethyl alcohol bath or in either a 70c or a 80c freezer overnight . the vials can also be placed in a programmable freezer and cooled by 1c per minute until a temperature of 80c is reached . once the vials are cooled , they are transferred into a liquid nitrogen freezer and stored at any of several recommended temperatures : 170c [ 15 , 16 ] , 180c [ 4 , 17 ] , 190c , or 196c . the vials containing the parathyroid tissue designated for delayed autotransplantation are removed from the liquid nitrogen freezer and placed in a warm water bath . the vials are shaken at 37c [ 11 , 17 ] to 42c until the parathyroid tissue fragments are thawed . the fragments are washed in rpmi 1640 solution at 37c three times [ 10 , 11 ] . other authors recommend rinsing the fragments five times in 1 ml of rpmi 1640 solution with a 20% autologous serum . the rpmi wash is performed to rinse the dmso , which is toxic to cells at room temperature . the patient 's nondominant forearm is chosen for the parathyroid tissue reimplantation under local anesthesia . dissection is continued until a flexor muscle , preferably the brachioradialis muscle , is exposed . one to three parathyroid graft fragments are transplanted into separate muscle pockets . to maximize the chances of graft function , care must be taken to not cause an intramuscular hematoma , which can compromise graft function . functionality is determined not only clinically but also biochemically ( by sampling blood from the grafted and nongrafted antecubital veins , to determine the pth level at both sites ) . the parathyroid graft is reported as fully functional , partially functional , or nonfunctional ( table 2 ) . clinically , the graft is considered fully functional when the patient remains eucalcemic and asymptomatic after all the calcium and vitamin d supplementation has been discontinued . a graft is also considered fully functional when the pth ratio between the grafted and nongrafted arms is greater than 1.5again , after all the calcium and vitamin d supplementation has been discontinued . the graft is considered partially functional when the patient continues to require calcium supplementation , with or without vitamin d supplementation , with a pth ratio of greater than 1.5 . the graft is considered nonfunctional when the patient is hypocalcemic and requires calcium supplementation , with or without vitamin d supplementation , with a pth ratio of less than 1.5 . in renal patients , the functionality of the parathyroid graft is determined by their pth levels , independent of calcium and vitamin d supplementation ( since most renal patients require supplementation ) ( table 2 ) . the potential benefits of cryopreservation are limited by the reduced functionality of cryopreserved grafts , as compared with immediately autotransplanted grafts . cryopreserved grafts retain functionality in < 70% of patients ; fresh autografts > 90% [ 2 , 15 ] . the cryopreservation process may induce cellular necrosis and impair cellular viability and , ultimately , cellular function . earlier studies found no difference in cell viability and secretory capacity between fresh and cryopreserved parathyroid tissue grafts [ 6 , 19 ] . yet other studies demonstrated that , even though the percentage of viable cells did not necessarily differ between fresh and cryopreserved tissue , cryopreservation decreased the total number of live cells by > 70% . that effect on live cell yield was the same whether the parathyroid tissue was frozen as tissue fragments or as dispersed cells . recently , other authors reported that the cryopreservation process decreased total cell viability and that decreased cell viability was associated with increased storage time . both viability and function were drastically reduced with a storage time of 22 months [ 15 , 17 ] . to counterbalance the effects of cellular necrosis , some authors routinely perform histologic examination of the cryopreserved tissue and autotransplant parathyroid tissue according to the percentage of necrotic cells . in addition , cryopreserved parathyroid tissue should be utilized as soon as it is deemed necessary , since a longer storage time limits delayed autotransplant success . the devastating effect of permanent aparathyroidism has been , for the most part , ameliorated by parathyroid gland cryopreservation and delayed autotransplants . an immediate autotransplant is preferred during thyroid surgery , yet cryopreservation of a portion of the parathyroid tissue can also greatly help patients at high risk of undergoing further surgery . cryopreservation allows the clinician to make appropriate decisions regarding the status of the remaining parathyroid glands . such enhanced decision making is important because most patients undergoing parathyroid and thyroid surgery experience only transient hypocalcemia . differentiating between transient and permanent hypocalcemia is critical , especially after a parathyroidectomy when hypocalcemia may result from bone hunger , rather than insufficiency of pth . cryopreservation facilitates appropriate surgical and clinical decisions , prevents unnecessary immediate parathyroid autotransplants , and offers a chance to cure aparathyroidism .

chiari malformation type i ( cm - i ) related to syndromic craniosynostosis in pediatric patients has been well - studied . the surgical management consists of cranial vault remodeling with or without posterior fossa decompression . there were also cases , in whom cm - i was diagnosed prior to the craniosynostosis in early childhood . we present a 16-year - old boy who admitted with symptoms related to cm - i . with careful examination and further genetic investigations , a diagnosis of crouzon syndrome was made , of which the patient and his family was unaware before . the patient underwent surgery for posterior fossa decompression and followed - up for crouzon 's syndrome . to our knowledge , this is the only case report indicating a late adolescent diagnosis of crouzon syndrome through clinical symptoms of an associated cm - i . the risk of permanent hypoparathyroidism following thyroid and parathyroid surgery is around 1% in the hands of experienced endocrine surgeons . although this complication is rare , rendering a patient permanently aparathyroid has significant consequences on the health and quality of life of the patient . immediate autotransplantation of parathyroid glands that are injured or unintentionally removed offers the best possibility of graft viability and functionality . however , since the majority of cases of hypoparathyroidism are transient , immediate autotransplantation can complicate postoperative surveillance in certain patients , especially those with primary hyperparathyroidism . cryopreservation of parathyroid tissue is an alternate technique that was developed to treat patients with permanent hypoparathyroidism . this method allows for parathyroid tissue to be stored and then autotransplanted in a delayed fashion once permanent hypoparathyroidism is confirmed . this article provides a contemporary review on cryopreservation of parathyroid tissue and its current role in thyroid and parathyroid surgery .

a major feature of the movement toward hospital cost containment in the last decade has been the replacement of expensive inpatient care with less costly outpatient care . most discussions about the increasing reliance on outpatient care focus on utilization and revenue measures . a more important issue for hospital payment policy , however , is how actual costs have been affected by this shift from inpatient to outpatient services . trends in relative cost changes are not easily identified because hospital accounting systems do not easily disaggregate total cost into inpatient and outpatient components . cost - finding methodologies are complex , allowing hospitals considerable discretion in cost - allocation patterns . furthermore , an incentive was created by the prospective payment system ( pps ) to allocate costs to centers incurring outpatient charges because medicare pays a fixed amount per inpatient discharge but continues to pay for outpatients on the basis of reasonable cost . changes in relative costs have important implications in the current environment of changing methods of payment for hospital outpatient services . concern over increases in medicare outpatient expenditures led to congressional legislation in 1986 that mandated the implementation of a pps for outpatient care . an understanding of true cost increases is critical to the adoption of a plan that accurately reflects outpatient costs . in this article , we provide a methodology for disaggregating total cost into inpatient and outpatient components to examine relative changes . this is accomplished through estimation of a multiple - output total - cost function for a sample of acute care hospitals for the years 1984 - 88 . the adoption of pps by medicare in 1983 changed the payment basis for hospital inpatient care from hospital - specific costs to diagnosis - related groups ( drgs ) . under pps , hospital payments are made according to prices that are determined by averaging historic costs for specific groups of diagnoses across hospitals . the force behind this change was the federal government , the largest third - party payer for inpatient care . an intended consequence of pps was the substitution of less expensive outpatient care for inpatient care , without compromise of quality . the change in the medicare system was not the only impetus behind the increased emphasis on outpatient care . hospitals have also been influenced by other third - party payers , who have also been trying to contain costs . managed care options have been expanded , as has the use of copayments and deductibles . many plans ( including the majority of blue cross and blue shield plans ) have adopted fee schedules similar to those of pps . new techniques in cataract extraction and cardiac catheterization , for example , have made it possible for people to undergo these procedures without an overnight stay . the magnitude of the increase in outpatient utilization over the period of this study is dramatic . the annual number of outpatient visits reported by community hospitals rose from approximately 210 million to 270 million from 1984 to 1988 . the surge occurred in the number of hospitals offering services as well as the types of services being performed . in 1984 , 49 percent of community hospitals reported having an organized outpatient department . the proportion of hospitals reporting the performance of ambulatory surgery increased from 91 to 95 percent during the same period . the substantial shift from inpatient to outpatient activity is also reflected in changes in inpatient and outpatient revenue components . whereas total revenues in community hospitals increased by 44 percent from 1984 to 1988 ( from $ 156 billion to $ 224 billion ) , outpatient revenues more than doubled during the same period ( from $ 22 to $ 46 billion ) . program payments for hospital outpatient services increased by $ 2.2 billion from 1984 through 1987 , or at an average annual growth rate of 18 percent . mounting medicare payments for outpatient care led to a call for the development of a prospective payment method for such care . in 1986 , congress passed the omnibus budget reconciliation act , which directed the secretary of health and human services to develop a prospective payment plan for all types of hospital outpatient care . implementation of this law requires a system for outpatient classification , and one grouping method currently under consideration and discussed later is ambulatory patient groups ( apgs ) ( lion et al . , this scheme is similar to that of drgs , which rely on charge data for calibration of payment weights . if there is a discrepancy between the true cost of outpatient visits and the charges made for those visits , distortion could occur in the establishment of their payment rates . in this section , we describe a procedure for disaggregating total costs into inpatient and outpatient components , which includes an estimable multiple - output cost function . to identify outpatient costs , we employ the concept of incremental costs as described in baumol , panzar , and willig ( 1988 ) . outpatient costs are the incremental costs incurred as a result of outpatient activity : where oc represents outpatient costs , tc represents total costs , dis and opv represent the number of discharges and outpatient visits , respectively , and x is a vector of exogenous variables . therefore , assuming total costs are the sum of outpatient costs and inpatient costs , inpatient costs are specifically , the cost function is evaluated at the actual level of outpatient visits and at zero outpatient visits . given this breakdown of total costs , hospital - specific cost components can be obtained . the discussion then turns to a multiple - output cost function that can be used to determine the disaggregated costs described in equations ( 1 ) and ( 2 ) . one type estimates average cost per patient or per patient day as a function of various regressors that are considered to affect costs . this widely used set of behavioral cost functions is often accused of being ad hoc and of lacking foundation in the assumptions of the usual production theory . another group of models , following the work of mcfadden ( 1978 ) , employs flexible functional forms that regress total cost on output levels and input prices and , hence , are more consistent with the characteristics of the standard economic theory of production . the advantage of these models is that they are better suited for the calculation of the scale and scope economy measures that have been developed for multiple - output production . however , these models have been criticized for the large numbers of parameters that must be estimated and for excluding many factors that are known to be significant in explaining variation in costs of complex , modern hospitals . some recent work estimates hybrid cost functions that incorporate a number of desirable features from both existing types of models ( grannemann , brown , and pauly , 1986 ; vita , 1990 ) . hadley and zuckerman ( 1990 ) expand the literature with a dynamic model designed to capture the process of adjustment to pps . however , a consensus has not been reached on the appropriate form of the hospital cost function . because our major objective is to disaggregate total costs into inpatient and outpatient components , the function to be estimated is a total - cost function . as the focus is not scale or scope economies nor substitution of inputs , we chose a form that , although not derived from any particular production technology , incorporates many factors likely to be important in explaining hospital cost variation . our approach draws from the work of grannemann , brown , and pauly ( 1986 ) . having no evidence that hospitals are in longrun equilibrium , our expression is that of shortrun total costs : geographic input price variation is a major determinant of cost variation . the only input price measure available was the index of local area wage rates that has been produced by the health care financing administration ( hcfa ) for use in determining prospective payments to hospitals . to impose the assumption of linear homogeneity in input prices , the dependent variable used in the equation is the logarithm of total cost minus the logarithm of the wage index . variation in the cost to the hospital of energy and food may be partially reflected in wage rates , which must compensate workers for higher costs of living . the second- and third - order terms for the variables for the number of inpatient discharges and outpatient visits are consistent with a cost function that exhibits u - shaped average and marginal cost curves . there are two aspects of inpatient care : the number of patients and the patient length of stay . these may be entered separately , as discharge and average - length - of - stay variables , or combined into one total - days - of - care variable . we chose the former approach , although the latter would likely yield a similar result . use of our functional form allows for outpatient levels to take the value of zero , which is the case for outpatient visits for some hospitals . calculation of the incremental cost of outpatient activity also requires that the cost function be evaluated at a level of zero outpatient visits for each hospital . the vector of remaining variables , which are described in a later section , was chosen based on the results of previous studies . we make the assumption that output is exogenous as has commonly been done in other studies ( conrad and strauss , 1983 ; grannemann , brown , and pauly , 1986 ; friedman and shortell , 1988 ; hadley and swartz , 1989 ; hadley and zuckerman , 1990 ) . the majority of data used in this analysis comes from two independent sources : the american hospital association ( 1984 - 88 ) ( aha ) annual survey of hospitals and the hcfa hospital cost reporting information system ( hcris ) data files . the hcris files are cycles one through five ( 1984 - 88 ) of pps supplemented by the tax equity and fiscal responsibility act ( tefra ) data set , which was used to complete the pps data for 1984 . the sample represents all hospitals for which both aha and pps data were available , after eliminating specialty hospitals , all - inclusive - rate payers , and hospitals with fewer than 100 beds . the data bases of those 68 hospitals subject to all - payer systems of payment were not comparable with those of the larger group ; the group of small hospitals exhibits cost structures that are distinctly different from those of hospitals having 100 or more beds . this latter point was verified using the chow test for structural difference between the sample that included and the sample that excluded small hospitals ( n = 3,961 and n = 2,235 , respectively ) . this unique data set consists of 2,235 hospitals , both non - profit and proprietary . total fixed assets , drawn from the hcris data , are used as a measure of fixed capital in estimating the shortrun cost function . in doing so , it is assumed that capital stock is exogenous . to test this assumption , i.e. , to supply evidence of whether or not hospitals are in longrun equilibrium , we performed the hausman ( 1978 ) specification test for exogeneity of the capital variable for the year 1988 . if capital is exogenous , it will be uncorrelated with the error term in the cost function . the null hypothesis of no misspecification ( no correlation with the error term ) is tested by comparing two sets of parameter estimates of the cost function : one using total fixed assets and one using an instrumental variable that is correlated with fixed assets but uncorrelated with the error term . the specification test is based on the statistic where 1 and m1 are the parameter estimates and the covariance matrix from the estimation using the instrumental variable , and 2 and m2 are similar estimates from the model using total fixed assets . the m - statistic has a (k ) distribution , where k is the number of unknown parameters . because the value of m is 2.29 , and the critical value at the 1-percent level is 40.29 , we fail to reject the null hypothesis and therefore incorporate the actual value of total fixed assets into the cost function . the cost and discharge variables just described were obtained from the hcris data set ; outpatient visits were obtained from the aha data . in addition to the cost , output , and capital variables , additional explanatory variables appear in the cost function . case mix ( measured using the medicare drg case - mix index ) is included to control for output variation among inpatients that is not captured by the discharge and length - of - stay variables . this estimate of the costliness of a particular hospital 's medicare patient load was unavailable prior to the adoption of pps by medicare . although still imperfect , it improves on many earlier cost studies that relied on cruder case - mix measures . the implications of market concentration for hospital costs have been addressed in a number of recent works . many of these studies have found evidence of various forms of non - price competition ( for example , by quality or range of service offerings ) , with the general conclusion that greater market competition is associated with higher costs ( joskow , 1980 ; robinson and luft , 1985 ; white , 1987 ; hadley and swartz , 1989 ) . zwanziger and melnick ( 1988 ) demonstrate that this effect is changing in california hospitals . this was constructed using the county as the market and the number of discharges as a measure of output from which to determine market shares . ( 1987 ) found that the county is an acceptable alternative to a uniform geographic area in defining markets . scope or range of services has not been measured precisely in most cost functions ; often the interaction terms for various outputs are examined in an attempt to establish the presence of economies or diseconomies of scope . we incorporate a scope - of - services index that is calculated by cluster analysis and verified using guttman scale statistics and that follows the methodology developed by klastorin and watts ( 1982 ) ( also henderson , defiore , and stefos , 1990 ) . we have grouped the hospitals ( which are ranked on a scale of 0 to 18 , where hospitals having a higher index offer more services ) into three categories of service availability ranging from lowest to highest : basic , community , and those offering the full range of services . our service - index approach to economies of scope differs from that of grannemann , brown , and pauly ( 1986 ) , who include interaction terms between output pairs . finally , dummy variables are included for teaching status , population size , and ownership . the level of teaching activity is classified into three groups : major teaching ( affiliation with a medical school and membership in the council of teaching hospitals ) , minor teaching ( medical school affiliation only ) , and non - teaching ( neither affiliation nor council of teaching hospitals membership ) . population of a hospital 's surrounding community was coded by collapsing the metropolitan statistical area ( msa ) size into one of three groups : large urban ( more than 1 million ) , small urban ( 100,000 to 1 million ) , and rural . ownership is categorized as non - profit , profit , and government ( city , county , or state facility ) . the teaching and population variables were obtained from the aha data set ; the ownership dummy was defined from hcris data . this allows for comparison of relative costs over time . as in most of the literature on cost functions , a potential hazard for estimation of total costs on cross - sectional data is the presence of heteroscedasticity associated with output levels . a useful procedure for detecting this violation of ols assumptions in the case of a multiple - output cost function is the park - glejser test ( see vitaliano , 1987 , for another application of this test to a hospital cost function . ) glejser ( 1969 ) generalizes the test to allow for the case of heteroscedasticity , in which the error term is proportional to more than one of the explanatory variables . in addition to testing for the failure of the assumption of a constant error term variance , the test supplies an estimate of the covariance matrix of the disturbance term , u : . the inverse of the diagonal matrix contains the weights to be used in a weighted least squares ( wls ) regression . the park - glejser test indicated the presence of heteroscedasticity for the years 1984 - 86 and 1988 . consequently , wls regressions were performed for those years and ols was applied to 1987 . the belsley , kuh , and welsch ( 1980 ) diagnostics were applied ; no problems due to multicollinearity were detected . the coefficients on the discharge , outpatient visit , and los variables exhibit a highly significant pattern of positive , negative , and positive for quantity , quantity squared , and quantity cubed , respectively . the sign on the herfindahl index is negative , which is supportive of the theory of non - price competition . small rural markets tend to have lower input prices , and because the only included input price is the wage index , it is possible that the herfindahl measure could be incorporating the effect of omitted input price measures . major teaching hospitals and those located in large urban areas are more expensive , as has been shown in previous empirical work . previous authors ( grannemann , brown , and pauly , 1986 ; vita , 1990 ) failed to find evidence of complementarities among their specific measured outputs . the signs of the coefficients on our scope - of - service dummy variables also fail to indicate the presence of economies of scope . however , these results are only suggestive , and the topic is one in need of further investigation . the results of the estimated cost functions were used to disaggregate total costs into inpatient and outpatient components . equations 1 and 2 were evaluated for each hospital using the estimated cost function to determine hospital - specific values . in particular , for hospital i , the means of inpatient and outpatient costs are listed for each year by various hospital categories in table 3 . as seen in the final column , inpatient costs grew at an average annual rate of 6.6 percent and outpatient costs at a rate of 7.4 percent . this result contrasts starkly with the relative change in inpatient and outpatient revenues already discussed . revenues for outpatient services rose much more rapidly than costs in the 5 years following the introduction of pps . the results of the cost - function disaggregation procedure indicate that the growth rate of outpatient costs did not differ substantially from that of inpatient costs , despite considerable differences in the relative utilization patterns and revenue components . to further explore this finding , it is useful to consider the relationship between total cost , output levels , and unit costs . the ratio between the two components of cost may be represented as that is , the ratio of costs is equal to the product of the ratio of output and the ratio of unit costs . because the ratio of outpatient to inpatient costs remained relatively steady , and the ratio of outpatient visits to discharges rose , it follows that the ratio of the average incremental cost of an outpatient visit to the average cost of a discharge fell . table 4 lists the outpatient visit and discharge unit costs by the same hospital strata . in 1984 , the overall average incremental cost of an outpatient visit was 2.3 percent of the average inpatient cost ; by 1988 , this percentage had fallen to 1.7 . the decline in the outpatient / inpatient unit cost ratio offset the increase in the ratio of outpatient visits to inpatient discharges . because the trend in unit costs for outpatient services is a finding of this analysis that was unanticipated , an interpretation of that result is in order . ( the mean of outpatient unit costs in 1988 is $ 57 in 1984 dollars , which is 11 percent lower than the 1984 unit cost . ) an economic effect that could partially account for this is economies of scale . the output - volume change for outpatient activity from 1984 to 1988 is considerable : the average number of visits rose from 58,000 to 74,000 or 28 percent . additional econometric evidence of economies of scale appears in a study of the determinants of 1987 medicare hospital outpatient department costs by miller ( 1992 ) . one change that accounts for an increase in the use of outpatient services is the rise in the number of ambulatory surgical procedures performed in hospitals . because these operations are relatively costly , it might be expected that they would drive up the average cost of a hospital outpatient visit . despite the focus on growth in ambulatory surgery , it should be noted that such surgery comprises a relatively small portion of total outpatient volume . in 1988 , less than 4 percent of all outpatient visits reported by community hospitals were for surgical procedures ( american hospital association , 1990 - 91 ) . the percentage of hospitals reporting ambulatory surgery rose from 91 to 95 during the period of this study , and the number of freestanding ambulatory surgical centers ( ascs ) rose 670 percent from 1983 through 1990 ( prospective payment assessment commission , 1992 ) . medicare changed the incentives for utilization of these facilities beginning in 1988 by bringing hospital outpatient surgery payments more in line with the lower rates already established for ascs . the increase in the number of outpatient visits in the sample of hospitals in this study is very large . the figures in table 1 indicate that the average hospital reported 16,000 more outpatient visits in 1988 than in 1984 . the question arises whether some of this increase is the result of unbundling on the part of hospitals . some states have instituted their own non - medicare cost - containment regulations that do not permit rate increases but do allow for volume adjustments . it has been suggested that hospitals have split services into multiple billable pieces in order to increase payments . this practice could be reflected in the aha number - of - visits measure , because each appearance by an outpatient to one unit of the hospital is counted as a visit . pre - admission testing on an outpatient basis has been another response by hospitals to fixed payments per discharge . this unbundling practice is a way of increasing the payment for the same workload . to the extent that the increase in number of visits from 1984 to 1988 is partially the result of overreporting or changes in the way visits are counted , unit costs for the outpatient component for the later years would be underestimated . ( the variation in total costs resulting from outpatient activity is spread out over a larger number of visits . ) although the estimated equation is a variable - cost function that excludes capital expenditures , it is questionable whether a remaining non - capital element of measured costs that is fixed with relation to number of discharges and number of outpatient visits could be significant . similarly , the incremental - cost approach can not properly allocate costs that vary jointly with inpatients and outpatients . if a considerable amount of joint costs is assigned to inpatients by this methodology , then our measure of outpatient costs is biased downward . however , the trends that are observed during the 4-year period of study would hold , assuming that the nature of fixed and joint costs is stable over time . another approach to disaggregation of cost components is that taken by aha in its method for representing multiple hospital outputs in a single measure . discharges are adjusted ( multiplied by an adjustment factor ) such that adjusted discharges refer to the number of discharges that the hospital could serve if it were offering no outpatient or other non - inpatient services . the desired adjustment factor is the ratio of total cost to inpatient cost , which converts non - inpatient services to discharge equivalents . ( aha adjusts inpatient days by the same adjustment factor . ) assuming that total costs are the sum of inpatient costs and outpatient costs , the desired adjustment factor may be expressed as adjusted discharges are then calculated as the latter is algebraically equivalent to dividing inpatient cost by unadjusted discharges ( ic / dis ) . however , aha uses the ratio of total revenue to inpatient revenue as a proxy for the ratio of total cost to inpatient cost . hence , the aha adjustment process is a revenue - based approach to calculation of unit costs . table 5 lists the aha costs per adjusted discharge along with the implied outpatient unit costs ( oc / opv ) . this revenue - based approximation to changes in unit costs substantially overestimates outpatient costs and cost increases and underestimates those of inpatient costs as observed by comparing the results listed in table 5 with those of table 4 . finally , the adjustment factors calculated using the cost - function approach to disaggregation of inpatient and outpatient components are listed with the aha adjustment factors in table 6 . the discrepancy between cost- and revenue - based unit costs and adjustment factors is most serious among hospitals that are smaller , less urban , and non - teaching , as seen by comparing the results listed in tables 4 and 5 as well as the results in table 6 . the growth rate of hospital outpatient costs has implications from the perspective of both policy and research . regarding policy , there is a current effort by both government and private insurers to control hospital payments for outpatient services . the results of our research indicate that hospital outpatient costs are not rising nearly as rapidly as are outpatient revenues . attempts at bringing payments more in line with actual cost increases will have a serious impact on hospitals with the largest discrepancies between costs and revenues . these tend to be the smaller , rural , and non - teaching facilities in the sample . this system is similar to that of drgs , which relies on charge data for calibration of payment weights . the appropriateness of using charge rather than cost data for the annual recalibration of drg weights has been the subject of some debate . work by cotterill , bobula , and connerton ( 1986 ) using 1981 data showed little difference between use of cost and charge data . the results of the study by rogowski and byrne ( 1990 ) showed that , by 1984 , cost- and charge - data - based drg weights were less congruent . however , the authors counsel the use of charge data . in addition to the timeliness of charge data , even the best cost data are partially based on charge data , so that many of the same biases are at work in either case . price ( 1989 ) updated the issue with a study using 1986 data , which showed much larger differences between cost- and charge - based weights than previously found . results of the present work indicate that the discrepancy between outpatient costs and revenues was significant and grew during the period 1984 - 88 . if historical charge data are used for weighting of outpatient payment groups , the system could be seriously distorted in favor of those procedures for which charges have been set well above costs . if outpatient rates in general are biased upward and inpatient rates downward , the smaller hospitals would be particularly vulnerable because of their relatively smaller outpatient departments . cost - allocation patterns also have research implications , particularly for the results of studies that rely on aha revenue - ratio adjusted output measures . if relatively high outpatient revenues are not reflective of true costs , then adjusted output measures will overstate true output levels . examination of changes in unit costs demonstrates the extent to which trends in the variables are misrepresented by the revenue - adjusted measures . from table 4 , it is seen that the cost - function measure of discharge unit cost rose 43 percent from 1984 through 1988 , while the revenue - ratio measure of this variable ( table 5 ) declined by 29 percent . measures of hospital labor productivity will also be understated if based on aha adjusted output measures , although these trends are more difficult to gauge because tracking measures of labor inputs over time is confounded by a number of factors . ( it should be noted that the results of much empirical research are dependent on the reliability of aha adjusted cost and output measures . numerous studies have used the aha cost per adjusted unit of output as the dependent variable in estimations of average cost functions . researchers should be aware of changing patterns of cost allocation and of how use of revenue or charge data as a proxy for costs may affect their conclusions . improvements in hospital accounting data would be beneficial for future research as well as in construction of drg and apg weights and payment levels . wilms tumor ( wt ) , also known as nephroblastoma , is the most frequent renal tumor occurring in children aged 0 to 15 years , especially among those younger than 6 years . wt accounts for roughly 6% of all childhood cancers ; 5% of whom had bilateral involvement , and 1% of whom had a family history . thanks to the prospective clinical trials performed by the international society of pediatric oncology renal tumor study group ( siop - rtsg , europe ) and the children s oncology group ( cog , formerly nwtsg , north america ) , most patients ( 85% ) affected by wt can be successfully cured . however , some studies released surveys reporting that there are still a few children who may have a poor prognosis or relapse with current therapies , 50% of whom will die despite intensive re - treatment . it has been proven that genetic variants are strongly associated with the development of wt . mutations in wt1 or epigenetic defects on chromosome 11p15 contribute to the major genetic susceptibility locus . previous studies have reported several non - wt1 loci with smaller effects on the genetic predisposition for wt , including p53 gene , insulin - like growth factor - ii gene , ctnnb1 gene , and h19 . however , less than half of wt cases are attributable to known genes that are associated with wt risk ; therefore , the exact pathogenesis of most wt cases remains unknown and identification of novel genetic loci is urgently needed . the reversion - inducing cysteine - rich protein with kazal motifs ( reck ) gene , known as a transformation suppressor gene , can restrain tumor invasion and metastasis through suppression of matrix metalloproteinases ( mmps ) , including mmp-4 and mmp-9 . mmps proteolytically degrade extracellular matrix proteins , which is critical for tumor metastasis and invasion . reck is expressed in a number of normal tissues , but it appears to be down - regulated in several types of cancers , including esophageal cancer , breast cancer , lung cancer , colorectal cancer , osteosarcoma , gastric carcinoma , prostatic cancer , oral cancer , pancreatic cancer , and cholangiocarcinoma . moreover , clinical investigations have demonstrated that high expression of reck in tumor tissues usually contributes to increasing survival rates and reducing tumor invasion . moreover , hawthorm et al . analyzed wt using single - nucleotide polymorphism ( snp ) mapping array - based comparative genomic hybridization and found chromosomal deletion in 9q ( reck gene locates on 9q13-p12 ) . although it is well documented that reck has an impact on metastasis and prognosis of human cancers , reck gene snps in wt susceptibility and clinical features remains poorly characterized . to obtain adequate power for assessing the potential association , we chose snps with minor allele frequencies of > 5% . furthermore , rs10972727 and rs11788747 snps both were previously identified to be potentially associated with several types of human cancers , including oral cancer , non - small cell lung cancer , and hepatocellular carcinoma . therefore , the purpose of our study was to determine whether the 2 snps ( rs10972727 and rs11788747 ) in reck gene were associated with susceptibility to wt in chinese children . this case - control study received approval from the human research ethics board of jinan children s hospital . a total of 291study participants 97 wt children and 194 healthy controls based on an age - sex - matched pair with 1 : 2 ratio were identified at the jinan children s hospital . all the participants were han chinese . written informed consent was obtained from parents of each participant before inclusion in the study . there were 41 male and 56 female patients , and the mean age at the time of surgery was 3.3 years . the patients histopathological types and cancer grading were determined according to classification of renal tumor of childhood defined by siop - rtsg . accordingly , 43 ( 44.3% ) out of 97 cases were characterized as wt stage i , 35 ( 36.1% ) as stage ii , 12 ( 12.4% ) as stage iii , and 7 ( 7.2% ) as stage iv . according to the prognostic group , moreover , no case had bilateral wt . according to the histologic type , there were 26 cases with blastemal type wt , 8 with diffuse anaplasia , 9 with focal anaplasia , 17 with epithelial , and 37 with mixed . genomic dna was extracted by qiaamp dna blood mini kits ( qiagen , valencia , ca ) according to the instructions of the manufacturer . reck genotype polymorphisms ( rs10972727 and rs11788747 ) were determined by polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) method . pcr was carried out in a reaction mixture of 10 l containing 58 l dna template , 0.5 u of taq biocatalysts , 1.8 mmol / l mg2 + , 2.4 l dntps ( promega , madison , wi ) , and 200 nm of each primer . the 2 snps were genotyped according to the methods described by chung et al . . primers for rs10972727 were 5-gtagaagaagtgactgatcc-3 and 5-atctgactccgaagataacc-3. the primers for the rs11788747 were 5-ttctatcaggtcatggaaca-3 and 5-tgcggttaagactggagaag-3. the pcr cycling conditions were : initial denaturation at 94c for 5 min , followed by 35 cycles of 1 min at 94c , at 60c for 30 s , and at 72c for 1min , with a final extension at 72c for 10 min . to verify results from pcr - ctpp , approximately 10% of the sample analysis was a duplication and each genotype was determined by the dna sequence analysis . , hardy - weinberg equilibrium was analyzed to compare the observed and expected genotype frequencies using the standard test or fisher s exact test . the differences in distributions of genotypes and alleles between cases and controls were assessed by tests . the estimated genotype - specific risks are presented as odds ratios ( ors ) . in all cases , this case - control study received approval from the human research ethics board of jinan children s hospital . a total of 291study participants 97 wt children and 194 healthy controls based on an age - sex - matched pair with 1 : 2 ratio were identified at the jinan children s hospital . all the participants were han chinese . written informed consent was obtained from parents of each participant before inclusion in the study . there were 41 male and 56 female patients , and the mean age at the time of surgery was 3.3 years . the patients histopathological types and cancer grading were determined according to classification of renal tumor of childhood defined by siop - rtsg . accordingly , 43 ( 44.3% ) out of 97 cases were characterized as wt stage i , 35 ( 36.1% ) as stage ii , 12 ( 12.4% ) as stage iii , and 7 ( 7.2% ) as stage iv . according to the prognostic group , moreover , no case had bilateral wt . according to the histologic type , there were 26 cases with blastemal type wt , 8 with diffuse anaplasia , 9 with focal anaplasia , 17 with epithelial , and 37 with mixed . genomic dna was extracted by qiaamp dna blood mini kits ( qiagen , valencia , ca ) according to the instructions of the manufacturer . reck genotype polymorphisms ( rs10972727 and rs11788747 ) were determined by polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) method . pcr was carried out in a reaction mixture of 10 l containing 58 l dna template , 0.5 u of taq biocatalysts , 1.8 mmol / l mg2 + , 2.4 l dntps ( promega , madison , wi ) , and 200 nm of each primer . the 2 snps were genotyped according to the methods described by chung et al . . primers for rs10972727 were 5-gtagaagaagtgactgatcc-3 and 5-atctgactccgaagataacc-3. the primers for the rs11788747 were 5-ttctatcaggtcatggaaca-3 and 5-tgcggttaagactggagaag-3. the pcr cycling conditions were : initial denaturation at 94c for 5 min , followed by 35 cycles of 1 min at 94c , at 60c for 30 s , and at 72c for 1min , with a final extension at 72c for 10 min . to verify results from pcr - ctpp , approximately 10% of the sample analysis was a duplication and each genotype was determined by the dna sequence analysis . , hardy - weinberg equilibrium was analyzed to compare the observed and expected genotype frequencies using the standard test or fisher s exact test . the differences in distributions of genotypes and alleles between cases and controls were assessed by tests . the estimated genotype - specific risks are presented as odds ratios ( ors ) . in all cases , five out of 97 cases had a family history of wt , 6 cases had relapse , and 16 had metastasis . in the present study , distributions of genotypes in cases and controls were in agreement with hardy - weinberg equilibrium ( rs10972727 , p=0.349 and p=0.121 ; and rs11788747 , p=0.519 and p=0.123 , respectively ) . table 2 shows the genotype and allele frequencies of the 2 snps ( rs10972727 and rs11788747 ) in reck gene . no significant difference between cases and controls was observed for distribution of rs10972727 genotype , rs11788747 genotype , and rs11788747 allele . however , we found that the allele g of rs11788747 snp was significantly associated with an increased risk of wt ( or=0.7 , 95%ci : 0.450.99 ; p=0.042 ) . moreover , although when taking the aa genotype as a reference , we found that ag + gg genotype was not statistically significantly associated with the risk of wt , there was a pronounced trend that carriers of 1 variant combined genotype had an increasing risk of wt ( or=0.6 , 95%ci : 0.381.03 ; p=0.065 ) . further analysis was conducted to investigate the associations between distribution of the genotype and allele of rs11788747 polymorphisms and clinicopathological features of wt patients . as shown in table 3 , although there was no significant difference in distribution of the genotype , allele g carriers with advanced tumor stage ( p=0.026 ) had an increased risk of wt , as did those who had metastasis ( p=0.002 ) . table 2 shows the genotype and allele frequencies of the 2 snps ( rs10972727 and rs11788747 ) in reck gene . no significant difference between cases and controls was observed for distribution of rs10972727 genotype , rs11788747 genotype , and rs11788747 allele . however , we found that the allele g of rs11788747 snp was significantly associated with an increased risk of wt ( or=0.7 , 95%ci : 0.450.99 ; p=0.042 ) . moreover , although when taking the aa genotype as a reference , we found that ag + gg genotype was not statistically significantly associated with the risk of wt , there was a pronounced trend that carriers of 1 variant combined genotype had an increasing risk of wt ( or=0.6 , 95%ci : 0.381.03 ; p=0.065 ) . further analysis was conducted to investigate the associations between distribution of the genotype and allele of rs11788747 polymorphisms and clinicopathological features of wt patients . as shown in table 3 , although there was no significant difference in distribution of the genotype , allele g carriers with advanced tumor stage ( p=0.026 ) had an increased risk of wt , as did those who had metastasis ( p=0.002 ) . the findings of this novel study provide evidence of the effects of snps of reck on wt susceptibility and clinicopathologic status association . various lines of evidence have found that tumor stage and metastasis are the risk factors for development of wt . in the current study , we explored the association of reck gene and wt risk , and subsequently analyzed the effects of combinations of functionally related polymorphisms and clinicopathological features of wt patients . to the best of our knowledge , this is the first study examining the association of reck gene polymorphism with wt risk , although several previous studies have reported on its association with several other types of human cancers . as the most frequent renal tumor occurring in children , although wt has a high survival rate , 25% of wt children may have a poor prognosis or relapse with current therapies , 50% of whom will die despite intensive re - treatment . therefore , it is urgent for us to identify novel genetic loci that might be associated with wt risk . it has been proven that the igf pathway plays a role in development of wt . the igf pathway is a complex signaling system stimulated by insulin - like growth factors ( igfs ) , which can be produced by almost any tissue in the body and has an important effect on growth , development , and survival in many different cell types . overexpression of igf2 , activating the insulin signaling pathway , results in disorder of protein synthesis , cell cycle and cell growth , and blocks apoptosis . furthermore , yamamoto et al . indicated that reduced reck could increase igf2 expression . the interaction between reck gene and igf gene might have a potential influence on susceptibility to wt . a study by huang et al . demonstrated down - regulation of reck gene expression in patients with wt . therefore , we conducted this study to determine whether reck gene is associated with wt risk . in the current study , our findings suggest that mutant of rs10972727 in reck had an impact on increasing wt risk . in addition , allele g carriers with advanced tumor stage involving metastasis had an increasing risk of wt . advanced tumor stage and metastasis are responsible for patient mortality in most tumors [ 3739 ] . found the level of reck expression determined the prognosis of pancreatic cancer and tumors , with overexpression of reck significantly decreasing invasiveness compared with reck - negative tumors , revealing the potential value of reck as a prognostic molecular marker for pancreatic cancer . zhang et al . reported that silenced reck gene was associated with poor survival in hepatocellular carcinoma . . showed that those who carried rs10814325 with at least 1 c allele had a higher risk of hepatocellular carcinoma . , after a follow - up of salivary adenoid cystic carcinoma patients , revealed that expression of reck and mmp-2 gene were correlated with tumor progression . in our study , we found a correlation between rs10972727 in reck and tumor stage and metastasis . however , the exact physiological mechanism by which reck influences progression of wt remains unclear . firstly , the primary weakness of this study is that the small sample size may affect the power in statistical analysis . secondly , the results were not replicated in additional individuals , and this might contribute to potential false - positive errors . moreover , the method used to select potentially functional snps as targets by using a web - based tool might have led to some positive or negative errors . finally , we did not perform a functional study to further reveal the mechanism by which the genetic polymorphisms in reck affect wt risk . data from the present data indicate that rs10972727 in reck gene is not associated with wt risk in chinese children , but the association between mutant g of rs11788747 in reck and wt risk was shown . g carriers with advanced tumor stage or with metastasis might have an increased risk of wt . therefore , further research is necessary to reveal the mechanism by which the genetic polymorphisms in reck affect the wt risk .

in this article , the authors estimate a multiple - output cost function for a sample of 2,235 hospitals during the period 1984 - 88 to disaggregate total costs into inpatient and outpatient components . the results suggest that outpatient cost growth is roughly proportional to that of inpatient cost , despite much higher relative growth in revenues and utilization on the outpatient side . the stability in the outpatient / inpatient cost ratio implies that the increase in the outpatient - to - inpatient utilization ratio was offset by a decline in their relative unit costs . backgroundwilms tumor ( wt ) is the most common malignant renal tumor in children . previous studies suggested the reversion - inducing , cysteine - rich protein with kazal motifs ( reck ) down - regulation might have a role in numerous human cancers . the current study was done to investigate the associations of reck single - nucleotide polymorphisms ( snps ) with the wt susceptibility in chinese children.material/methodswe analyzed 2 snps ( rs10972727and rs11788747 ) in a total of 97 wt children and 194 healthy matched controls ( 1:2 ratio ) by real - time pcr and pcr - rflp genotyping analysis.resultswe found that the g allele of rs11788747 in the reck gene was significantly associated with wt in chinese children ( or=0.7 , 95% ci : 0.450.99 ; p=0.042 ) ; as with another snp rs10972727 , however , no statistically significant difference was detected . further analysis showed there was also a statistically significant difference in genotype frequencies between terminal tumor stage ( p=0.026 ) and metastatic groups ( p=0.002).conclusionsthe present data indicate that there is a significant association between mutant g of rs11788747 in reck and wt risk . g carriers with advanced tumor stage or with metastasis might have an increased risk of wt .

type 2 diabetes is a public health concern across the united states , with certain ethnic groups bearing a disproportionate burden [ 1 , 2 ] . native hawaiians and other pacific islanders ( nh / pi ; e.g. , samoan , chuukese ) have higher type 2 diabetes incidence and prevalence compared to other ethnic groups [ 3 , 4 ] . they are two times more likely to die from diabetes than the general population and suffer from high rates of diabetes - related medical complications and preventable hospitalization [ 5 , 6 ] . addressing the burden of type 2 diabetes is a priority in eliminating health disparities among nh / pi . culturally relevant , diabetes self - management interventions are important in treating type 2 diabetes among nh / pi [ 4 , 8 , 9 ] . found that a culturally adapted diabetes self - management intervention , called partners in care ( pic ) , significantly improved glycemic control and diabetes self - care behaviors in nh / pi compared to a wait - list control . despite the effectiveness of diabetes self - management education intervention , the maintenance of improved glycemic control continues to be a challenge across ethnic groups . the long - term , postintervention maintenance of optimal glycemic control is important in judging an intervention 's effectiveness . diabetes - related social support groups for those with type 2 diabetes have shown promise as a maintenance component for diabetes self - management interventions to improve long - term glycemic control and diabetes - related psychosocial functioning , self - care activities , and quality of life [ 13 , 14 ] . diabetes - related social support can include four types : appraisal support ( e.g. , alternative perspectives of stressors ) , informational support ( e.g. , knowledge ) , emotional support ( e.g. , expression of care ) , and tangible support ( e.g. , providing material help ) . the incorporation of a diabetes - related social support group for nh / pi as a maintenance component to a diabetes self - management intervention is also consistent with their shared ethnocultural values and preferences for group - based interactions . they often rely on their immediate and extended family network ( e.g. , friends and neighbors ) for emotional , physical , and spiritual support and daily decision - making . group participation with other nh / pi offers a safe and supportive environment that can increase the cultural relevance of activities and participation and enhances diabetes self - care . to examine the effects of a diabetes - specific social support maintenance component , the community - academic partnership , the pili 's study conducted another study of pic with an added social support component that emphasized the four types of support . the pop partnership designed a 3-month , 6-session , semistructured support group ( ssg ) to reinforce positive changes made during the 3-month pic intervention . specifically , the maintenance effects of a novel ssg on hba1c control and diabetes self - care behaviors were examined against a control group in a sample of nh / pi with type 2 diabetes who were randomized into these conditions following their completion of pic . the institutional review boards of the native hawaiian health care systems and university of hawaii at mnoa approved this study . community researchers recruited nh / pi from their respective communities and the larger nh / pi population on the island of oahu . eligibility criteria were hba1c > 7% , nh / pi ethnicity , age 18 years , and physician - diagnosed type 2 diabetes . eligible participants provided consent and baseline assessments ( t1 ) were done just prior to starting pic . pic involves 12 , 1-hour weekly group meetings , providing information on diabetes self - management and encouraging participants to work with their diabetes team that includes the individual , their family , physician , and other diabetes experts ( e.g. , certified diabetes educator ) . the intervention is based on the american diabetes association and the national diabetes education program guidelines . pic was culturally adapted for nh / pi based on information from focus groups with nh / pi living with diabetes and nh / pi community leaders as described in sinclair et al . . the community partners included kula no na poe hawaii ( a nonprofit serving urban hawaiian homesteads ) , hawaii maoli ( a nonprofit serving the hawaiian civic clubs ) , ke ola mamo ( the native hawaiian health care system for oahu ) , and kkua kalihi valley ( a health clinic serving low - income pi ) . the community partners recruited participants , delivered the intervention , and conducted the baseline and outcomes assessments at their respective organizations . all participants completed a baseline assessment ( t1 ) , received pic , and underwent a second assessment at 3 months ( t2 ) . the protocol used at each assessment and measures were the same as used by sinclair et al . . following assessment at t2 , participants were randomized , based on a 1 : 1 randomization by site , to either the 3-month ssg or standard follow - up control group . participants randomized to the ssg attended six bimonthly , semistructured group meetings , lasting for about 1 hour , to reinforce skills taught in pic . trained community facilitators ( cf ) led two of the sessions and health professionals ( i.e. , pharmacist , nutritionist , physician , and psychologist ) led the remaining four sessions . community facilitators were instructed to provide appraisal and emotional support ( e.g. , talking through difficulties and encouraging connection between group members ) on how to garner additional support from family / friends for diabetes self - management activities ( i.e. , healthy eating , physical activity , and medication adherence ) . the health professionals concentrated on providing informational and appraisal support around managing diet , medications , diabetes - related complications , and maintaining self - care activities . the control group received only six bimonthly postcards reminding them of performing diabetes self - management activities . all participants underwent a final assessment at t3 after the 3-month maintenance component ( i.e. , six months after t1 ) . clinical measures included hba1c , measured with the bayer dca 2000 via a fingerstick sample of whole blood . the same blood sample was used to measure total cholesterol , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) and triglycerides with the cholestech ldx lipid profile system . blood pressure , weight ( kg ) , and height ( cm ) were measured twice at each assessment with the average of the two values used in the analysis . the understanding subscale of the diabetes care profile ( dcp ) measured understanding of diabetes self - care activities . it consists of 12 items with a 1 ( poor understanding ) to 5 ( excellent understanding ) likert - type response scale . the scores for the 12 items were averaged to yield a total score between 1 and 5 . seven of the 11 items from the summary of diabetes self - care activities ( sdsca ) were used to measure the frequency with which participants conducted self - care activities ( e.g. , checked their feet ) during the previous week . the scoring for each item was as follows : 1 ( not at all during the past 7 days ) , 2 ( 2 - 3 days ) , 3 ( 46 days ) , and 4 ( 7 days ) . higher scores indicate greater frequency of self - care activities . the 20-item problem areas in diabetes ( paid ) assessed quality of life such as physical / social functioning and mental / emotional well - being specific to living with diabetes . the possible responses to each item ranged from 0 ( not a problem ) to 4 ( serious problem ) . the total score was the sum of all items multiplied by 1.25 so that scores ranged from 0 to 100 . demographic and clinical measures were summarized by frequencies and percentages for categorical variables and means ( m ) and standard deviations ( sd ) for continuous variables . support and control groups were compared using chi - square or fisher 's exact test when appropriate for continuous and categorical variables . analysis of covariance ( ancova ) was used to test between group differences at t2 and t3 , adjusting for between - group differences at t1 and t2 , respectively . the baseline characteristics for the 47 nh / pi receiving the pic intervention are summarized in table 1 . it indicates that , among the participants , slightly over half were female , married , and native hawaiian and had a high school diploma or its equivalent . participants on average had bmi in the severely obese category ( m = 36.01 6.77 ) , blood pressure in the prehypertensive range ( sbp m = 129.59 mmhg 15.77 ; dbp m = 76.46 mmhg 11.00 ) , and mean hba1c of 9.98 2.23 . although mean total cholesterol ( m = 183.45 mg / dl 43.77 ) and ldl cholesterol ( m = 93.36 mg / dl 38.49 ) were within the recommended range , participants had low hdl cholesterol ( m = 40.72 mg / dl 13.40 ) and high triglyceride levels ( m = 240.59 171.07 ) . table 1 also summarizes participant characteristics by group at 3-month assessment ( t2 ) . at t2 , both the ssg and control group had mean bmis that remained in the severely obese category ( m = 37.27 7.66 and m = 35.42 4.63 , resp . ) . the ssg had slightly higher mean systolic ( m = 137.48 24.81 ) and diastolic blood pressure ( m = 81.72 14.22 ) but lower hba1c ( m = 8.96 1.82 ) compared to the control group ( m = 132.03 21.43 , m = 76.50 12.96 , m = 9.47 2.69 , resp . ) . however , none of these differences between groups at t1 or t2 were statistically significant . data in table 2 shows the mean changes in behavioral and biological measures across three assessment periods and for the combined sample for both the complete case and the intent - to - treat analysis . in the complete case analysis , there were significant improvements in the following variables from t1 to t2 : hba1c ( m = 0.76 1.86 , p < 0.01 ) , dcp ( m = 0.73 0.97 , p < 0.001 ) , paid ( m = 11.1 21.87 , p < 0.001 ) , and sdsca ( m = 2 5.12 , p < 0.01 ) . except for hba1c , significant improvements in these variables were also found from t1 to t3 . examining change between t2 and t3 shows a significant increase in ldl ( m = 13.55 mg / dl 26.42 , p < 0.05 ) , decrease in sbp ( m = 7.62 mmhg 16.6 , p < 0.05 ) , and increase in sdsca ( m = 1.7 4.67 , p < 0.05 ) . the intent - to - treat analysis provided similar results , with the exception of change in hba1c from t1 to t3 , which showed a significant decrease ( m = 0.53 1.80 , p < 0.05 ) . a comparison of the mean changes in variables between t2 and t3 by group is presented in table 3 . at t2 , 25 participants were randomized to the ssg and 22 to the control group , with 22 and 12 participants being retained at t3 , respectively . there were no significant differences in the changes in variables between the ssg and control group from t2 to t3 , controlling for t2 values . there was a statistically significant reduction in sbp in the ssg ( m = 8.36 mmhg 16.22 , p = 0.025 ) but not in the control group ( m = 6.25 mmhg 17.93 , p = 0.253 ) . there were marginally significant improvements in dcp ( m = 0.24 0.55 , p = 0.054 ) and sdsca ( m = 1.41 3.49 , p = 0.072 ) scores in the ssg but not in the control group ( m = 0.12 0.80 , p = 0.621 , and m = 2.27 6.62 , p = 0.281 , resp . ) . type 2 diabetes is a serious threat to the health and well - being of nh / pi as culturally tailored , diabetes self - management interventions , such as pic , can help attenuate . the 12-week pic intervention led to significant improvements in hba1c , diabetes self - care knowledge and activities , and emotional well - being . however , we did not find significant differences in the maintenance of these improvements between participants randomized to either the ssg or control group following completion of pic . participants ' glycemic control at 6 months was not significantly different from their control immediately after pic . this suggests that participants were able to maintain initial improvements from pic with or without the ssg . while not significantly different between groups , the ssg group had a significant within - group decrease in systolic blood pressure from t2 to t3 while the control group did not . the ssg also had improvements in understanding of diabetes and frequency of self - care activities that were marginally significant . although this study did not support the hypothesis that ssg can improve the maintenance of glycemic control after intervention , we did find some improvements in other outcomes ( e.g. , systolic blood pressure ) . to date our results are consistent with other studies that found modest improvements in diabetes understanding and self - care activities but no change in hba1c [ 14 , 22 ] . our results indicate that the social support provided to the ssg may have helped to improve their systolic blood pressure . a similar study in african americans found that despite no improvements in hba1c after a 3-month diabetes self - management intervention , participants randomized to a 12-month social support group had significant improvements in systolic blood pressure while the control group did not . this finding is important given that over time cardiovascular disease risk factors , such as systolic blood pressure , tend to worsen . additionally , the ukpds study found that maintaining blood pressure in the normal range resulted in an 11% decrease in diabetes complications over 10 years . other studies have found that intensive blood pressure control can save approximately $ 2,000 per quality - adjusted life - year in patients with type 2 diabetes . despite mixed findings in the research on the impact of social support on hba1c in patients with diabetes , based on communication with community researchers , there is a belief that social support groups can help to build relationships among community members and encourage interaction outside of the intervention . this could provide participants with a sense of accountability and opportunities to learn from each other , which may increase motivation to maintain positive behavior changes and improve psychosocial functioning [ 14 , 28 ] . also , participants in the control group received bimonthly postcards reminding them of the skills they learned in the pic intervention . these postcards may have been effective at helping participants maintain the self - care activities they initiated during the intervention , lessening any between group differences at t3 . as a rct , participants were randomized after the 12-week pic intervention . due to the fact that several of these groups were small ( e.g. , 8 people ) , the number of people randomized to ssgs was very small , which may have limited the amount of support each group was able to provide . additionally , some participants formed relationships in pic but were separated by randomization into different groups , which possibly decreased the motivation of these participants . the structure of the ssg was set a priori ; however some participants expressed an interest in diabetes - related topics not included and/or in an order different from what was scheduled , which may have caused participants to lose interest . , in which they state that future research on the use of social support groups in improving hba1c and blood pressure is necessary , a belief with which the community agrees . the use of rcts in which participants are randomized at the individual level after intervention may not be an appropriate design in testing support group components . future designs could randomize by community site , allowing relationships built during the intervention to continue during support groups . other recommendations include the use of support groups that occur on an ongoing basis facilitated by health professionals with diabetes expertise . consequently , participants could attend as they feel necessary and exercise control in determining topics discussed . in conclusion , the pic diabetes self - management intervention is effective at decreasing participants ' hba1c and improving their self - management skills . chest pa showed superior mediastinal widening and bulging of the right paraspinal interface ( fig . 1 ) . increased opacity of the infrahilar window and thickening of the posterior wall of the bronchus intermedius were seen on the lateral chest x - ray . an oval - shaped mass measuring 9.73.86.0 cm was seen in the left superior mediastinum . a bizarre - shaped vascular structure that was connected to left innominate vein was seen in central area of the mass ( fig . the mass showed insinuating appearance without significant compression of adjacent structures . only the thymus was slightly compressed by the mass . another similarly natured mass was seen in the right paraspinal area , which measured about 4.23.06.6 cm . this mass also contained a bizarre - shaped vascular structure that communicated with the left atrium directly through an anomalous vascular structure ( fig . 2 ) . multiplanar reconstruction ( mpr ) and 3d reformation showed that the contrast directly entered the vascular components of the masses via an abnormal vein originating from the left innominate vein ( fig . the mediastinal window setting of contrast - enhanced ct showed additional multiple small cystic lesions in the middle mediastinum . a two - stage operation was planned due to the fact that the tumors were located bilaterally . first , for the left side tumor resection , posterolateral thoracotomy was performed and a large cystic mass was found in the anterosuperior mediastinum . the content of the cyst was bloody and a large communicating vein to left innominate vein and few small communicating veins to left intercostal veins were found , which were then ligated and divided . the second operation was performed for the right mediastinal mass 45 days after the first surgery . the right paraspinal mass was successfully resected and an anomalous communicating vein to left atrium was identified and divided . the patient recovered completely without any complications such as chylothorax or hemothorax . on gross pathologic examination , the cut surface of the tumor revealed numerous cystic spaces and a cavernous sinus containing bloody contents ( fig . histology of the mass showed combined proliferation of venous vascular channels ( ve ) and lymphatic channels ( ly ) that exhibited infiltration into mediastinal fat pad ( fig . mediastinal lymphangiohemangioma is a very rare venolymphatic malformation containing both vascular and lymphatic elements . direct vascular communication between the lesion and the vena cava system ct images of the mediastinal lymphangiohemangioma are very rarely found in the english literature [ 1 - 4 ] . moreover , neither a case with multiple masses and more than one vascular communication nor a case directly connected to the heart chamber through an anomalous vessel was found in the literature . we report a case of surgicopathologically confirmed mediastinal lymphangiohemangioma that communicated with the left innominate vein and left atrium directly via anomalous vascular structures ( fig . vascular malformations are subcategorized as lymphatic , capillary , venous , arteriovenous , and mixed malformations on the basis of their histologic nature . . therefore , our case can be categorized as low - flow vascular malformations with mixed lymphatic and venous components . slow - flowing blood appears as high signal intensity on t2-weighted images and as intermediate signal intensity on t1-weighted images . most lesions present early in childhood and the most common locations for lymphatic malformations are the neck and axilla . a less common location is the mediastinum , and the anterior mediastinum is the most commonly found mediastinal site . ct scan , the mass showed diffuse low attenuation without calcification , although previous reports showed scattered phleboliths in the mass , which represent calcified emboli in the venous channels . the anomalous vascular structures were enhanced at the same intensity as the systemic venous system and was connected directly to the left innominate vein and left atrium . the peripheral portion of the lesion was not enhanced on all phases of dynamic ct scan , which may be related to the lymphatic components or fibroconnective tissues of the mass . a previous report postulated that the anomalous vein acts not only as a draining vessel but also as a feeding vessel . in our case , only the proximal part of the vascular structure was enhanced on the pulmonary arterial phase , whereas all of the large bizarre - shaped vascular spaces were enhanced on the systemic venous phase ( fig . 2 ) . a delayed ct scan may show delayed clearance of contrast within the anomalous venous channels because of the lack of smooth muscle in these channels . lymphangiohemangiomas are not considered an congenital anomaly but are frequently associated with other vascular malformations , most commonly in the left superior vena cava . in our case , the anomalous vessels connected to the left innominate vein may be considered a left superior vena cava remnant . a lymphangiohemangioma should be included in the list of differential diagnoses of an incidentally found low - attenuating mediastinal mass , especially in young patients . another therapeutic option is percutaneous sclerotherapy using agents such as absolute ethanol , bleomycin , cyclophosphamide , doxycycline , alcohol solution of zein , or ok-432 . recognizing that the lesion is a low - flow vascular malformation is more important than determining whether the lesion is predominantly venous or lymphatic when making treatment decisions . we planned surgical excision of the lesions and successfully excised them without any complications such as hemothorax or chylothorax . for successful surgery , the anomalous vascular pedicle of the mass communicating with the systemic vein or heart chamber should be carefully identified and divided to prevent massive hemothorax .

native hawaiians and other pacific islanders ( nh / pi ; e.g. , samoan and chuukese ) have higher type 2 diabetes prevalence compared to other groups in hawaii . partners in care ( pic ) , a culturally tailored , community - based , diabetes self - management education intervention ( dsme ) , is effective at improving participants ' glycemic control and self - care behaviors . maintenance of improvements is challenging . diabetes - related social support groups ( ssg ) are a promising maintenance component for dsme . this study examined the effects of a diabetes - specific ssg component relative to a control group , after the receipt of the 3-month pic intervention , which was delivered to 47 adult nh / pi with type 2 diabetes . participants were then randomized to either a 3-month , 6-session ssg or a control group . hemoglobin a1c ( hba1c ) , blood pressure , triglycerides , cholesterol , and diabetes self - management knowledge and behaviors were assessed at baseline , 3 months , and 6 months . results indicated significant improvements in hba1c , diabetes - related self - management knowledge , and behaviors from baseline to 3-month assessment . however , no differences between the ssg and control group from 3-month to 6-month assessment suggest that all participants were able to maintain initial improvements . the ssg group had a significant decrease in systolic blood pressure from 3-month to 6-month assessment while the control group did not . study limitations and future directions are discussed . lymphangiohemangiomas of the mediastinum are exceedingly rare and few cases have been published in the english literature . this report may be the only reported case in which lymphangiohemangiomas were found bilaterally . we report a case of a 7-year - old boy with an incidental finding of an abnormal mediastinal shadow on a chest x - ray . the chest ct showed a large mass in the left superior mediastinum and another in the right posterior mediastinum . the left mass had anomalous venous channels connected to the left innominate vein , and the right mass to the left atrium . we performed an excision of the mass in the left side first and then the right side one month later . anomalous venous channels were dissected carefully and ligated . there were no complications and no signs of recurrence 30 months after the operation .

parkinson disease ( pd ) is a neurodegenerative disease which its prevalence varies by location and ethnicity ( 1 , 2 ) . the correlation between depression and parkinson disease has remained unclear among iranian population . the roles of demographic and disease - related variables are not fully described in the association between depression and pd . depression is highly prevalent among patients with parkinson disease ( 3 , 4 ) and has been demonstrated to reduce quality of life among affected individuals ( 5 , 6 ) . factors like male gender ( 7 ) , cognitive impairment ( 8) , motor symptoms ( left - sided onset tremor ) ( 9 ) , and impulsivity ( 10 ) are shown to be associated with higher risk of developing depression among patients with pd . studies have demonstrated that the association between depression and parkinson disease is not merely a reaction to chronic disease , but a consequence of the neurodegenerative process ( 11 , 12 ) . therefore , multifactorial disease - related variables along with non - pd specific components may affect the prevalence of depression in parkinson disease ( 13 ) . the prevalence of depression and related variables has not yet been described among iranian patients with pd living in kermanshah province . since depression disruptively affect quality of life among patients with pd , determination of its prevalence and appropriate treatment are essential . there is some evidence indicating that depression has been underdiagnosed and undertreated in patients with pd ( 14 ) . studies have shown that depression in pd is more commonly accompanied with somatic and cognitive symptoms ( 15 , 16 ) which may contribute to complexity and underdiagnoses of depression among this population . determination of predictive factors for depression may help clinicians to cautiously consider susceptible patients for treatment . in this investigation , the prevalence of depression and the effect of demographic and disease - related variables were assessed in patients with pd living in kermanshah province , iran . patients with confirmed parkinson disease who were referred to clinics of kermanshah university of medical sciences were invited to participate in this cross - sectional study . exclusion criteria were as follows : history of depression or other psychiatric disorders before onset of pd , existence of other chronic medical conditions , including cancer , endocrinology disease , coronary vascular disease , history of myocardial infarct , and spinal cord injury . patients under treatment with special medications such as glucocorticoid , thyroid hormones , anticonvulsive drugs , heparin , lithium , blood glucose reducing agents , and atorvastatin were also excluded . those with the history of smoking , alcohol , or drug abuse were excluded as well . participation was voluntarily and patients were assured about the confidentiality of their information . during interviews , private locations were provided to respect patient privacy . although an assistant was present at the interviews , the final judgements of subjective assessment of depression were indexed according to the psychologist s interpretations . patients age , gender , educational level , daily medication treatment schedules were asked directly from each individual and indexed in prepared forms during interviews . pd - related variables , including tremor , rigidity , impaired posture , loss of autonomic movement , mask face , speech and writing changes , excessive salvia , and urinary urgency were examined by a neurologist and also indexed . the farsi version of snaith - hamilton pleasure scale has 14 items and each of the items has a set of 5 response categories ( definitely agree , agree , no idea , disagree , and strongly disagree ) which are scored from 1 to 5 . 1 ) i would enjoy my favorite television or radio program , 2 ) i would enjoy being with family or close friends , 3 ) i would find pleasure in my hobbies and pastimes , 4 ) i would be able to enjoy my favorite meal , 5 ) i would enjoy a warm bath or refreshing shower , 6 ) i would find pleasure in the scent of flowers or the smell of a fresh sea breeze or freshly baked bread , 7 ) i would enjoy seeing other people s smiling faces , 8) i would enjoy looking smart when i have made an effort with my appearance , 9 ) i would enjoy reading a book , magazine , or newspaper , 10 ) i would enjoy a cup of tea or coffee or my favorite drink , 11 ) i would find pleasure in small things ; e.g. , bright sunny day , telephone call from a friend , 12 ) i would be able to enjoy a beautiful landscape or view , 13 ) i would get pleasure from helping others , and 14 ) i would feel pleasure when i receive praise from other people ( 18 ) . these items cover 4 domains of hedonic experience : interest / pastimes , social interaction , sensory experience , and food / drink . studies have demonstrated that snaith - hamilton pleasure scale has an internal consistency of 0.91 which shows its validity and reliability to assess hedonic capacity ( 19 ) . beck depression inventory - ii consists of 21 items and the score ranges from 0 to 63 . interpretations of the scores are as follows : 1 - 10 , normal ; 11 - 16 , mild mood disturbance ; 17 -2 0 , borderline clinical depression ; 21 - 30 , moderate depressive mood ; 31 - 40 , severe depressive mood ; and over 40 , extreme depression ( 20 ) . the persian version of bdi - ii has been shown to have acceptable internal consistency ( cronbach = 0.87 ) and test - retest reliability ( r = 0.74 ) ( 21 ) . bdi - ii was checked to be a valid and reliable instrument to assess depression among patients with parkinson disease ( 22 ) . because a weak association between diabetes mellitus type 2 and depression has been reported , the history of diabetes in all patients were recorded and blood samples were taken to measure fasting plasma glucose ( fpg ) . cochran s formula was used to estimate sample size . according to sadeghirad et al . ( 23 ) , the approximate prevalence of major depression in iran is 4.1% . by considering the total population of kermanshah which is about 2000000 , the approximate number of major depression in kermanshah is estimated to be 82000 . using cochran s sample size formula with a margin of error of 5% and confidence interval of 95 , the formula was as follows : similar sample size was determined using morgan s table . we interviewed 412 patients with pd and 62 subjects were excluded according to exclusion criteria . finally , a total of 350 patients participated in this study which is close to estimated sample size . all statistical analysis was performed using spss software version 21 ( ibm corporation , usa ) . categorical variables were described by numbers and percentages whereas continuous variables by mean and standard deviation . comparison of means between groups was done by t test and 1-way analysis of variance ( anova ) . patients with confirmed parkinson disease who were referred to clinics of kermanshah university of medical sciences were invited to participate in this cross - sectional study . exclusion criteria were as follows : history of depression or other psychiatric disorders before onset of pd , existence of other chronic medical conditions , including cancer , endocrinology disease , coronary vascular disease , history of myocardial infarct , and spinal cord injury . patients under treatment with special medications such as glucocorticoid , thyroid hormones , anticonvulsive drugs , heparin , lithium , blood glucose reducing agents , and atorvastatin were also excluded . those with the history of smoking , alcohol , or drug abuse were excluded as well . participation was voluntarily and patients were assured about the confidentiality of their information . during interviews , private locations were provided to respect patient privacy . although an assistant was present at the interviews , the final judgements of subjective assessment of depression were indexed according to the psychologist s interpretations . patients age , gender , educational level , daily medication treatment schedules were asked directly from each individual and indexed in prepared forms during interviews . pd - related variables , including tremor , rigidity , impaired posture , loss of autonomic movement , mask face , speech and writing changes , excessive salvia , and urinary urgency were examined by a neurologist and also indexed . the farsi version of snaith - hamilton pleasure scale has 14 items and each of the items has a set of 5 response categories ( definitely agree , agree , no idea , disagree , and strongly disagree ) which are scored from 1 to 5 . 1 ) i would enjoy my favorite television or radio program , 2 ) i would enjoy being with family or close friends , 3 ) i would find pleasure in my hobbies and pastimes , 4 ) i would be able to enjoy my favorite meal , 5 ) i would enjoy a warm bath or refreshing shower , 6 ) i would find pleasure in the scent of flowers or the smell of a fresh sea breeze or freshly baked bread , 7 ) i would enjoy seeing other people s smiling faces , 8) i would enjoy looking smart when i have made an effort with my appearance , 9 ) i would enjoy reading a book , magazine , or newspaper , 10 ) i would enjoy a cup of tea or coffee or my favorite drink , 11 ) i would find pleasure in small things ; e.g. , bright sunny day , telephone call from a friend , 12 ) i would be able to enjoy a beautiful landscape or view , 13 ) i would get pleasure from helping others , and 14 ) i would feel pleasure when i receive praise from other people ( 18 ) . these items cover 4 domains of hedonic experience : interest / pastimes , social interaction , sensory experience , and food / drink . studies have demonstrated that snaith - hamilton pleasure scale has an internal consistency of 0.91 which shows its validity and reliability to assess hedonic capacity ( 19 ) . beck depression inventory - ii consists of 21 items and the score ranges from 0 to 63 . higher scores indicate more severe depressive moods . interpretations of the scores are as follows : 1 - 10 , normal ; 11 - 16 , mild mood disturbance ; 17 -2 0 , borderline clinical depression ; 21 - 30 , moderate depressive mood ; 31 - 40 , severe depressive mood ; and over 40 , extreme depression ( 20 ) . the persian version of bdi - ii has been shown to have acceptable internal consistency ( cronbach = 0.87 ) and test - retest reliability ( r = 0.74 ) ( 21 ) . bdi - ii was checked to be a valid and reliable instrument to assess depression among patients with parkinson disease ( 22 ) . because a weak association between diabetes mellitus type 2 and depression has been reported , the history of diabetes in all patients were recorded and blood samples were taken to measure fasting plasma glucose ( fpg ) . et al . ( 23 ) , the approximate prevalence of major depression in iran is 4.1% . by considering the total population of kermanshah which is about 2000000 , the approximate number of major depression in kermanshah is estimated to be 82000 . using cochran s sample size formula with a margin of error of 5% and confidence interval of 95 , the formula was as follows : similar sample size was determined using morgan s table . we interviewed 412 patients with pd and 62 subjects were excluded according to exclusion criteria . finally , a total of 350 patients participated in this study which is close to estimated sample size . all statistical analysis was performed using spss software version 21 ( ibm corporation , usa ) . categorical variables were described by numbers and percentages whereas continuous variables by mean and standard deviation . comparison of means between groups was done by t test and 1-way analysis of variance ( anova ) . a total of 412 patients selected based on inclusion criteria were interviewed and 62 subjects were excluded according to exclusion criteria . finally , 350 patients ( 52.9% men and 47.1% women ) participated in this investigation . mean age of the study sample and mean age by the time of pd onset were 64.07 5.08 and 58.46 4.47 years , respectively . the majority of patients were illiterate ( n = 183 , 52.3% ) and only 4.6% of participants had higher education . tremor was observed in 325 ( 92.9% ) patients and rigidity was detected in 291 ( 83.1% ) patients . impaired posture , loss of autonomic movements , changes in speech and handwriting were found in 217 ( 62.0% ) , 256 ( 73.1% ) , 217 ( 62.0% ) , and 163 ( 46.6% ) patients , respectively . only 10.6% of the patients had hyposmia whereas other pd - related variables , including masked face , excessive salvia , and urinary urgency were detected in a higher proportion of patients ( 55.7% , 48% , and 41.1% , respectively ) . anhedonia was assessed by farsi version of snaith - hamilton pleasure scale , which determines hedonic tone in 4 domains . obtained scores in each domain were as follows : interest / pastimes ( 7.89 2.01 ) , social interaction ( 14.0 3.94 ) , sensory experience ( 11.53 3.39 ) , food / drink ( 5.47 1.93 ) ( table 1 ) . female patients were significantly more susceptible to severe and profound depression ( p < 0.0001 ) . high school educational level and academic educations were related to higher incidence of profound depression ( p < 0.0001 ) , whereas 12.5% of illiterate patients and 14.3% of patients with primary educational level had normal mood . patients with familial history of pd experienced more severe depression ( p = 0.019 ) . surprisingly , 40% of patients without tremor had profound depression whereas only 22.7% of individuals with tremor suffered from profound depression ( p = 0.004 ) . however , since only 7.1% of patients did not demonstrate tremor as a pd clinical sign , results on tremor should be interpreted cautiously . rigidity was also a determinant of increased risk of depression ( p < 0.0001 ) . similarly , patients with impaired posture were more likely to have profound depression whereas those with intact posture most commonly experienced borderline clinical depression . furthermore , changes in speech and handwritings were not determinants of depression ( p = 0.095 and 0.062 , respectively ) . profound depression was detected among 40% of patients with masked face whereas only 5% of individuals without masked face showed profound depressive mood . this finding demonstrates that masked face is a significant determinant of depression among patients with pd ( p < 0.0001 ) . hyposmia was also related to higher susceptibility to depression and 50% of patients with hyposmia experienced profound depression ( p = 0.001 ) ( table 2 ) . abbreviation : pd : parkinson disease . patients with diabetes mellitus type 2 ( dm ii ) were more likely to have mild disturbed mood compared with those individuals without history of dm ii ( 25.7% vs. 6.7% ; p < 0.0001 ) . similarly , patients with mild disturbed mood had the highest mean value for fpg ( p = 0.007 ) . mean age of the patients with mild mood disturbance was low compared to other groups ( p < 0.0001 ) . lower scores of all domains of farsi version of snaith - hamilton pleasure scale were related to more severe depression ( p < 0.0001 for all subscales ) ( table 3 ) . p values stand for 1-way analysis of variance ( anova ) for comparison of means between groups . forty - three patients ( 12.3% ) had normal mood ; however , mild disturbance mood was detected in 36 ( 10.3% ) patients ; 70 ( 20.0% ) patients experienced borderline clinical depression ; and 47 ( 13.4% ) patients had moderate depression . severe and profound depression was detected in 70 ( 20.0% ) and 84 ( 24.0% ) patients . the overall estimation of prevalence of severe and profound depression among iranian individuals with pd was 44% . although the association between depression and parkinson disease ( pd ) has been described ( 11 , 12 ) , the role of pd - related variables on the severity of depression is yet unclear . ( 8) demonstrated that increased severity of pd is associated with higher susceptibility to depression . in the present study , we assessed pd - related variables , including clinical features to identify major determinants of depression among iranian individuals with pd . our results show that rigidity , impaired posture , masked face , and hyposmia are commonly related to more severe depression . anhedonia assessed by farsi version of snaith - hamilton pleasure scale was also a major determinant of depression . ( 24 ) showed that prevalence of major depression among patients with pd is about 8.1% . estimation of prevalence of depression in patients with pd varies among different populations according to study methodology , assessment tool , and community characteristics ( 24 ) . our estimation of depression prevalence among iranian patients with pd in kermanshah province is about 44% . using different assessment instrument to assess depression may also contribute to different estimations of depression prevalence in patients with pd . in this regard , williams et al . ( 25 ) compared scales for detection of depression in patients with pd ( beck depression inventory - ii , center for epidemiologic studies depression rating scale - revised , 30-item geriatric depression scale , inventory of depressive symptoms - patient , patient health questionnaire-9 , and unified parkinson disease rating scale - part i ) . their results showed that all mentioned instruments , except for the unified parkinson disease rating scale - part i , were valid screening tools ( 25 ) . however , it seems that proper comparison of depression among nations requires utilization of similar methods . our results demonstrated impaired posture as a determinant of depression . among pd - related variables , hassan et al . ( 26 ) showed that postural instability was correlated with depression among patients with pd which was in line with our findings . furthermore , letvinenko et al . ( 27 ) showed that back pain was correlated with the extent of posture disturbances . since the relationship between pain and depression is also well - documented ( 27 ) , we concluded that impaired posture increases the risk of depression through raising the level of pain . according to recent ninds recommendation ( 28 ) , anhedonia is a specific component of depression in pd . our study illustrates that anhedonia is significantly correlated with more severe depression among patients with pd which is consistent with ninds recommendations ( 28 ) . similarly , the close relationship between anhedonia and depression has been reported in general population ( 29 ) and in adolescents with major depression disease ( 30 ) . this evidence , in line with our study , supports the predictive value of anhedonia in determining depression . on the other hand , there is evidence demonstrating weak discriminating value of anhedonia compared to negative affect and apathy in determining depression in patients with pd ( 16 , 31 ) . in the present study , apathy was not assessed and further investigations are required to compare the discriminating value of apathy and anhedonia as determinants of depression in iranian population with pd in kermanshah . studies have shown that involvement of ventral striatum and prefrontal cortical regions may contribute to the development of anhedonia in depression ( 32 ) . deficits in dopamine generating cells in the substantia nigra of the brain are the underlying pathophysiology of pd . further studies are required to determine whether coincidental involvement of ventral striatum and substantia nigra in pd results in anhedonia among affected individuals . our investigation showed that women with pd are more susceptible to major depression compared to men which is contrary to the results of hsu et al . ( 33 ) reported that depression is more prominent among women with pd in the united kingdom which supports our results . since multifactorial components , including community characteristics should be considered when addressing depression based on gender distribution , further investigations with controlling social and environmental factors are required to clarify the role of gender in the incidence of depression among iranian people with pd . some studies have demonstrated the association of depression with diabetes mellitus ( 34 - 37 ) . however , riederer et al . ( 38 ) showed the association between depression and diabetes mellitus ( dm ) is relatively week among patients with pd . our study did not approve higher severity of depression in patients with coexistence of pd and diabetes . these findings show that dm and fpg may not be direct determinant of depression in pd but they have predictive value in determining a specific subgroup of mild depressive mood . identification of these variables can help give priorities to each patient based on their vulnerability to depression which is the strong point of this study . however , the weak point of this study is the lack of follow - up process . therefore , patients could not be assessed with regard to changes in depressive mood through time . in this study , the prevalence of depression among iranian patients with pd in kermanshah province and the association between depression severity and pd - related variables were assessed . it is estimated that 44% of patients with pd suffer from severe and profound depression . major determinants of depression were female gender , rigidity , impaired posture , masked face , hyposmia , and anhedonia . fatigue is the most frequent symptom in multiple sclerosis ( ms).1 the multiple sclerosis council defines ms related fatigue as a subjective lack of physical and/or mental energy that is perceived by the individual or caregiver to interfere with usual and desired activities,2 and has recommended evaluation with the 21-item modified fatigue impact scale ( mfis ) to measure fatigue,2 which is a smaller version of the 40-item fatigue impact scale ( fis).3 krupp et al,4 in 1989 , published the unidimensional fatigue severity scale ( fss ) , which is widely used not only in ms , but also in stroke,5 parkinson s disease,5 lung diseases,6 myotonic dystrophy,7 neuroborreliosis,8 hiv infection,9 and cancer,10 and in sleep disorders such as insomnia,11 shift working,12 restless legs syndrome ( rls),13,14 and obstructive sleep apnea.15 the international classification of sleep disorders second edition ( icsd-2 ) mentions exhaustion as a minimal criterion of sleep apnea,16 but in general there are no objective measurements to differentiate fatigue from exhaustion . complaints of fatigue may be as important as those of sleepiness are to obstructive sleep apnea syndrome ( osas ) patients,17 and consequently , continuous positive airway pressure ( cpap ) improves fatigue substantially in osas patients.18 ( interestingly , women suffering from osas appear to have complaints of fatigue more frequently than men with osas).17 hossain et al19 investigated 283 patients by polysomnography in a sleep laboratory . sixty - four percent of referred patients reported pathological fatigue without overlap of sleepiness ( mean fss 5.1 ; mean epworth sleepiness scale [ ess]20 6.9 ) , 19% were highly fatigued and sleepy ( mean fss 5.4 ; mean ess 17.2 ) , 13% did not feel either fatigue or sleepiness ( mean fss 2.1 ; mean ess 5.4 ) , and only 4% reported pathological sleepiness without overlap of fatigue ( mean fss 1.9 ; mean ess 14.5 ) . in summary , fatigue and sleepiness can occur together , but fatigue and sleepiness can be independent manifestations of a sleep disorder;21 there are many patients suffering from sleep disorders with fatigue but without sleepiness . in all likelihood this underemphasis may contribute to the overlooking of treatable sleep disorders , with enormous consequences for misdiagnosed patients . veauthier et al22 performed the first cross - sectional polysomnographic trial in the context of ms - related fatigue . they found a significant relationship between relevant sleep disorders ( including sleep - related breathing disorders [ srbds ] , rls , periodic limb movement disorder [ plmd ] , insomnia , and others ) on the one hand and fatigue measured with the mfis and the fss on the other hand ( mean mfis in fatigued ms patients 42.8 [ standard deviation { sd } 18.3 ] and 20.5 [ sd 17 ] in non - fatigued patients [ p < 0.001 ] ) . after performing a receiver operating characteristic analysis,23 they recommended polysomnographic investigations in all fatigued ms patients , when mfis > 34 or pittsburgh sleep quality index ( psqi)24 > 5 , so as not to overlook treatable sleep disorders.23 two different sleep studies have shown a significant relationship between ms - related fatigue and sleep disorders,22,25 and , in the corresponding follow - up trials , medical sleep treatment led to an improvement of ms - related fatigue.26,27 aside from the abovementioned studies,10,11,19,21 there are only a few polysomnographic studies investigating fatigue measured with the fss,28,29 and there are no other polysomnographic studies investigating fatigue measured with the mfis in the general population . hossain et al described fss mean values from 4.4 ( sd 1.6 ) to 5.1 ( sd 1.0 ) in osas , rls , plmd , depression , narcolepsy , parasomnia , delayed sleep phase syndrome , and insomnia,19 but as there have been , to date , no pathophysiological explanations for fatigue caused by sleep disorders , the question remains : is it caused by reduced sleep efficacy , disrupted sleep , suppressed deep sleep , intensified desaturation , or an increased number of hypopnea / apnea episodes ? the aims of this study were : to describe the mfis values in patients admitted to a sleep laboratory and to investigate which polysomnographic parameters can predict fatigue measured with the mfis and the fss . in this retrospective observational study , all consecutive patients who were admitted to the sleep laboratory of the hanse - klinikum , stralsund , germany between february 1 , 2011 and january 31 , 2012 were included . all patients admitted to the sleep laboratory completed the mfis , fss , ess , and psqi . these whole - night measurements were made after an impedance test and a biosignal test over a period of 8 hours , comprising : c3/c4 electroencephalograph ( eeg ) electrodes to the contralateral mastoid electrode ; ground electrode ; electrooculogram on the ipsilateral mastoid electrode ; bipolar chin electromyogram ( emg ) of the muscle mentalis or muscle submentalis ( according to biosignal testing and anatomical conditions ) ; nasal airflow ( pressure sensor ) ; thoracic and abdominal breathing ( piezoelectric measurement ) ; position sensor ; snoring signal ; pulse oximetry ; pulse , electrocardiogram ; and bipolar two - point emg electrodes on both anterior tibial muscles . the polysomnograph readings were scored according to the criteria of rechtschaffen and kales,30 using 30-sec - ond epochs . the following polysomnographic parameters were included in the analysis : apnea / hypopnea index ( ahi ) per hour of sleep , according to the american academy of sleep medicine;31 minimal desaturation ( spo2% min ) ; sleep efficacy ( ratio total sleep time / time spent in bed ) ; deep sleep ( ratio non - rapid eye movement sleep stage 3 and 4 [ nrem-3/4]/time spent in bed ) ; number of awakenings ; arousal index per hour of sleep ; and sleep latency ( time to first sleep epoch in minutes ) . sleep diagnoses were made according to the icsd-2.16 patients were divided for statistical reasons into different subgroups : ( 1 ) untreated srbds ; ( 2 ) treated srbds ( patients were treated with cpap or bilevel therapy ) ; ( 3 ) plmd and rls ; ( 4 ) insomnia ; ( 5 ) parasomnia ; ( 6 ) other neurological diseases such as narcolepsy , hypersomnia , and ms ( due to a low number of patients and small sample sizes , these patients were classified into one subgroup even though these diseases are quite different ) ; and ( 7 ) patients admitted to the sleep laboratory for tiredness or sleepiness but without pathological findings in the medical sleep investigations ( exclusion of a sleep disorder ) . the results were expressed as mean , sd , and range . following an exploratory analysis of the data and after a ( negative ) check for normality of the underlying distributions , differences between subgroups with respect to mfis , ess , psqi , and fss were ( univariately ) analyzed using the mann spearman correlation coefficients were calculated to assess the relationship between questionnaire results , and polysomnographic parameters and age , and scatter plots were performed to demonstrate these relationships . we defined spearman s rank correlations ( spearman s rho ) as follows : for values between 0.9 and 1 , the correlation is very strong ; for values between 0.7 and 0.89 , the correlation is strong ; values between 0.5 and 0.69 show a moderate correlation ; for values between 0.3 and 0.49 , the correlation is moderate to low ; and for values < 0.3 , the correlation is weak and not important . patients were classified into subgroups by mfis ( mfis greater versus less than or equal to mfis mean ) and fss ( fss greater versus less than or equal to fss mean ) values . afterwards , a logistic regression was performed in order to predict the outcome of these two dependent variables ( greater versus less than or equal to fss mean / mfis mean ) with age , sex , and polysomnographic parameters , including ahi , spo2% min , sleep efficacy , nrem-3/4 , number of awakenings , arousal index , and sleep latency , as predictor variables . this analysis was performed in two steps : first , a univariate analysis with all predictor variables ; subsequently , all significant variables remained in the final model of multiple logistic . all tests should be understood as constituting exploratory data analysis , such that neither previous power calculations nor subsequent adjustments for multiple testing have been made . analysis was performed with spss software ( v19 ; ibm corporation , armonk , ny , usa ) . in this retrospective observational study , all consecutive patients who were admitted to the sleep laboratory of the hanse - klinikum , stralsund , germany between february 1 , 2011 and january 31 , 2012 were included . all patients admitted to the sleep laboratory completed the mfis , fss , ess , and psqi . these whole - night measurements were made after an impedance test and a biosignal test over a period of 8 hours , comprising : c3/c4 electroencephalograph ( eeg ) electrodes to the contralateral mastoid electrode ; ground electrode ; electrooculogram on the ipsilateral mastoid electrode ; bipolar chin electromyogram ( emg ) of the muscle mentalis or muscle submentalis ( according to biosignal testing and anatomical conditions ) ; nasal airflow ( pressure sensor ) ; thoracic and abdominal breathing ( piezoelectric measurement ) ; position sensor ; snoring signal ; pulse oximetry ; pulse , electrocardiogram ; and bipolar two - point emg electrodes on both anterior tibial muscles . the polysomnograph readings were scored according to the criteria of rechtschaffen and kales,30 using 30-sec - ond epochs . the following polysomnographic parameters were included in the analysis : apnea / hypopnea index ( ahi ) per hour of sleep , according to the american academy of sleep medicine;31 minimal desaturation ( spo2% min ) ; sleep efficacy ( ratio total sleep time / time spent in bed ) ; deep sleep ( ratio non - rapid eye movement sleep stage 3 and 4 [ nrem-3/4]/time spent in bed ) ; number of awakenings ; arousal index per hour of sleep ; and sleep latency ( time to first sleep epoch in minutes ) . sleep diagnoses were made according to the icsd-2.16 patients were divided for statistical reasons into different subgroups : ( 1 ) untreated srbds ; ( 2 ) treated srbds ( patients were treated with cpap or bilevel therapy ) ; ( 3 ) plmd and rls ; ( 4 ) insomnia ; ( 5 ) parasomnia ; ( 6 ) other neurological diseases such as narcolepsy , hypersomnia , and ms ( due to a low number of patients and small sample sizes , these patients were classified into one subgroup even though these diseases are quite different ) ; and ( 7 ) patients admitted to the sleep laboratory for tiredness or sleepiness but without pathological findings in the medical sleep investigations ( exclusion of a sleep disorder ) . the results were expressed as mean , sd , and range . following an exploratory analysis of the data and after a ( negative ) check for normality of the underlying distributions , differences between subgroups with respect to mfis , ess , psqi , and fss were ( univariately ) analyzed using the mann spearman correlation coefficients were calculated to assess the relationship between questionnaire results , and polysomnographic parameters and age , and scatter plots were performed to demonstrate these relationships . we defined spearman s rank correlations ( spearman s rho ) as follows : for values between 0.9 and 1 , the correlation is very strong ; for values between 0.7 and 0.89 , the correlation is strong ; values between 0.5 and 0.69 show a moderate correlation ; for values between 0.3 and 0.49 , the correlation is moderate to low ; and for values < 0.3 , the correlation is weak and not important . patients were classified into subgroups by mfis ( mfis greater versus less than or equal to mfis mean ) and fss ( fss greater versus less than or equal to fss mean ) values . afterwards , a logistic regression was performed in order to predict the outcome of these two dependent variables ( greater versus less than or equal to fss mean / mfis mean ) with age , sex , and polysomnographic parameters , including ahi , spo2% min , sleep efficacy , nrem-3/4 , number of awakenings , arousal index , and sleep latency , as predictor variables . this analysis was performed in two steps : first , a univariate analysis with all predictor variables ; subsequently , all significant variables remained in the final model of multiple logistic . all tests should be understood as constituting exploratory data analysis , such that neither previous power calculations nor subsequent adjustments for multiple testing have been made . analysis was performed with spss software ( v19 ; ibm corporation , armonk , ny , usa ) . in sum , 410 patients 20 to 86 years ( 290 men and 120 women ) were investigated . table 1 displays the demographic and polysomnographic data and table 2 shows the questionnaire results . there was a moderate to strong correlation between mfis and fss values in the whole cohort and in all subgroups except for the plmd / rls subgroup . there was a moderate correlation between mfis and psqi in the whole cohort and in srbd patients ( treated and untreated ) and in patients without sleep disorders or with narcolepsy / hypersomnia / ms ( but not in plmd / rls patients nor insomnia patients ) . there was a moderate correlation between fss and psqi in srbd patients ( treated and untreated ) . there was a low correlation between ess values and mfis in untreated srbd patients and a moderate correlation between ess values and fss in patients without sleep disorders . higher age was related to increased psqi values in plmd / rls patients and with increased ess values in patients with narcolepsy / hypersomnia / ms ( table 3 ) . with respect to polysomnographic data , a low correlation could be found between increased ahi and lower minimal desaturation on the one hand and increased psqi values on the other in treated srbd patients . furthermore , there was a low correlation between the number of awakenings and psqi in all patients and untreated srbd patients . similarly , there was a low correlation between an increased arousal index and higher psqi values in treated srbd and insomnia patients . figures 1 and 2 show the relationships between mfis / fss values and polysomnographic parameters by scatter plots . whereas the graphical visualization did not show any correlation between mfis / fss values and ahi , spo2% min , sleep efficacy , sleep latency , or deep sleep , the scatter plots did visualize a correlation between mfis values and the number of awakenings and between fss values and a high arousal index . in the subgroup without sleep disorders , we did find a moderate nonparametric correlation between increased deep sleep ( nrem-3/4 ) and higher mfis and fss values and , similarly , between deep sleep and mfis in plmd / rls patients . in plmd / rls patients , lower sleep latency was correlated with increased ess values . in insomnia patients , higher fss values we introduced the dichotomous variable mfis higher than mfis mean ( 34.2 ) ( yes or no ) and the dichotomous variable fss higher than fss mean ( 4.0 ) the univariate logistic regression showed no significant relationship with spo2% min , sleep efficacy , sleep latency , or deep sleep . a significant relationship could be found with age , sex , and number of awakenings for mfis as well as fss ( table 4 ) . furthermore , there was a significant relationship with arousal index and ahi only for fss . the multiple logistic regression ( table 4 ) investigating the fss , including age , sex , arousal index , number of awakenings , and ahi , demonstrated a significant relationship with age , sex , and arousal index , whereas ahi and number of awakenings were not significant . younger age , female sex , and high arousal index were highly predictive of increased fss scores . in regards to mfis , the multiple logistic regression ( table 4 ) investigating the mfis , including age , sex , and number of awakenings , confirmed the significant relationship with age , sex , and number of awakenings . younger age , female sex , and high numbers of awakenings predicted high mfis values . in sum , 410 patients 20 to 86 years ( 290 men and 120 women ) were investigated . table 1 displays the demographic and polysomnographic data and table 2 shows the questionnaire results . there was a moderate to strong correlation between mfis and fss values in the whole cohort and in all subgroups except for the plmd / rls subgroup . there was a moderate correlation between mfis and psqi in the whole cohort and in srbd patients ( treated and untreated ) and in patients without sleep disorders or with narcolepsy / hypersomnia / ms ( but not in plmd / rls patients nor insomnia patients ) . there was a moderate correlation between fss and psqi in srbd patients ( treated and untreated ) . there was a low correlation between ess values and mfis in untreated srbd patients and a moderate correlation between ess values and fss in patients without sleep disorders . higher age was related to increased psqi values in plmd / rls patients and with increased ess values in patients with narcolepsy / hypersomnia / ms ( table 3 ) . with respect to polysomnographic data , a low correlation could be found between increased ahi and lower minimal desaturation on the one hand and increased psqi values on the other in treated srbd patients . furthermore , there was a low correlation between the number of awakenings and psqi in all patients and untreated srbd patients . similarly , there was a low correlation between an increased arousal index and higher psqi values in treated srbd and insomnia patients . figures 1 and 2 show the relationships between mfis / fss values and polysomnographic parameters by scatter plots . whereas the graphical visualization did not show any correlation between mfis / fss values and ahi , spo2% min , sleep efficacy , sleep latency , or deep sleep , the scatter plots did visualize a correlation between mfis values and the number of awakenings and between fss values and a high arousal index . in the subgroup without sleep disorders , we did find a moderate nonparametric correlation between increased deep sleep ( nrem-3/4 ) and higher mfis and fss values and , similarly , between deep sleep and mfis in plmd / rls patients . in plmd / rls patients , lower sleep latency was correlated with increased ess values . in insomnia patients , higher fss values we introduced the dichotomous variable mfis higher than mfis mean ( 34.2 ) ( yes or no ) and the dichotomous variable fss higher than fss mean ( 4.0 ) the univariate logistic regression showed no significant relationship with spo2% min , sleep efficacy , sleep latency , or deep sleep . a significant relationship could be found with age , sex , and number of awakenings for mfis as well as fss ( table 4 ) . furthermore , there was a significant relationship with arousal index and ahi only for fss . the multiple logistic regression ( table 4 ) investigating the fss , including age , sex , arousal index , number of awakenings , and ahi , demonstrated a significant relationship with age , sex , and arousal index , whereas ahi and number of awakenings were not significant . younger age , female sex , and high arousal index were highly predictive of increased fss scores . in regards to mfis , the multiple logistic regression ( table 4 ) investigating the mfis , including age , sex , and number of awakenings , confirmed the significant relationship with age , sex , and number of awakenings . younger age , female sex , and high numbers of awakenings predicted high mfis values . this is the first study , to our knowledge , to investigate the relationship between sleep disorders and mfis values , and we observed a number of important findings . firstly , this study clearly shows that sleep disorders can lead to high mfis values in sleep - disordered patients who are not suffering from ms . secondly , our results show both high mfis and fss values in sleep - disordered patients without ms and , above all , a strong correlation between mfis and fss for all patients and all subgroups except for plmd / rls patients . ( the fact that this correlation could not be demonstrated in plmd / rls patients could be due to small sample size ) . thirdly , the correlation between psqi and mfis values in all patients ( except for plmd / rls and insomnia patients ) and between psqi and fss in srbd patients , suggests causation of fatigue by sleep disorders ( the lack of correlation in some subgroups may be caused by small sample size ) . this could be confirmed by multiple logistic regressions , showing that a high number of awakenings were in line with a higher risk of developing fatigue measured with the mfis and that a high arousal index increased the risk of developing fatigue measured with the fss . this possible causative role of awakenings and arousals in the pathophysiology of fatigue is of particular interest , and further studies should be performed to elucidate its etiology . guilleminault et al used cyclic alternating pattern ( cap ) to investigate the relationship between sleep instability and fatigue in patients with upper airway resistance syndrome ( uars ) compared with healthy controls.32 they found higher arousal indexes and sleep disturbances measured with cap analysis in patients with uars that correlated with sleepiness and fatigue . feige et al summarized the hyperarousal concept in their review about sleep microstructural differences in chronic insomnia.33 they recommended spectral analysis of the sleep eeg , microarousal , and cap analysis as a research instrument of sleep in insomnia . it would be promising to investigate fatigue as well with these methods in patients without known sleep disorders , in order to find out what drives fatigue . on the other hand , little is known about the impact of arousal on fatigue in sleep disorders . yue et al34 found a significant relationship between emotional fatigue measured with the multidimensional fatigue symptom inventory short form35 and spontaneous movement arousals in untreated osas patients . it is worth noting that previous studies have not shown a significant association between the severity of osas ( ahi , spo2% min ) and fatigue scores.36,37 in our study , there was also no significant relationship between fatigue and ahi or spo2% min after multiple logistic regressions . previous polysomnographic investigations of consecutive ms patients showed a significant association of sleep disorders with fatigue ( fss / mfis),22 but not with sleepiness ( ess ) . supporting this finding , the works by hossain et al19 and aguillard et al21 showed that , in sleep - disordered patients without ms , fatigue and sleepiness can occur independently as well as together . in the present study , there was not a significant relationship between mfis values and ess values ( only untreated srbd patients showed a low correlation between ess values and mfis ) . the fact that we found a strong correlation between ess values and fss values in patients without sleep disorders remains without explanation . we have to note that these patients were not healthy controls , but rather fatigued and sleepy patients without pathological findings either in the primary health care setting or in the sleep laboratory . overall , as we performed multiple logistic regressions only for mfis and fss values , we can not interpret the correlations between ess and psqi values and age or sex or polysomnographic parameters . our study was focused on the relationship between sleep disorders and fss and mfis . in our study , women had a higher risk of suffering from fatigue than men . our results are in line with a previous study,17 suggesting that , in srbd patients , fatigue may be particularly correlated with female sex : in our study , female sex was predictive of high fss and mfis values . currently , we have no explanation for this relationship and further studies are needed to investigate the pathophysiological mechanisms . similarly , we have no explanation for the fact that younger age is predictive of fatigue and further studies are needed to understand this relationship as well . the present study has some methodological limitations : due to the retrospective design and owing to the lack of exclusion criteria , we can not exclude that some patients have other reasons for fatigue as a possible confounder . on the other hand , this study reflects the daily clinical praxis with consecutive patients over 1 year . younger age , female sex , and high number of awakenings and arousals are predictive of fatigue in sleep - disordered patients . this study proves that high mfis and fss values can occur in sleep - disordered patients .

background : the factors affecting the correlation between parkinson disease ( pd ) and depression have remained unclear.objectives:we assessed the prevalence of depression among patients with pd and the association between pd - related variables and depression severity.patients and methods : this is a cross - sectional study performed in kermanshah province of iran . sampling was based on recruitment of subjects according to inclusion and exclusion criteria . patients with confirmed parkinson disease who were referred to clinics of kermanshah university of medical sciences participated in this study . depression was evaluated with beck depression inventory - ii ( bdi - ii ) . clinical characteristics of pd , including tremor , rigidity , impaired posture , loss of autonomic movement , changes in speech and handwriting , masked face , and hyposmia were indexed . anhedonia was assessed with farsi version of snaith - hamilton pleasure scale . data were collected between april 2010 and march 2014.results:a total of 350 patients ( 52.9% men and 47.1% women ) participated in this investigation . female gender ( 36.5% in women vs. 13.0% in men , p < 0.0001 ) , impaired posture ( 27.2% in affected individuals vs. 18.8% , p = 0.002 ) , masked face ( 39.0% vs. 5.2% , p < 0.0001 ) , and hyposmia ( 48.7% vs. 21.0% , p = 0.001 ) were associated with higher susceptibility to profound depression . lower scores of all domains of farsi version of snaith - hamilton pleasure scale ( including interest / pastimes , social interaction , sensory experience , and food / drink ) were related to more severe depression ( p < 0.0001 for all subscales ) . severe and profound depression was found in 44% of the participants.conclusions:this study estimated that the prevalence of major depression among iranian individuals with pd living in kermanshah as 44% . major determinants of depression were female gender , rigidity , impaired posture , masked face , hyposmia , and anhedonia . backgroundthe fatigue severity scale ( fss ) is widely used to assess fatigue , not only in the context of multiple sclerosis - related fatigue , but also in many other medical conditions . some polysomnographic studies have shown high fss values in sleep - disordered patients without multiple sclerosis . the modified fatigue impact scale ( mfis ) has increasingly been used in order to assess fatigue , but polysomnographic data investigating sleep - disordered patients are thus far unavailable . moreover , the pathophysiological link between sleep architecture and fatigue measured with the mfis and the fss has not been previously investigated.methodsthis was a retrospective observational study ( n = 410 ) with subgroups classified according to sleep diagnosis . the statistical analysis included nonparametric correlation between questionnaire results and polysomnographic data , age and sex , and univariate and multiple logistic regression.resultsthe multiple logistic regression showed a significant relationship between fss / mfis values and younger age and female sex . moreover , there was a significant relationship between fss values and number of arousals and between mfis values and number of awakenings.conclusionyounger age , female sex , and high number of awakenings and arousals are predictive of fatigue in sleep - disordered patients . further investigations are needed to find the pathophysiological explanation for these relationships .

currently , chronic kidney disease ( ckd ) is one of the most serious public health problems . some recent epidemiological studies have suggested that approximately one million people with end stage renal disease ( esrd ) undergo replacement therapy worldwide . the increased prevalence of ckd in the developed and developing countries threatens to become a global epidemic , as exemplified by the increased number of cases of diabetes mellitus and the increased life expectancy of the population1 . ckd severely affects patients health , lifestyle and wellbeing , compromising their quality of life2 . according ponngeon et al . , ckd patients had sedentary and inactive levels of physical activity on dialysis and non - dialysis days , respectively3 . silva and marinho observed that the level of physical activity of most patients esrd undergoing hemodialysis is poor and that most do not perform exercise regularly4 . the most common comorbidities observed in patients with ckd are diabetes mellitus type 2 , dyslipidemia , coronary heart disease , heart failure5 , 6 , hypertension7 , 8 , respiratory disorders8 , 9 , stress10 , depression10,11,12 , anxiety13 , and obstructive sleep apnea ( osa)14 . osa is a major medical problem , estimated to affect up to 1530% of male adults and up to 515% of female adults15 , 16 . it is characterized by repetitive obstructions of the upper airway during sleep , frequently causing oxygen desaturation . this induces frequent awakenings ( arousals ) , resulting in fragmented sleep and excessive daytime sleepiness . in brazil , according to a recent epidemiological study conducted in so paulo , an osa prevalence of 24.8% in men and 9.6% in women was observed , based on an apnea - hypopnea index ( ahi)15 events / h ; for an ahi5 events / h , this prevalence increased to 40% in men and 26% in women17 . in a recent study involving 2,121 individuals conducted in the city of lausanne in switzerland , a prevalence of 50% in men and 23% in women was observed , based on an ahi15 events / h18 . in another study conducted by oliveira et al . , patients with neuromuscular disorders such as myasthenia gravis were observed to have a poor quality of sleep , excessive daytime sleepiness , presence of restless syndrome , and a high incidence of sleep - disordered breathing ( sdb)19 . the prevalence of sleep disorders among patients with ckd is 4080% , which is higher than that among the general population . among these sleep disorders , the presence of periodic leg movements during sleep and osa has been highlighted20 . the high prevalence ( 50% ) of sdb in patients undergoing hemodialysis ( hd ) can be attributed to compromised upper airway stability ( extracellular fluid volume overload)21,22,23 , ventilatory control instability ( altered central and peripheral chemosensitivity ) , and reduced upper airway muscle tone ( uremia)24,25,26,27,28 . risk factors of sdb in the general population , such as older age , male gender , obesity , smoking , increased neck circumference , and diabetes , are also prevalent in the ckd population29 . sdb has been associated with increased cardiovascular risk , and may contribute to the morbidity and mortality of patients with advanced ( stages 4 to 5 ) ckd or those undergoing hd30 . although numerous studies have shown that a substantial proportion of patients undergoing hd have sleep apnea , these studies were limited by the following factors : very small sample sizes27 , 31,32,33,34 ; the use of partial channel polysomnography ( psg)34,35,36 ; the study of populations with limited generalizability to patients undergoing hd37 ; a selected subpopulation of patients undergoing dialysis , without sleep symptoms38 ; or study samples that were largely composed of symptomatic patients26 , 31 , 39 . hence , a better understanding of the prevalence and risk factors of sdb in ckd patients undergoing hd is critical . therefore , a systematic review that can demonstrate the status of the knowledge is justified . this knowledge may enable health professionals , especially physiotherapists , to devise new strategies to reduce morbidity and mortality and improve the quality of life of ckd patients . the present study aimed to provide a thorough overview of the literature regarding the occurrence of sleep disorders in patients with esrd undergoing hd . two independent reviewers performed a computer - assisted search of the medline , lilacs , and scielo virtual health library medical databases from their inception to november 2015 . a combination of the following medical subject headings ( mesh ) were used in the search ( sleep or sleep disorders or obstructive sleep apnea ) and ( kidney dialysis or hemodialysis or dialysis ) . the first stage of the search identified articles according to the title , abstract , and key words . in the second stage , articles that involved patients > 18 years old , having esrd and undergoing hd , and that used overnight standard psg for the diagnosis of sleep disorders , were included in the final analysis . one thousand one hundred twenty - six articles were identified that met the inclusion criteria . a total of 796 articles were excluded because they were not in english , the patients did not undergo hd , or the studies were not cross - sectional or clinical trials . after an evaluation of the full text , 312 studies were excluded , either because psg was not performed or they were duplicated . the remaining 18 studies , comprising 8 clinical trials and 10 cross - sectional studies , were included for further examination ( fig . 1fig . the results and outcomes of all included studies are summarized in tables 1table 1.cross-sectional studies that used overnight standard polysomnography for the diagnosis of sleep disorders in chronic kidney diseaseauthor . 200136improvement of sleep apnea in patients with chronic renal failure who undergo nocturnal hemodialysispatients who underwent both conventional hd and nocturnal hd ( n=14)ahi decreased during the nocturnal hd phase.nocturnal hd corrects sa associated with chronic renal failure.sanner et al . 200230sleep - related breathing disorders impair quality of life in hemodialysis patientsesrd patients who underwent hd ( n=46)twenty - one patients had clinically significant sleep - related breathing disorders with a median ahi of 13.3 events / h.twenty - seven ( 22.7% ) of the 119 patients had sa with subjective symptoms such as daytime somnolence and snoring.parker et al . 200337daytime sleepiness in stable hemodialysis patientsesrd patients who underwent hd ( n=46)higher indices of sa and brief arousals correlated significantly with increased physiological , but not subjective , sleepiness.daytime sleepiness is common in hd patients , and may be severe despite the absence of obvious clinical risk factors.parker et al . 200333nocturnal sleep , daytime sleepiness , and quality of life in stable patients on hemodialysisesrd patients who underwent hd ( n=46)better sleep quality and less daytime sleepiness were associated with improved qol in stable hd subjects.poor nocturnal sleep and increased daytime sleepiness are associated with decreased qol in hd patients.jung et al . 200532sleep apnea , coronary artery disease , and antioxidant status in hemodialysis patientsesrd patients who underwent hd ( n=26)coronary artery calcification is associated with the severity of sa.oxygen desaturation triggered by sa is associated with severe coronary artery disease.miskowiec et al . 200638prevalence of sleep apnea syndrome in hemodialyzed patients with end stage renal diseaseesrd patients , assessed using psg , on the night before hd ( n=17 ) and on the night after hd ( n=11)seven patients presented with sa on the 2 nights during the psg.hd does not change the prevalence of osa in ckd.tada et al . 200739the predictors of central and obstructive sleep apnoea in haemodialysis patientsesrd patients who underwent hd ( n=30)forty - one patients presented with sa ; 27 had sa with symptoms such as daytime somnolence and snoring.there is a high prevalence of sa in hd patients . 201016nocturnal hypoxemia and periodic limb movement predict mortality in patients on maintenance hemodialysisesrd patients who underwent hd ( n=30)the median ahi and the plm index were 22 and 36.9 events / h , respectively.nocturnal hypoxemia and periodic limb movement during sleep were associated with an increased risk of death in patients with esrd.elias et al . 201240rostral overnight fluid shift in esrd renal disease : relationship with obstructive sleep apneaesrd patients who underwent hd ( n=26)the change in the leg fluid volume was correlated with the apnea hypopnea time and neck circumference.nocturnal rostral fluid shift is associated with the severity of osa in esrd.elias et al . 201341relationship of pharyngeal water content and jugular volume with severity of obstructive sleep apnea in renal failureesrd patients who underwent hd ( n=20)a positive correlation was found among the ahi , the internal jugular vein volume , and the upper airway mucosal water content.fluid overload increases the internal jugular vein volume and the upper airway mucosal water content , contributing to the pathogenesis of osa in esrd patients.ahi : apnea - hypopnea index ; hd : hemodialysis ; esrd : end stage renal disease ; sa : sleep apnea ; qol : quality of life ; osa : obstructive sleep apnea ; ckd : chronic kidney disease ; plm : periodic leg movementand 2table 2.clinical trials that used overnight standard polysomnography for the diagnosis of sleep disorders in chronic kidney diseaseauthor . 200542polysomnographic measures of nocturnal sleep in patients on chronic , intermittent daytime hemodialysis vs those with chronic kidney diseaseesrd patients who underwent hd ( n=16 ) and the ckd patients ( n=8)both groups had reduced total sleep time and sleep efficiency.sleep disorders of ckd patients and those receiving chronic , intermittent daytime hd may have different etiologies.lee et al . 200643a secondary analysis of racial differences in periodic leg movements in sleep and ferritin in hemodialysis patientsesrd african american patients who underwent hd ( n=36 ) ; esrd caucasian patients who underwent hd ( n=10)african american hd patients had higher ferritin and lower plm than caucasians.plm was less common in the african american population , suggesting a differential genetic vulnerability.unruh et al . 200644sleep apnea in patients on conventional thrice - weekly hemodialysis : comparison with matched controls from the sleep heart health studyesrd patients who underwent hd ( n=46 ) and the normal renal function group ( n=137)esrd patients had a higher frequency of arousals per hour and ahi , and greater percentage of the tst below an oxygen saturation of 90%.there was a strong association of hd with severe sdb and nocturnal hypoxemia independent of age , bmi , and a higher prevalence of chronic disease.beecroft et al . 200711pharyngeal narrowing in end - stage renal disease : implications for obstructive sleep apnoeaesrd patients who underwent hd ( n=44 ) and the normal renal function group ( n=41)the pharynx was narrower in patients who underwent hd than in subjects with normal renal function.a narrower upper airway can contribute to the pathogenesis of sleep apnea in dialysis - dependent patients.unruh et al . 200845subjective and objective sleep quality in patients on conventional thrice - weekly hemodialysis : comparison with matched controls from the sleep heart health studyesrd patients who underwent hd ( n=46 ) and the normal renal function group ( n=137)there was no association between psg sleep time and self - reported sleep time or between the ess and the severity of sa in the hd population.kidney failure treated with thrice - weekly hd is significantly associated with poor subjective and objective sleep quality.enomoto et al . 200846clinical characteristics of restless legs syndrome in end - stage renal failure and idiopathic rls patientsuremic rls patients ( n=15 ) and idiopathic rls patients ( n=20)the plm index was significantly higher in the uremic rls group.uremic rls appears to deteriorate faster and to become more severe than idiopathic rls.loewen et al . 200947sleep disruption in patients with sleep apnea and end - stage renal diseaseesrd patients who underwent hd ( n=12 ) and a normal renal function group ( n=18)the prevalence of rls lm - related arousals was higher in esrd patients.the co - existence of plm is an additional source of sleep disruption in patients with esrd and sa.roumelioti et al . 201148sleep - disordered breathing and excessive daytime sleepiness in chronic kidney disease and hemodialysisesrd patients who underwent hd ( n=75 ) , the ckd group ( n=89 ) , and the normal renal function group ( n=224)nocturnal hypoxemia was significantly elevated in the hd group compared with the ckd group.severe sdb and excessive daytime sleepiness are common among advanced ckd and hd patients.esrd : end - stage renal disease ; hd : hemodialysis ; ckd : chronic kidney disease ; plm : periodic leg movement ; sdb : sleep disordered breathing ; psg : overnight standard polysomnography ; ess : epworth sleepiness scale ; sa : sleep apnea ; rls : restless legs syndrome ; tst : total sleep time and are discussed below . thus , a detailed analysis of 18 articles that used overnight psg , which is considered the gold standard method for the diagnosis of sleep disorders , was conducted . flowchart of the study ahi : apnea - hypopnea index ; hd : hemodialysis ; esrd : end stage renal disease ; sa : sleep apnea ; qol : quality of life ; osa : obstructive sleep apnea ; ckd : chronic kidney disease ; plm : periodic leg movement esrd : end - stage renal disease ; hd : hemodialysis ; ckd : chronic kidney disease ; plm : periodic leg movement ; sdb : sleep disordered breathing ; psg : overnight standard polysomnography ; ess : epworth sleepiness scale ; sa : sleep apnea ; rls : restless legs syndrome ; tst : total sleep time the aim of this study was to conduct a systematic review of studies showing the presence of sleep disorders in patients with esrd undergoing hd . the presence of sleep disorders , including sdb , periodic limb movement disorder ( plmd ) and/or restless legs syndrome ( rls ) , which compromise the quality of life and increase the morbidity and mortality rates of patients with ckd , has been documented in the literature50,51,52,53 . seventy percent of esrd patients are believed to have some form of sdb26 , 39 . rls is a common and extremely distressing problem experienced by many patients undergoing hd . it is a neurological condition characterized by an irresistible urge to move the legs and occurs more frequently during inactivity and at night54 , 55 . the prevalence of rls in patients who are under treatment with hd varies across studies performed in broad geographical regions ( 6.668% ) , and recent studies using the international rls study group criteria have suggested a prevalence rate of 33% in esrd patients , which is greater than that in the general population56 . sleep fragmentation and sleep deprivation caused by rls may contribute to cardiovascular complications and infections , often with poor prognosis in patients undergoing hd . according to the literature , patients undergoing hd have a poorer quality of sleep compared to the normal population . the presence of rls and associated sleep disorders , such as daytime sleepiness , insomnia , and poor sleep quality , should be considered a problem that further impairs the quality of life of patients . studies have demonstrated that 4080% of esrd patients undergoing hd suffer from poor sleep quality associated with a poor quality of life57 , 58 . sleep disorders are usually associated with physical , behavioral , and psychological disorders that predispose to cognitive impairment , decline in social performance and interpersonal interactions , and depression59 . sdb is very common among patients with ckd , especially those undergoing hd36 , 60 . some studies have suggested osa has a prevalence of 5060% in esrd patients12 , 61 . depending on the methodology applied , approximately 5080% of ckd patients have some form of sdb26 , 39 . osa is 4 to 10 times more prevalent among ckd patients than in the general population22 , 49 . a more recent study conducted by nicholls et al . involving 254 ckd patients showed a significant increase in the occurrence of sdb ( predominantly osa ) which was related to impairment of renal function , with a prevalence of 57%62 . this study utilized only studies performed using psg , considered the gold standard for the diagnosis of sleep disorders . despite the high prevalence of sleep disorders in the ckd population , there is limited scientific research on the topic , as evidenced by only as shown by 18 studies ( 10 cross - sectional studies and 8 controlled clinical trials ) having been retrieved in this review . this is perhaps explained by the high cost of psg examination , or by a lack of knowledge about the presence of sleep disorders in ckd population . other more viable diagnostic methods , such as pulse oximetry and specific validated sleep questionnaires , could be used as screening tools for the presence of sdb , aiding in the early diagnosis and choice of appropriate treatment with non - invasive ventilatory support . esrd contributes to the emergence and/or development of osa due to factors such as metabolic acidosis , compromised sensitivity of chemoreceptors , uremic toxins , generalized muscle weakness , particularly of the dilator muscles of the pharynx , and narrowing of the upper airway due to fluid accumulation in the interdialytic period . it is known that osa induces ckd due to increased blood pressure , oxidative stress , and repetitive hypoxia . this systematic review verified the status of knowledge regarding the prevalence of sleep disorders in esrd patients undergoing hd . according to the studies analyzed , a high prevalence of plmd , rls , and osa affecting the quality of sleep was observed , which in turn undermined the quality of life of these patients , leading to increased morbidity and mortality . thus , it is very important for healthcare professionals to recognize the signs and symptoms of sleep disorders in ckd patient population , both for accurate diagnosis and an appropriate therapeutic approach . for physical therapists , it is extremely important to identify sdb , particularly osa , so they can institute treatment with continuous positive airway pressure . autosomal dominant polycystic kidney disease ( adpkd ) is an inherited disorder characterized by the development and growth of cysts in the kidneys and other organs . in adpkd patients , nephrotic range proteinuria is unusual and needs to be investigated further to exclude coexisting glomerular disease . among the anecdotal case reports of adpkd associated with nephrotic syndrome , we report the case of a 26-year - old male with adpkd and concomitant nephrotic syndrome , in which an ultrasound ( us)-guided renal biopsy showed a mesangioproliferative glomerulonephritis . we treated the patient with prednisone 1 mg / kg / day , because of the failure of treatment with angiotensin - converting enzyme inhibitor / angiotensin receptor blocker association . after 6 months of steroid treatment , we observed a stability of his gfr and a reduction of proteinuria . this case report and other cases of the literature underline the importance of a renal biopsy in patients with adpkd and nephrotic syndrome in order to make an accurate diagnosis and an appropriate treatment / prevention of renal function deterioration . autosomal dominant polycystic kidney disease ( adpkd ) , the most common heritable renal disease , with an estimated incidence of 1:800 live births , is a disorder characterized by the development and growth of cysts in the kidneys and other organs . this disease is genetically heterogeneous ; in 85% of the cases , the disease is caused by a mutation localized on chromosome 16 ( pkd1 ) and in 15% by a mutation localized on chromosome 4 ( pkd2 ) , while a few families have been identified in which the disease is caused by a mutation in an unmapped locus . however , within the two identified forms of the disease , there is a remarkable variability in clinical features . proteinuria and microalbuminuria ( ma ) also occur with a highly variable severity and are associated with a more progressive course of the disease [ 3 , 4 ] . mild proteinuria , usually < 2 g/24 h , is a common finding on routine examination in adpkd patients ; however , the association of nephrotic syndrome with adpkd is considered rare [ 4 , 5 ] and needs to be investigated further to exclude coexisting glomerular disease . among the anecdotal case reports of adpkd associated with nephrotic syndrome , we report the case of a 26-year - old male with adpkd and concomitant nephrotic syndrome , in which the renal biopsy showed a mesangioproliferative glomerulonephritis in april 2009 , a 24-year - old man was referred to our hospital with a history of adpkd . the patient 's father had a diagnosis of adpkd and the mother was affected by a membranous nephropathy . at first observation , laboratory studies showed a daily urinary protein excretion of 3.19 g , serum creatinine 106.08 mol / l ( 1.2 mg / dl ) , and egfr ( estimated glomerular filtration rate ) 84.7 ml / min/1.73 m. we thus started therapy with an angiotensin - converting enzyme inhibitor ( acei ) , ramipril 5 mg / day . after 6 months , his proteinuria decreased to 1.13 g / day , so we added an angiotensin receptor blocker ( arb ) , losartan potassium 50 mg / day . his proteinuria remained about 1.82 g / day until the end of 2010 . in july 2011 , urine analysis showed a daily protein excretion of 7.4 g and 15 red blood cells per high power field ; the patient had neither peripheral leg oedema nor other symptoms ; urine culture was sterile ; tests for hbsag and hcvab and anti - nuclear antibodies were negative ; igg , iga , igm , c3 , c4 were normal ; there were no monoclonal bands on immunoelectrophoresis of the serum and no monoclonal light chains were detected in the urine . an abdomen ultrasound ( us ) analysis showed the right kidney measuring 11.4 cm in length with multiple cysts ranging in diameter from 1.6 to 3.2 cm , and the left kidney measuring 13.4 cm in length with multiple cysts . due to the persistent presence of nephrotic - range proteinuria , a us - guided biopsy was performed , the diagnosis of which was mesangioproliferative glomerulonephritis . because of the failure of treatment with acei / arb association , we added prednisone to the antiproteinuric agents at the initial dose of 1 mg / kg / day . after 6 months of steroid treatment , we observed an egfr stability , a gradual reduction of proteinuria until 2 g/24 h and an increase of the serum albumin . proteinuria and ma occur with a highly variable severity in adpkd patients and proteinuria is usually < 2 g/24 h. the frequency of occurrence of proteinuria in adpkd ranges from 14 to 34% in non - uraemic adults to 80% in adults with advanced renal failure , even if a high prevalence of ma was found in normotensive adults and children with adpkd [ 7 , 8 ] . chapman et al . have demonstrated that both overt proteinuria and ma in adpkd patients were associated with a higher mean arterial pressure , lower gfr , larger renal volume , worse renal prognosis and with an increased cardiovascular morbidity . the association of nephrotic syndrome with adpkd is considered rare [ 4 , 5 ] and , when possible , should be investigated by histological studies to exclude the possibility of a superimposed glomerular disease . in 1957 , dalgaard described three instances of nephrotic range proteinuria ( > 5 g / day ) in a report of 122 cases with adpkd ; but renal biopsy data are not available in these series . subsequently , four other adpkd patients with nephrotic syndrome were described without histopatologic diagnosis [ 1113 ] . in 1972 , kida et al . reported the first case of adpkd with nephrotic syndrome due to biopsy - proven minimal change nephrotic syndrome . in 1995 , contreras et al . reviewed 14 cases of adpkd in which the renal lesions had been evaluated by histopathological studies . in 2006 , hiura et al . expanded contreras review adding seven more japanese cases our review of the literature reveals that since 1972 to the present there have been only 29 cases ( including this report ) of adpkd , associated with nephrotic syndrome , in which the renal lesions were evaluated by histopathological studies ( table 1 ) ; but it is reasonable to assume that many other similar cases were not investigated or published . this may be due in part to the reluctance of nephrologists to perform an open renal biopsy in adpkd patients and in part to the real risk and complexity of percutaneous renal biopsy in these patients . since the presence of multiple bilateral cysts has been listed as relative contraindication to percutaneous renal biopsy , due to the presumed risk of complications and difficulties in obtaining suitable tissue for diagnosis , a majority of adpkd patients received an open surgical biopsy . in fact , of the 26 patients whose methodological data were available , only 5 ( including our patient ) received a percutaneous renal biopsy , 1 patient received a computerized tomography ( ct)-guided renal biopsy , in 1 patient the diagnosis of amyloidosis was supposed on the basis of a gingival and intestinal biopsy . table 1.renal histology in patients with adpkd and nephrotic range proteinuriafirst authoragesexrenal biopsyrenal histopathologycontreras et al . 51momes - pgnvisciano ( this report)26mpmes - pgnp , percutaneus biopsy ; o , open surgical biopsy ; ct , computed tomography - guided biopsy ; gr , gingival rectum biopsy ; fsgs , focal segmental glomerulosclerosis ; mcd , minimal change disease ; mn , membranous nephropathy ; igan , iga nephropathy ; cregn , crescentic glomerulonephritis ; idgs , intercapillary diabetic glomerulosclerosis ; mpgn i , membranoproliferative glomerulonephritis type i ; post - inf gn , post infectious mesangial proliferative glomerulonephritis ; mln , membrabous lupus glomerulonephritis ; d - pgn , diffuse proliferative glomerulonephritis ; mespgn , mesangial proliferative glomerulonephritis . renal histology in patients with adpkd and nephrotic range proteinuria p , percutaneus biopsy ; o , open surgical biopsy ; ct , computed tomography - guided biopsy ; gr , gingival rectum biopsy ; fsgs , focal segmental glomerulosclerosis ; mcd , minimal change disease ; mn , membranous nephropathy ; igan , iga nephropathy ; cregn , crescentic glomerulonephritis ; idgs , intercapillary diabetic glomerulosclerosis ; mpgn i , membranoproliferative glomerulonephritis type i ; post - inf gn , post infectious mesangial proliferative glomerulonephritis ; mln , membrabous lupus glomerulonephritis ; d - pgn , diffuse proliferative glomerulonephritis ; mespgn , mesangial proliferative glomerulonephritis . of the 29 cases evaluated by biopsy procedures , focal segmental glomerulosclerosis ( fsgs ) [ 6 , 1720 ] ( 6 patients ) , minimal change nephropathy [ 14 , 2124 ] ( 5 patients ) and membranous nephropathy [ 2529 ] ( 5 patients ) were the dominant diagnoses . next were non - iga mesangial proliferative glomerulonephritis [ 30 , 31 ] , ( with three patients including ours ) , iga nephropathy [ 15 , 32 ] ( with two patients ) and amyloidosis [ 16 , 33 ] ( with two patients ) . other types of glomerulonephritis diagnosed were crescentic glomerulonephritis , diabetic nephropathy , membranoproliferative glomerulonephritis , membranous lupus glomerulonephritis , diffuse proliferative glomerulonephritis and postinfectious mesangial proliferative glomerulonephritis ( each with one patient ) . of the 27 patients whose data were available , 18 were male and 9 were female and only 1 female had a diagnosis of focal segmental glomerulosclerosis ; the mean age was 44 16 years ( 40 15 years for male and 53 15 years for female ) . it is difficult to be certain whether these associations are coincidental or whether they demonstrate a specific pathogenetic relationship with adpkd . the frequency of focal segmental glomerulosclerosis ( 6/29 , 20% ) is higher than the 15% frequency of focal segmental glomerulosclerosis found in the general adult population . in contrast , membranous nephropathy , the most common cause of idiopathic nephrotic syndrome in adults , with a frequency of 25% , was found in 17% ( 5/29 ) of the adpkd patients with nephrotic syndrome , which suggests that fsgs may be more than a coincidental finding and glomerular hyperfiltration could play an important role in the development of fsgs and heavy proteinuria in patients with adpkd . moreover , the coincidence of adpkd and fsgs can be caused by two independent concurrent genetic mutations which are not necessarily related or one single mutation , which is unknown yet . it is possible that different mutations in these patients could clarify the nature of this coincidence . however , in a histological study of kidneys of 12 adpkd patients without nephrotic syndrome , montoyo et al . reported that interstitial fibrosis and tubular atrophy were found to be the main determinants of the development of chronic renal failure in adpkd . in a study of 18 cases , zeir et al . reported interstitial fibrosis and arteriolar sclerosis to be the most important lesions in the kidney of adpkd patients , whereas fsgs was observed in < 5% of the glomeruli . interestingly , in our patient , the severe increase of proteinuria after about 2 years of acei / arb association and the peculiar family history ( father with a diagnosis of adpkd and mother affected by membranous nephropathy ) induced us to perform a us - guided biopsy . we chose the percutaneous biopsy because the lower pole of the left kidney of our patient still had a good representation of the renal parenchyma . although the treatment of mesangioproliferative glomerulonephritis is not well defined , we decided to use corticosteroids for our patient with a good clinical response . in addition to our report , there are two other cases of mesangioproliferative glomerulonephritis in adpkd patients in the literature . in the first one , the data are not available , and in the second one , seyrek et al . described a case of a 56-year - old woman with flank pain , haematuria , proteinuria ( 0.5 g / day ) and normal renal function , in which a kidney biopsy revealed the presence of mesangioproliferative glomerulonephritis . the patient received prednisone 0.5 mg / kg / day and her haematuria and proteinuria improved and during the following years , urinary sediments occasionally showed microscopic haematuria . at the time of kidney biopsy , five years later , a ct diagnosed adpkd , showing two kidneys and the pancreas occupied by numerous cysts ; moreover , the family history revealed that her sister was a haemodialysis patient . of the patients with follow - up information , six were on dialysis therapy from 3 months to 6 years after the nephrotic syndrome was discovered ; six patients presented a reduction of the proteinuria and a stability or an improvement of the renal function after steroid and/or cytotoxic therapy in a follow - up from 2 months to 5 years ; one patient had reduced proteinuria and normalized renal function after salt restriction and antihypertensive medications ( losartan potassium and amlopdipine ) ; two patients showed worsened renal function . this evidence supports the need for performing a renal biopsy in patients with adpkd and nephrotic syndrome . a kidney biopsy is an invasive examination procedure , and should only be indicated on an individual basis , depending on the patient 's characteristics and after careful consideration of the risks and benefits for each particular case , such as the case of an adpkd patient with a nephrotic syndrome . with the use of real - time ultrasonography for guiding the biopsy procedure and the use of automatic biopsy needles , the success rate has improved in 95% of cases . a ct - guided percutaneous renal biopsy is an alternative when the kidneys can not be properly visualized . alternative methods have been attempted for obtaining samples of kidney tissue in patients with contraindications for the percutaneous approach . although open or surgical renal biopsies have been performed for over 40 years as a standard procedure in patients with contraindications for the percutaneous approach , there are other less invasive alternatives . recently , a technique has been proposed which combines the laparoscopic approach with a percutaneous needle biopsy . however , a kidney biopsy allows an accurate diagnosis and an appropriate treatment / prevention of renal function deterioration . the treatment for various histopathological subtypes leading to nephrotic syndrome is different , with corticosteroids beneficial in some conditions and useless in other cases . reaching a firm diagnosis based on histopathology and immunofluorescence studies will help the physician to give an appropriate treatment and to avoid empirical therapy with potentially toxic agents . the enlargement of cysts by compressing normal parenchyma is a central factor in the pathogenesis of chronic renal failure in this disorder . at present , it is impossible to assess the potential benefit of measures leading to a decrease in proteinuria in the progression of adpkd toward end - stage renal disease . the data related to follow - up of the adpkd patients treated with immunosuppressive therapy are too few to evaluate if steroid or other cytotoxic agents may influence the course of the adpkd and if the progression of this disease may depend on the histological subtype of associated glomerulonephritis .

the purpose of this study was to conduct a systematic review of the available evidence on sleep disorders in patients with end stage renal disease ( esrd ) undergoing hemodialysis ( hd ) . [ subjects and methods ] two independent reviewers performed a computer - assisted search of the medline , scielo , lilacs , and bireme virtual health library medical databases from their inception to november 2015 . [ results ] one thousand one hundred twenty - six articles were found that met the inclusion criteria . articles were excluded if they were not in english , the patients did not undergo hd , or the studies were not cross - sectional or clinical trials . after reading the full text , a further 300 studies were excluded because they did not use polysomnography . the remaining 18 studies with esrd patients undergoing hd comprised 8 clinical trials and 10 cross - sectional studies . this systematic review followed the criteria outlined by the prisma declaration . [ conclusion ] in this systematic review , a high prevalence of sleep disorders was observed in esrd , including sleep - disordered breathing . this knowledge may enable health professionals to devise new strategies for the diagnosis and treatment of these patients , in order to reduce morbidity and mortality and improve their quality of life . backgroundautosomal dominant polycystic kidney disease ( adpkd ) is an inherited disorder characterized by the development and growth of cysts in the kidneys and other organs . in adpkd patients , nephrotic range proteinuria is unusual and needs to be investigated further to exclude coexisting glomerular disease . among the anecdotal case reports of adpkd associated with nephrotic syndrome , focal segmental glomerulosclerosis occurs most frequently.methodswe report the case of a 26-year - old male with adpkd and concomitant nephrotic syndrome , in which an ultrasound ( us)-guided renal biopsy showed a mesangioproliferative glomerulonephritis . we treated the patient with prednisone 1 mg / kg / day , because of the failure of treatment with angiotensin - converting enzyme inhibitor / angiotensin receptor blocker association.resultsafter 6 months of steroid treatment , we observed a stability of his gfr and a reduction of proteinuria.conclusionthis case report and other cases of the literature underline the importance of a renal biopsy in patients with adpkd and nephrotic syndrome in order to make an accurate diagnosis and an appropriate treatment / prevention of renal function deterioration .

it consists of a pair of cylindrically shaped centrioles surrounded by fibrous pericentriolar material . before or during dna replication in s phase , the centrioles split , and each cylinder serves as a template for the assembly of a new daughter centriole . before mitosis , when cells contain two pairs of centrioles , each pair serves as a nucleation center for microtubules of the spindle apparatus . defects in centrosome assembly or in centrosome separation can result in defective nucleation of spindle microtubules , and in several cases , in the formation of monopolar spindles and mitotic arrest ( sunkel et al . several years ago , evidence was published that defective centrosome assembly can prevent cells from entering s phase . in particular , removal of the centrosome by microsurgery or by laser ablation resulted in a cell cycle arrest , as did inhibition or silencing of several centrosome - associated proteins , such as dynactin , parp-3 , centriolin , or akap450 ( hinchcliffe et al . , 2001 ; khodjakov and rieder , 2001 ; quintyne and schroer , 2002 ; augustin et al . , 2003 ; gromley et al . , 2003 ; keryer et al the mechanism leading to this centrosome - dependent cell cycle arrest in g1 phase has been unclear ; it was proposed that a checkpoint control would prevent those cells with imperfect centrosomes from continuing the cell cycle , to prevent the assembly of defective spindles later in mitosis ( murray , 2001 ) . in this study , we followed cell cycle progress after inhibition of centrosome assembly by depleting the pericentriolar proteins pericentriolar material 1 ( pcm-1 ) and pericentrin . these proteins have been shown to be necessary for the assembly of other centrosomal constituents ( dictenberg et al . , 1998 ; dammermann and merdes , 2002 ; kubo and tsukita , 2003 ) . we found that depletion of pcm-1 or pericentrin activates the p38-dependent stress pathway and the p53-dependent cell cycle checkpoint . we have previously shown that depletion of the protein pcm-1 leads to defects in the assembly of the centrosomal components centrin , ninein , and pericentrin , and to an altered organization of the microtubule network in interphase cells ( dammermann and merdes , 2002 ) . to investigate the consequences of pcm-1 depletion on the cell cycle , we performed rna silencing experiments in primary human fibroblasts , mrc-5 . depleted cells were tested for incorporation of brdu into the nucleus , as an indicator of dna synthesis ( fig . we determined that in pcm-1depleted cells only 15 4% incorporated brdu , as compared with 35 3% in controls , as expected for a normal cycling population ( fig . this is consistent with previous reports on microinjection of pcm-1inhibiting antibodies ( balczon et al . , 2002 ) and on centrosome removal by microsurgery or laser ablation , which prevent cells from entering s phase ( hinchcliffe et al . several years ago , experiments on cells treated with the microtubule drugs colcemid , nocodazole , and taxol indicated that untransformed cells are arrested in g1 phase , when microtubules are depolymerized or when microtubule dynamics are altered ( trielli et al . , 1996 ; di leonardo et al . , 1997 this raises the question of whether dna replication in pcm-1depleted cells is inhibited because of an altered microtubule network , or whether defects at the centrosome itself suffice to induce a cell cycle arrest . 1 a ) , which in contrast to pcm-1 , only slightly reduces microtubule density but seems to have no significant effect on microtubule anchoring at the centrosome ( dammermann and merdes , 2002 ) . consistently , ( a ) immunofluorescence of mrc-5 fibroblasts treated with control rna or sirna against pcm-1 and pericentrin , respectively . graphs depict the percentages of cells lacking pcm-1 or pericentrin expression after treatment with control or silencing rnas . cells were scored as negative if they lacked centrosomal staining of pcm-1 or pericentrin , only displaying diffuse background fluorescence . ( b ) percentages of cells incorporating brdu after treatment with control rna or silencing rna against pcm-1 or pericentrin . data were obtained from cell cultures that were double labeled for brdu and pcm-1 or pericentrin . among cell cultures treated with silencing rna , only cells that were visibly depleted of pcm-1 or pericentrin were scored . ( a ) immunoblots of mrc-5 cells treated with control rna or sirna against pcm-1 , prb , or both pcm-1 and prb simultaneously . blots were probed with antibodies against pcm-1 , cyclin a , the licensing factor mcm3 , the dna replication factor pcna , prb , and -tubulin . ( b ) immunoblots of cells treated with control rna or sirnas against pcm-1 , pericentrin , or both simultaneously . ( c ) mrc-5 cells treated with control rna , sirna against pcm-1 , or sirna against pcm-1 and prb simultaneously . the percentages of cells staining positively for the proliferation marker ki-67 or for incorporated brdu are shown . ( d ) flow - cytometric analysis of mrc-5 cells treated with control rna or sirna against pcm-1 . ( e ) immunoblots of mrc-5 cells treated with control rna or sirna against pcm-1 for 48 or 120 h or with sirna against pericentrin for 72 h. blots were probed with antibodies against p53 , -tubulin , or p21 . because these data indicated that cells failed to undergo s phase dependent dna replication when missing the full complement of centrosome proteins , we wanted to test in more detail at what stage cells are arrested . immunoblotting of cell extracts of pcm-1depleted cultures indicated drastic reduction of cyclin a ( fig . 2 a ) , which is normally found expressed in cells during late g1 , s , and g2 phases . furthermore , we detected that pcm-1depleted cells also showed reduced amounts of the proteins minichromosome maintenance deficient 3 ( mcm3 ) and proliferating cell nuclear antigen ( pcna ) acting in licensing and dna replication ( fig . 2 a ) and reduced percentages of cells expressing the cell proliferation marker ki-67 ( fig . altogether , these data indicated that depletion of centrosome proteins reduces the number of cells entering s phase . we verified this hypothesis by comparing profiles of cell cultures analyzed by flow cytometry ( fig . 2 d ) : consistent with our data on brdu incorporation we then wanted to determine whether s phase entry was blocked because of checkpoint activation in cells depleted of pcm-1 or pericentrin . we found that the overall levels of the retinoblastoma protein ( prb ) were reduced to 38 17% and that most of the remaining prb , normally hyperphosphorylated during late g1 and s phase , was present in its faster migrating , hypophosphorylated form ( 77 13% in depleted vs. 38 13% in control cells ; fig . however , was found up - regulated , especially after prolonged depletion of pcm-1 or pericentrin ( fig . in depleted cells , the cdk2 inhibitor p21 was found equally up - regulated ( fig . these data suggested that depletion of the two centrosome - associated proteins pcm-1 and pericentrin leads to the activation of the p53-dependent checkpoint . in the next step , we wanted to determine whether cell cycle progress would be affected if the p53-dependent checkpoint control was abrogated for this purpose , we attempted simultaneous depletion of p53 and pcm-1 by cotransfecting sirna oligomers against both . 3 a shows that p53 levels could not be reduced when pcm-1 was missing . we tried to refine this experiment by sequential depletion of mrc-5 cells , by first depleting > 90% of p53 after 72 h , followed by simultaneous sirna treatment against p53 and pcm-1 . however , we observed that under these conditions , p53 levels increased back to 4050% in three different experiments ( unpublished data ) . we concluded that p53 turnover is altered and that the residual p53 protein might be stabilized in the absence of an intact centrosome . we therefore changed our experimental strategy and compared cell cycle progress after pcm-1 depletion in several cell lines lacking p53 . we used mouse embryonic fibroblasts from p53 knockout mice ( unpublished data ) as well as the human lung carcinoma cell line h1299 . because of the loss of p53 checkpoint control , both lines displayed a relatively high basic rate of dna synthesis ( fig . we found that in both p53/ cell lines , pcm-1 depletion did not inhibit cell cycle progress . , we also tested the effect of pcm-1 depletion in the p53+/+ and p53/ lines of hct116 cells . brdu incorporation in these was reduced from 42% in controls to 33% in partially depleted p53 + cells , whereas p53 cells showed brdu incorporation in 49% after partial pcm-1 depletion . consistently , hela cells with functionally suppressed p53 checkpoint control do not arrest in the absence of centrosomes ( la terra et al . , in contrast to p53 , the removal of prb did not cause resumption of the cell cycle in pcm-1depleted mrc-5 cells , because the percentages of brdu - incorporating cells and ki-67expressing cells remained low , as did the expression of mcm3 protein ( fig . on the other hand , the amounts of cyclin a and pcna were restored to nearly control levels . a possible scenario would be that centrosome defects activate both prb and p53 in parallel , with p53 having feedback effects on prb phosphorylation and prb protein levels but not vice versa . this would be consistent with our observation that prb levels drop after pcm-1 or pericentrin depletion and that depletion of prb itself does not fully restore s phase activity , which is probably blocked because of checkpoint control mechanisms directly dependent on p53 . eventually , loss of prb might be compensated by rb - related pocket proteins , such as p107 or p130 . cell cycle arrest after pcm-1 depletion depends on the checkpoint protein p53 . ( a ) immunoblots of mrc-5 cells treated with control rna or sirnas against pcm-1 , p53 , or both pcm-1 and p53 combined . ( b ) graph depicts brdu incorporation in p53/ h1299 cells treated with control rna or sirna against pcm-1 . ( c ) immunoblots of h1299 and mrc-5 cells treated with control rna or sirna against pcm-1 ( pcm1d ) for 72 h. blots were probed with antibodies against pcm-1 , p53 , pcna , mcm3 , prb , or -tubulin . finally , we addressed the question of how defects in centrosome assembly could activate the p53-dependent checkpoint . it has been reported that p38 mapk is involved in the cell 's response to a range of stress factors , such as uv irradiation , osmotic shock , heat shock , starvation , and cytokine treatment ( zarubin and han , 2005 ) . the response to these stress factors is mediated partly through p53 phosphorylation , which is believed to stabilize p53 and to increase the expression of the cdk inhibitor p21 , thereby blocking the cell cycle ( agarwal et al . , 1998 ) . to assess the involvement of stress - activated p38 mapk in response to centrosome defects , we have treated pcm - depleted mrc-5 cells with the p38 mapk specific inhibitor sb203580 . we found that the inhibitor prevented cell cycle arrest , as indicated by resumption of dna synthesis ( fig . p38 inhibition in pcm-1depleted cells also restored regular levels of cyclin a and hyperphosphorylated prb , as well as expression of ki-67 and mcm3 ( fig . further , the inhibition of p38 led to decreased levels of p21 but only slightly decreased p53 ( fig . this could be explained by a stabilization of p53 after centrosome inactivation , as discussed in the previous paragraph , which might occur independently of p38-dependent p53 activation . alternatively , the centrosome - dependent cell cycle arrest might not simply be a linear consequence of p53 activation via p38 . for example , p53 might be activated by other kinases in addition to p38 . moreover , although p38 has been shown in multiple experiments to mediate cell cycle arrest by phosphorylating various sites of p53 ( bulavin et al . , 1999 ; huang et al . , 1999 ) , p38 is also known to phosphorylate and thereby inactivate cdc25a and cyclin d , which in turn arrests the cell cycle ( lavoie et al . , 1996 ; casanovas et al . , 2000 however , p38 mapk activation in the absence of p53 seems to be insufficient for centrosome - dependent cell cycle arrest because pcm-1 depletion in p53/ cells did not stop the cell cycle . ( a ) immunoblots of mrc-5 cells treated with control rna , sirna against pcm-1 , or sirna against pcm-1 together with the p38 inhibitor sb203580 ( pcm-1 + inh ) . blots were probed with antibodies against prb , cyclin a ( cyc a ) , p53 , the cdk inhibitor p21 , active p38 , or -tubulin . ( b ) graphs depicting mrc-5 cells treated with control rna , sirna against pcm-1 , or sirna against pcm-1 together with the p38 inhibitor sb203580 ( pcm-1 + inh ) . the staining of brdu incorporation ( after rna treatment for 72 or 96 h ) , ki-67 immunofluorescence ( after rna treatment for 96 h ) , or mcm3 immunofluorescence ( after rna treatment for 72 h ) is shown . in our pcm-1depletion experiments , we observed a significant decrease in the expression of proteins associated with cell proliferation , such as mcm3 , pcna , or ki67 . however , because about half of the depleted cells were still entering s phase , we wanted to test whether centrosome defects only slowed down cell cycle activity , or whether depleted cells were predisposed to a stable arrest that did not affect all cells simultaneously . immunofluorescence of p53 after pcm-1 depletion revealed that p53 levels varied between individual cells , with some cells showing a very high increase but a small increase or no obvious response in others ( fig . 5 a ) . we then tested whether the cells ' response to centrosome inhibition induces exit from the cell cycle via senescence . senescence is marked by the acquisition of a permanent g0 state and an increase of cellular -galactosidase activity ( dimri et al . , 1995 ) . a cytochemical analysis of pcm-1depleted cultures revealed a 10-fold increase of -galactosidase positive cells ( fig . 5 b ) , compared with a basal level of 3% in controls . consistently , we previously reported that cultures of u2os cells died after silencing of pcm-1 for long periods ( dammermann and merdes , 2002 ) , although the experimental conditions varied from the protocol used here . senescence has been characterized as a cellular program activated as a result of physiological stresses preventing further cell proliferation ( ben - porath and weinberg , 2004 ) . instead of a specific centrosome - dependent cell cycle control as proposed by murray ( 2001 ) , the idea of the centrosome as a center for stress - related signaling seems plausible , considering its central location in the focus of the microtubule network and its ability to bind various molecules of signaling pathways and of cell cycle regulation . for example , significant amounts of protein kinase a , polo - like kinase , and protein phosphatases 1 and 2a , as well as cyclin e and p53 , have all been localized to the centrosome ( fry et al . , 2000 ; morris et al . , 2000 ; is mediated by large coiled - coil proteins such as pericentrin , akap450 , or akap220 ( diviani et al . , 2000 ; reinton et al . , 2000 ; diviani and scott , 2001 ) . absence or inhibition of anchoring proteins could therefore disrupt cellular signaling pathways and elicit a stress response . further research will be necessary to investigate the multiple interactions between centrosome proteins , cell signaling , and the complex regulation of the cell cycle . ( a ) fibroblasts treated with control and pcm-1 sirna , stained for pcm-1 ( red ) , dna ( blue ) , and p53 ( green ) . ( b ) -galactosidase assay on control and pcm-1depleted cells 96 h after transfection using 5-bromo-4-chloro-3-indolyl -d - galactopyranoside as a chromogenic substrate . human fetal lung fibroblasts mrc-5 ( european collection of cell cultures ) and h1299 cells ( a gift from b. vojtesek , masaryk memorial cancer institute , brno , czech republic ) were cultured in dme ( invitrogen ) , supplemented with 10% fetal bovine serum ( perbio science ) , 2 mm l - glutamine , and antibiotics . hct116 cells ( a gift from b. vogelstein , johns hopkins university , baltimore , md ) were cultured in mccoy 's medium containing the same supplements . about 3 10 cells from early passages of mrc-5 and 0.5 10 cells of h1299 or hct116 were transfected with 9 or 18 g of sirna oligonucleotides , respectively , in a nucleofector electroporation device , using the appropriate transfection kit ( amaxa ) . the cells were then plated onto two 10-cm petri dishes to obtain a confluency of 3040% on the next day . routinely , sirna treatment was for a total of 72 h before fixation or preparation of cell extracts , unless indicated otherwise . p38 mapk inhibition was performed in cells that were transfected with oligos and cultured overnight before the specific inhibitor sb203580 ( calbiochem ) was added at a concentration of 10 m . inhibitor - treated cells were left in culture for another 48 h and then fixed in methanol or extracted for sds - page . cells were harvested using trypsin - edta , counted , and washed in ice - cold pbs . amounts of extract equal to 200,000 cells were loaded and run on 7.5 , 10 , or 12.5% sds - page gels and blotted onto nitrocellulose . the following antibodies were used for western blot analysis or immunofluorescence : affinity - purified rabbit anti pcm-1 ( dammermann and merdes , 2002 ) , anti - mcm3 mab clone 3a2 ( mbl international corporation ) , anti - prb mab clone 4h1 ( cell signaling ) , anti - pcna mab clone pc-10 ( sigma - aldrich ) , anti cyclin e mab clone he12 ( zymed laboratories ) , anti cyclin a mab clone cy - a1 ( sigma - aldrich ) , anti p53 mab clone do-1 ( novocastra ) , anti - p21 mab clone sx118 ( bd biosciences ) , rabbit anti - activep38mapk polyclonal antibody ( promega ) , anti-tubulin mab clone dm1a ( sigma - aldrich ) , goat anti rabbit igg polyclonal antibody hrp ( ge healthcare ) , rat anti - brdu mab ( harlan ) , anti ki-67 mab clone mm1 ( novocastra ) , rabbit anti - pericentrin polyclonal antibody ( covance ) , donkey anti rabbit or anti mouse igg conjugated with alexa 488 or alexa 594 ( invitrogen ) . quantification of protein levels was performed by scanning immunoblots and analyzed using the photoshop ( adobe ) histogram tool . quantification of cells expressing specific proteins was performed by counting sirna - treated cells that were double stained with pcm-1 antibodies to verify depletion . the following sirna oligomers with dtdt overhangs ( qiagen ) were used : pcm-1.2 , corresponding to human pcm-1 ( ucagcuucgugauucucag ) ; peric , corresponding to human pericentrin ( gcagcugagcugaaggaga ; dammermann and merdes , 2002 ) ; prb ( gcccuuacaaguuuccuag ) ; and p53 ( cuacuuccugaaaacaacg ) . all cells in a culture dish were harvested by trypsinization , washed in ice - cold pbs , and fixed in 80% ice - cold ethanol in pbs . before staining , the cells were spun down in a cooled centrifuge and resuspended in the cold . bovine pancreatic rnase ( sigma - aldrich ) was added at a final concentration of 2 g / ml , and cells were incubated at 37c for 30 min , followed by an incubation in 20 g / ml of propidium iodide ( sigma - aldrich ) for 20 min at room temperature . cells grown on coverslips were fixed in ice - cold methanol and stored at 20c until use . antibody staining was performed using the reagents listed in the previous paragraphs , according to standard protocols . the percentage of cells in s phase was assessed by adding 100 m brdu ( sigma - aldrich ) to the cultures 30 min before fixation . double labeling of brdu and pcm-1 was performed by probing first for pcm-1 , using rabbit anti pcm-1 and fluorescent anti - rabbit antibody , and coverslips were postfixed in pbs containing 3.7% paraformaldehyde , treated with 2 m hcl for 30 min , and stained with rat anti - brdu and fluorescent secondary anti - rat antibody . cells were viewed with a fluorescence microscope ( axioskop 2 ; carl zeiss microimaging , inc . ) equipped with a camera ( axiocam ; carl zeiss microimaging , inc . ) and software ( axiovision ; carl zeiss microimaging , inc . ) . human fetal lung fibroblasts mrc-5 ( european collection of cell cultures ) and h1299 cells ( a gift from b. vojtesek , masaryk memorial cancer institute , brno , czech republic ) were cultured in dme ( invitrogen ) , supplemented with 10% fetal bovine serum ( perbio science ) , 2 mm l - glutamine , and antibiotics . hct116 cells ( a gift from b. vogelstein , johns hopkins university , baltimore , md ) were cultured in mccoy 's medium containing the same supplements . about 3 10 cells from early passages of mrc-5 and 0.5 10 cells of h1299 or hct116 were transfected with 9 or 18 g of sirna oligonucleotides , respectively , in a nucleofector electroporation device , using the appropriate transfection kit ( amaxa ) . the cells were then plated onto two 10-cm petri dishes to obtain a confluency of 3040% on the next day . routinely , sirna treatment was for a total of 72 h before fixation or preparation of cell extracts , unless indicated otherwise . p38 mapk inhibition was performed in cells that were transfected with oligos and cultured overnight before the specific inhibitor sb203580 ( calbiochem ) was added at a concentration of 10 m . inhibitor - treated cells were left in culture for another 48 h and then fixed in methanol or extracted for sds - page . cells were harvested using trypsin - edta , counted , and washed in ice - cold pbs . amounts of extract equal to 200,000 cells were loaded and run on 7.5 , 10 , or 12.5% sds - page gels and blotted onto nitrocellulose . the following antibodies were used for western blot analysis or immunofluorescence : affinity - purified rabbit anti pcm-1 ( dammermann and merdes , 2002 ) , anti - mcm3 mab clone 3a2 ( mbl international corporation ) , anti - prb mab clone 4h1 ( cell signaling ) , anti - pcna mab clone pc-10 ( sigma - aldrich ) , anti cyclin e mab clone he12 ( zymed laboratories ) , anti cyclin a mab clone cy - a1 ( sigma - aldrich ) , anti p53 mab clone do-1 ( novocastra ) , anti - p21 mab clone sx118 ( bd biosciences ) , rabbit anti - activep38mapk polyclonal antibody ( promega ) , anti-tubulin mab clone dm1a ( sigma - aldrich ) , goat anti mouse polyclonal antibody hrp ( promega ) , donkey anti rabbit igg polyclonal antibody hrp ( ge healthcare ) , rat anti - brdu mab ( harlan ) , anti ki-67 mab clone mm1 ( novocastra ) , rabbit anti - pericentrin polyclonal antibody ( covance ) , donkey anti rabbit or anti mouse igg conjugated with alexa 488 or alexa 594 ( invitrogen ) . quantification of protein levels was performed by scanning immunoblots and analyzed using the photoshop ( adobe ) histogram tool . quantification of cells expressing specific proteins was performed by counting sirna - treated cells that were double stained with pcm-1 antibodies to verify depletion . the following sirna oligomers with dtdt overhangs ( qiagen ) were used : pcm-1.2 , corresponding to human pcm-1 ( ucagcuucgugauucucag ) ; peric , corresponding to human pericentrin ( gcagcugagcugaaggaga ; dammermann and merdes , 2002 ) ; prb ( gcccuuacaaguuuccuag ) ; and p53 ( cuacuuccugaaaacaacg ) . all cells in a culture dish were harvested by trypsinization , washed in ice - cold pbs , and fixed in 80% ice - cold ethanol in pbs . before staining , the cells were spun down in a cooled centrifuge and resuspended in the cold . bovine pancreatic rnase ( sigma - aldrich ) was added at a final concentration of 2 g / ml , and cells were incubated at 37c for 30 min , followed by an incubation in 20 g / ml of propidium iodide ( sigma - aldrich ) for 20 min at room temperature . cells grown on coverslips were fixed in ice - cold methanol and stored at 20c until use . antibody staining was performed using the reagents listed in the previous paragraphs , according to standard protocols . the percentage of cells in s phase was assessed by adding 100 m brdu ( sigma - aldrich ) to the cultures 30 min before fixation . double labeling of brdu and pcm-1 was performed by probing first for pcm-1 , using rabbit anti pcm-1 and fluorescent anti - rabbit antibody , and coverslips were postfixed in pbs containing 3.7% paraformaldehyde , treated with 2 m hcl for 30 min , and stained with rat anti - brdu and fluorescent secondary anti - rat antibody . cells were viewed with a fluorescence microscope ( axioskop 2 ; carl zeiss microimaging , inc . ) equipped with a camera ( axiocam ; carl zeiss microimaging , inc . ) and software ( axiovision ; carl zeiss microimaging , inc . ) . a male neonate born to g2 p1l1 mother at term by spontaneous vaginal delivery to iii degree consanguineous marriage was found to have proximal shortening of both upper and lower limbs [ figure 1 ] . the antenatal period was uneventful and antenatal ultrasound was reportedly not done during pregnancy and the mother was referred to our hospital after the onset of labor . apart from rhizomelic shortening , the neonate also had coronal clefts of thoracic vertebrae and stippled epiphysis of femur tibia and humerus on skeletal survey radiograph [ figure 2 ] . based on the above features a provisional diagnosis of rhizomelic chondro - dysplasia punctata ( rcdp ) was made and the prognosis was explained to the parents . the baby developed progressively severe respiratory distress and was discharged at request on day 3 of life as the parents were unable to come to terms with the diagnosis . rhizomelic shortening of upper limb punctate calcification and epiphyseal abnormalities chondrodysplasia punctata is a radiological diagnosis characterized by punctate or stippled calcifications in epiphyseal cartilage and seen in peroxisomal disorders such as zellweger syndrome , neonatal adrenoleukodystrophy , and infantile refsum disease . it may also be inherited as x - linked dominant , x - linked recessive , and autosomal recessive forms . it is classically associated with pex7 gene ( peroxin family of genes ) mutation and has been reported in indian patients too . rcdp is characterized by proximal shortening of the humerus and to a lesser degree the femur , punctate calcifications in cartilage with epiphyseal and metaphyseal abnormalities , radiolucent defects ( coronal clefts ) of the vertebral bodies which represents cartilage that are not ossified , cataracts , contractures , microcephaly , characteristic skin changes of icthyosis , facial dysmorphism ( depressed nasal bridge , hypertelorism , hypoplastic midface , anteverted nostrils , full cheeks ) , and developmental impairment . this condition is considered to be lethal and most of the affected fetuses die in utero or soon after birth . only few of them survive beyond infancy with severe physical disability and profound mental retardation in whom , death usually occurs in the first decade of life . diagnosis of rcdp is based on clinical findings and confirmed by clinically available biochemical or molecular genetic testing which includes biochemical tests of peroxisomal function like red cell plasmologen concentration , plasma phytanic acid , and very long chain fatty acid estimation . this case is presented due to its rarity and failure to detect such an abnormality in utero resulting in a wasted pregnancy . the lack of resources ( both money and manpower ) is probably responsible for this tragedy to the parents which could have been prevented by early diagnosis and appropriate counseling . establishing regional genetic labs which are connected with district level hospitals can be of immense help in reducing the burden of genetic diseases by appropriate prenatal diagnosis and counseling .

previous evidence has indicated that an intact centrosome is essential for cell cycle progress and that elimination of the centrosome or depletion of individual centrosome proteins prevents the entry into s phase . to investigate the molecular mechanisms of centrosome - dependent cell cycle progress , we performed rna silencing experiments of two centrosome - associated proteins , pericentriolar material 1 ( pcm-1 ) and pericentrin , in primary human fibroblasts . we found that cells depleted of pcm-1 or pericentrin show lower levels of markers for s phase and cell proliferation , including cyclin a , ki-67 , proliferating cell nuclear antigen , minichromosome maintenance deficient 3 , and phosphorylated retinoblastoma protein . also , the percentage of cells undergoing dna replication was reduced by > 50% . at the same time , levels of p53 and p21 increased in these cells , and cells were predisposed to undergo senescence . conversely , depletion of centrosome proteins in cells lacking p53 did not cause any cell cycle arrest . inhibition of p38 mitogen - activated protein kinase rescued cell cycle activity after centrosome protein depletion , indicating that p53 is activated by the p38 stress pathway . a male neonate was born with rhizomelic shortening of limbs . skeletal radiograph showed punctate calcification of epiphysis of humerus , femur , and tibia . the diagnosis and a brief review of literature pertaining to the condition with emphasis on antenatal diagnosis and counseling are being reported .

radioactive iodine thyroid ablation has been shown to be an inexpensive , safe , and effective treatment for hyperthyroidism . it is the most commonly used method for treating adult patients with graves ' disease . special precautions must be used in women of child - bearing age because of the possible detrimental side effects of fetal exposure . the american college of radiology ( acr ) practice guideline for the performance of therapy with unsealed radiopharmaceutical sources states that pregnancy should be ruled out using one of the following four criteria : ( 1 ) a negative hcg test obtained within 72 hours prior to administration of the radiopharmaceutical , ( 2 ) documented history of hysterectomy , ( 3 ) a postmenopausal state with absence of menstrual bleeding for two years , and ( 4 ) premenarche in a child age of 10 years or younger . the society of nuclear medicine ( snm ) procedure guideline for therapy of thyroid disease with i-131 states that females of child - bearing age should routinely be tested for pregnancy within 72 hours or less before i-131 treatment . when the patient 's history clearly indicates that pregnancy is impossible , the treating physician may omit the pregnancy test . tj ( not the patient 's initials ) is an adult female diagnosed with graves ' disease , which was treated with i-131 therapy in december 2004 . she had been doing well until june 2006 when she presented to the pediatric endocrinology clinic with reoccurrence of her disease . her thyroid function tests showed tsh < 0.03 ( normal 0.325.0 munit / ml ) , free t4 2.80 ( normal 0.711.85 ng / dl ) , and t4 19.1 ( normal 5.012.0 mcg / dl ) . tj was scheduled for a radioactive iodine uptake and scan with subsequent ablation with i-131 . a qualitative urine pregnancy test was performed 24 hours prior to ablation and was negative . a radioiodine uptake and scan revealed a 24-hour uptake of 100% ( normal 10%30% ) . informed consent for i-131 therapy was obtained . tj verified that she was not pregnant and was given an activity of 19.8 mci of i-131 . approximately four months later , after slipping and falling at work , tj presented to her physician with a complaint of low back pain . magnetic resonance imaging ( mri ) of her lumbar spine revealed an intrauterine pregnancy . it was estimated that the fetus was eight to ten days old at the time of i-131 treatment . neonatal thyroid function tests revealed suppressed tsh ( < 0.02 , normal 0.325.0 munit / ml ) , elevated t4 ( 24.4 , normal 5.012.0 mcg / dl ) , and free t4 ( 4.54 , normal 0.581.64 ng / dl ) . in addition , the infant had an elevated thyroid receptor antibody ( trab ) titer of 82% ( negative < 10% , intermediate 10%15% , positive > 15% ) . based on the laboratory evaluation , because he was asymptomatic , a decision was made not to treat but to monitor him closely . over the next six months his thyroid function tests normalized with tsh 0.73 munit / ml , t4 10.3 mcg / dl , free t4 1.11 ng / dl , and trab < 15% . thyroid embryogenesis is largely completed by 1012-week gestation . at 10-week gestation , the thyroid gland is able to trap and concentrate iodine and synthesize thyroid hormones thyroxine and triiodothyronine . after 10-week gestation , significant exposure to the fetal thyroid can occur from therapeutic doses to the mother , resulting in hypothyroidism and cretinism . there have been no reports of birth defects or childhood malignancy in children born to the mothers who received radioactive iodine for graves ' disease before the 10th week of pregnancy . we believe the infant 's thyroid gland was not affected by the i-131 therapy because he was exposed prior to 10-week gestation . quantifying the exact absorbed dose by the embryo in this case is difficult and depends on many variables . this calculation likely overstates the actual absorbed dose , as the model assumes that i-131 crosses the placenta . in this case , due to the placenta being in the early stages of development during the second week of gestation it is estimated that the embryo absorbed dose with i-131 treatment in early pregnancy is 0.072 mgy / mbq . our patient received a dose of 19.8 mci ( 732.6 mbq ) , which corresponds to an absorbed dose of approximately 5.3 rads after converting from gy to rads . deterministic effects are those that are known to occur at a given radiation threshold . for example , if a patient were to receive an acute dose of 2 gy to the lens of the eye , the patient will develop a cataract . stochastic effects are those that can theoretically occur at any radiation dose . for example , any radiation dose could potentially induce a cancer in the future . the probability of a stochastic effect increases as dose increases , but there is no radiation threshold dose below which one can say a stochastic effect will not occur . therefore , stochastic effects can theoretically occur with any radiation dose . the only known deterministic effect of radiation at an absorbed dose of less than 10 rads is possible spontaneous abortion at 114 days postconception . based on the data above , this dose level ( 10 rads ) was not reached in this patient . on the other hand , thus , the radiation dose to any developing embryo should be minimized , and it is reasonable to ascertain an accurate pregnancy test prior to the use of i-131 therapy . studies have demonstrated that the risk of congenital effects have been negligible at doses of 5 rads or less when compared to other risks of pregnancy . in addition , the risk of malformation only significantly increases at doses above 15 rads . mental retardation has been shown to occur in the offspring of pregnant mothers ; however , it is at doses greater than 20 rads and generally after the eighth week of gestation . a screening study of thyroid cancer among individuals exposed to in utero i-131 from the chernobyl fallout , however , did demonstrate an increased risk of thyroid carcinoma approximately 20 years after the accident . thus , our estimation that the infant was exposed to approximately 5 rads supports that no deterministic effects should have occurred , but one can not exclude the future potential for stochastic effects . as previously stated , the acr practice guideline for the performance of therapy with unsealed radiopharmaceutical sources states that pregnancy should be ruled out using one of the following four criteria : ( 1 ) a negative hcg test obtained within 72 hours prior to administration of the radiopharmaceutical , ( 2 ) documented history of hysterectomy , ( 3 ) a postmenopausal state with absence of menstrual bleeding for two years , and ( 4 ) premenarche in a child age 10 or younger . the snm procedure guideline for therapy of thyroid disease with i-131 states that females of child - bearing age should routinely be tested for pregnancy within 72 hours or less before i-131 treatment . when the patient 's history clearly indicates that pregnancy is impossible , the treating physician may omit the pregnancy test . neither of the above guidelines specifies whether the pregnancy tests should be quantitative or qualitative . typically , serum pregnancy tests are more sensitive than standard urine pregnancy tests because the serum concentration of hcg is significantly higher than its urine concentration . detection of hcg in maternal serum is evident only after implantation and vascular communication has been established with the decidua by the syncytiotrophoblast , which occurs 810 days following conception . serum hcg can be detected in about 5% of patients eight days after conception and about 98% of patients by 11 days postconception . in contrast , urine pregnancy tests will be positive 1517 days after conception in 98% of patients [ 14 , 16 ] . this rule suggests that radioactive iodine therapy only be administered during the 10 days after the onset of the menstrual period . however , this protocol may not be effective if the patient has irregular menstrual cycles . one possible suggestion is to incorporate the ten day rule protocol into the current guidelines for patients who have regular 28-day menstrual cycles . for patients with irregular cycles or who require immediate ablation , quantitative serum hcg pregnancy tests on the day of the treatment should be considered . because serum hcg is detected in 98% of pregnant patients by day 11 , recommending the patients to abstain from sexual activity for at least two weeks prior to i-131 therapy may be suggested . this abstinence would cover the gap from the time of conception to the time the serum hcg test becoming positive . however , the physician would have to rely on the patient for providing an accurate menstrual cycle and sexual history . it is imperative to rule out pregnancy prior to the administration of radioactive iodine therapy due to the potential detrimental side effects of fetal exposure . the dose of i-131 in this particular case would be unlikely to result in any deterministic events . however , under the premise that any unnecessary exposure to radiation is important in preventing any potential stochastic events , an accurate pregnancy screening protocol may be warranted in preventing inadvertent i-131 treatment in early pregnancies of women with graves ' disease . the current acr and snm guidelines only mandate a hcg test being obtained within 72 hours prior to the treatment . this approach may miss a small number of pregnancies as a serum or urine pregnancy test does not become positive until implantation occurs , which happens 810 days postconception . though we recommend re - evaluating the acr and snm guidelines for future clarifications , it is possible that despite combining the patient 's history , the ten day rule and the hcg screening , an unsuspected pregnancy may be missed . ischemic heart disease is the first leading cause of death among the world and caused 62.6 million disability adjusted life years in 2004 . cardiovascular diseases ( cvds ) accounts for 17.7 million annual deaths in world - wide . cvds are increasing in developing countries as they half of deaths and 80% of the related global burden occur in these countries . cvds are the main cause of death and its covers 38% of total mortality in iran as well . recent studies have indicated that primary prevention to reduce cvds death is four times more effective than other levels of prevention . knowledge of the magnitude of cvds related avoidable burden resulting from different risk factors regarding their own significance in codifying the prevention and priority setting by policy makers is absolutely essential . the contribution of every risk factor to the avoidable / attributable burden of diseases can be calculated by a measure entitled potential impact fraction ( pif ) . the pif ( also called the generalized attributable fraction ) was introduced by walter in 1980 and morgenstern and bursic in 1982 as a measure that generalizes the population attributable fraction ( attributable risk ) . it is defined as the fractional reduction of a disease resulting from changing the current distribution of a risk factor to some modified distribution or to incomplete elimination of exposure . the concept of avoidable / attributable burden and such modified levels , which considered to some alternative distribution of exposure in the counterfactual analysis have previously been reported elsewhere . there are according to the authors knowledge , few published studies that consider the joint effect of multiple risk factors on avoidable burden of cvds . accordingly , this study was aimed to estimate the joint effect of avoidable burden of multiple risk factors to cvds in iran . estimates of the avoidable burden were made using world health organization comparative risk assessment ( cra ) methodology . the reason for choosing the cra methodology was the ability of considering the effects of intervention on observed exposure distribution to other distribution , rather than a single reference level such as non - exposed . pif is an epidemiological measure of effect that calculates the proportional change in average disease incidence ( or prevalence or mortality ) after a change in the exposure of a related risk factor . the pif is given by the following equation 1 : where rr is the measure of effect at a given exposure level , p is the prevalence of risk factor , and n is the maximum exposure level . in this study , scenario 1 corresponds to the theoretical minimum risk levels and based on the reduction of the prevalence of a specific risk factor to zero . plausible minimum risk level indicates the distribution of a risk factor at imaginable level in iran . more details about this methodology and the pif as a measure to estimate avoidable / attributable burden is explained elsewhere . authors used aggregates data and reported above and mentioned reference for achievement the objectives of the study ; hence ethical considerations in performance of the project were observed . accordingly to estimate the pif measure , the prevalence of each risk factor , the corresponding measures of effect and alternative prevalence of counterfactual levels is required as follows . diabetes is defined as either newly diagnosed diabetes mellitus ( ndm ) and known diabetes mellitus ( kdm ) . ndm is defined as individuals who had fasting plasma glucose 126 mg / dl . those people who if a health - care professional had ever told them to have diabetes were considered as kdm . hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or use of anti - hypertensive drugs . central obesity and hypercholesterolemia were defined as waist circumstance 88 cm in females and 102 cm in males according to adult treatment panel iii criteria , total cholesterol 240 mg / dl , respectively . prevalence of above mentioned risk factors among iranian adults were obtained from the third national surveillance of risk factors of non - communicable diseases-2007 , conducted in 2007 . in this study , the available and updated prevalence rates of risk factors in the iranian population have been presented . in present study , theoretical minimum risk levels for selected risk factors were considered as zero in the 1 scenario . plausible minimum risk level , the 2 scenario , for selected risk factors were determined different levels . contribution of the selected risk factors to the avoidable burden of cvds by sex data on corresponding measures of effect were derived from the tehran lipid and glucose study ( tlgs ) . the tlgs is a long - term integrated community - based program for prevention of non - communicable disorders ( ncd ) by development of a healthy life - style and reduction of ncd risk factors . the study begun in 1999 , is ongoing , to be continued for at least 20 years . corresponding rr , which measured association between cvds and selected risk factors were shown in table 1 . in our work , we used multivariate adjusted hazard ratios for estimating pifs . since , obesity did not have a significant hazard ratio for cvds , so its contribution was not estimated in the present study . uncertainties for the pifs were considered using calculation their own values based on lower and upper levels of the prevalence of each risk factor and its related measures of effects . estimates of the avoidable burden were made using world health organization comparative risk assessment ( cra ) methodology . the reason for choosing the cra methodology was the ability of considering the effects of intervention on observed exposure distribution to other distribution , rather than a single reference level such as non - exposed . pif is an epidemiological measure of effect that calculates the proportional change in average disease incidence ( or prevalence or mortality ) after a change in the exposure of a related risk factor . the pif is given by the following equation 1 : where rr is the measure of effect at a given exposure level , p is the prevalence of risk factor , and n is the maximum exposure level . in this study , scenario 1 corresponds to the theoretical minimum risk levels and based on the reduction of the prevalence of a specific risk factor to zero . plausible minimum risk level indicates the distribution of a risk factor at imaginable level in iran . more details about this methodology and the pif as a measure to estimate avoidable / attributable burden is explained elsewhere . authors used aggregates data and reported above and mentioned reference for achievement the objectives of the study ; hence ethical considerations in performance of the project were observed . accordingly to estimate the pif measure , the prevalence of each risk factor , the corresponding measures of effect and alternative prevalence of counterfactual levels is required as follows . diabetes is defined as either newly diagnosed diabetes mellitus ( ndm ) and known diabetes mellitus ( kdm ) . ndm is defined as individuals who had fasting plasma glucose 126 mg / dl . those people who if a health - care professional had ever told them to have diabetes were considered as kdm . hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or use of anti - hypertensive drugs . central obesity and hypercholesterolemia were defined as waist circumstance 88 cm in females and 102 cm in males according to adult treatment panel iii criteria , total cholesterol 240 mg / dl , respectively . prevalence of above mentioned risk factors among iranian adults were obtained from the third national surveillance of risk factors of non - communicable diseases-2007 , conducted in 2007 . in this study , the available and updated prevalence rates of risk factors in the iranian population have been presented . prevalence of selected risk factors in both sexes is shown in table 1 . in present study , theoretical minimum risk levels for selected risk factors plausible minimum risk level , the 2 scenario , for selected risk factors were determined different levels . data on corresponding measures of effect were derived from the tehran lipid and glucose study ( tlgs ) . the tlgs is a long - term integrated community - based program for prevention of non - communicable disorders ( ncd ) by development of a healthy life - style and reduction of ncd risk factors . the study begun in 1999 , is ongoing , to be continued for at least 20 years . corresponding rr , which measured association between cvds and selected risk factors were shown in table 1 . in our work , we used multivariate adjusted hazard ratios for estimating pifs . since , obesity did not have a significant hazard ratio for cvds , so its contribution was not estimated in the present study . uncertainties for the pifs were considered using calculation their own values based on lower and upper levels of the prevalence of each risk factor and its related measures of effects . in addition , table 2 shows the 95% uncertainty interval for all of the risk factors related pifs at both theoretical ( scenario 1 ) and plausible minimum risk level ( scenario 2 ) . about 9.3% of attributable burden to cvds ( 95% uncertainty interval : 6.07 - 12.81 ) in males and 14.74% ( 95% uncertainty interval : 9.31 - 20.20 ) in females are avoidable after changing the current prevalence of hypertension to 14% in both sexes . uncertainty intervals for pifs based on estimated uncertainty ranges around point estimate of selected risk factors the pifs for other risk factors at theoretical minimum risk level are shown in table 1 . moreover , modifying the current distribution of the diabetes , central obesity and hypercholesterolemia to plausible minimum risk lead to avoided 3.4 ( 95% uncertainty interval : 2.0 - 4.4 ) , 3.1 ( 95% uncertainty interval : 0.9 - 5.5 ) and 2.9% ( 95% uncertainty interval : 1.4 - 4.8 ) of burden , which attributed to cvds in males and 6.6 ( 95% uncertainty intervals : 4.3 - 9.2 ) , 14.6 ( 95% uncertainty interval : 7.7 - 21.53 ) , and 2.9% ( 95% uncertainty interval : 0.09 - 6.25 ) in females , respectively . contribution of the diabetes , hypertension , central obesity , and hypercholesterolemia to the avoidable burden of cvds at both theoretical and plausible minimum risk levels has been depicted in figure 1 . contribution of the selected risk factors to the avoidable burden of cardiovascular diseases at both theoretical and plausible minimum risk levels ( a ) theoretical minimum risk level ( b ) plausible minimum risk level table 3 shows the joint effect of the contribution of the selected risk factors including diabetes , hypertension , central obesity , and hypercholesterolemia to the avoidable burden of cvds by sex . as it has been shown , at the theoretical minimum risk level the joint effect of diabetes , hypertension , central obesity , and hypercholesterolemia to the cvds avoidable burden equals to 37.1% among iranian men and 59.4% in adult women . the corresponding values at the plausible minimum risk level were 17.8% and 34.0% , respectively [ table 3 ] . joint effect of the contribution of the selected risk factors to the avoidable burden of cvds by sex our findings indicated the highest portion of pif among females was central obesity and after that hypertension , diabetes , and hypercholesterolemia . however , among adult men hypertension , diabetes , hypercholesterolemia , and central obesity were ranked with importance respectively at theoretical minimum risk level . the corresponding values at plausible minimum risk level were different among both males and females . moreover , the attributable burden to the joint effects of the above risk factors in females is also higher than in males . central obesity , unrelated to bmi , increases the risk of the prevalence of cvds ; and previous studies indicated that it is a strong predictor in the prevalence of cvds . recent studies have shown that the prevalence of central obesity is increasing in the united states . moreover , the estimated prevalence of central obesity in iran is high , too ; and among females , it is higher that of males . in this study , central obesity for females is the most important risk factor at theoretical minimum risk level and secondary risk factor at plausible minimum risk level . these researchers in a case - control studied an estimated 15152 cases and 14820 controls in 52 countries and calculated the odds ratio and population 's attributable risk for tobacco usage , hypertension , diabetes , waist - hip ratio , dieting , physical activity , and alcohol usage . high prevalence of central obesity in iranian females can be caused by changes in lifestyle pattern , inactivity , and modernization of the community . by reducing this factor , which has the first priority among females another important risk factor which was assessed in this study is hypertension , which results showed that it is of particular importance in both sexes . with the increase of 10 unit of diastolic hypertension or 20 unit of the systolic hypertension , we will have a double increase in the risk of cvds . a study conducted in australia in 2003 showed that 17% of deaths and 7.6% of burden of cvds attributed to hypertension . yusuf et al . estimated that 17.9% of burden of myocardial infarction attributed to hypertension . nilsson et al . suggested that 14% of females cvds and 23% of males cvds are attributable to hypertension . in the present study , hypertension is the most important factor among males and the secondary factor among females at theoretical minimum risk level . the pif for hypertension among females is higher comparing to males in both levels , which is consistent with other studies . a study in spain showed that 2800 deaths attributed to cvds ( about 6% of the total mortality of cvds ) attributed to diabetes in spanish adults . in addition , 2% of deaths in males and 1.6% of deaths in females due to cvds and 10.4% and 3.4% due to stroke are attributed to diabetes for females and males respectively . yusuf et al . indicated that the contribution of diabetes is 9.9% of burden of cvds . the results of a study , which was conducted to quantify population - level effects of all higher - than - optimum concentrations of blood glucose on mortality from ischemic heart disease and stroke world - wide found that higher - than - optimum blood glucose is a leading cause of cardiovascular mortality in most world regions and reported that in addition to 959,000 deaths directly assigned to diabetes , 1,490,000 deaths from ischemic heart disease and 709,000 from stroke were attributable to high blood glucose , accounting for 21% and 13% of all deaths from these conditions . 792,000 ( 53% ) of deaths from ischemic heart disease and 345,000 ( 49% ) from a stroke that were attributable to high blood glucose were in men . largest numbers of deaths attributable to this risk factor from ischemic heart disease were in low- and - middle - income countries of south asia ( 548,000 ) and europe and central asia ( 313,000 ) , and from stroke in south asia ( 215,000 ) and east asia and pacific ( 190,000 ) . bradshaw et al . that of south africans aged equals or greater than 30 years , 5.5% had diabetes which increased with age and about 14% of ischaemic heart disease , 10% of stroke , 12% of hypertensive disease and 12% of renal disease burden in adult males and females ( 30 + years ) were attributable to diabetes . furthermore , diabetes was estimated to have caused 22,412 ( 95% uncertainty interval 20,755 - 24,872 ) or 4.3% ( 95% uncertainty interval 4.0 - 4.8% ) of all deaths in south africa in 2000 . our results showed females approximately twice as much avoidable burden as males due to diabetes , which is consistent with other studies . different criteria were used for hypercholesterolemia in different studies , so comparing their results are difficult . reported 11.6% of death and 6.2% of burden of cvds attributed to hypercholesterolemia . in this study , hypercholesterolemia has been forth and lesser importance than among other risk factors in both sexes at theoretical and plausible minimum risk levels . whereas , european studies estimated attributable contribution for this risk factors higher than of hypertension that this variation may be caused to different prevalence in variation within communities . furthermore , it should be consider to prescription of the statin drug , which is one of the lipid lowering drugs . according to higher avoidable burden to cvds in females , policy makers should be more focused on women for preventive interventions toward remove or reduce these risk factors , particularly central obesity and hypertension that itself would be the key strategy to reducing morbidity and mortality of cvds .

graves ' disease is a thyroid - specific autoimmune disorder in which the body makes antibodies to the thyroid - stimulating hormone receptor leading to hyperthyroidism . therapeutic options for the treatment of graves ' disease include medication , radioactive iodine ablation , and surgery . radioactive iodine is absolutely contraindicated in pregnancy as exposure to i-131 to the fetal thyroid can result in fetal hypothyroidism and cretinism . here we describe a case of a female patient with recurrent graves ' disease , who inadvertently received i-131 therapy when she was estimated to be eight days pregnant . this was despite the obtaining of a negative history of pregnancy and a negative urine pregnancy test less than 24 hours prior to ablation . at birth , the infant was found to have neonatal graves ' disease . the neonatal graves ' disease resolved spontaneously . it was suspected that the fetal thyroid did not trap any i-131 as it does not concentrate iodine until 10 weeks of gestation . background : there are few published studies that consider the joint effect of multiple risk factors on avoidable burden of cardiovascular diseases ( cvds ) . this study aimed to estimate the joint effect of avoidable burden of multiple risk factors to cvds.methods:estimates of avoidable burden to cvds were made using potential impact fraction ( pif ) . in order to calculate pif , data on the prevalence of the risk factors include diabetes , hypertension , central obesity , and hypercholesterolemia were obtained from 3rd national surveillance of risk factors of non - communicable diseases-2007 in iran and data on corresponding measures of effect were derived from a cohort study with multivariate adjusted hazard ratios . then , joint effect of risk factors was calculated.results:about 37% ( 95% uncertainty interval : 21.7 - 50.2 ) of attributable disability adjusted life years ( dalys ) to cvds in adult males and 59.4% ( 95% uncertainty interval : 30 - 76 ) in adult females due to selected risk factors are avoidable in theoretical minimum risk levels . after changing the current prevalence of these risk factors to the plausible minimum risk levels , 17.8% ( 95% uncertainty interval : 10.1 - 25.1 ) of cvds attributable dalys among adult males and 34% ( 95% uncertainty interval : 20 - 46.7 ) in adult females can be avoided.conclusions:to better priority setting as well as reporting the magnitude of avoidable dalys rather than the percentage of avoidable burden , pif should be applied to updated and revised burden of cvds .

a 56-year - old female presented with best corrected visual acuity ( bcva ) of 20/120 and nuclear sclerosis ( nuclear opacity 3 , nuclear color 2 using lens opacification classification system iii ) in the left eye ( le ) . the surgery was performed using proparacaine drops ( paracain ophthalmic solution 0.5% , sunways pvt . ltd . , a foldable iol of + 22.5 d of the sensar ar40e variety [ abbott medical optics inc ( amo ) , 1700 e. st . andrew place , santa ana , ca 92705 usa ] was loaded into the emerald c cartridge ( lot ch00841 of amo inc . , usa ) by the first assistant outside the field of the operating microscope to save the surgical time . the cartridge was inserted in the injector and the loaded injector was handed to the surgeon . the tip of the cartridge was just inserted snugly by slight rotatory motion into the anterior chamber through the 2.8-mm incision . however , while removing the injector system , it was noticed that there was a gross downward beaking of the bevelled anterior end of the cartridge [ fig . immediately , the incision site was inspected under the operating microscope and a descemet 's tear was detected with a rolled out flap of about 2 mm in length . the viscoelastic was meticulously washed out and the main incision wound and the two side port entries were carefully hydrated and an air bubble was injected into the anterior chamber . downward beaking of the beveled anterior end of the emerald c cartridge ( lot ch00841 of advanced medical optics , inc . , usa ) on the first postoperative day , the patient 's le had an uncorrected va of 20/60 with a small rolled out descemet 's flap and adjacent descemet 's striae at the site of the main clear corneal incision ( temporally ) [ fig . a corneal opacity remained at the deeper corneal layers with mild surrounding edema , even at the time of last check - up at 4 weeks post - op , with a bcva of 20/30 [ fig . a small rolled out descemet 's flap and adjacent descemet 's striae ( left eye of the reported case on the first postoperative day ) corneal opacity at the deeper corneal layers with mild surrounding edema ( left eye of the reported case at 4 weeks post - op ) damage to iols as a consequence of passage through various injector systems includes marks or scratches , stress fractures , cracks and tear lines . damage to descemet 's membrane can occur due to various factors during cataract surgery , including engaging of descemet 's membrane by the leading haptic during iol implantation or with the irrigation / aspiration device ( when mistaken as an anterior capsular remnant ) or due to inadvertent injection of viscoelastic between descemet 's membrane and corneal stroma . repair techniques include manual repositioning , repositioning with viscoelastic or air , suturing of descemet 's membrane to the peripheral cornea or use of sf6 or c3f8 . our case report describes injury to the corneal endothelium and descemet 's membrane intraoperatively due to frayed and beaked tip of the amo emerald c cartridge . the case has been followed up for a period of about 4 weeks as on the day of reporting , and as the visual axis was not completely involved , the vision is maintained , although the descemet 's stria and mild surrounding edema remains . we have reported only a single case ; however , a damaged amo emerald c cartridge has been found in a few subsequent cases in our institute . in cases where a back - up cartridge was not available , the size of the clear corneal incision was increased to 3.2 mm at least to protect the entry wound architecture and the corneal endothelium , while injecting the iol using the damaged cartridge . the cause of the damaged nature of the amo emerald c cartridges appears to be a manufacturer 's oversight which has been duly informed to the concerned authorities , who have assured speedy correction of the defect . these cartridges are delivered in sterile transparent cases and the tip can be easily examined under the slit lamp beforehand without opening the casing . this should be made a routine practice as this will allow any damaged cartridge to be replaced before starting the surgery by ordering a fresh one from the manufacturer . we suggest loading of the foldable iol should be done by the surgeon himself under the operating microscope . the speed of the surgery should never compromise the quality of the surgery and/or the final visual outcome . with the advent of newer techniques like the microincision cataract surgery ( mics ) , surgeons also can not compromise on the incision size or wound integrity and architecture . the onus is on the various manufacturing companies to provide surgeons with precision instruments that are both safe and durable for the patient 's eyes . and more importantly , every instrument entering the patient 's eye should undergo careful preoperative microscopic inspection by the operating surgeon himself so that a microscopic manufacturing defect can be identified and immediate rectification of the situation can be done . cytoscape 1 , 2 provides an environment for the visualization and analysis of networks and associated annotations . the primary audience for cytoscape is the biological community and cytoscape supports a number of standard use cases for analyzing and visualizing biological data . many of these use cases involve the selection of a number of nodes or edges based on some analysis or annotation and either performing an action on that selection or comparing those nodes or edges to a different set of nodes or edges that resulted from alternative analyses or analyses based on alternative annotations . the core capabilities for cytoscape provide some tools to facilitate these types of comparisons but they can be counterintuitive or complicated to use . setsapp is a cytoscape 3 application that provides a general set of tools for users and developers to define and maintain sets of nodes or edges and compare those sets using the standard set operations of union , intersection , and difference . in this paper , we present the implementation of setsapp , in particular , how setsapp integrates with the cytoscape command system , and then present a sample biological workflow using setsapp . cytoscape provides two approaches to implementing apps : a simple app and a bundle app . simple apps are implemented using the same general approach as in cytoscape 2.8 , but at the cost of significant flexibility . bundle apps utilize open service gateway initiative ( osgi ) interfaces through apis provided by cytoscape to interact with the cytoscape core functionality . there are three main components to the setsapp implementation : the user interface , the command interface , and the underlying data model for maintaining sets of nodes and edges . the setsapp user interface consists of menu items in the main apps menu , node and edge context menus , and a panel added to the control panel ( left or west ) section of the cytoscape user interface . the main feature of the sets panel is the list of currently defined sets . each set can be expanded to see all of the nodes or edges within that set , and context menus provide the ability to select , deselect , rename , or remove sets . when multiple sets are selected , the set operations buttons are enabled . this allows users to create new sets based on the union , intersection , or difference of other sets . note that the results of a union or intersection are well - defined for multiple sets , but the difference operation is order dependent . if more than two sets are selected , the order is the order of selection , so care must be taken when attempting to create a difference set of more than two sets . sets can be created from the currently selected nodes or edges , or based on a particular node or edge attribute . when creating sets from attributes , the user will need to supply a prefix for the sets to be created and choose the attribute ( currently only string attributes are supported ) from a list . in addition to having menu items to manage sets , the user may select all set members in the network , or if the set is expanded , individual members . this functionality presents an easy way for users to visualize the results of set operations and to perform interactive exploratory analysis . the menus provided through the top - level apps menu offer the same functionality as the create set from menu in the setsapp control panel with the addition of a menu to import a set from a file . node and edge context menus provide the user with the ability to add or remove the corresponding node or edge from sets . in addition to the standard user interface described above , setsapp provides a number of commands . these commands may be used for scripting purposes or by other cytoscape apps that wish to take advantage of the setsapp functionality . a command is made available to cytoscape by creating a standard cytoscape taskfactory with two new properties in the org.cytoscape.work package : serviceproperties.command_namespace , which is always set to setsapp ; and serviceproperties.command , which is the command name ( e.g. addto ) . the command arguments are implemented as tunables within the task called by the designated taskfactory . because there is no guarantee that the task will be executed within the context of a gui , care should be taken to make sure that the appropriate tunable types are used . for example , the nodelist tunable allows the command - line user to enter a list of nodes rather than assuming that the user will have selected nodes interactively . for another cytoscape app to use any of these commands , it would need to call one of the cytoscape taskmanagers and provide it org.cytoscape.command.commandexecutortaskfactorys createtaskiterator method with the appropriate argument map , command , and command namespace . getservice ( synchronoustaskmanager . class ) ; commandexecutortaskfactory cetf = serviceregistrar . getservice ( commandexecutortaskfactory . class ) ; map < string , object > argmap = new hashmap < string , object > ( ) ; argmap.put ( " name " , " newset " ) ; // selected is a special keyword for the nodelist tunable argmap.put ( " nodelist " , " selected " ) ; // the current network argmap.put ( " network " , " current " ) ; // assumes that this implements taskobserver tm.execute(cetf.createtaskiterator ( " setsapp " , " createset " , argmap , this ) , this ) ; the main model object for a node or edge set is the set object , which stores a map of all of the nodes or edges in this set . the setsmanager also serves the critical function of serializing the information about sets to the default hidden table ( see cynetwork.hidden_attrs ) for nodes or edges ( depending on the type of the set ) . each set is created as a boolean column in the hidden table which is set to true if the corresponding node or edge is in that set . by storing values in the default hidden tables , the information about sets is automatically saved in cytoscape sessions and restored when sessions are reloaded . setsmanager implements sessionreloadedlistener and recreates the sets from the information stored in the hidden table columns . the setsapp user interface consists of menu items in the main apps menu , node and edge context menus , and a panel added to the control panel ( left or west ) section of the cytoscape user interface . the main feature of the sets panel is the list of currently defined sets . each set can be expanded to see all of the nodes or edges within that set , and context menus provide the ability to select , deselect , rename , or remove sets . when multiple sets are selected , the set operations buttons are enabled . this allows users to create new sets based on the union , intersection , or difference of other sets . note that the results of a union or intersection are well - defined for multiple sets , but the difference operation is order dependent . if more than two sets are selected , the order is the order of selection , so care must be taken when attempting to create a difference set of more than two sets . sets can be created from the currently selected nodes or edges , or based on a particular node or edge attribute . when creating sets from attributes , the user will need to supply a prefix for the sets to be created and choose the attribute ( currently only string attributes are supported ) from a list . in addition to having menu items to manage sets , the user may select all set members in the network , or if the set is expanded , individual members . this functionality presents an easy way for users to visualize the results of set operations and to perform interactive exploratory analysis . the menus provided through the top - level apps menu offer the same functionality as the create set from menu in the setsapp control panel with the addition of a menu to import a set from a file . node and edge context menus provide the user with the ability to add or remove the corresponding node or edge from sets . in addition to the standard user interface described above , setsapp provides a number of commands . these commands may be used for scripting purposes or by other cytoscape apps that wish to take advantage of the setsapp functionality . a command is made available to cytoscape by creating a standard cytoscape taskfactory with two new properties in the org.cytoscape.work package : serviceproperties.command_namespace , which is always set to setsapp ; and serviceproperties.command , which is the command name ( e.g. addto ) . the command arguments are implemented as tunables within the task called by the designated taskfactory . because there is no guarantee that the task will be executed within the context of a gui , care should be taken to make sure that the appropriate tunable types are used . for example , the nodelist tunable allows the command - line user to enter a list of nodes rather than assuming that the user will have selected nodes interactively . for another cytoscape app to use any of these commands , it would need to call one of the cytoscape taskmanagers and provide it org.cytoscape.command.commandexecutortaskfactorys createtaskiterator method with the appropriate argument map , command , and command namespace . getservice ( synchronoustaskmanager . class ) ; commandexecutortaskfactory cetf = serviceregistrar . getservice ( commandexecutortaskfactory . class ) ; map < string , object > argmap = new hashmap < string , object > ( ) ; argmap.put ( " name " , " newset " ) ; // selected is a special keyword for the nodelist tunable argmap.put ( " nodelist " , " selected " ) ; // the current network argmap.put ( " network " , " current " ) ; // assumes that this implements taskobserver tm.execute(cetf.createtaskiterator ( " setsapp " , " createset " , argmap , this ) , this ) ; the main model object for a node or edge set is the set object , which stores a map of all of the nodes or edges in this set . the setsmanager also serves the critical function of serializing the information about sets to the default hidden table ( see cynetwork.hidden_attrs ) for nodes or edges ( depending on the type of the set ) . each set is created as a boolean column in the hidden table which is set to true if the corresponding node or edge is in that set . by storing values in the default hidden tables , the information about sets is automatically saved in cytoscape sessions and restored when sessions are reloaded . setsmanager implements sessionreloadedlistener and recreates the sets from the information stored in the hidden table columns . a simple example use case might be the exploration of the data set from ideker et al . , 2001 , which measured the change in expression for 331 genes after a systematic deletion of genes known to be involved in the saccharomyces cerevisiae switch to galactose metabolism . this data was combined with known protein - protein and protein - dna interactions to explore the biological response to deletions in the presence ( the g in the column names ) or absence of galactose in the medium . this data set is now included as a sample with cytoscape downloads ( galfiltered.cys ) . in our workflow we use cytoscape s select panel to select all proteins that are underexpressed ( gal1rgexp < -0.5 fold change ) in the deletion of gal1 ( figure 2 ) . that selection is saved as a set named gal1- ( apps setsappcreate node set ) . we then select all of the proteins that are overexpressed ( gal1rgexp > 0.5 fold change ) in the deletion of gal1 and save that selection as a set named gal1 + . repeating this for gal4 ( column gal4rgexp ) and gal80 ( column gal80rexp ) results in 6 sets altogether : gal1 + , gal1- , gal4 + , gal4- , gal80 + , gal80- ( figure 3 ) . note that the data for gal1 and gal4 is in the presence of galactose , but the data for gal80 is in the absence of galactose since gal80 is a known repressor of gal4 . given those six sets of genes , we can explore the data sets by looking at combinations of the sets . for example , we could look at the intersection of all of the underexpressed proteins by selecting each of gal1- , gal4- , and gal80- in the sets panel and pressing the intersection button in the set operations box near the bottom of the panel . if we name the resulting set gal- we see that it contains a single gene : arg1 . in this data set of 331 genes , only this one gene is repressed for all three of the deleted gal genes . in the absence of galactose when gal80 is deleted , arg1 is underexpressed , and in the presence of galactose when either gal1 or gal4 are deleted the gene is also underexpressed . looking at the expression significance values in the node table panel of cytoscape ( gal1rgsig , gal4rgsig , and gal80rsig ) this is a highly significant result , although there is no direct correlation between the galactose switch and arginine biosynthesis regulation that we were able to find in the literature . on the other hand , arg1 is regulated by the gcn4 activator which is known to repress protein synthesis during periods of stress or starvation , which explains the significant down - regulation of arg1 . we can perform a similar analysis to understand the consistent up - regulation of the five genes in the gal+ set : gip1 , nce103 , yig1 , pot1 , and icl1 . gal+ and gal- were created by performing the intersection of all of the + and - sets , respectively . there are many cytoscape workflows that could take advantage of the features of setsapp . any workflow that might want to look for groups of nodes or edges that share multiple traits , or that explicitly do not share those traits . while it is possible to duplicate many of the final results enabled by setsapp by using cytoscape 3.1 s new select panel , a user would need to know in advance exactly the combination of features that were biologically interesting . setsapp provides an alternative that allows users to explore various combinations of nodes and edges and to save such selections for later uses . in the workflow we developed above , we combined the functionality of cytoscape s select panel with setsapp to explore combinations of sets of genes based on shared properties . there are many more sophisticated apps available to cytoscape users that could be used to do a more thorough analysis of this data set including jactivemodules , clustermaker and rinalyzer , however , the workflow above demonstrates the utility of a simple set - oriented approach to exploring networks . source code as at the time of publication : https://github.com/f1000research/setsapp archived code as at the time of publication : http://www.dx.doi.org/10.5281/zenodo.10424 license : lesser gnu public license 3.0 : https://www.gnu.org/licenses/lgpl.html

foldable intraocular lens ( iol ) implantation using an injector system through 2.8-mm clear corneal incision following phacoemulsification provides excellent speedy postoperative recovery . in our reported case , a sensar ar40e iol ( abbott medical optics , usa ) was loaded into emerald c cartridge , outside the view of the operating microscope , by the first assistant . the surgeon proceeded with the iol injection through a 2.8-mm clear corneal incision after uneventful phacoemulsification , immediately following which he noted a descemet 's tear with a rolled out flap of about 2 mm near the incision site . gross downward beaking of the bevelled anterior end of the cartridge was subsequently noticed upon examination under the microscope . we suggest careful preoperative microscopic inspection of all instruments and devices entering the patient 's eyes to ensure maximum safety to the patient . setsapp ( http://apps.cytoscape.org/apps/setsapp ) is a relatively simple cytoscape 3 app for users to handle groups of nodes and/or edges . it supports several important biological workflows and enables various set operations . setsapp provides basic tools to create sets of nodes or edges , import or export sets , and perform standard set operations ( union , difference , intersection ) on those sets . the sets functionality is also exposed to users and app developers in the form of a set of commands that can be used for scripting purposes or integrated in other cytoscape apps .

each year , 200 000 inhospital cardiac arrests ( ihca ) occur in the united states , with overall survival rates of 18% to 20% . however , the extent to which hospitals vary in survival rates for ihca remains poorly understood , as prior studies have largely reported aggregate survival rates at the patient level or have had a limited sample of hospitals , and most studies have lacked detailed information on patient factors to ensure adequate casemix adjustment . furthermore , the extent of sitelevel variation in neurologic status among survivors an outcome of great importance to many patients , their family members , and providers remains unknown . defining variability in survival rates , and examining the degree to which variation persists even after adjusting for patient casemix , would provide important insights into which hospitals could improve resuscitation care and offer opportunities to identify best practices at these hospitals . if significant variability in survival exists even after adjusting for patient casemix , this may suggest that resuscitation process factors ( eg , time to defibrillation , quality of cardiopulmonary resuscitation ) and quality improvement initiatives may be the primary reasons for substantially higher survival rates in topperforming hospitals . moreover , it would suggest a greater urgency to develop appropriate research methodology to identify which factors are associated with best practices at hospitals with the highest ihca survival . accordingly , within the american heart association 's get with the guidelinesresuscitation ( gwtgr ) , a large national inhospital cardiac arrest registry , we examined the extent to which hospitals varied in rates of survival to discharge among patients with ihca . gwtgr is an ideal data source , as it contains ihca data from over 400 us hospitals and collects detailed information on a number of patient and cardiac arrest characteristics to ensure robust casemix adjustment in a specific patient cohort . this was a multicenter cohort study using data from gwtgr to calculate hospital riskadjusted survival rates for ihca . gwtgr is sponsored by the american heart association ( aha ) and is a large , comprehensive , prospective national database of ihca in the us . this dataset also has information from outcome , a quintiles company which is the data collection and coordination center for the gwtgr program . data are collected from participating acute care hospitals according to standardized utstein definitions for ihca . specially trained research coordinators at each facility routinely abstract detailed data from the medical chart about resuscitation events if the event elicits an inhospital emergency resuscitation response and a code record is generated . these data include pre , intra , and postarrest variables , as well as survival at discharge . the aha provides rigorous quality control and oversight for all gwtgr data collection , analysis , reporting , and research studies . the study population included all adult patients 18 years of age or older enrolled within the registry between january 1 , 2001 and december 31 , 2010 . we excluded ihca events in individuals who were not hospital patients , including hospital visitors or employees ( n=293 ) . since patients may have had recurrent ihca events , we focused on the index ihca and excluded 26 190 recurrent arrests . as our study objective was to examine hospital variation in survival outcomes , we excluded 872 ihca events from 94 hospitals with a case volume of fewer than 20 ihcas . this figure illustrates the exclusion and inclusion criteria applied to the get with the guidelinesresuscitation dataset . these factors are important to control for , as older and sicker patients are known to have worse outcomes after inhospital arrest . therefore , the following patientlevel factors were evaluated for model inclusion : age , sex , race , residence prior to hospitalization , and prearrest comorbidities ( neurologic conditions , arrhythmia , myocardial infarction , congestive heart failure , diabetes , pneumonia , respiratory insufficiency , hepatic insufficiency / failure , renal insufficiency / failure , hypotension / hypoperfusion , hematology / oncology conditions , major trauma , and septicemia ) . additionally , available arrest characteristics were collected in gwtgr and included arrest etiology , presenting rhythm , event location , prearrest inplace interventions , and time and day of arrest . several hospital factors have previously been identified as important predictors of ihca outcomes yet these factors are not often accounted for in multicenter studies of ihca . modifiable hospitallevel factors that are associated with outcomes are particularly important as they can reveal opportunities for improvement . structural variables included in our analysis were : bed size ( small ; 6 to 99 beds , medium ; 100 to 400 , and large ; greater than 400 ) , academic status , and urban compared with rural location . these variables are included in gwtgr and are adapted from the american hospital association annual survey . the primary outcome measure was survival at hospital discharge . in this study , we calculated riskadjusted hospital rates for these outcomes . moreover , since several hospital factors have previously been identified as important predictors of ihca outcomes , we examined how ( patientlevel ) riskadjusted survival rates varied by hospital bed size , academic status , and urban designation . the unadjusted ihca survival rate for each hospital was calculated , and hospital variation in unadjusted rates of survival was examined by deciles . we then used multivariable hierarchical regression models to calculate riskadjusted hospital rates of survival to discharge . these included the previously described demographic variables ( age , race , gender , residence prior to admission ) , comorbidities , intraarrest factors ( eg , initial rhythm , inplace interventions ) , and arrest factors ( eg , etiology , location , time ) . the year of arrest was also included to control for temporal trends in survival . from the model , we used the model coefficients to calculate the predicted survival probability for each patient . these probabilities were summed within each hospital to then produce a hospital survival rate . for illustrative purposes , the survival rates for hospitals in each decile were compared . moreover , we examined survival rates ( riskadjusted for patient level factors ) across hospital deciles after stratifying the sample by bed size , academic status , and urban designation . finally , we quantified the extent of variation in riskadjusted survival across hospitals in the registry using the median odds ratio ( mor ) , which was derived from the hierarchical model after adjusting for patient factors as follows : the mor is estimated from hierarchical models and includes only patientlevel factors . the mor describes the likelihood that patients with similar measured covariates would have different outcomes at randomly chosen hospitals . a mor of 1.5 suggests that a patient would have a 50% greater odds of a particular outcome at a randomly identified hospital when compared with a similar patient . this was a multicenter cohort study using data from gwtgr to calculate hospital riskadjusted survival rates for ihca . gwtgr is sponsored by the american heart association ( aha ) and is a large , comprehensive , prospective national database of ihca in the us . this dataset also has information from outcome , a quintiles company which is the data collection and coordination center for the gwtgr program . data are collected from participating acute care hospitals according to standardized utstein definitions for ihca . specially trained research coordinators at each facility routinely abstract detailed data from the medical chart about resuscitation events if the event elicits an inhospital emergency resuscitation response and a code record is generated . these data include pre , intra , and postarrest variables , as well as survival at discharge . the aha provides rigorous quality control and oversight for all gwtgr data collection , analysis , reporting , and research studies . the study population included all adult patients 18 years of age or older enrolled within the registry between january 1 , 2001 and december 31 , 2010 . we excluded ihca events in individuals who were not hospital patients , including hospital visitors or employees ( n=293 ) . since patients may have had recurrent ihca events , we focused on the index ihca and excluded 26 190 recurrent arrests . as our study objective was to examine hospital variation in survival outcomes , we excluded 872 ihca events from 94 hospitals with a case volume of fewer than 20 ihcas . this figure illustrates the exclusion and inclusion criteria applied to the get with the guidelinesresuscitation dataset . these factors are important to control for , as older and sicker patients are known to have worse outcomes after inhospital arrest . therefore , the following patientlevel factors were evaluated for model inclusion : age , sex , race , residence prior to hospitalization , and prearrest comorbidities ( neurologic conditions , arrhythmia , myocardial infarction , congestive heart failure , diabetes , pneumonia , respiratory insufficiency , hepatic insufficiency / failure , renal insufficiency / failure , hypotension / hypoperfusion , hematology / oncology conditions , major trauma , and septicemia ) . additionally , available arrest characteristics were collected in gwtgr and included arrest etiology , presenting rhythm , event location , prearrest inplace interventions , and time and day of arrest . several hospital factors have previously been identified as important predictors of ihca outcomes yet these factors are not often accounted for in multicenter studies of ihca . modifiable hospitallevel factors that are associated with outcomes are particularly important as they can reveal opportunities for improvement . structural variables included in our analysis were : bed size ( small ; 6 to 99 beds , medium ; 100 to 400 , and large ; greater than 400 ) , academic status , and urban compared with rural location . these variables are included in gwtgr and are adapted from the american hospital association annual survey . the primary outcome measure was survival at hospital discharge . in this study , we calculated riskadjusted hospital rates for these outcomes . moreover , since several hospital factors have previously been identified as important predictors of ihca outcomes , we examined how ( patientlevel ) riskadjusted survival rates varied by hospital bed size , academic status , and urban designation . the unadjusted ihca survival rate for each hospital was calculated , and hospital variation in unadjusted rates of survival was examined by deciles . we then used multivariable hierarchical regression models to calculate riskadjusted hospital rates of survival to discharge . these included the previously described demographic variables ( age , race , gender , residence prior to admission ) , comorbidities , intraarrest factors ( eg , initial rhythm , inplace interventions ) , and arrest factors ( eg , etiology , location , time ) . the year of arrest was also included to control for temporal trends in survival . from the model , we used the model coefficients to calculate the predicted survival probability for each patient . these probabilities were summed within each hospital to then produce a hospital survival rate . for illustrative purposes , the survival rates for hospitals in each decile were compared . moreover , we examined survival rates ( riskadjusted for patient level factors ) across hospital deciles after stratifying the sample by bed size , academic status , and urban designation . finally , we quantified the extent of variation in riskadjusted survival across hospitals in the registry using the median odds ratio ( mor ) , which was derived from the hierarchical model after adjusting for patient factors as follows : the mor is estimated from hierarchical models and includes only patientlevel factors . the mor describes the likelihood that patients with similar measured covariates would have different outcomes at randomly chosen hospitals . a mor of 1.5 suggests that a patient would have a 50% greater odds of a particular outcome at a randomly identified hospital when compared with a similar patient . we evaluated a total of 135 896 inhospital cardiac arrest events at 468 hospitals ( figure 1 ) . of these , 78 787 ( 58% ) were men , 93 945 ( 74% ) were of white race , and 112 877 ( 83% ) were over 50 years of age ( table s1 ) . two in 5 ihca events had ventricular tachycardia or ventricular fibrillation ( vf / vt ) as the presenting rhythm and 86 672 ( 64% ) occurred in an intensive care unit . half , 52% ( 245 ) of all study hospitals were academic teaching hospitals ( 245 ) , 89% ( 419 ) were in urban settings , and 278 ( 59% ) had at least 100 beds . unadjusted rates of survival to discharge varied markedly across hospitals , with a median unadjusted rate for the bottom decile of 8.3% ( range : 0% to 10.7% ) and for the top decile of 31.4% ( range : 28.6% to 51.7% ) ( figure 2 ) . using hierarchical regression , we identified 36 predictors of inhospital survival to discharge ( table 1 ) . among the predictors were key demographic factors including age , female sex ( or 1.10 ; 95% ci : 1.06 to 1.13 , p<0.01 ) , and black race ( or 0.93 ; 95% ci : 0.87 to 0.99 , p=0.03 ) , comorbidities such as arrhythmia ( or 1.24 ; 95% ci : 1.20 to 1.28 , p<0.01 ) and myocardial infarction ( or 1.19 ; 95% ci : 1.14 to 1.24 , p<0.01 ) , and intraarrest factors including cardiac etiology ( or 1.18 ; 95% ci : 1.14 to 1.23 , p<0.01 ) and initial rhythm of vf or vt ( or 3.14 ; 95% ci : 3.02 to 3.27 ) . model predictors for survival to discharge ci indicates confidence interval ; icd , implanted cardioverter defibrillator ; or , odds ratio ; vf / vt , ventricular fibrillation / ventricular tachycardia . this figure illustrates inhospital cardiac arrest ( ihca ) rates ( yaxis ) across hospitals . rates are adjusted for the patientlevel factors identified in table 1 . after applying the model coefficients to patients in the sample , we calculated riskadjusted survival rates for ihca for each hospital . the median adjusted rate was 12.4% ( range : 0% to 15.6% ) for the bottom decile and 22.7% ( range : 21.0% to 36.2% ) for the top decile . of note , of the 46 hospitals in the top decile for ihca survival before risk adjustment , only 24 ( 52% ) remained in the top decile after risk adjustment . to illustrate the extent of hospital variation in rates of survival to discharge , we determined that the median or was 1.42 ( 95% ci : 1.37 to 1.46 ) , which suggests that there was a 42% greater odds of patients with identical covariates surviving to hospital discharge at one randomly selected hospital compared with another . after multivariable adjustment , the median adjusted survival rate was 11.9% ( range : 0% to 14.8% ) for the bottom decile of academic hospitals and 22.8% ( range : 20.8% to 36.2% ) for the top decile . similar patterns of hospital variation were observed for nonacademic hospitals and by hospital bed size and rural versus urban status ( table 2 ) . stratified ( bottom and top decile ) survival rates by hospital type max indicates maximum ; min , minimum . unadjusted rates of survival to discharge varied markedly across hospitals , with a median unadjusted rate for the bottom decile of 8.3% ( range : 0% to 10.7% ) and for the top decile of 31.4% ( range : 28.6% to 51.7% ) ( figure 2 ) . using hierarchical regression , we identified 36 predictors of inhospital survival to discharge ( table 1 ) . among the predictors were key demographic factors including age , female sex ( or 1.10 ; 95% ci : 1.06 to 1.13 , p<0.01 ) , and black race ( or 0.93 ; 95% ci : 0.87 to 0.99 , p=0.03 ) , comorbidities such as arrhythmia ( or 1.24 ; 95% ci : 1.20 to 1.28 , p<0.01 ) and myocardial infarction ( or 1.19 ; 95% ci : 1.14 to 1.24 , p<0.01 ) , and intraarrest factors including cardiac etiology ( or 1.18 ; 95% ci : 1.14 to 1.23 , p<0.01 ) and initial rhythm of vf or vt ( or 3.14 ; 95% ci : 3.02 to 3.27 ) . model predictors for survival to discharge ci indicates confidence interval ; icd , implanted cardioverter defibrillator ; or , odds ratio ; vf / vt , ventricular fibrillation / ventricular tachycardia . this figure illustrates inhospital cardiac arrest ( ihca ) rates ( yaxis ) across hospitals . . rates are adjusted for the patientlevel factors identified in table 1 . after applying the model coefficients to patients in the sample , we calculated riskadjusted survival rates for ihca for each hospital . the median adjusted rate was 12.4% ( range : 0% to 15.6% ) for the bottom decile and 22.7% ( range : 21.0% to 36.2% ) for the top decile . of note , of the 46 hospitals in the top decile for ihca survival before risk adjustment , only 24 ( 52% ) remained in the top decile after risk adjustment . to illustrate the extent of hospital variation in rates of survival to discharge , we determined that the median or was 1.42 ( 95% ci : 1.37 to 1.46 ) , which suggests that there was a 42% greater odds of patients with identical covariates surviving to hospital discharge at one randomly selected hospital compared with another . after multivariable adjustment , the median adjusted survival rate was 11.9% ( range : 0% to 14.8% ) for the bottom decile of academic hospitals and 22.8% ( range : 20.8% to 36.2% ) for the top decile . similar patterns of hospital variation were observed for nonacademic hospitals and by hospital bed size and rural versus urban status ( table 2 ) . stratified ( bottom and top decile ) survival rates by hospital type max indicates maximum ; min , minimum . in a large national registry of ihca , we found substantial variation in survival across hospitals . this variability was not explained by differences in patient casemix or hospital characteristics . given that survival outcomes are of immense importance to patients with ihca , these findings raise questions about the quality of resuscitation care at hospitals in the lower deciles and offer important opportunities to identify best practices at hospitals in the upper deciles . our study extends the work of prior studies of ihca , which largely focused on patientlevel aggregate rates of survival . while important , most of these studies have not examined the influence of the hospital in affecting survival . by employing multilevel hierarchical models to identify patient predictors in this study , we were able to derive robust riskadjusted survival rates for each hospital , which allowed us to examine the extent of sitelevel variation . indeed , we found that the hospital at which one is admitted may influence one 's odds of survival by 42% , an enormous effect considering that this is a hard clinical end points . however , the reason for these large variations in outcomes across hospitals is less clear . these variations in survival may suggest that significant differences in highquality resuscitation care exist among hospitals . top performing hospitals may have better response times for defibrillation , chest compressions , and initiation of acute cardiac life support medications ( eg , epinephrine , vasopressin ) , or may provide higherquality cardiopulmonary resuscitation ( eg , adequate depth , positioning and rate , minimal interruptions ) . these hospitals may also excel in postresuscitation care ( eg , therapeutic hypothermia ) , which arguably is an equally important determinant of survival after ihca . moreover , top performing hospitals may differ in structural variables ( eg , nursing staff ratios ) , training variables ( eg , routine resuscitation simulations , debriefing after ihca events ) , and hospital culture ( eg , leadership , emphasis on quality improvement , differences in duration of cpr)all of which may improve ihca survival outcomes . a second reason for significant variation in hospital survival may be related to differences in care prior to cardiac arrest . it is possible that top performing hospitals were able to achieve high rates of survival because their prearrest systems of care were able to detect physiological decline in patients earlier rather than later . as a result , their ability to respond and treat patients who developed ihca was more timely and effective . presence or absence of rapid response teams and the structure and function of these teams may also be a contributing factor to acrosshospital variation in outcomes . alternatively , it is plausible that hospitals with the highest rates of ihca survival do so because they have a high cardiac arrest incidence rate . therefore , they only appear to perform well with cardiac arrest survival because some patients go on to develop ihca when the event could have been prevented . as the relationship between incidence and outcomes is likely nonlinear , and there are multiple ways that incidence could impact outcomes , this variable was not included in this analyses but could be modeled in future work . finally , hospital differences in ihca survival and may be due to differential application of donotresuscitate orders for all admitted patients . thus , it is possible that the top performing hospitals have higher rates of donotresuscitate orders which would be a good outcome in the right patient and therefore would only appear to have better resuscitation outcomes . in order to better understand the determinants of hospital variation in ihca outcomes , these approaches will require onsite qualitative interviews with hospital administrators and medical and nursing staff to better understand a given site 's resuscitation practices , strategies to monitor and prevent ihca , and hospital culture and leadership . moreover , these analyses can provide insights into whether the success of higher performing hospitals is largely due to more liberal use of donotresuscitate orders . in the past , a mixedmethods approach was critical in differentiating hospitals with better doortoballoon times for stelevation myocardial infarction and identifying core components of top performing hospitals . needed are clear guidelines and policies that hospitals can implement to measure their performance , provide feedback to providers , and modify hospitalwide practices . in order to move the field of inhospital resuscitation science forward , future studies will need to shift from describing hospital variation to identifying and proscribing best practices at top performing hospitals . although our riskadjusted rates were able to control for multiple confounders , the presence of residual confounding in affecting ihca outcomes exists . if rates of unmeasured confounders differed markedly across hospitals , this could affect the magnitude of our hospital variation findings . there may also be additional , more granular patientlevel differences ( eg , laboratory values : prearrest creatinine , prearrest hematocrit , liver function tests ) or differences in data collection between hospitals that are not measurable in the gwtgr registry but may be important to consider in interpreting our reported results . patient and family preferences regarding endoflife care were not collected in gwtgr , nor did we have rates of donotresuscitate orders ( and timing or reason for these orders ) for all hospital admissions . therefore , some of the observed variation in ihca survival may be due to systematic differences in how aggressively hospitals offered guidance to patients on endoflife care . additionally , systematic validated information about neurologic status is not available for all patients in the gwgtr dataset . there may be significant variability in hospitals ' survival to discharge rates and survival to discharge with favorable neurologic outcomes , which could be identified . a better understanding of this important clinical outcome would be important for future work evaluating hospital variability in ihca outcomes . finally , gwtgr is a voluntary registry of hospitals ; therefore , our findings may not be generalizable to all us hospitals . although our riskadjusted rates were able to control for multiple confounders , the presence of residual confounding in affecting ihca outcomes exists . if rates of unmeasured confounders differed markedly across hospitals , this could affect the magnitude of our hospital variation findings . there may also be additional , more granular patientlevel differences ( eg , laboratory values : prearrest creatinine , prearrest hematocrit , liver function tests ) or differences in data collection between hospitals that are not measurable in the gwtgr registry but may be important to consider in interpreting our reported results . patient and family preferences regarding endoflife care were not collected in gwtgr , nor did we have rates of donotresuscitate orders ( and timing or reason for these orders ) for all hospital admissions . therefore , some of the observed variation in ihca survival may be due to systematic differences in how aggressively hospitals offered guidance to patients on endoflife care . additionally , systematic validated information about neurologic status is not available for all patients in the gwgtr dataset . there may be significant variability in hospitals ' survival to discharge rates and survival to discharge with favorable neurologic outcomes , which could be identified . a better understanding of this important clinical outcome would be important for future work evaluating hospital variability in ihca outcomes . finally , gwtgr is a voluntary registry of hospitals ; therefore , our findings may not be generalizable to all us hospitals . although ihca is associated with low survival , significant variation in survival exists across hospitals , even after adjustment for patient casemix . further research is needed to investigate additional patientlevel factors and identify best practices at top performing hospitals so that ihca outcomes can improve at all hospitals . according to the world health organization ( who ) classification of tumors , a grading scheme , which represents a malignancy scale and a key factor influencing the choice of therapies , has been successfully applied to astrocytomas , the most common type of glioma . glioblastoma is the most frequent type of brain tumor and the median survival time is 2 years after diagnosis . at present , no effective treatment has been developed for glioblastoma patients . molecular biomarkers are typically indicators of exposure , effect , or susceptibility . known risk factors , high - dose ionizing radiation , and smoking , account for only a small proportion of cases . recently , genome - wide association studies determined that inherited variants in some chromosomal regions , such as chromosomes 20q13.3 , 5p15.3 , and 11q23.3 , have a significant association with the risk of glioma . although genome - wide association studies ( gwas ) found that some sites have relationships with glioma , these studies are mainly limited to the european populations and there were significant differences between europeans and chinese in genetic background . therefore , we investigated whether the gene polymorphisms contribute to glioblastoma risk in a chinese han population from northwestern china . from october 2011 to september 2012 we recruited 72 gbm patients into an on - going molecular epidemiological study at the department of neurosurgery of the tangdu hospital affiliated with the fourth military medical university in xian , china . tumor histological type and grade were determined based on the who criteria and we successfully genotyped 72 gbm cases for further study . as controls we randomly selected 320 unrelated healthy individuals from the medical center of tangdu hospital from june 2011 to july 2012 according to standard recruitment and exclusion criteria . detailed recruitment and exclusion criteria were used . subjects with chronic diseases and conditions involving vital organs such as the heart , lung , liver , kidney , and brain , and/or had severe endocrinological , metabolic , or nutritional diseases were excluded from this study . all of the control subjects were generally healthy without diseases related to the vital organs and serum levels of alpha - fetoprotein and plasma carcinoembryonic antigen were within normal range . we excluded 18 samples because of missing information , resulting in successful genotyping of 302 healthy control subjects . we obtained demographic and personal data through a face - to - face interview via a standardized epidemiological questionnaire , which including age , sex , ethnicity , residence , smoking status , alcohol drinking , education status , and family history of cancer . in addition , patient clinical information was obtained through a medical record review or consulting treating physicians to understand the patient s condition . the use of human blood sample and the protocol in this study strictly conformed to the principles expressed in the declaration of helsinki and were approved by the institutional ethics committees of tangdu hospital and northwest university . written informed consent was obtained from all participants before their participation in the study . according to the previous glioma association analysis and snps with minor allele frequency ( maf ) greater than 0.05 in the hapmap chb ( han chinese in beijing , china ) population , we picked 29 snps from 21 genes . in genome - wide association studies , if the maf < 5% , some loci variants could not be detected in the samples , so the snps with minor allele frequency ( maf ) greater than 0.05 were used . we isolated genomic dna samples from the whole blood with goldmag - mini purification kit ( goldmag co. ltd . xian city , china ) , and concentrations were measured using a nanodrop 2000 device ( thermo scientific , waltham , massachusetts , usa ) . massarray assay design 3.0 software ( sequenom , san diego , ca , usa ) was used to design the pcr assay and iplex single - base extension primers for the multiplexed snp massextend assay . the snp genotypes were obtained according to the iplex protocol provided by sequenom massarray rs1000 ( sequenom . san diego , california , usa ) and the sequenom typer 4.0 software was used for data analysis . the chi - squared test was used to compare the differences in frequency distributions of genotypes and alleles between cases and controls . hardy - weinberg equilibrium was assessed using a pearson chi - squared test only among controls at the 1% level . odds ratios ( ors ) and corresponding 95% confidence intervals ( 95% ci ) were obtained by binary logistic regression analysis , which adjusted for age and sex . the possibility of sex differences was evaluated by a genotype test for each tsnp in males and females separately . we adopted the snp stats ( website software from http://bioinfo.iconcologia.net/snpstats/start.htm ) to analyze the association of certain single - nucleotide polymorphism loci contributed to the glioblastoma risk under variant models . we used the akaike s information criterion ( aic ) and bayesian information criterion ( bic ) to select the best - fit model for each snp . all p values presented were calculated based on a 2-sided test , and p<0.05 was considered significant . from october 2011 to september 2012 we recruited 72 gbm patients into an on - going molecular epidemiological study at the department of neurosurgery of the tangdu hospital affiliated with the fourth military medical university in xian , china . tumor histological type and grade were determined based on the who criteria and we successfully genotyped 72 gbm cases for further study . as controls we randomly selected 320 unrelated healthy individuals from the medical center of tangdu hospital from june 2011 to july 2012 according to standard recruitment and exclusion criteria . detailed recruitment and exclusion criteria were used . subjects with chronic diseases and conditions involving vital organs such as the heart , lung , liver , kidney , and brain , and/or had severe endocrinological , metabolic , or nutritional diseases were excluded from this study . all of the control subjects were generally healthy without diseases related to the vital organs and serum levels of alpha - fetoprotein and plasma carcinoembryonic antigen were within normal range . we excluded 18 samples because of missing information , resulting in successful genotyping of 302 healthy control subjects . we obtained demographic and personal data through a face - to - face interview via a standardized epidemiological questionnaire , which including age , sex , ethnicity , residence , smoking status , alcohol drinking , education status , and family history of cancer . in addition , patient clinical information was obtained through a medical record review or consulting treating physicians to understand the patient s condition . the use of human blood sample and the protocol in this study strictly conformed to the principles expressed in the declaration of helsinki and were approved by the institutional ethics committees of tangdu hospital and northwest university . written informed consent according to the previous glioma association analysis and snps with minor allele frequency ( maf ) greater than 0.05 in the hapmap chb ( han chinese in beijing , china ) population , we picked 29 snps from 21 genes . in genome - wide association studies , if the maf < 5% , some loci variants could not be detected in the samples , so the snps with minor allele frequency ( maf ) greater than 0.05 were used . we isolated genomic dna samples from the whole blood with goldmag - mini purification kit ( goldmag co. ltd . xian city , china ) , and concentrations were measured using a nanodrop 2000 device ( thermo scientific , waltham , massachusetts , usa ) . massarray assay design 3.0 software ( sequenom , san diego , ca , usa ) was used to design the pcr assay and iplex single - base extension primers for the multiplexed snp massextend assay . the snp genotypes were obtained according to the iplex protocol provided by sequenom massarray rs1000 ( sequenom . san diego , california , usa ) and the sequenom typer 4.0 software was used for data analysis . the chi - squared test was used to compare the differences in frequency distributions of genotypes and alleles between cases and controls . hardy - weinberg equilibrium was assessed using a pearson chi - squared test only among controls at the 1% level . odds ratios ( ors ) and corresponding 95% confidence intervals ( 95% ci ) were obtained by binary logistic regression analysis , which adjusted for age and sex . the possibility of sex differences was evaluated by a genotype test for each tsnp in males and females separately . we adopted the snp stats ( website software from http://bioinfo.iconcologia.net/snpstats/start.htm ) to analyze the association of certain single - nucleotide polymorphism loci contributed to the glioblastoma risk under variant models . we used the akaike s information criterion ( aic ) and bayesian information criterion ( bic ) to select the best - fit model for each snp . all p values presented were calculated based on a 2-sided test , and p<0.05 was considered significant . table 1 shows the basic information of candidate snps in our study such as chromosome position and minor allele frequency in case and control groups . we used the chi - squared test to assess the influence of gene polymorphism of gbm risk in the allele model , and found that 2 snps significantly increased gbm risk : rs2297440 [ rtel1 ( regulator of telomere elongation helicase 1 ; omim 608833 ) , or=1.72 , 95% ci : 1.172.52 , p<0.01 ] and rs6010620 [ rtel1 , or=1.72 , 95% ci : 1.182.52 , p<0.01 ) ] ( table 1 ) . genetic model analysis found that the variant genotype tt of rs17748 in treh ( a , a - trehalose-1-d - glucohydrolase ; trehalase ) phldb1 ( pleckstrin homology - like domain , family b , member 1 ) decreased the risk of gbm in the recessive model ( or=0.24 , 95% ci : 0.061.05 , p=0.02 ) ( table 2 ) . the variant genotype tt of rs498872 in phldb1 also correlated with a decreased risk in the recessive model ( or=0.14 , 95% ci : 0.021.09 , p=0.01 ) ( table 2 ) . conversely , individuals carrying the variant cc genotype of rs2297440 in rtel1 had higher gbm risk than those carrying tt and tc genotype in the recessive model ( or=7.46 , 95% ci : 2.9119.12 , p<0.01 ) . the genotype gg of rs6010620 in rtel1 also showed an increased risk in the recessive model ( or=7.72 , 95% ci : 3.0619.51 , p<0.01 ) ( table 2 ) . we further sought to determine whether any of the 29 snps had a sex - specific effect on gbm risk , and found that allele c of rs17748 in treh increased gbm risk in males ( or=4.10 , 95% ci : 1.968.59 , p=0.04 ) in the dominant model ( table 3 ) . in this case - control study we genotyped 29 snps in the han chinese population and identified rtel1rs2297440 and rs6010620 , trehrs17748 and phldb1 rs498872 are potentially associated with gbm . c of rs17748 in the treh gene showed an increased risk in males in the dominant model . rtel1 encodes a dna helicase that plays a crucial role in regulating telomere length in mice . based on these observations , rtel1 dysfunction appears to be closely related to the incidence of cancer . moreover , rtel1 plays an important role in maintaining genomic stability by suppressing homologous recombination and is a key protein in the repair of double - strand breaks ( dsbs ) through direct involvement in the dsb repair ( dsbr ) pathway . dsbr plays a prominent role in cell survival , maintenance of genomic integrity , and prevention of tumorigenesis . our results suggest that polymorphisms of the rtel1 gene may influence the risk of gbm in the han chinese population . moreover , genome - wide association studies have shown that rs6010620 and rs2297440 in 20q13.33 ( rtel1 ) are related to glioma risk in the european population . reported that rs6010620 is associated with susceptibility to high - grade glioma , and egan et al . showed that snps of rtel1 are associated with both low- and high - grade astrocytic tumors . thus , rtel1 may play complex roles in the development of gliomas of different origins . 20 phldb1 was expressed in all tissues examined , with the highest expression in ovary , brain , lung , and kidney . phldb1 protein contains an n - terminal phosphorylation - dependent forkhead - associated protein interaction domain , a central chromosome segregation atpase domain , and a c - terminal pleckstrin homology ( ph ) domain . studies have shown that the ph domain can bind pi(3,4,5)p3 and that phldb1 functions in adipocytes as a positive regulator of akt activation , where it is required for optimal insulin - induced glucose transport and glut4 translocation . it has been reported that phldb1 is an insulin - responsive protein and enhances akt activation . however , there are few studies of its potential regulatory or growth promoting activities with respect to glioma genesis . this snp was also reported to be associated with a change in diastolic blood pressure . in our study , we also found that phldb1 rs498872 is associated with gbm in the han chinese population . in support of this , association between glioma risk and rs498872 was recently identified in a genome - wide association study . this association was confirmed in other studies , including a previous study in a chinese han population [ 2224 ] . therefore , although phldb1 is associated with low - grade glioma , there is currently no direct functional evidence for a role of phldb1 in initiation of gbm . research on the properties of renal and urinary human trehalase found that expression of trehalase increased damage to renal tubes . therefore , it is a useful marker of renal proximal tubular damage . to the best of our knowledge , the present study is the first to show a relationship between treh rs17748 and gbm susceptibility . our findings and those of previous studies suggest that polymorphisms of particular genes play a role in gbm development . these findings should be taken into consideration in future research of genes causing disease susceptibility . due to the low incidence , based on the limitation of the present study , larger - sample studies are warranted to confirm our findings . the exact functions of these genes in gbm and the regulatory mechanisms for gene expression have not been elucidated and need to be further investigated .

backgroundinhospital cardiac arrest ( ihca ) is common and often fatal . however , the extent to which hospitals vary in survival outcomes and the degree to which this variation is explained by patient and hospital factors is unknown.methods and resultswithin get with the guidelinesresuscitation , we identified 135 896 index ihca events at 468 hospitals . using hierarchical models , we adjusted for demographics comorbidities and arrest characteristics ( eg , initial rhythm , etiology , arrest location ) to generate riskadjusted rates of inhospital survival . to quantify the extent of hospitallevel variation in riskadjusted rates , we calculated the median odds ratio ( or ) . among study hospitals , there was significant variation in unadjusted survival rates . the median unadjusted rate for the bottom decile was 8.3% ( range : 0% to 10.7% ) and for the top decile was 31.4% ( 28.6% to 51.7% ) . after adjusting for 36 predictors of inhospital survival , there remained substantial variation in rates of inhospital survival across sites : bottom decile ( median rate , 12.4% [ 0% to 15.6% ] ) versus top decile ( median rate , 22.7% [ 21.0% to 36.2% ] ) . the median or for riskadjusted survival was 1.42 ( 95% ci : 1.37 to 1.46 ) , which suggests a substantial 42% difference in the odds of survival for patients with similar casemix at similar hospitals . further , significant variation persisted within hospital subgroups ( eg , bed size , academic).conclusionsignificant variability in ihca survival exists across hospitals , and this variation persists despite adjustment for measured patient factors and within hospital subgroups . these findings suggest that other hospital factors may account for the observed sitelevel variations in ihca survival . backgroundglioblastoma ( gbm ) is a highly invasive , aggressive , and incurable brain tumor . genetic factors play important roles in gbm risk . the aim of this study was to elucidate the influence of gene polymorphism on gbm susceptibility.material/methodsin this case - control study , we included 72 gbm patients and 320 healthy controls to analyze the association between 29 single - nucleotide polymorphisms and gbm cancer risk in the chinese han population . the single - nucleotide polymorphisms were determined by sequenom massarray rs1000 and statistical analysis was performed using spss software and snpstats software.resultsusing the 2 test , we found that rs2297440 and rs6010620 in rtel1 increased risk of gbm . in the recessive model , we also found that the genotypes cc of rs2297440 and gg of rs6010620 in rtel1 significantly increased gbm risk . the variant tt genotype of treh rs17748 and the variant tt genotype of phldb1 rs498872 decreased gbm risk in the recessive model . we also found that the treh rs17748 variant c allele showed an increased risk in males in the dominant model.conclusionsour results suggest a significant association between the retl1 , treh , and phldb1 genes and gbm development in the han chinese population .

caregiver is an individual who has the responsibility of meeting the physical and psychological needs of the dependent patient . psychiatric patients need assistance or supervision in their daily activities and this often places a major burden on their caregivers , thereby placing the caregiver at a great risk of mental and physical health problems . the term caregiver burden is used to describe the physical , emotional and financial toll of providing care . as the disease progresses , it carries with it a tremendous increase of burden on the caregiver who does the caregiving . the burden perceived by caregivers of patients with psychiatric illness is a fundamental prognostic aspect as the caregiver burden is reportedly a critical determinant for negative caregiving outcomes . the burden upon caregivers for a mentally ill patient living at home was first acknowledged by grad and sainbury in the early 1960s . the most common mental health consequences identified are depression , anxiety and burnout which occurs when a caregiver slips beyond exhaustion or depression . studies conducted showed that caregivers reported burden in different areas including effects on family functioning , social isolation , financial problems , and health . most of the notable community - based studies proved that 1847% of caregivers land in depression . it is also known that caring for someone with psychiatric illness is associated with a higher level of stress than caring for someone with functional impairment from other chronic medical illnesses . many authors opine that the level of burden does not correlate with the duration of illness , but has enough variability with age , gender and educational status . a previous study concluded that poor social support and severity of illness have major role in determining the amount of burden on a caregiver . the burden perceived by caregivers of patients with psychiatric illness is a fundamental prognostic aspect in the history of the disease , and the caregiver burden is reportedly a critical determinant for negative caregiving outcomes . caregiver burden is categorized in terms of objective burden ( ob ) , subjective burden ( sb ) and demand burden ( db ) . ob is defined as the extent of disruptions or changes in various aspects of the caregivers life and household . sb is defined as the caregivers attitude or emotional reactions to the caregiving experience . db measures the extent to which the caregiver feels the responsibilities are overly demanding . the assessment of burden has become a challenging task for most researchers because cultural , ethical , religious and other personal values may influence perceptions of meaning and consequences of burden . there is a paucity of data comparing the caregiver burden of psychiatric patients to that of chronic medical illness . the objective of this study is to evaluate the contrast between the caregiver burden of psychiatric and chronic medical illness patients and to study the association between demographic factors like age , gender , duration of care - giving and caregiver burden . the study sample consisted of caregivers of psychiatric inpatients admitted in the government hospital for mental care who were diagnosed as per the guidelines given in icd-10 . the psychiatric sample included schizophrenia ( 22 cases ) , mood disorder ( 16 cases ) and substance abuse ( 14 cases ) , and caregivers of patients suffering from chronic medical illnesses such as refractory tuberculosis , chronic bronchial asthma , diabetes mellitus , rheumatoid arthritis , and lodged in the king george hospital and tuberculosis , chest disease hospitals located in visakhapatnam . the caregivers were classified as group 1 ( g1 ) consisting of 50 caregivers of psychiatric patients and group 2 ( g2 ) consisting of appropriately matched caregivers of patients suffering from chronic medical illness . caregivers aged more than 20 years and who were staying with the patient since the onset of illness were included . the caregivers aged less than 20 years and those who were not blood related to the patients were excluded from the study . the caregivers were subjected to the montgomery borgatta caregiver burden scale questionnaire which was applied as a self - administered questionnaire in english to those who could understand it , and to the rest of the participants it was administered by interview technique in which the interviewer translated the questionnaire into the vernacular language in which both the patient and the interviewer were well versed . this is a 14-item questionnaire with 5 item responses to each question ; each response was given a score from 0 to 5 . of the 14 items in the questionnaire , a set of 6 questions measure ob , 4 questions measure sb and 4 questions measure db . montgomery borgatta caregiver burden scale the obtained data were tabulated and analyzed with descriptive statistics , levene 's test for equality of variance and t test ( spss software version 13.0 ) . caregivers aged more than 20 years and who were staying with the patient since the onset of illness were included . the caregivers aged less than 20 years and those who were not blood related to the patients were excluded from the study . the caregivers were subjected to the montgomery borgatta caregiver burden scale questionnaire which was applied as a self - administered questionnaire in english to those who could understand it , and to the rest of the participants it was administered by interview technique in which the interviewer translated the questionnaire into the vernacular language in which both the patient and the interviewer were well versed . this is a 14-item questionnaire with 5 item responses to each question ; each response was given a score from 0 to 5 . of the 14 items in the questionnaire , a set of 6 questions measure ob , 4 questions measure sb and 4 questions measure db . the obtained data were tabulated and analyzed with descriptive statistics , levene 's test for equality of variance and t test ( spss software version 13.0 ) . of the 100 caregivers included in the study , 44 were males and 56 were females , with an age range of 2060 years . tables 2 and 3 show the distribution of sample in demographic variables such as literacy level and duration of care giving respectively . the two samples that is psychiatric illness and chronic medical illness have almost similar characteristics in the literacy rate and duration of care giving . literacy level of care givers duration of care giving the montgomery borgatta caregiver burden scale scores in the caregivers of psychiatric patients were significantly higher than that of chronic medical illness caregivers with a p value of less than 0.0001 ( p<0.0001 ) in all the categories of caregiver burden , i.e. ob , sb and db [ table 4 and figure 1 ] . comarision of various care giver burdens categories psychiatric illness burden categories caregiver burden in psychiatric illness was observed to increase with the age of the caregiver except in db which increased up to a certain age and then decreased . the difference in caregiver burden among different age groups of psychiatric patient and chronic medical illness caregivers is also statistically significant with p value of less than 0.0001 ( p<0.0001 ) [ table 5 and figure 2 ] . association of care giver burden with age of care giver psychiatric illness caregiver burden with age of caregiver there was a minor caregiver burden variation with the gender of caregiver , with the burden being higher among females ( n=44 ) for sb and ob and among males for db ( n=56 ) . the difference observed is not statistically significant with p=0.4694 for ob , p=0.3851 for sb and p=0.7319 for db [ table 6 ] . association of care giver burden with gender the caregiver burden in objective domain for psychiatric illness patients was observed to increase with the duration of care - giving till a certain period and then decreased . the difference in caregiver burden for various duration categories among psychiatric illness and chronic medical illness is statistically significant with p value less than 0.0001 ( p<0.0001 ) [ table 7 and figure 3 ] . our analysis shows that caregiver burden in our population is less as the ob which measures the disruption of the caregiver 's life and the db which measures the extent to which the caregiver feels care responsibilities are overly demanding do not cross the reference high value in the given scale , whereas sb which measures the emotional impact of caregiving responsibilities on a caregiver is on the higher side . a study conducted in 2004 by erie county department of senior service caregiver resource centre showed very high values of burden than those in this study . these observations can be explained by the presence of intimate family relations and social support in the indian culture . the results of this study comparing caregiver burden for caregivers of psychiatric illness patients and chronic medical illness patients are in agreement with those of other similar studies and show more caregiver burden for caregivers of psychiatric illness patients . these observations may be due to the varied nature of psychiatric illness and the bizarre and inappropriate behavior of the psychiatrically ill , as these factors disrupt the relation between the caregiver and the patient . in spite of the adequate care , affection and support given by the caregiver moreover , in some cases , the patient may even suspect the motives of the caregiver . these aspects of psychiatric illness further take a toll and worsen the burden on the caregiver . the varied mood swings , disturbed sleep pattern , aggressive and violent outbursts are some other aspects of illness that may further worsen the burden of caregiver . lastly , the personal , social and occupational deterioration in the patient 's life may reflect negatively on the burden experienced by the caregiver . surprisingly , the level of burden did not correlate with the gender of caregiver , but had enough variability with the age of caregiver and duration of caregiving . in this study , the results show that caregiver burden in objective and subjective domains increases with increase in age of the caregiver . the ability of the caregiver to cope up with the physical and mental stress starts diminishing with increase in age due to added responsibilities of the caregivers related to their career , family and their own health , whereas the db increases to a certain age and then decreases as the caregiver understands the disease process better after a certain age and is better adapted to the demands of the patient and his / her illness . this study shows that there is only little difference in caregiver burden among female and male caregivers , with the ob and sb being higher in females while the db is slightly higher in males , but these observations are not statistically significant . the caregiver burden in terms of ob was observed to increase with the duration of caregiving up to a certain extent but subsequently it decreased as the caregivers became less sensitive and began to modify their lifestyle . the sb which mainly measures the emotional impact on caregivers increased with the duration of illness , and db also showed a similar pattern of increase with the duration of illness . as all people in the sample were not educated and there was no rating scale measuring caregiver burden in vernacular language , the scale had to be translated into local language , which can be a confounding factor . the sample did not include dementia in which the caregiver burden is very high . as all people in the sample were not educated and there was no rating scale measuring caregiver burden in vernacular language , the scale had to be translated into local language , which can be a confounding factor . the caregiver burden in families of psychiatric clients is statistically higher than that of caregivers of other medical illnesses . the caregiver burden increases with the duration of illness as well as with the age of caregivers . the caregiver burden in our population was less as the ob and db did not cross the reference higher value in the given scale , whereas the emotional impact measured by sb was on the higher side . the various means to reduce ob are to improve home care , adult day care , routine friendly visits and use of assistive equipment . sb can be reduced by professional counselling and by the assistance of support groups , while db can be lowered by professional counselling and educating more about the disease . anesthesia providers routinely care for patients undergoing magnetic resonance imaging ( mri ) , and the implications for patient care are well known . however , there is scant literature on the occupational hazards of mri for health care workers . it is well known in the radiology community that exposure to a magnetic field of sufficient strength from an mri scanner can cause vertigo , though the practical considerations for health care workers involved in direct patient care are not well established.1 in the 3 months following introduction of a 3 t mri scanner ( replacing a 1.5 t scanner ) at our institution , there were three incidents of vertigo reported by our anesthesia providers , unseen during years of standard mri anesthesia practice . herein , we describe one such case of an anesthetist experiencing acute vertigo while caring for a patient undergoing a scan in a 3 t mri machine . we then discuss the incidence , mechanism , risk factors , and practical implications of this phenomenon . a 52-year - old male nurse anesthetist with no significant past medical history was caring for a patient undergoing an mri in a 3 t scanner ( magnetom skyra ; siemens healthcare , erlangen , germany ) under sedation with a propofol infusion . on two occasions , he had to approach and lean toward the mri bore to assess the patient s airway patency . during these events , there was no headache , ocular complaint , tinnitus , or other subjective neurologic deficit . he did not lose his balance or become nauseated though the vertigo was so intense that he felt he needed to sit down . the sensation lasted for 1520 seconds and resolved when he stopped moving and sat down in a chair . the anesthetist had no history of vertigo , inner or middle ear dysfunction , or recent respiratory infection . the vertigo was attributed to exposure to the mri , and no further evaluation or treatment was required . the anesthetist felt that the intensity of the vertigo was such that it impaired his ability to provide safe care and he requested that he not be assigned to the mri in the future . multiple authors have reported transient sensory effects , including vertigo , nausea , dizziness , metallic taste , and visual phosphenes , in subjects exposed to static magnetic fields.1 despite being a well - known phenomenon , there are only limited data about the incidence and contributing factors of mri - induced vertigo . in a survey of workers in an mri scanners manufacturing department , 22% reported experiencing vertigo while at work.2 wilen and de vocht3 published the results of a questionnaire , about health complaints , given to nurses working with mri scanners . in that study , 47% of the nurses surveyed reported at least one health complaint , but only 15% attributed the symptoms to mri exposure . seven percent reported vertigo or dizziness specifically , 12% reported an illusion of movement , and 14% reported a ringing sensation in the head . in a recent observational study of 361 clinical and research employees working in mri facilities , 5.6% of study subjects exposed to a static magnetic field reported vertigo.4 the vestibular system in the inner ear is the peripheral organ that is primarily responsible for the sensation of balance as well as movement in one s environment . it is composed of the six ( three on each side ) semicircular canals that detect angular acceleration , in addition to the connected structures known as the utricle and the saccule that detect linear acceleration ( figure 1 ) . these structures are filled with fluid known as endolymph that moves or flows when the head moves in space . this fluid movement causes deflections of tiny stereocilia attached to specialized neural structures within the vestibular apparatus , known as hair cells , which respond to these movements by firing neural impulses to the brain that in turn are ultimately interpreted as the perception of movement ( figure 2 ) . as a result , the vestibular system can become activated through hydrodynamic pressure changes in the endolymph , direct deflection of stereocilia , or neurostimulation via electrical currents . however , the precise mechanism through which magnetic vestibular stimulation occurs is unclear , with some proposed mechanisms including electromagnetic induction ( ie , voltage induced by a changing magnetic field ) and magnetic susceptibility differences between vestibular organs and surrounding fluid.5 in 2011 , roberts et al6 published compelling evidence that the static magnetic field produced by the mri scanner is the primary cause of vertigo caused by interaction with the vestibular system in the inner ear . the authors contend that the magnetic field induces an electrical perturbation in the potassium - rich endolymph within the semicircular canals , which stimulates the hair cells in the vestibular system , thereby causing an abnormal sensation of movement . this work was later refined by providing more - detailed calculations of the lorentz forces and resulting pressures within the vestibular system in strong static magnetic fields ( figure 3).7 more recently , stationary exposures to a static 7 t mri field were found to be associated with the presence of vertigo and nystagmus , and the reversal of symptoms following withdrawal from the field was taken as evidence for adaptation to continuous vestibular input caused by the static magnetic field.8 a separate body of work further confirms that these effects should be more pronounced with stronger magnetic fields that are encountered with higher - field - strength mri scanners and in closer proximity to the epicenter of the mri scanner bore ( ie , on the patient undergoing the mri ) . de vocht et al2 reported an increase in the incidence of symptoms with higher - strength magnets ( 1.0 t and higher ) and increased exposure time ( > 20 minutes ) . wilen and de vocht3 reported that the majority of symptoms in the study subjects were observed after exposure to higher - strength magnets ( 1.5 t and 3.0t).3 there is evidence that the measured velocity of nystagmus in mri - exposed volunteers increases in proportion to the strength of the magnetic field,9 and schaap et al4 have found a positive correlation between scanner strength and reported symptoms in health care and research workers using 1.5 t , 3 t , and 7 t systems . given that the fringe magnetic field declines exponentially with distance from the mri scanner , the anesthetist in the current case would have experienced the largest static magnetic field when peering into the end of the bore to evaluate the sedated patient , which corresponded to symptom onset ( figure 4 ) . although variable depending on the exact position of the anesthetist s head relative to the magnetic flux isolines of the 3 t scanner , it is likely that the anesthetist s head was experiencing a magnetic field on the order of 2 t . although the static magnetic field appears to play the strongest role in magnetic vestibular stimulation , the induction of electrical current within a time - varying magnetic field may still be contributory . however , these are much smaller in magnitude than the static magnetic field , so the net changes in the fringe magnetic field felt by anesthesia staff outside of the bore are rather small . the second form of time - varying magnetic field occurs when moving through a magnetic field , such as when the patient is advanced into and out of the mri scanner bore . this becomes applicable to health care workers as they move through the magnetic field in the mri scanner room , which includes both linear ( walking ) and angular ( head rotation ) movement . laakso et al10 modeled head movement in the vicinity of a 3 t mri scanner and found that induced eddy currents are within the same order of magnitude as currents used to electrically stimulate the vestibular apparatus . furthermore , faster motion through the magnetic field will produce stronger electrical currents and thus , in theory , more frequent or intense vertigo . in fact , de vocht et al11 reported a much higher incidence of vertigo in subjects who were defined as fast movers as opposed to slower - moving subjects . in addition , head position in relation to the mri bore is an important factor , mainly because the strength of the static magnetic field is stronger within the mri bore . furthermore , if a person s head is within the bore , he or she may be exposed to the gradient and radiofrequency magnetic fields that are usually confined to the patient , and this may in theory increase the likelihood of vertigo.12 it is unknown whether there are any subject - specific factors , such as age , gender , body weight , or other medical conditions ( in particular preexisting vertigo disorders ) , that predispose a person to experiencing vertigo when exposed to an mri scanner . currently , there are no known long - term sequelae from mri - induced vertigo or , for that matter , from mri exposure in general . clinical experience suggests that the symptoms of vertigo are mild and transient , disappearing when exposure to the magnetic field ceases . preliminary data also suggest that low - dose diphenhydramine may help lessen symptoms,13 which may be useful for awake and symptomatic patients undergoing mri but is not practical for workers because of the drug s sedating effects . interestingly , there is substantial evidence that exposure to mri can adversely affect measurable neurobehavioral functions such as hand eye coordination and visual tracking tasks when subjects move their heads within a static magnetic field of 1.53.0 t . there is some theoretical concern that higher - strength magnets , in particular , could pose some safety and performance issues for health care providers by causing disequilibrium , poor hand eye coordination , nausea , or falls.1 regulations regarding occupational exposure to high - field - strength mri scanners have been deficient in some countries and misleading in others . the european union issued the physical agents ( emf ) directive in 2004 , which was primarily intended to limit the exposure of electrical power and telecommunications workers to electromagnetic fields , or emfs as they are known . however , this legislation had the unintended consequence of potentially constraining the clinical use of mri , particularly at 3 t field strengths , which have become part of standard - of - care imaging.15 according to the guidelines on the limits of exposure to static magnetic fields , published in 2009 by the international commission on non - ionizing radiation protection ( icnirp ) , head and neck exposure to static magnetic fields should not exceed 2 t due in large part to concerns about symptoms of vertigo and nausea.1 the most recent icnirp guidelines published in 2014 further distinguish between controlled and uncontrolled exposures.16 in general , health care workers operating in the vicinity of an mri scanner would be considered to be in a controlled environment because they have training on the biologic effects of high - field - strength mri and they typically can control their movements when exposed to the magnetic field . as such , the workers can decrease the likelihood of experiencing vertigo by maximizing the distance between themselves and the mri scanner ( to reduce the effect of the static magnetic field ) and limiting their motion to maintain the magnetic flux density below 2 t for any 3-second period ( ie , to decrease the potential for motion - induced electrical currents ) . in directive 2013/35/eu , the european union established new standards for exposure to static and time - varying magnetic fields but largely exempted the use of mri equipment in the health sector . this begs the question of what are the occupational exposure limits for health care workers in the us ? anesthesiologists are exposed to mri more than most other clinicians , but the major anesthesiology organizations have not declared an official stance on mri exposure . the american society of anesthesiologists practice advisory on anesthetic care for magnetic resonance imaging , published in 2009 , does not address vertigo or other potential health effects of mri exposure.17 in fact , occupational exposure is not addressed at all in those guidelines . in a letter to the editor of anesthesiology in 2010 , bryan et al18 expressed concerns about the lack of both adequate tracking and standard exposure limits to electromagnetic fields among health care personnel . clearly , as scanners become stronger , anesthesia providers and other health care personnel may be exposed to much stronger magnetic fields . in the absence of clear guidelines for exposure limits and safe practices , we suggest that local institutions educate anesthesia providers about the risks of vertigo induced by mri exposure and formulate standard practices to prevent and manage those symptoms . recent data from schaap et al19 confirm that workers with the same job demonstrate significant variability in exposure levels to both static magnetic fields and motion - induced time - varying magnetic fields . this inconsistency suggests that education and behavioral modification have the potential to reduce exposure and potentially decrease the incidence and severity of vertiginous symptoms . in our institution obviously , limiting exposure altogether is the ideal practice , so if it is feasible , the anesthesia provider should simply remain outside of the mri room and remain inside the mri room for as little time as possible . in cases where the anesthesia provider must be close for monitoring or needs to actively attend to a patient , the practitioner should be advised to avoid rapid head movements and to avoid leaning directly into the scanner bore . furthermore , if the symptoms become too intense or begin to interfere with safe work practice , the anesthetist should notify the mri technician and call for immediate assistance . finally , our anesthetists have been encouraged to obtain an evaluation by occupational medicine and can opt out of practicing in the mri environment if health concerns are significant . since the initiation of an educational program and these practice recommendations , we have had no problems with vertigo among our anesthesia providers .

background : caregivers of individuals suffering from psychiatric illness are at risk of being subjected to mental health consequences such as depression , anxiety and burnout . community - based studies proved that 1847% of caregivers land in depression . the caregiver burden can be quantified into objective , subjective and demand burdens . there is paucity of data comparing the caregiver burden of psychiatric patients and that of chronic medical illness patients.aims and objectives:(1 ) to compare the caregiver burden in psychiatric illness and chronic medical illness . ( 2 ) to study the association of caregiver burden with demographic factors like age , gender , duration of caregiving.materials and methods : the study included two groups of caregivers , each of 50 members . group 1 consisted of caregivers of psychiatric patients and group 2 consisted of caregivers of chronic medical illness patients . the montgomery borgatta caregiver burden scale was used to assess the burden in terms of objective , subjective and demand burdens.results and conclusion : the caregiver burden scores in the caregivers of psychiatric patients were significantly higher than that of chronic medical illness ( p<0.0001 ) . the caregiver burden was found to increase with the duration of illness as well as with the age of caregiver . the caregiver burden in the sample population was less as the objective and demand burden did not cross the reference higher value in the given scale , whereas the emotional impact given by the subjective burden was on higher side . vertigo induced by exposure to the magnetic field of a magnetic resonance imaging ( mri ) scanner is a well - known phenomenon within the radiology community but is not widely appreciated by other clinical specialists . here , we describe a case of an anesthetist experiencing acute vertigo while providing sedation to a patient undergoing a 3 tesla mri scan . after discussing previous reports , and the evidence surrounding mri - induced vertigo , we review potential etiologies that include the effects of both static and time - varying magnetic fields on the vestibular apparatus . we conclude our review by discussing the occupational standards that exist for mri exposure and methods to minimize the risks of mri - induced vertigo for clinicians working in the mri environment .

this has caused widespread concern , considering the associations between obesity and a range of adverse health conditions . there is a widely held view that the increasing rates of obesity will lead to an epidemic of diabetes , other chronic conditions , and a subsequent reduction in life expectancy . however the picture is complicated . since the 1960s all - cause and cardiovascular - specific mortality rates have decreased steadily in most developed countries , and life expectancy has consistently increased . the aim of this paper is to suggest several reasons for the discrepancy between increasing levels of obesity and gains in life expectancy , and those factors may be masking the effects of obesity on life expectancy . a better understanding of the way in which obesity affects health and longevity will help determine the most appropriate response to increasing levels of excess body weight and assist our understanding of the likely impact of obesity on the health of individuals and the future burden on the health care system . this is considered to have led to an epidemic of type ii diabetes . the progression of this epidemic , in tandem with cardiovascular disease and several other morbidities associated with obesity , is predicted to slow or reverse the decline in mortality that has been noted in most western countries over the past 3040 years . the data accumulated to date have provided relatively little evidence in support of this view . levels of obesity have been increasing since the 1950s ( albeit slowly , initially ) in the usa and other developed countries . over the same period , life expectancy has continued to increase at an undiminished rate , and cardiovascular - specific mortality rates have also decreased continually . ( 1 ) improvement in other risk factorsit is possible that the deleterious impact of obesity is outweighed by other factors favourably influencing life expectancy . ( 2010 ) have reported that in the united states three of the six major risk factors for chd total cholesterol , prevalence of smoking , and physical activity levels improved between 1988 and 2003 . the rate of decline of all - cause and cvd mortality might be faster still if it was not for the increasing prevalence of diabetes [ 5 , 6 ] , for which there is a clear association with heart disease . examples of factors driving mortality down include population - wide changes such as reductions in the prevalence and intensity of smoking 2009 ) have predicted that over the next decade the negative effects of increasing levels of obesity will outweigh the benefits from reductions in the prevalence of smoking . however , peto et al . ( 2010 ) critiqued this finding , suggesting that stewart et al . ( 2009 ) have overestimated the hazards of obesity and underestimated the hazards of smoking . it is possible that the deleterious impact of obesity is outweighed by other factors favourably influencing life expectancy . ( 2010 ) have reported that in the united states three of the six major risk factors for chd total cholesterol , prevalence of smoking , and physical activity levels improved between 1988 and 2003 . the rate of decline of all - cause and cvd mortality might be faster still if it was not for the increasing prevalence of diabetes [ 5 , 6 ] , for which there is a clear association with heart disease . examples of factors driving mortality down include population - wide changes such as reductions in the prevalence and intensity of smoking . ( 2009 ) have predicted that over the next decade the negative effects of increasing levels of obesity will outweigh the benefits from reductions in the prevalence of smoking . however , peto et al . ( 2010 ) critiqued this finding , suggesting that stewart et al . ( 2009 ) have overestimated the hazards of obesity and underestimated the hazards of smoking . ( 2 ) pharmacological treatmentthere is the possibility of improved medical interventions in some of the pathways linking obesity to cvd and all - cause mortality . for example , improved control of hypertension and better management of dyslipidaemia may blunt the impact of obesity on adverse health outcomes [ 3 , 9 ] . hypertension has been fairly well controlled in recent years , and there has been increased use of statins , angiotensin pathway inhibitors , and aspirin , all of which may be contributing to the limited effect of rising obesity levels . there is the possibility of improved medical interventions in some of the pathways linking obesity to cvd and all - cause mortality . for example , improved control of hypertension and better management of dyslipidaemia may blunt the impact of obesity on adverse health outcomes [ 3 , 9 ] . hypertension has been fairly well controlled in recent years , and there has been increased use of statins , angiotensin pathway inhibitors , and aspirin , all of which may be contributing to the limited effect of rising obesity levels . ( 3 ) prevalence of more extreme obesitythe impact of obesity may have been overestimated because its principal adverse effects are experienced by a minority of the population . the most robust estimates of the association between bmi and mortality , from the prospective studies collaboration of 900,000 adults in 57 prospective studies , suggests that the mortality risk from excess body weight increases from a bmi of 25 but is not substantial until bmi exceeds 3235 . it is a significant proportion but nevertheless a clear minority of the population , at least currently . although significant increases in the prevalence of severe obesity ( bmi > 40 ) have been reported in a number of countries [ 11 , 12 ] , the risk for this group is outweighed by the considerably less significant risks associated with body weight in the majority of the population ( figure 1 ) . the impact of obesity may have been overestimated because its principal adverse effects are experienced by a minority of the population . the most robust estimates of the association between bmi and mortality , from the prospective studies collaboration of 900,000 adults in 57 prospective studies , suggests that the mortality risk from excess body weight increases from a bmi of 25 but is not substantial until bmi exceeds 3235 . it is a significant proportion but nevertheless a clear minority of the population , at least currently . although significant increases in the prevalence of severe obesity ( bmi > 40 ) have been reported in a number of countries [ 11 , 12 ] , the risk for this group is outweighed by the considerably less significant risks associated with body weight in the majority of the population ( figure 1 ) . ( 4 ) reversal of relationship in old agein old age those of low body weight are at higher risk of disability ( limitations to activities of daily living ) and mortality . the relationship between obesity and health appears to reverse in old age . 1988 ) found little relationship between bmi and mortality in older adults , except for those of very low body weight , who had a higher mortality than others . it is likely , however , that this reversal of the obesity and health relationship in older people is due to weight loss in old age due to health problems or disease . in old age those of low body weight are at higher risk of disability ( limitations to activities of daily living ) and mortality . ( 1988 ) found little relationship between bmi and mortality in older adults , except for those of very low body weight , who had a higher mortality than others . it is likely , however , that this reversal of the obesity and health relationship in older people is due to weight loss in old age due to health problems or disease . ( 5 ) body weight per se not associated with mortalityit is possible that body weight is not a significant risk factor for mortality . body weight may act simply as a surrogate for a constellation of attributes of lifestyle , in particular diet , physical activity level , and genetic factors . if this were the case , obese individuals would represent a heterogeneous group of high body weight due to a variety of reasons , some of which may not be so strongly related as others to morbidity or mortality . body weight may act simply as a surrogate for a constellation of attributes of lifestyle , in particular diet , physical activity level , and genetic factors . if this were the case , obese individuals would represent a heterogeneous group of high body weight due to a variety of reasons , some of which may not be so strongly related as others to morbidity or mortality . ( 6 ) latency period and cohort effectit is possible that the deleterious impact of recent obesity trends has not yet affected life expectancy due to a considerable latency period between onset of obesity and disease . recent cohorts of the us population are experiencing a greater magnitude and duration of obesity over their lifetime . the duration of exposure to obesity has been reported to have an important impact on disease and mortality . thus , it is possible that the adverse impact of obesity is a function of both severity and duration , and that with more recent cohorts the deleterious impact of obesity on life expectancy will be observed . indeed , recent mortality statistics ( 2008 ) in the us demonstrate , for the first time in over 25 years , a slight decline in us life expectancy . nevertheless , in this same report diabetes and cardiovascular mortality rates maintain a continual decline . furthermore , in an assessment of overweight and obesity on the risk of cardiovascular disease and mortality in middle - aged men , arnlov et al . additionally , the positive effects of weight loss after bariatric surgery on conditions such as diabetes , hypertension , and dyslipidemia have occurred in a much shorter period ( less than two years ) . it is possible that the deleterious impact of recent obesity trends has not yet affected life expectancy due to a considerable latency period between onset of obesity and disease . recent cohorts of the us population are experiencing a greater magnitude and duration of obesity over their lifetime . the duration of exposure to obesity has been reported to have an important impact on disease and mortality . thus , it is possible that the adverse impact of obesity is a function of both severity and duration , and that with more recent cohorts the deleterious impact of obesity on life expectancy will be observed . indeed , recent mortality statistics ( 2008 ) in the us demonstrate , for the first time in over 25 years , a slight decline in us life expectancy . nevertheless , in this same report diabetes and cardiovascular mortality rates maintain a continual decline . furthermore , in an assessment of overweight and obesity on the risk of cardiovascular disease and mortality in middle - aged men , arnlov et al . additionally , the positive effects of weight loss after bariatric surgery on conditions such as diabetes , hypertension , and dyslipidemia have occurred in a much shorter period ( less than two years ) . ( 7 ) the obesity paradoxthere is the obesity paradox in which survival from acute events such as myocardial infarction , heart failure , and dialysis for renal failure is improved in patients with overweight and obesity [ 2022 ] . the mechanism for this observation is not known but is unlikely to be simply that more severely ill patients have lost weight on account of the severity of their illness . it is also possible that diseases such as hypertension and diabetes may be more aggressive and of a different origin in lean individuals than when they are associated with obesity . for example , among patients with a history of hypertension and coronary artery disease , uretsky et al . ( 2007 ) found that overweight and obesity ( classes i to iii ) were associated with a lower risk of morbidity and mortality compared with normal - weight patients , despite poorer blood pressure control . there is the obesity paradox in which survival from acute events such as myocardial infarction , heart failure , and dialysis for renal failure is improved in patients with overweight and obesity [ 2022 ] . the mechanism for this observation is not known but is unlikely to be simply that more severely ill patients have lost weight on account of the severity of their illness . it is also possible that diseases such as hypertension and diabetes may be more aggressive and of a different origin in lean individuals than when they are associated with obesity . for example , among patients with a history of hypertension and coronary artery disease , uretsky et al . ( 2007 ) found that overweight and obesity ( classes i to iii ) were associated with a lower risk of morbidity and mortality compared with normal - weight patients , despite poorer blood pressure control . thus , considerations such as lag time , duration of exposure to obesity , and increasing proportions of the population with severe obesity suggest that obesity may in the future have a considerable impact on life expectancy . however there are also important reasons improved control of hypertension , shape of bmi - mortality relationship , and body weight as a surrogate marker of lifestyle why obesity may not be strongly linked to life expectancy , except at the extremes of the weight distribution . rather , the principle impact of obesity may be on disability - free life expectancy . several studies suggest an association between body weight and a higher risk of becoming disabled [ 2325 ] , although others dispute this [ 3 , 26 ] . thus , considerations such as lag time , duration of exposure to obesity , and increasing proportions of the population with severe obesity suggest that obesity may in the future have a considerable impact on life expectancy . however there are also important reasons improved control of hypertension , shape of bmi - mortality relationship , and body weight as a surrogate marker of lifestyle why obesity may not be strongly linked to life expectancy , except at the extremes of the weight distribution . rather , the principle impact of obesity may be on disability - free life expectancy . several studies suggest an association between body weight and a higher risk of becoming disabled [ 2325 ] , although others dispute this [ 3 , 26 ] . several reasons explain why the impact of obesity on life expectancy may be more complex than is commonly recognised . it is possible that the principal impact of obesity is on disability - free life expectancy rather than on life expectancy itself . it is also possible that health and life expectancy gains could be even greater if it was not for the increasing prevalence of extreme obesity . it is important to understand why life expectancy is generally continuing to improve despite a high and increasing prevalence of obesity . further research in this area and careful monitoring of recent cohorts and future trends will assist policy makers with determining the most appropriate response to increasing levels of obesity . if only those with more extreme obesity are facing substantial health and mortality risks , this will help inform as to the appropriate mix of population and high - risk ( targeting people already obese , or at risk of significant weight gain , e.g. ) approaches to obesity reduction . if the principle impact of obesity is on disability - free life expectancy rather than on life expectancy itself , this has substantial implications for the heath of individuals and the future burden on the health care system . although the peripheral nervous system has the ability to regenerate axons following injury , when nerve gaps are larger than 12 cm , bridging strategies are required for repair . in such cases , autologous nerve grafts have been used as the gold standard ( deumens et al . , 2010 ; pfister et al . , . however , there are limited numbers of suitable sites for harvesting nerve grafts , because of the adverse effects to donor sites , such as the loss of motor and sensory function . in order to overcome these issues , scaffold bridging materials that may be synthetic , absorbable or non - absorbable have been studied vigorously . through these studies , several criteria were suggested to be necessary for better results , as follows : 1 ) the bridging conduit is necessary to maintain adequate mechanical support of separated nerve ends , in addition to preventing the diffusion of neurotrophic and neurotropic factors secreted by transected stumps ( deumens et al . , 2010 ) ; 2 ) nerve scaffolds should be biocompatible in order to eliminate undesirable influences on neural cells / tissues , such as immune rejection ( avitable et al . , 2001 ) ; 3 ) it should be degradable and/or absorbable in the appropriate duration in vivo ( de ruiter et al . , 2009 ) ; 4 ) it must have sufficient permeability for nutrient and gas exchange ( she et al . , 2008 ) ; and 5 ) it must have a biomechanical flexibility to allow bending at the graft site , without kinking the transected nerve stumps ( gu et al . , 2011 ) . for longer nerve gap injuries , scaffolds with a single hollow lumen represent the basic structure , but this has been refined using various biosynthetic materials in order to mimic the nerve - graft structure and to facilitate axonal re - extension . the multi - luminal substructure ( de ruiter et al . , 2008b ) and the filament - filled structure ( bunting et al . ; hu et al . , 2008 ) are typical cases ; the former shows no significant benefit , but the latter shows improved nerve regeneration , as compared to the single lumen scaffold . intraluminal surface coating with extracellular matrix ( ecm ) components , such as laminin , fibronectin and collagen , has been also attempted to promote active axonal re - growth . first , this was attempted using a single hollow conduit , and several positive effects were obtained ( kauppila et al . , 1993 ; the use of conduits of biological origin , such as acellular conduits , which have preserved ecm components , but are decellularized and immunosuppressed , was another approach ( frerichs et al . , 2002 ; hudson et al . , 2004a ) . however , use of this hollow - type conduit shows non - significant effects on nerve regeneration across long gaps ( pfister et al . , 2011 ) . in contrast , conduits including laminin - coated collagen fibers were able to support the regeneration of long sciatic nerve gaps of over 80 mm in dogs ( matsumoto et al . , 2000 ) . at this point , the filament - filled conduit with laminin coating is likely to be the best alternative method . importantly , these efforts have been performed to expect alternative effects of laminin - coated fibers in the conduit , similar to the basal lamina sheet in the individual endoneurium of the healthy nerve fibers . laminin coating may exert supportive effects for the migration , proliferation and orientation of preserved schwann cells , similarly to formation of bands of bungner , which normally occurs in the case of axonotmesis ( deumens et al . furthermore , application of growth factors into the conduit is another method to mimic native conditions . for example , supply of several factors into the conduit , such as nerve growth factor ( ngf ) , brain - derived neurotrophic factor ( bdnf ) , glial cell line - derived neurotrophic factor ( gdnf ) , ciliary neurotrophic factor ( cntf ) , basic fibroblast growth factor ( bfgf ) and insulin - like growth factor-1 ( igf-1 ) , has also been performed , and favorable effects on axonal growth and nerve regeneration have been confirmed ( rich et al . , 1989 ; nachemson et al . , 1990 ; fine et al . , 2002 ; all of the applications described above were based on the notion of simulating the healthy nerve graft condition . in this regard , transplantation of schwann cells with the conduit is considered to be the best method as the alternative for nerve grafting . acellular conduits have thus been used with several cell sources , including schwann cells and/or schwann - like cells induced from cultivated bone morrow stromal cells ( dezawa et al . , 2005 ) , olfactory ensheathing cells ( radtke et al . , 2011 ) and adipose tissue - derived cells ( kingham et al . , 2007 ) , but it is unlikely that these cells would match or exceed the performance of auto nerve grafts . although schwann cells play a central role in peripheral nerve regeneration , the formation of endoneurium and/or perineurium by endoneurial fibroblasts and perineurial cells is also important because of their protective role in axons with schwann cells and myelin sheath . in particular , the perineurium plays an important role in preventing the passage of large molecules from the epineurium into perineurial fascicles , which is also known as the blood - nerve - barrier system ( weerasuriya and mizisin , 2011 ) . furthermore , reconstitution of vascular networks is also an inevitable factor in tissue reconstruction . in this regard , cell sources that can give rise to schwann cells and cells associated with the formation of perineurium / endoneurium and blood vessels are considered to be the best source for peripheral nerve regeneration . recently , we reported preferential and comprehensive reconstitution of severely damaged sciatic nerve using 7-day cultivated murine skeletal muscle - derived multipotent stem cells ( sk - mscs ; tamaki et al . , 2014 ) . in this treatment , engrafted donor cells preferentially differentiated into schwann cells and perineurial / endoneurial cells , and formed myelin sheath and perineurium / endoneurium , encircling the regenerated axons . donor cell - derived perineurium also had tight - junctions , which play a key role in the blood - nerve - barrier system ; thus , suggesting functional reconstitution . stem cells showing suitable multipotency to cover all peripheral nerve support cells in damaged nerve - specific niche have not been reported previously , although cultivated bone morrow stromal cells ( mbcss ; dezawa et al . , 2005 ) , olfactory ensheathing cells ( radtke et al . , 2011 ) and adipose tissue - derived cells ( kingham et al . , 2007 ) we directly compared the contribution to damaged nerve reconstitution among cultivated sk - mscs and bmscs , and freshly isolated cells derived from damaged sciatic nerve ( sndc - d ) ( tamaki et al . , the results showed that sk - mscs have a significantly greater engraftment ratio when compared with the other two groups , and the values in the latter two groups were similar . in addition , there was no definitive formation of peripheral nerve supporting cells or incorporation into blood vessels in bmsc transplantation , whereas sk - mscs and sndc - d showed typical differentiation into all support cells . in addition , engrafted sk - mscs contributed to increased vascular formation , which is favorable for blood supply and waste product excretion . facilitation of blood vessel formation in sk - mscs was also confirmed in damaged skeletal muscle ( tamaki et al . , 2005 ; 2007b ; 2013 ) and urethra ( hoshi et al . , 2008 ) , as well as in and around the bladder wall ( nitta et al . , 2010 expression of key neurotrophic and nerve / vascular growth factor mrnas was also confirmed ; in particular , ngf ( rich et al . , 1989 ) , bdnf ( lewin et al . , 2009 ) , gdnf ( fine et al . , 2002 ) , galectin-1 ( horie et al . , 1999 ) , ninjurin ( araki and milbrandt , 1996 ) , cntf ( dubovy et al . , 2011 ) , lif ( wang et al . , 2009 ) , sox10 ( bremer et al . , 2011 ; britsch et al . , 2001 ; finzsch et al . , 2010 ) , bfgf ( timmer et al . , 2003 ) and igf-1 ( nachemson et al . , 1990 ) are important for nerve regeneration , while vgef ( ferrara , 2004 ) and hgf ( schroder et al . , 2011 ) are important for blood vessels . some of these are redundant and/or primarily involved in the recipient nerve regeneration process ( campbell , 2008 ) ; however , sufficient expression of these factors in the damaged / transplantation site may induce paracrine effects on both recipient and donor cells as an adjuvant for nerve regeneration . using sk - mscs , we examined the therapeutic capacity in a long transected nerve gap on the mouse sciatic nerve via an acellular conduit ( 1.52.0 10 cells / nerve ) , and demonstrated 94% reconstitution of the number of axons , 60% recovery of myelin , and a 9.1-fold increase in blood vessels through the conduit ( tamaki et al . , 2014 ) . features and immunohistochemical properties of regenerated nerve at 8 weeks after bridging with sk - mscs transplantation are shown in figure 1 . thick gfp tissues were evident throughout the conduit ( figure 1a ) , and a large number of gfp multi - luminal substructures , such as perineurium / endoneurium , were also apparent in histological sections ( figure 1b and c ) . importantly , regenerated axons in the conduit were encircled by engrafted gfp perineurium / endoneurium ( figure 1b ) , having myelination ( figure 1c ) , thus suggesting that regenerating axons extended along the endoneurial / perineurial substructures with supporting schwann cells . the bridging acellular conduit also includes a large number of blood vessels ( figure d ) ; thus , favorable blood supply and waste product excretion are expected . these relationships between gfp tissues and regenerated axons were confirmed in longitudinal sections ( figure 1e ) , and gfp multi - luminal substructures also possessed the basal lamina sheath ( figure 1f and g ) . these results clearly indicated that our nerve treatment method using the sk - mscs with acellular conduits meet all of the above criteria , making it useful for nerve regeneration . in addition , we also demonstrated the greater therapeutic potential of sk - msc treatment as compared to case of healthy nerve grafts using the same mouse model ( tamaki et al . , 2014 ) . typical structure of engrafted gfp skeletal muscle - derived multipotent stem cells ( sk - mscs ) in acellular bridging conduit at 8 weeks after transplantation . ( a ) macroscopic features of regenerated sciat - ic nerve with engrafted gfp tissues . ( b ) cross - sectional profile of the bridging conduit filled with donor - derived gfp perineurium / endoneu - rium . ( c ) cross - sectional profile of bridging conduit filled with do - nor - derived gfp perineurium / endoneurium . ( d ) same portion of panel ( c ) is stained with cd31 ( vascular endothelial cell marker ) . gfp perineurium / endoneu - rium show continuously elongated features having axons ( red ) inside . ( f ) longitudinal view of gfp peri - neurium / endoneurium in the conduit , and ( g ) laminin staining ( red ) . gfp perineurium / en - doneurium also has basal lamina sheets , which are detected by anti - laminin staining . skeletal muscle is the largest organ in the body , comprising approximately 4050% of total body mass , and presumably allowing donor cells to be obtained with relative ease and safety . therefore , sk - mscs represent a novel / suitable alternative cell source for healthy nerve autografts . skeletal muscle - derived stem cells have been used by many researchers for various purposes ; however , our isolation method of sk - mscs differs from others ( tamaki et al . , 2002 ; 2010 ) , and this is potentially crucial to our results after transplantation ( tamaki et al . , 2005 , 2007a , b ) . in future research , the potential of human skeletal muscle - derived cells needs to be clarified . we are currently investigating the optimal isolation , fractionation and expansion culture conditions for human cells in order to maximize the nerve regeneration potential . in vivo evidence of the differentiation and reconstruction capacities of human sk - mscs is also important . we used the acellular conduit from the wild - type mice esophageal submucous membrane ( mainly longitudinal muscle layer ) separated after 3 days of 70% ethanol dehydration . in the human case , use of an appropriate size vein may be a candidate cellular or acellular conduit . however , from the viewpoint of minimizing invasiveness , biosynthetic tubes that are a good match for sk - mscs treatment need to be identified . application of sk - msc treatment for the total regeneration of multi - branched nerve transections may also be useful in the case of surgical nerve damage .

background . increasing levels of obesity over recent decades have been expected to lead to an epidemic of diabetes and a subsequent reduction in life expectancy , but instead all - cause and cardiovascular - specific mortality rates have decreased steadily in most developed countries and life expectancy has increased . methods . this paper suggests several factors that may be masking the effects of obesity on life expectancy . results . it is possible that health and life expectancy gains could be even greater if it was not for the increasing prevalence of extreme obesity . it is also possible that the principal impact of obesity is on disability - free life expectancy rather than on life expectancy itself . conclusion . if the principal impact of obesity were through disability - free life expectancy rather than on life expectancy itself , this would have substantial implications for the health of individuals and the future burden on the health care system . long gap peripheral nerve injuries usually reulting in life - changing problems for patients . skeletal muscle derived - multipotent stem cells ( sk - mscs ) can differentiate into schwann and perineurial / endoneurial cells , vascular relating pericytes , and endothelial and smooth muscle cells in the damaged peripheral nerve niche . application of the sk - mscs in the bridging conduit for repairing long nerve gap injury resulted favorable axonal regeneration , which showing superior effects than gold standard therapy -- healthy nerve autograft . this means that it does not need to sacrifice of healthy nerves or loss of related functions for repairing peripheral nerve injury .

when the gallbladder is not visualized in its normal location , the possibility of its ectopic location should be considered . a case of incidentally detected anomalous position of gall bladder causing confounding problem in interpretation of pet - ct is described . a 70-year - old man , with h / o chronic liver disease and suspected of hepatocellular carcinoma [ serum alpha - fetoprotein ( afp ) 5024 ng / ml ] was subjected to fluorine-18 fluorodeoxyglucose positron emission tomography ( f-18 fdg pet)/computed tomography ( ct ) imaging . rest of the liver revealed non - fdg avid lesions in segments iii and viii . on viewing the fused pet / ct images , the radiotracer accumulation was localized to the anomalously placed suprahepatic gallbladder . magnetic resonance ( mr ) images of the same patient confirmed the presence of the suprahepatic gallbladder [ figure 1 ] . ( a ) transaxial view of pet image showing suprahepatic subdiaphragmatic gallbladder with tracer uptake ; ( b ) coronal view of pet image ; ( c ) sagittal view of pet image ; ( d ) transaxial view of ct image ; ( e ) coronal view of ct image ; ( f ) sagittal view of ct image ; ( g ) transaxial view of post - contrast t1-weighted mri image ; ( h ) coronal view of post - contrast t1-weighted mri image ; ( i ) transaxial view of t2 fat saturated mri image shows gallbladder as a bright structure routine imaging of the gallbladder demonstrates a wide array of imaging variants , including anomalies in location , number , and configuration . an awareness of these normal variants would prevent misdiagnosis and aid in the assessment of differential diagnostic possibilities . normally , the gallbladder is situated adjacent to the inferior surface of the liver , in the plane of the interlobar fissure , with the gallbladder neck maintaining a constant relationship to porta hepatis . the gallbladder is generally found in the right upper quadrant , but may be seen in other parts of the abdomen . while anomalous positions are rare , the most common of these are ( 1 ) under the left hepatic lobe , ( 2 ) intrahepatic , ( 3 ) transverse , and ( 4 ) retroplaced ( retrohepatic or retroperitoneal ) . the lesser common of these are ( 1 ) supradiaphragmatic and ( 2 ) suprahepatic . gallbladder is intrahepatic during the embryonic period and becomes extrahepatic only later . an intrahepatic gallbladder ( usually a congenital anomaly ) this poses a problem for scintigraphy , as an intrahepatic gallbladder can cause a focal defect ( pseudo space - occupying lesion ) ; ultrasonography can be helpful in these cases . the suprahepatic region is among the rarest sites , and very few reports have appeared in either the surgical or radiological literature.[35 ] of the very few reports on the suprahepatic gallbladder , one refers to a normally inserted organ that rotated 180 upward to an intrathoracic position after eventration of the diaphragm . in two other cases , an abnormally mobile gallbladder was found trapped between the chest wall and the upper border of the liver ; this became symptomatic and caught the attention of the clinician and the imageologist . faintuch et al . reported three cases of suprahepatic gallbladder with hypoplasia of the right hepatic lobe and upward migration of the gallbladder . gansbeke reported a case of suprahepatic gallbladder which was associated with hepatomegaly due to macronodular cirrhosis complicating existing hepatitis . kabaroudis reported a case of floating gallbladder associated with hypoplasia of the right hepatic lobe , whereas maeda had reported a similar case associated with hypoplasia of left hepatic lobe . pet - ct is found to be useful in diagnosing this rare anatomical variant of ectopically located gall bladder and predicting its functional implication . digital intraoral radiographic systems have several advantages compared with conventional film - based radiography . these advantages , in endodontics , include the potential of a lower radiation exposure to the patient and faster image display . moreover , it was introduced the opportunity of evaluating the progression of periapical bone loss by digital tools as the pixel values . radiographs do not always reflect the extent of the destructive process in the periapical tissues and generally under - represent the size of a lesion . studies have suggested that pathologic involvement of the cortical plate , or at least junctional trabeculae , is a prerequisite for radiographic detection of periapical pathoses . nevertheless , periapical lesions confined to cancellous bone could be detected radiographically using pixel values using one of the tools of the digital image , the histogram . only a few studies employing animal models or human biopsies have compared the diagnostic and quantifying accuracy of radiographs to the gold standard represented by the histology . the aim of this study was to induce periapical disease in rats to assess the pixel values in different experimental periods to evaluate the bone loss progression . this study was approved by the animal research ethics committee of the bauru school of dentistry , university of so paulo . the experimental groups were composed of 27 wistar rats ( rattus novergicus ) , divided into three groups with nine animals each , according to the sacrifice date , group 1 : 2 days , group 2 : 15 days , group 3 : 30 days . , a cavity was made on the occlusal face of the mandibular first molar on the right side with a spherical burr size 1/4 , at high speed without cooling . the material for study was obtained after euthanizing the animal with an anesthetic overdose . the right and left hemi - mandibles of each rat were separated and radiographed with two phosphor plates ( psps/ digora - soredex - tuusula , finland ) . the exposition was performed according to the parameters : 70 kvp , 7 ma , focus - film distance of 30 cm , exposure time of 0.09 s. a standard aluminum stepwedge was set in each image , and an acrylic plate measuring 0.3x0.4x0.2 cm was positioned over the hemi - mandibles to simulate soft tissue . the stepwedge was used as a standard image technique , which is essential for control of day - to - day variations in the sensitivity of the detector . the radiographic evaluation was performed using the public domain software image j ( version 1.33 , national institute of health , washington , dc ) a region of interest ( roi/252 pixels ) was opened in the periapical region of the every tooth , closest to the mesial root , without touching it . only the periapical bone was enclosed in the roi . using the histogram tool , the normalized pixel values of the images ( npi ) were then obtained by using the following equation : npi = pi / cr . where pi is the mean pixel intensity of the roi , and cr is the pixel intensity of the roi of the standard aluminum stepwedge . the pixel value of zero corresponded to black and the pixel value of 255 to white . hemi - mandibles were fixed in formalin 10% , decalcified in edta and processed histologically . five semi - serial sections of each hemi - mandible with 5 m thickness were cut and stained with hematoxylin and eosin . all histological sections were identified with a random numerical sequence and periapical tissues were histomorphometrically analyzed for both test and control sites . in the test site it was noted whether the tooth had pulpitis , necrosis or presented any sign of inflammation , and whether or not there was presence of periapical bone resorption . the percentages of bone tissue in the apical region of the root , soft tissue ( connective tissue and inflammatory infiltrate , when present ) , bone marrow and inferior alveolar nerve were obtained in a digital image analysis system , composed of a zeiss axioskop ii microscope , ccd - iris rgb - sony camera ( sony dxc-151a rgb video camera - sony corporation , tokyo , japan ) and kontron ks300 software ( kontron electronic gmbm , munich , germany ) . for this purpose , an image was captured at 10x objective from each histologic slice , which contained the apical portion of the mesial root perpendicular to the alveolar nerve , positioned centrally . the area ( in pixels ) was measured to quantify the area occupied by bone tissue in the apical region of the root , soft tissue ( connective tissue and inflammatory infiltrate , when present ) , bone marrow and inferior alveolar nerve . this study was approved by the animal research ethics committee of the bauru school of dentistry , university of so paulo . the experimental groups were composed of 27 wistar rats ( rattus novergicus ) , divided into three groups with nine animals each , according to the sacrifice date , group 1 : 2 days , group 2 : 15 days , group 3 : 30 days . , a cavity was made on the occlusal face of the mandibular first molar on the right side with a spherical burr size 1/4 , at high speed without cooling . the material for study was obtained after euthanizing the animal with an anesthetic overdose . the right and left hemi - mandibles of each rat were separated and radiographed with two phosphor plates ( psps/ digora - soredex - tuusula , finland ) . the exposition was performed according to the parameters : 70 kvp , 7 ma , focus - film distance of 30 cm , exposure time of 0.09 s. a standard aluminum stepwedge was set in each image , and an acrylic plate measuring 0.3x0.4x0.2 cm was positioned over the hemi - mandibles to simulate soft tissue . the stepwedge was used as a standard image technique , which is essential for control of day - to - day variations in the sensitivity of the detector . the radiographic evaluation was performed using the public domain software image j ( version 1.33 , national institute of health , washington , dc ) a region of interest ( roi/252 pixels ) was opened in the periapical region of the every tooth , closest to the mesial root , without touching it . only the periapical bone was enclosed in the roi . using the histogram tool , the normalized pixel values of the images ( npi ) were then obtained by using the following equation : npi = pi / cr . where pi is the mean pixel intensity of the roi , and cr is the pixel intensity of the roi of the standard aluminum stepwedge . the pixel value of zero corresponded to black and the pixel value of 255 to white . hemi - mandibles were fixed in formalin 10% , decalcified in edta and processed histologically . five semi - serial sections of each hemi - mandible with 5 m thickness were cut and stained with hematoxylin and eosin . all histological sections were identified with a random numerical sequence and periapical tissues were histomorphometrically analyzed for both test and control sites . in the test site it was noted whether the tooth had pulpitis , necrosis or presented any sign of inflammation , and whether or not there was presence of periapical bone resorption . the percentages of bone tissue in the apical region of the root , soft tissue ( connective tissue and inflammatory infiltrate , when present ) , bone marrow and inferior alveolar nerve were obtained in a digital image analysis system , composed of a zeiss axioskop ii microscope , ccd - iris rgb - sony camera ( sony dxc-151a rgb video camera - sony corporation , tokyo , japan ) and kontron ks300 software ( kontron electronic gmbm , munich , germany ) . for this purpose , an image was captured at 10x objective from each histologic slice , which contained the apical portion of the mesial root perpendicular to the alveolar nerve , positioned centrally . the area ( in pixels ) was measured to quantify the area occupied by bone tissue in the apical region of the root , soft tissue ( connective tissue and inflammatory infiltrate , when present ) , bone marrow and inferior alveolar nerve . the radiographic analysis showed that the pixel values in the three experimental periods increased on the test side in comparison with the control side , indicating bone loss in a similar manner both psps plates used . the student 's t test showed a statistically significant difference between control and test sites for the three groups tested ( p<0.05 ) ( table 1 ) . the influence of the time on periapical disease was studied using one - way anova and tukey 's tests . anova showed no statistically significant variation for any of the time intervals in plate 1 ( p>0.05 ) , as well as for the control side of plate 2 ( p>0.05 ) . this variation was only shown to be significant in plate 2 ( p<0.05 ) between the test groups 2 and 3 ( figures 1a , d , g , j ) . means and standard deviation of the pixel values on the test and control sides for the digora system phosphorus plates 1 and 2 , in the rats of groups i , ii and iii mean pixel value from 9 animal samples . control group : a ) radiographic image of hemi - mandible ; b - c ) photomicrograph of the left first molar showing healthy pulp ( black arrow ) and periapical tissues with cementum ( ce ) , fibers of periodontal ligament ( li ) and alveolar bone ( ab ) . group 1 : d ) radiographic image of hemi - mandible similar at the control group ; e - f ) photomicrograph of the right first molar exhibiting inflammatory cells ( black arrow ) in the periapical region . group 2 : g ) radiographic image of hemi - mandible showing radiolucent area in the periapical region ; h - i ) photomicrograph of the right first molar showing extensive bone ( b ) resorption by osteoclasts ( red arrow ) and connective tissue ( ct ) . group 3 : j ) radiographic image of hemi - mandible exhibiting extensive radiolucent area in the periapical region ; k - l ) photomicrography of the right first molar showing abscess ( red arrow ) , increase of connective tissue area ( ct ) and decrease of bone tissue ( b ) in relation to the other groups the histological analysis of the left first molars of the rats , which served as control , showed no alterations in the pulp or periapical tissues , and evidenced that 100% of these teeth presented healthy pulp and periapical tissues ( figures 1b , c ) . the teeth submitted to coronal opening in group 1 , showed in 100% of the cases , necrosis in the coronal region and pulp inflammation with the presence of inflammatory cells and increased vascularization in the medial and apical thirds . it was possible to observe inflammatory cells and small bone and cementum resorption in the periapical region ( figures 1e , f ) . group 2 showed necrosis in the coronal and middle radicular pulp and extensive inflammatory process in the apical third . in the periapical region exhibited increase of bone resorption and replaced by conjunctive tissue rich in inflammatory cells ( figures 1h , i ) . the group 3 showed total pulp necrosis , extensive area of abscess and bone , cementum and dentine resorption and few bone trabeculae surrounded by conjunctive tissue in the apical portion ( figures 1k , l ) . the total numbers of pixels obtained for each of the measured regions in the control and tests sides , in the various experimental periods are presented in table 2 . two - way anova and tukey 's tests showed statistically significant difference between the test and control sides and among the experimental periods as well . mean and standard deviation of the total number of pixels of each structure present in the periapical region of the mesial root of the mandibular first molar in the control and test groups in the experimental periods mean pixel value from 9 animal samples . the radiographic analysis showed that the pixel values in the three experimental periods increased on the test side in comparison with the control side , indicating bone loss in a similar manner both psps plates used . the student 's t test showed a statistically significant difference between control and test sites for the three groups tested ( p<0.05 ) ( table 1 ) . the influence of the time on periapical disease was studied using one - way anova and tukey 's tests . anova showed no statistically significant variation for any of the time intervals in plate 1 ( p>0.05 ) , as well as for the control side of plate 2 ( p>0.05 ) . this variation was only shown to be significant in plate 2 ( p<0.05 ) between the test groups 2 and 3 ( figures 1a , d , g , j ) . means and standard deviation of the pixel values on the test and control sides for the digora system phosphorus plates 1 and 2 , in the rats of groups i , ii and iii mean pixel value from 9 animal samples . control group : a ) radiographic image of hemi - mandible ; b - c ) photomicrograph of the left first molar showing healthy pulp ( black arrow ) and periapical tissues with cementum ( ce ) , fibers of periodontal ligament ( li ) and alveolar bone ( ab ) . group 1 : d ) radiographic image of hemi - mandible similar at the control group ; e - f ) photomicrograph of the right first molar exhibiting inflammatory cells ( black arrow ) in the periapical region . group 2 : g ) radiographic image of hemi - mandible showing radiolucent area in the periapical region ; h - i ) photomicrograph of the right first molar showing extensive bone ( b ) resorption by osteoclasts ( red arrow ) and connective tissue ( ct ) . group 3 : j ) radiographic image of hemi - mandible exhibiting extensive radiolucent area in the periapical region ; k - l ) photomicrography of the right first molar showing abscess ( red arrow ) , increase of connective tissue area ( ct ) and decrease of bone tissue ( b ) in relation to the other groups the histological analysis of the left first molars of the rats , which served as control , showed no alterations in the pulp or periapical tissues , and evidenced that 100% of these teeth presented healthy pulp and periapical tissues ( figures 1b , c ) . the teeth submitted to coronal opening in group 1 , showed in 100% of the cases , necrosis in the coronal region and pulp inflammation with the presence of inflammatory cells and increased vascularization in the medial and apical thirds . it was possible to observe inflammatory cells and small bone and cementum resorption in the periapical region ( figures 1e , f ) . group 2 showed necrosis in the coronal and middle radicular pulp and extensive inflammatory process in the apical third . in the periapical region exhibited increase of bone resorption and replaced by conjunctive tissue rich in inflammatory cells ( figures 1h , i ) . the group 3 showed total pulp necrosis , extensive area of abscess and bone , cementum and dentine resorption and few bone trabeculae surrounded by conjunctive tissue in the apical portion ( figures 1k , l ) . the total numbers of pixels obtained for each of the measured regions in the control and tests sides , in the various experimental periods are presented in table 2 . two - way anova and tukey 's tests showed statistically significant difference between the test and control sides and among the experimental periods as well . mean and standard deviation of the total number of pixels of each structure present in the periapical region of the mesial root of the mandibular first molar in the control and test groups in the experimental periods mean pixel value from 9 animal samples . in the present study , the periapical inflammation was induced by coronal opening of the mandibular right first molar and confirmed that only coronal opening and pulp contact with the oral cavity were sufficient for inducing periapical disease . various other studies have described this technique for induction of pulpal and periapical inflammatory reactions in rat teeth . the inflammatory process was observed in the pulp region in the experimental period of 2 days . this is in agreement with the findings of a previous study that showed that pulp inflammation is already observed 6 h after the operative procedure . in the present study , pulp necrosis was observed in the experimental periods of 15 and 30 days . this is also in agreement with a previous study that showed the presence of complete pulp necrosis between the 8 and 20 days . ( 1995 ) affirmed that bone resorption is present from the 3 day after the operative procedure . bone resorption in the present study , although very subtle , could already be observed in the group 1 . resorptive bone activity was actively present on the 15 day ( group 2 ) . some authors affirmed that this activity begins to diminish from then on , with a decrease in the number of osteoclasts . although the lesion appeared to be stabilized , with diminished bone resorption , new bone formation did not occur due to the presence of aggressive factors . in the 30-day experimental period , a large area of resorption the amount of periapical bone was minimal , and it was replaced by inflammatory tissue . statistical analysis showed a significant difference between test and control sides in the 3 experimental periods , except for the root region , in which this difference was not significant between the test and control sides , in the 2-day period , and between the experimental periods of 15 and 30 days . this was probably because resorption in periapical disease is greater in the bone region than in the root region , which was not very evident . the present study confirmed radiographically the presence and intensity of bone resorption observed by the histological study . ( 2003 ) removed medullar bone from cadaver mandibles , radiographed the areas to measure the pixel value respectively . these studies showed that it is possible to detect bone loss in the mandibular region by analyzing the pixel value , even if it is not yet visible radiographically . conversely , studies that compared images obtained by conventional radiographs ( without pixel value analysis ) and bone resorption seen microscopically , showed that this relationship is not established with precision de rossi , et al . ( 2007 ) concluded that although image digitization could not improve the detection of the early stages of periapical lesions , it provides valuable quantitative assessment of extensive periapical lesions . in order to diminish the noise level in the images captured directly , the phosphorus plates were covered by a protective envelope , as they are sensitive to light . the images were always downloaded immediately after the radiograph was taken , as the time between taking the radiograph and downloading the plates also influences the pixel values . in the direct photostimulable phosphorus plate systems , such as digora , there is a correlation between the radiation dose and the quantity of capture plate luminescence . the capture plates present less noise and a better quality image than the other direct systems , in addition to being more sensitive to small variations of the exposure source in comparison with conventional films , and should be used when small differences in contrast are important . the noise and coefficient of variability of pixel value calculations showed low noise and small variation in the images in this study . in spite of resistance by many professionals , the high cost of the technology and the ethical implications because of the risk of manipulating the images , the use of digital imaging has increased and is constantly being perfected . the methodology applied was efficient for causing pulpitis and pulpal necrosis in the studied rats . teeth with pulpitis microscopically presented periapical bone resorption , but the teeth with necrosis presented greater resorption . the control teeth presented healthy pulp , without signs of periapical bone resorption . the means of the pixel values in the areas of periapical disease induced in rats indicated greater bone resorption than the means of these values on the control side for the three experimental periods ( p<0.05 ) . even small periapical bone resorption was already sufficient for determining changes in the pixel values of that area in the direct digital method , when compared to their respective controls .

the purpose of this study was to appraise the imageologists of a possible mislocalization of tracer accumulation to anomalously placed gallbladder during positron emission tomography - computed tomography ( pet / ct ) examination . pet / ct is increasingly playing an important role in staging and restaging of the disease process in cancer patients . with the advent of fusion imaging , the tracer accumulation can be correctly localized to a structure or lesion on ct . we did a staging pet / ct scan of a patient with hepatocellular carcinoma for liver transplant evaluation . fluorine-18 fluorodeoxyglucose ( f-18 fdg ) was used as a tracer and the scan was performed on seimens biograph - mct pet / ct machine . we noted the tracer accumulation at the superior surface of liver , which was localized to the anomalously placed gallbladder in suprahepatic subdiaphragmatic location . the anomalously placed gallbladder can create localization confusion . keeping the possibility of ectopically placed gallbladder in mind , the imageologist can better localize the tracer uptake . objectivethis study evaluated experimentally - induced periapical bone loss sites using digital radiographic and histopathologic parameters . material and methodstwenty - seven wistar rats were submitted to coronal opening of their mandibular right first molars . they were radiographed at 2 , 15 and 30 days after the operative procedure by two digital radiographic storage phosphor plates ( digora ) . the images were analyzed by creating a region of interest at the periapical region of each tooth ( imagej ) and registering the corresponding pixel values . after the sacrifice , the specimens were submitted to microscopic analysis in order to confirm the pulpal and periapical status of the tooth . resultsthere was significant statistically difference between the control and test sides in all the experimental periods regarding the pixel values ( two - way anova ; p<0.05 ) . conclusionsthe microscopic analysis proved that a periapical disease development occurred during the experimental periods with an evolution from pulpal necrosis to periapical bone resorption .

carcinoid tumors develop from stem cells of the bronchial epithelium known as kulchitsky cells , which have neuroendocrine activity . these neuroendocrine cells can develop tumors in many different organs with most common being the lungs , the appendix , the small intestine ( duodenum ) , the rectum and the pancreas.1 the most recent classification system from the world health organisation categorized neuroendocrine pulmonary tumors in four types : typical carcinoid ; atypical carcinoid ( atc ) ; large cell neuroendocrine carcinoma ; and small cell lung cancer.2 carcinoid bronchopulmonary tumors represent approximately 25% of all carcinoid tumors and 1%2% of all lung neoplasms.3 approximately 70% of these tumors are located centrally in the large bronchial tubes leading to the lung , while 10%20% , known as peripheral carcinoids , develop in the pulmonary periphery.4 typical carcinoid pulmonary tumors manifest nine to ten times more often than atc tumors . typical carcinoid tumors are seen in younger patients more often than atcs and appear to be the most frequent pulmonary neoplasms during childhood and puberty.5,6 atc lung tumors have a more aggressive histologic appearance and a greater tendency to metastasize than typical carcinoid lung tumors . these tumors are more frequent in males , and the average age of onset is 46 years.7 unlike most lung cancers , the development of carcinoid pulmonary tumors is not related to smoking or other environmental causes ( eg , tobacco smoke , air pollution , or asbestos ) . the detection of carcinoid tumorlets in lung biopsies led to speculation that carcinoid tumors may grow from these cells.1 however , further research needs to be conducted as this association has not been yet determined . a 26-year - old female presented with wheezing episodes , cough and mild dyspnea . after a chest x - ray revealed normal findings and spirometry showed mild restriction ( table 1 ) , she was commenced on bronchodilators and inhaled corticosteroids . two years later , the symptoms were not entirely relieved and she had developed hemoptysis of approximately 10 ml/24 hours . therefore , she was referred to the pulmonary department for further examinations . during clinical examination the patient had reduced respiratory whistling and mild wheezing in the upper left pulmonary field . the results of the exams revealed normal blood tests , a mild restrictive spirometry ( table 1 ) , a negative mantoux and a left perihilar shadow in the chest radiograph ( figure 1a and b ) . a chest high resolution computed tomography was performed showing a nodule in the left primary bronchus causing its partial obstruction ( figure 2 ) . the suspicion of malignancy was established and the patient underwent a bronchoscopy ( olympus bf - p240 ; 6 mm insertion tube , biopsy channel 2.6 mm , 55 cm working length , [ olympus corp , tokyo , japan ] ) . the findings showed exophytic damage with increased vascularization in the left primary bronchus 5 cm from the carina . the biopsy demonstrated a typical carcinoid and the patient underwent pelvic and cerebral computed tomography , which both showed normal findings . in an effort to avoid pneumonectomy , and to produce rapid palliation of hemoptysis and immediate tumor debulking , a rigid bronchoscopy was inserted into the trachea under general anesthesia using a 7.5 mm storz rigid bronchoscope ( karl storz gmbh and co , tuttlingen , germany ) . electrocautery ( power setting of 40 watts , blend mode , normal coagulation , electrosurgical monopolar unit psd-20 , with foot switch , flexible monopolar electrocautery blunt probe olympus cd-6c-1 , loop snare , [ olympus , tokyo , japan ] ) was performed on the exophytic tumor tissue followed by mechanical removal . this intervention fully re - opened the obstructed left main bronchus and the upper subsegment of the left upper lobe . the remaining tissue in the obstructed lower subsegment of the left upper lobe was treated by surgical resection , performing a lobectomy to remove the tissue entirely . during this procedure , the final report stated that there was no expanding malignancy affecting the pleural cavity or the lymph nodes . the patient was not given chemotherapy but was followed up with regular medical monitoring combined with imaging studies ( ie , chest x - ray , chest computed tomography ) . five years after surgical treatment , the patient was asymptomatic with normal chest radiographs and normal spirometry ( table 1 ; figure 1c ) . 8 the severity and variety of symptoms depend on the size of the carcinoid pulmonary tumor and the production of hormones . in symptomatic patients with central carcinoids , the most common symptoms are : persistent cough , asthma - like wheezing , chest pain , dyspnea , hemoptysis and obstructive pneumonitis.9 a number of studies have reported that a mistaken diagnosis of asthma is frequent in these cases , delaying early diagnosis . occasionally , carcinoid pulmonary tumors may initiate an excessive hormonal secretion causing various neuroendocrine syndromes such as carcinoid syndrome and cushing syndrome.9,10 the most common symptoms of carcinoid syndrome are : facial flushing , sweating , diarrhea , rapid cardiac beats and wheezing . even more rarely , patients with cushing syndrome caused by the abnormal overproduction of cortisol can develop muscle weakness , obesity in the upper body , high blood pressure , high blood sugar and increased body and facial hair . almost 90% of typical carcinoid tumors are diagnosed at an early stage while more than 50% of atc tumors are diagnosed at stage ii or iii.11 the diagnosis of carcinoid pulmonary tumor is established on the basis of the patient s symptoms combined with imaging examination such as chest radiography , computed tomography and magnetic resonance imaging.12 to verify the histologic type of the pulmonary tumor detected , there are several biopsy options including bronchoscopic biopsy , needle biopsies and surgical biopsies ( eg , thoracotomy).13 however , it has been reported that bronchoscopic biopsy has a risk of hemorrhage , especially in cases of partial or total removal of vascular tumors.14 the primary and most effective treatment for all pulmonary carcinoid tumors is surgical resection as long as no contraindications to surgery exist ( eg , widespread metastatic disease).15 chemotherapy and radiotherapy have no therapeutic contribution and there is no verified optimal therapy for unresectable carcinoid lung tumors.16 there are a variety of resection techniques that treat lung carcinoids effectively.13 presently , the most commonly utilized resection procedure is lobectomy . for smaller tumors located in the periphery or within a pulmonary segment , surgeons may perform segmental resection or wedge resection . , a bilobectomy or pneumonectomy may be required due to the size or the location of the tumor . postoperatively , the most common complications are excessive bleeding , atelectasis and prolonged air leak.13 interventional therapeutic bronchoscopies such as laser bronchoscopy , electrocautery therapy , cryotherapy , endobronchial brachytherapy , photodynamic therapy and airway stents may also be performed in certain cases.17,18 nd - yag lasers and electrocautery , together with mechanical tumor removal , are more appropriate for rapid palliation and immediate tumor debulking.19 in particular , electrocautery therapy , as applied in our case , can achieve immediate relief of the symptoms resulting from obstructed airways in 55%75% of patients . its advantages over laser treatment include cost effectiveness , higher availability , and faster removal of tumors and components resistant to laser coagulation . 18 moreover , in a study evaluating the degree of damage and bronchial wall healing after photodynamic , nd - yag laser , and electrocautery therapy , electrocautery produced the least amount of airway scarring and subepithelial fibrosis.19 in another study , the amount of mucosal damage visualized after electrocautery was correlated with histologic tissue damage . the investigators concluded that this is a potential advantage of electrocautery compared to other therapies , where the real histologic damage may be more severe than what is visualized after the therapy.20 side effects of electrocautery therapy include burn , haemorrhage , and inadvertent electrical shock to the endoscopist or the patient , but there are no reports of treatment - related deaths or respiratory failure episodes.18,21 these bronchoscopic techniques are usually used for palliative management of bronchial obstruction , for mass reduction prior to surgery , or for patients unsuitable for formal surgery.18 a number of studies of endobronchial resection have confirmed that bronchoscopic techniques are successful modalities of treatment.22 several therapeutic modalities used in conjunction may be essential to achieve the best management of the disease , rather than a single invasive approach.23 in a recent review , even though 50% of patients underwent a lung resection after a single bronchoscopic resection , the authors reported that the inability to remove the remaining tumor was entirely due to the inaccessibility of the tumor.2426 some experts believe that tumor reduction by this method may result in a more conservative surgical resection.15 our study confirms these findings as the use of bronchoscopic electrocautery followed by surgical lobectomy not only removed the tumor entirely but also avoided pneumonectomy . among pulmonary malignancies , carcinoid tumors have the highest prognostic rates . the 5-year survival rate reaches 92%100% for typical lung carcinoids and only 61%88% for atcs.27 previous studies have shown that lymph node involvement as well as the presence of tumorlets , have significant negative effects on prognoses.28 early suspicion , combined with imaging examination and biopsy , is the key to diagnosis . improved surgical techniques allow smaller incisions resulting in shorter hospitalization and less postoperative pain.19 many studies suggest that the use of endobronchial management techniques not only improve clinical symptoms and quality of life , but also increase overall survival.21,23 therefore , performing less invasive procedures should be considered throughout the management of lung cancer patients , provided no poor prognostic factors exist.29 series utilizing this mode of therapy must be reported in order to determine long - term outcomes . it is very important that scientists continue to investigate and publish results from such cases so that new information about the etiology and treatment of the disease can be made available . familial mediterranean fever ( fmf ) is a genetic , autosomal recessive disease affecting primarily non - ashkenazi jews , turks , arabs , and armenians . this disease is characterized by acute , short episodes of serosal membrane inflammation , and fever . the fmf gene , called as mediterranean fever ( mefv ) gene that encodes pyrin / marenostrin , is mapped to the short arm of chromosome 16 . although il-1 plays a fundamental role in pathogenesis of fmf , tumor necrosis factor - alpha ( tnf- ) , soluble il-2 receptor , il-6 , and il-8 have also been shown to stimulate this process . patients have an increased erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) , serum amyloid a ( saa ) , and fibrinogen levels during these attacks and usually , this increase returns to normal values in attack - free periods . however , it is now known that subclinical inflammation may continue in attack - free periods of fmf patients . this type of inflammation may lead to developing of amyloidosis , which is the most devastating complication of fmf . neutrophil to lymphocyte ratio ( nlr ) and mean platelet volume ( mpv ) were used to show the inflammation in both cardiac and noncardiac disorders before . , we aimed to investigate whether nlr and mpv could be used to indicate the subclinical inflammation in pediatric fmf patients . ninety of the patients were in attack - free period , and seventy were in attack period . criteria . attack - free period was described as at least 2 weeks from the end of an fmf attack according to the symptoms and acute phase reactants levels . control group was selected from the patients who had no acute or chronic disease that could cause inflammation and who were attended to pediatric clinics of our hospital for other reasons . patients and controls having any other systemic disease or infection and using medication other than colchicine were excluded from the study . laboratory data such as esr , crp , hemoglobin level , white blood cell count , platelet count , and mpv were noted using the electronic patient database of the last visit . the analyses were performed with the same analyzer ( beckman coulter , lh 780 ) in our center . nlr was calculated as a simple ratio between the absolute neutrophil count and absolute lymphocyte count . as this study represents a retrospective chart review , however , all patients signed an informed consent that allows our institution to use their clinical data . chi - square statistics was used for the comparisons of categorical variables between independent groups . the normal distributions of the numerical variables were evaluated by the shapiro wilk test . wallis nonparametric analysis of variances test was used for the comparisons of numerical variables between more than two independent groups , and mann whitney u - test was used for the comparisons between two groups . results for qualitative variables were presented as percentages , and quantitative variables were presented as mean and standard deviation . when a statistically significant difference was found by kruskal wallis test , then , post hoc pairwise comparisons were performed using mann whitney u - test . the associations between the numerical variables of the study were evaluated using spearman nonparametric correlation analysis . the type - i error level was accepted as 5% in the study , and all statistical analyses were performed as two - tailed . for the statistical analysis of the data , the statistical package for the social sciences 17.0 ( spss inc . , ninety of the patients were in attack - free period , and seventy were in attack period . criteria . attack - free period was described as at least 2 weeks from the end of an fmf attack according to the symptoms and acute phase reactants levels . control group was selected from the patients who had no acute or chronic disease that could cause inflammation and who were attended to pediatric clinics of our hospital for other reasons . patients and controls having any other systemic disease or infection and using medication other than colchicine were excluded from the study . laboratory data such as esr , crp , hemoglobin level , white blood cell count , platelet count , and mpv were noted using the electronic patient database of the last visit . the analyses were performed with the same analyzer ( beckman coulter , lh 780 ) in our center . nlr was calculated as a simple ratio between the absolute neutrophil count and absolute lymphocyte count . as this study represents a retrospective chart review , however , all patients signed an informed consent that allows our institution to use their clinical data . chi - square statistics was used for the comparisons of categorical variables between independent groups . the normal distributions of the numerical variables were evaluated by the shapiro wilk test . wallis nonparametric analysis of variances test was used for the comparisons of numerical variables between more than two independent groups , and mann whitney u - test was used for the comparisons between two groups . results for qualitative variables were presented as percentages , and quantitative variables were presented as mean and standard deviation . when a statistically significant difference was found by kruskal wallis test , then , post hoc pairwise comparisons were performed using mann whitney u - test . the associations between the numerical variables of the study were evaluated using spearman nonparametric correlation analysis . the type - i error level was accepted as 5% in the study , and all statistical analyses were performed as two - tailed . for the statistical analysis of the data , the statistical package for the social sciences 17.0 ( spss inc . , chicago , il , usa ) there were 160 participants ( 68.4% ) in the patient group and 74 participants ( 31.6% ) in the control group . ninety of patients ( 38.5% ) were in attack - free period , and 70 ( 29.9% ) were in attack period . the male / female distribution in the groups was 29 ( 41.4%)/41 ( 58.6% ) in fmf attack group , 35 ( 38.9%)/55 ( 61.1% ) in attack - free group , and 38 ( 51.4%)/36 ( 48.6% ) in control group . the statistical analyses revealed that the sex distribution between groups was similar ( p = 0.25 ) . ages according to the groups were 11.56 4.3 years in attack period , 12.81 3.88 years in attack - free period , and 10.74 4.01 years in the control group . the general group comparisons revealed that hemoglobin ( p < 0.001 ) , leukocyte ( p < 0.001 ) , thrombocyte ( p = 0.016 ) , crp ( p < 0.001 ) , mpv ( p < 0.001 ) , neutrophil ( p < 0.001 ) , and lymphocyte ( p < 0.001 ) levels were significantly differed between study groups . we found that mpv values of attack patients and attack - free patients had no significant difference ( p = 0.07 ) . mpv values of attack patients were significantly higher than the control group ( p < 0.001 ) , and mpv values of attack - free patients were also significantly higher than the control group ( p < 0.001 ) [ table 1 ] . we also found a negative correlation between mpv and thrombocyte count in all groups and it was significant in attack and attack - free periods ( p = 0.002 and p < 0.001 , respectively ) . complete blood count parameters in study groups the values of nlr in study groups are presented in table 2 . according to this , general group comparisons revealed a statistically significant difference between groups ( p < 0.001 ) , and post hoc pairwise comparisons showed that the highest values of nlr were found in the patients at attack period . the patients in attack - free period and the participants in control group had similar levels of nlr ( p = 0.45 ) , and they had lower ratios than the patients did at attack period ( p < 0.001 for both ) . correlations between the neutrophil / lymphocyte ratio and the other parameters in the study groups are summarized in table 3 . there were positive weak correlation between crp and nlr levels in attack ( r = 0.049 , p = 0.68 ) and attack - free patients ( r = 0.086 , p = 0.07 ) , but they were not statistically significant . neutrophil / lymphocyte ratios in study groups correlations between neutrophil / lymphocyte ratio and the other parameters in study groups the distribution of mefv gene mutations are summarized in table 4 . patients carrying m694v ( either homozygous or heterozygous ) were compared with patients carrying other mutations . nlr and mpv values did not have significant difference between different mutation groups ( p = 0.30 and p = 0.37 ) . only patients with m694v homozygous and m694v / m680i compound heterozygous mutations have higher crp levels ( p < 0.001 ) . mediterranean fever gene mutation analysis the colchicine usage was evaluated in attack and attack - free periods . accordingly , the patients in attack period used colchicine with a mean dose of 1.03 0.34 mg / day , and the patients in attack - free period used colchicine with a mean dose of 1.09 0.29 mg / day . the difference of colchicine usage between groups was not statistically significant ( p = 0.25 ) . the main aim of our study is to determine the subclinical inflammation in pediatric fmf patients using mpv and nlr . fmf is an autoinflammatory disease caused by abnormalities confined to the innate immune system and the result of reduced or complete loss of pyrin function . pyrin mutations and fmf link have been well established before . both pro- and anti - inflammatory roles pyrin is an important element of inflammasome and mutations in pyrin result in increased inflammation . this activates caspase-1 and the processing and release of active il-1. il-1 with other cytokines il-6 and tnf- results in high levels of acute phase reactants . esr , crp , fibrinogen , and saa are all used as markers for acute phase response in fmf . these acute phase proteins increase during attack period and usually return to normal in attack - free period . subclinical inflammation continues in up to 30% of the fmf patients during attack - free period . at this point , persistent elevation of acute phase proteins is important as it reflects the subclinical inflammation , which has the key role in the development of amyloidosis and other complications such as anemia , splenomegaly , and osteopenia . as a result , nlr was shown as a useful indicator of clinical outcome and disease severity in diseases having malign and inflammatory components . they also found that nlr was significantly higher in patients with amyloidosis than in amyloidosis - free patients . concluded that in fmf patients nlr can be used as an acute phase response such as crp . they found that nlr of fmf patients during attack period was significantly higher than those of attack - free patients and control group . uluca et al . found that nlr levels were higher in patients in attack - free period and they concluded that nlr may be an indicator for attack period but not attack - free period . zer et al . also reported nlr values of pediatric symptom - free fmf patients and healthy controls . however , in this study , they did not compare the nlr values of patients in attack period and attack - free period . our results provide evidence which supports the thought that nlr may be a parameter to show the inflammation in attack period . however , it may not be useful to define the subclinical inflammation according to our study groups since there was no significant difference between attack - free patients and control group . as it is cost - effective , available and can be calculated easily , nlr could be used to predict systemic inflammation in pediatric fmf patients during attack period . mpv has been investigated many times before to show disease activity , inflammatory load , and systemic inflammation in diseases . mpv is an easily available and cost - effective test that can show thrombocyte activation and function . most of them have found higher mpv levels in fmf patients , while others have reported lower mpv values . published two studies discussing the relationship between mpv and atherosclerosis in fmf patients . in the first study , they found that mpv values were similar in fmf patients and healthy controls . they concluded that mpv did not predict atherosclerosis risk in pediatric fmf patients . in the later study , they compared the mpv values and epicardial adipose tissue thickness in children with fmf . they found significantly greater epicardial adipose tissue thickness and higher mpv values in children with fmf . they concluded that mpv values might indicate an increased risk of atherosclerosis in fmf . in our study , both attack and attack - free patients had significantly higher mpv values compared to control group similar to most of the literature results . however , there were no significant differences between mpv values of attack and attack - free periods . therefore , we concluded that mpv is not useful to show the attack periods in fmf , but it may be valuable to show the risk of atherosclerosis and subclinical inflammation in pediatric fmf patients . many studies have reported that m694v mutation is related with high disease activity and amyloidosis in fmf . although we could not find any significant difference of nlr and mpv values between fmf patients carrying m694v mutation , patients with m694v homozygous and heterozygous mutations had higher crp values . a possible limitation of our study is that all of our study groups were under colchicine treatment , which may reduce inflammation . in addition , our study was performed in one center , so our results do not reflect all pediatric fmf population . the mean age of the patients differ significantly between study groups ; however , this should be accepted as a drawback rather than a positive finding as we did not perform an age - matched analysis . in addition , parameters were evaluated by cross - section , and no other follow - up values were measured . however , our results did not support that mpv might show attack and attack - free period . there were conflicting results in the literature about the role of mpv and nlr in inflammation and subclinical inflammation . ob contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work.nu contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work.bac contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work.fa contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work.nc contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work ob contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . nu contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . bac contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . fa contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . nc contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work

carcinoid bronchopulmonary tumors represent approximately 25% of all carcinoid tumors and 1%2% of all lung neoplasms . the most common symptoms are : persistent cough , asthma - like wheezing , chest pain , dyspnea , hemoptysis and obstructive pneumonitis . we present a case of a young adult diagnosed with a typical carcinoid tumor . the diagnosis was established on the basis of imaging examination and bronchoscopic biopsy . the patient was treated with bronchoscopic electrocautery therapy to relieve the obstructed airway , followed by surgical lobectomy in order to entirely remove the exophytic damage . this approach was not only a palliative management to bronchial obstruction but also avoided pneumonectomy . recent studies support the use of such interventional resection methods , as they may result in a more conservative surgical resection . background : blood neutrophil to lymphocyte ratio ( nlr ) and mean platelet volume ( mpv ) both have been used as a simple marker of inflammation in many disorders . here , we aimed to investigate the relationship between nlr , mpv , and familial mediterranean fever ( fmf).materials and methods : in this retrospective study , the files of fmf patients in pediatric rheumatology outpatient clinic were reviewed . there were 160 participants ( 68.4% ) in the fmf patient group and 74 participants ( 31.6% ) in the control group . ninety of patients were in attack - free period , and 70 were in attack period.results:the highest values of nlr were found in the patients at attack period . patients in attack - free period and the participants in control group had similar levels of nlr ( 1.71 0.83 and 1.91 1.86 respectively ) ( p = 0.457 ) , and they had lower ratios than the patients did at attack period ( 4.10 3.11 ) ( p < 0.001 for both ) . there was no significant difference between mpv values of attack patients ( 8.35 4.91 ) and attack - free patients ( 8.43 1.15 ) ( p = 0.074 ) . mpv values of attack patients and attack - free patients were significantly higher than control group ( 7.99 0.81 ) ( p < 0.001 for both).conclusion : nlr ratio may indicate fmf attack period . since there was no significant difference between attack - free patients and control groups , nlr ratio can not be used as a subclinical inflammation marker . however , nlr could be a useful predictor of inflammation in fmf patients . on the other hand , since our attack and attack - free patients have similar mpv values and both had greater mpv values than control group , we suggest that mpv may be used to show subclinical inflammation .

cone beam computed tomography ( cbct ) was introduced to the dental field to replace the cumbersome , expensive , and high - radiation producing medical ct scans around a decade ago suomalainen et al . the american association of oral and maxillofacial radiology has stated that cross - sectional views are recommended for planning dental implants , and this in combination with the easy accessibility , easy handling , and low - radiation dose of cbct imaging will lead to the widespread use of cbct imaging in implantology . considering the dose of radiation from other image acquisition modalities such as multislice computed tomography ( msct , e.g. , maxmand ct scan : effective dose of 2100 according to the 1990 recommendations of the international commission on radiological protection ( icrp ) ) and panoramic radiography ( e.g. , panoramic orthophos plus : effective dose of 6.3 and 13.3 according to icrp 1990 and 2005 , respectively ) , cbct has lesser radiation than msct and 10 times more radiation than a panoramic x - ray . the dose of radiation should be reported in millisievert ( msv ) or microsievert ( sv ) to express the effective dose ( e ) . as ludlow stated , the e of radiation has been recommended by the icrp as a means of comparing the detriment of different exposures to ionizing radiation to an equivalent detriment produced by a full - body dose of radiation . the e should be calculated using the equation e = pwt_ht , where wt = weighting factor , ht = equivalent dose . the ht should be calculated using the equation ht = pwr_dt , where wr = radiation weighting factor ( which is 1 in the case of x - ray radiation ) , dt = absorbed dose . benefits of cbct are three - dimensional ( 3d ) dataset , real - size data , the potential for generating all 2d images ( e.g. , orthopantomogram , lateral cephalogram , imaging of the temporomandibular joint ( tmj ) ) , potential for vertical scanning in a natural seated position , isotropic voxel size , high - resolution ( e.g. , bone trabeculae , periodontal ligament ( pdl ) , root formation ) imaging , lower dose of radiation than msct , less disturbance from metal artifacts , reduced costs compared with msct , easier accessibility , in - office imaging , easier handling , small footprint , digital imaging and communications in medicine ( dicom ) compatiblity , user - friendly postprocessing and viewing software , and better saving of energy compared with msct . limitations are low contrast range , limited detector size causing limited field of view and limited scanned volume , limited inner soft tissue information , increased noise from scatter radiation and concomitant loss of contrast resolution , movement of artifacts affecting the whole dataset , truncation artifacts ( caused by the fact that projections acquired with the region of interest selection do not contain the entire object ) , and that they can not be used for estimation of hounsfield units ( hus ) . hus represent the relative density of body tissues according to a calibrated gray - level scale , based on normalized hu values for air ( 1000 hu ) , water ( 0 hu ) , and dense bone ( + 1000 hu ) . hus are standard numbers originating from conventional msct imaging . despite the advantages of cbct compared with msct imaging , the latter is still considered a better choice for the analysis of bone density using an hu scale . the fan - shaped x - ray beam associated with less scattering and artifact production may explain the better accuracy of intensity values in an msct than in a cbct scanner which is a cone - shaped x - ray beam , and its higher amount of scattering and artifacts may explain the inaccuracies of their intensity values . in both technologies , the accuracy of intensity values can be affected by the beam - hardening phenomenon , which causes artifacts on the reconstructed images . in the posterior mandibular region , a deep lingual undercut is a common finding and can be difficult to manage , especially when a lingual plate perforation is suspected . it is essential to check the angulations and positioning of the drills or implant fixtures via radiographs and clinical detection of a possible perforation in the osteotomy site . for preoperative implants , cts are preferred because cross - sectional views bring a clearer visualization of the anatomy of the surgical site [ figure 1 ] . the major potential risks of encountering a lingual plate perforation are massive hemorrhage of submental and sublingual arteries , airway obstruction , and a perforation above the mylohyoid ridge might injure the lingual nerve . if the extruded implant is left unattended , the infection might spread to the parapharyngeal and retropharyngeal space , leading to more severe complications , such as mediastinitis , mycotic aneurysm formation with possible subsequent rupture of the internal carotid artery , and internal jugular vein thrombosis with septic pulmonary embolism or upper airway obstruction . the experimental site has to have sufficient vertical bone height ( 12 mm from the alveolar crest to the superior border of the inferior alveolar nerve canal ( ian ) ) to possibly place a 10 mm implant and a minimum gap of 2 mm between the tip of implant and mandibular canal . mandibular cross - sectional imaging at the edentulous first molar region shows three types of morphologies . the undercut ridge type ( type u , 66% ) is a ridge with a narrow base that expands bucco - lingually to a wider crest with a prominent point ( point p ) on the lingual plate , giving rise to a lingual undercut . the parallel ridge type ( type p , 20.4% ) ridge generally has a more or less parallel ridge form ; no lingual undercut is seen . the convergent ridge type ( type c , 13.6% ) ridge is one where the base of the ridge is wider than its crest ; no obvious undercut is seen [ figure 2 ] . cone beam computed tomography image demonstrating the possibility of lingual plate perforation by an implant three types of cross - sectional posterior mandibular morphology : ( a ) c type , ( b ) p type , and ( c ) u type ; line a represented a reference line 2 mm coronal to the inferior alveolar nerve canal watanabe et al . classified the cross - sectional mandibular morphology based on the outlines of the lingual and buccal plates , round on the buccal side and concave on the lingual side ( type a ) , concave on the buccal side and round on the lingual side ( type b ) , and round shape on both sides ( type c ) . they reported that at the posterior region , type c ( round ) was the most commonly found ( 59 - 61% ) , followed by type a ( lingual concavity ) ( 36 - 39% ) . the width of the mandible 5 - 20 mm from the inferior border of the mandible ranged from 10.5 to 15.8 mm , with no significant differences between genders . panoramic radiographs have an inherent magnification ranging from 10 to 30% , with the horizontal magnification being more variable and thus less reliable . marginal loss of bone and loss of bone - to - implant contact ( e.g. , by marsupuilization ) may indeed negatively influence success of the implant . most of these studies suggest an acceptable average of marginal loss of bone ( bone loss of 2 mm after the first year ) and the absence of a peri - implant radiolucency as a criterion of radiological success . the mandibular canal and mental foramen involve the inferior alveolar artery and inferior alveolar nerve . because images of the accessory mental foramina and bony canal to the accessory mental foramen overlap in various trabecular bone patterns . it was reported that the presence of the bifid mandibular canal in the mandibular ramus region was observed more frequently with cbct images , in 65% of patients compared with rotational panoramic radiographs , with a range from 0.08 to 0.95% . the accessory mental nerve communicated with branches of the facial and buccal nerves . so , it was indicated that surgical complications might be attributed to the existence of a mandibular incisive canal with a true neurovascular supply , and potential risks might also be related to the presence of the lingual foramen and anatomic variations , such as an anterior looping of the mental nerve [ figure 3 ] . measurement between the accessory mental foramen and point of bifurcation from the mandibular canal : ( a ) two - dimensional cone beam computed tomography image of the accessory mental foramen and point of bifurcation from the mandibular canal ; ( b ) linear distance between the accessory mental foramen and point of bifurcation from the mandibular canal ; ( c ) schematic drawing of 2d cbct image ( a ) the nasopalatine canal is usually described as being located in the midline of the palate , posterior to the central maxillary incisors . the funnel - shaped oral opening of the canal in the midline of the anterior palate is known as the incisive foramen , and is usually located immediately below the incisive papilla . the canal divides into two canaliculi on its way to the nasal cavity , and terminates at the nasal floor with an opening ( known as the foramina of stenson ) at either side of the septum . the canal contains the nasopalatine ( incisive ) nerve and the terminal branch of the descending nasopalatine artery , as well as fibrous connective tissue , fat , and even small salivary glands . contact of the implant with neural tissue may result in failure of osseointegration or lead to sensory dysfunction . the anatomic variants of the canal are differentiated into three groups [ figures 4 and 5 ] . classification of anatomic variations of the nasopalatine canal:(a ) a single canal ; ( b ) two parallel canals ; ( c ) variations of the y type of canal , with one oral / palatal opening ( incisive foramen ) and two or more nasal openings ( foramina of stenson ) type a nasopalatine canal ( a single canal ) type b nasopalatine canal ( two separate canals ) as evaluated in a coronal cone beam computed tomography image type c nasopalatine canal ( y configuration of canal ) with one oral / palatal opening and two nasal openings dimensional alterations occur on the alveolar process following tooth extraction . after the healing process is completed , loss of bone at the facial aspect of the marginal one - third of the socket is more pronounced than in the palatal / lingual aspect . this difference in the healing outcome maybe related to the fact that the buccal bone wall is thinner than its palatal counterpart . the thinner the facial bone wall , the more extensive the loss of facial bone . following tooth removal / loss , the entire marginal , buccal bone plate be lost , but an additional 2 mm of the original socket dimension may disappear during the process of socket healing and site adaptation . in the posterior mandibular region , a deep lingual undercut is a common finding and can be difficult to manage , especially when a lingual plate perforation is suspected . it is essential to check the angulations and positioning of the drills or implant fixtures via radiographs and clinical detection of a possible perforation in the osteotomy site . for preoperative implants , cts are preferred because cross - sectional views bring a clearer visualization of the anatomy of the surgical site [ figure 1 ] . the major potential risks of encountering a lingual plate perforation are massive hemorrhage of submental and sublingual arteries , airway obstruction , and a perforation above the mylohyoid ridge might injure the lingual nerve . if the extruded implant is left unattended , the infection might spread to the parapharyngeal and retropharyngeal space , leading to more severe complications , such as mediastinitis , mycotic aneurysm formation with possible subsequent rupture of the internal carotid artery , and internal jugular vein thrombosis with septic pulmonary embolism or upper airway obstruction . the experimental site has to have sufficient vertical bone height ( 12 mm from the alveolar crest to the superior border of the inferior alveolar nerve canal ( ian ) ) to possibly place a 10 mm implant and a minimum gap of 2 mm between the tip of implant and mandibular canal . mandibular cross - sectional imaging at the edentulous first molar region shows three types of morphologies . the undercut ridge type ( type u , 66% ) is a ridge with a narrow base that expands bucco - lingually to a wider crest with a prominent point ( point p ) on the lingual plate , giving rise to a lingual undercut . the parallel ridge type ( type p , 20.4% ) ridge generally has a more or less parallel ridge form ; no lingual undercut is seen . the convergent ridge type ( type c , 13.6% ) ridge is one where the base of the ridge is wider than its crest ; no obvious undercut is seen [ figure 2 ] . cone beam computed tomography image demonstrating the possibility of lingual plate perforation by an implant three types of cross - sectional posterior mandibular morphology : ( a ) c type , ( b ) p type , and ( c ) u type ; line a represented a reference line 2 mm coronal to the inferior alveolar nerve canal watanabe et al . classified the cross - sectional mandibular morphology based on the outlines of the lingual and buccal plates , round on the buccal side and concave on the lingual side ( type a ) , concave on the buccal side and round on the lingual side ( type b ) , and round shape on both sides ( type c ) . they reported that at the posterior region , type c ( round ) was the most commonly found ( 59 - 61% ) , followed by type a ( lingual concavity ) ( 36 - 39% ) . the width of the mandible 5 - 20 mm from the inferior border of the mandible ranged from 10.5 to 15.8 mm , with no significant differences between genders . panoramic radiographs have an inherent magnification ranging from 10 to 30% , with the horizontal magnification being more variable and thus less reliable . marginal loss of bone and loss of bone - to - implant contact ( e.g. , by marsupuilization ) may indeed negatively influence success of the implant . most of these studies suggest an acceptable average of marginal loss of bone ( bone loss of 2 mm after the first year ) and the absence of a peri - implant radiolucency as a criterion of radiological success . the mandibular canal and mental foramen involve the inferior alveolar artery and inferior alveolar nerve . because images of the accessory mental foramina and bony canal to the accessory mental foramen overlap in various trabecular bone patterns . it was reported that the presence of the bifid mandibular canal in the mandibular ramus region was observed more frequently with cbct images , in 65% of patients compared with rotational panoramic radiographs , with a range from 0.08 to 0.95% . the accessory mental nerve communicated with branches of the facial and buccal nerves . so , it was indicated that surgical complications might be attributed to the existence of a mandibular incisive canal with a true neurovascular supply , and potential risks might also be related to the presence of the lingual foramen and anatomic variations , such as an anterior looping of the mental nerve [ figure 3 ] . measurement between the accessory mental foramen and point of bifurcation from the mandibular canal : ( a ) two - dimensional cone beam computed tomography image of the accessory mental foramen and point of bifurcation from the mandibular canal ; ( b ) linear distance between the accessory mental foramen and point of bifurcation from the mandibular canal ; ( c ) schematic drawing of 2d cbct image ( a ) the nasopalatine canal is usually described as being located in the midline of the palate , posterior to the central maxillary incisors . the funnel - shaped oral opening of the canal in the midline of the anterior palate is known as the incisive foramen , and is usually located immediately below the incisive papilla . the canal divides into two canaliculi on its way to the nasal cavity , and terminates at the nasal floor with an opening ( known as the foramina of stenson ) at either side of the septum . the canal contains the nasopalatine ( incisive ) nerve and the terminal branch of the descending nasopalatine artery , as well as fibrous connective tissue , fat , and even small salivary glands . contact of the implant with neural tissue may result in failure of osseointegration or lead to sensory dysfunction . the anatomic variants of the canal are differentiated into three groups [ figures 4 and 5 ] . classification of anatomic variations of the nasopalatine canal:(a ) a single canal ; ( b ) two parallel canals ; ( c ) variations of the y type of canal , with one oral / palatal opening ( incisive foramen ) and two or more nasal openings ( foramina of stenson ) type a nasopalatine canal ( a single canal ) type b nasopalatine canal ( two separate canals ) as evaluated in a coronal cone beam computed tomography image type c nasopalatine canal ( y configuration of canal ) with one oral / palatal opening and two nasal openings dimensional alterations occur on the alveolar process following tooth extraction . after the healing process is completed , loss of bone at the facial aspect of the marginal one - third of the socket is more pronounced than in the palatal / lingual aspect . this difference in the healing outcome maybe related to the fact that the buccal bone wall is thinner than its palatal counterpart . the thinner the facial bone wall , the more extensive the loss of facial bone . following tooth removal / loss , the entire marginal , buccal bone plate be lost , but an additional 2 mm of the original socket dimension may disappear during the process of socket healing and site adaptation . male young adult ( 4 - 6 weeks of age ) c57bl/6 ( charles river ) , drd1a tdtomato bac transgenic ( d1-tdtomato , gift of n. calakos , duke university , durham , nc ) , drd2egfp bac transgenic ( d2-egfp ) , oxtrtm1.1wsy homozygous ( cotr ko , jackson laboratory ) , or otr venus neo/+ ( otr - venus , gift of l.j . young , emory university , atlanta , ga ) mice backcrossed to c57bl/6 were used for all experiments . all procedures complied with the animal care standards set forth by the national institutes of health and were approved by stanford university s administrative panel on laboratory animal care . experimenters were blind to the treatment condition when subjective criteria were used as a component of data analysis , and control and test conditions were interleaved for all experiments . the protocol for social conditioned place preference ( scpp ) was shortened to 2 days of conditioning ( fig . animals were weaned ( or delivered from charles river ) at 3 weeks of age into home cages containing 3 - 5 cage - mates , and housed on corncob bedding ( bed - ocobs , 1/8 , pharmaserv ) . one to two weeks later , animals were subjected to experimental manipulations and returned to their home cage ( all cage - mates were of the same genotype and received the same experimental manipulation ) . animals were then placed in open field activity chamber ( env-510 , med associates ) equipped with infrared beams and a software interface ( activity monitor , med associates ) that monitors the position of the mouse . the apparatus was divided into two equally sized zones using a clear plastic wall , with a 5 cm diameter circular opening at the base ; each zone contained one type of novel bedding ( alpha - dri , pharmaserv , alpha chip , pharmaserv ; bed - ocobs , 1/4 , pharmaserv ; or kaytee soft granule , petco ) . the amount of time spent freely exploring each zone was recorded during 30-minute test sessions . after an initial test ( pre - conditioning trial ) to establish baseline preference for the two sets of bedding cues , mice were assigned to receive social conditioning ( with cage - mates ) for 24 hours on one type of bedding , followed by 24 hours on the isolate bedding cue ( without cage - mates ) on the other type of bedding . 24 hours later , animals received a 30-minute post - conditioning trial to establish preference for the two conditioned cues . animals were excluded ( pre - established criteria ) if they exhibited a pre - conditioning preference score > 1.5 or < 0.5 ( for an unbiased procedure ) ; pre - conditioning versus post - conditioning social preference scores were considered significant if paired student s t - test p values were < 0.05 . comparisons between experimental conditions were made using both normalized social preference scores ( time spent in social zone post over pre ) , and subtracted social preference scores ( time spent in social zone post minus pre ) ; these were considered significant if unpaired student s t - test ( two conditions ) , or anova ( three conditions , supplementary fig . 6 ) p values were < 0.05 . for cocaine - conditioned place preference ( ccpp ) , the apparatus was divided into two equally sized zones using plastic floor tiles with distinct visual and tactile cues ( grey & smooth or white & rough ) . after 5 days b.i.d . saline injections for habituation in the home cage , the amount of time spent freely exploring each zone was recorded during 30-minute test sessions . after an initial test to establish baseline preference for the two sets of cues , mice in each of the two treatment groups ( i.p . otr - a ) were randomly assigned in a counterbalanced fashion to receive cocaine ( 20 mg / kg ) or saline in the presence of one set of cues ( i.e. , an unbiased design ) . the second conditioning session was conducted 24 hours later in the presence of the other set of cues . the post - conditioning test session was conducted 24 hours after the second conditioning session to determine time spent in the presence of the cocaine versus saline associated cue . isolation and socially housed animals were not different in terms of ccpp so they were pooled for further analysis . pre - conditioning , post - conditioning , subtracted , and normalized cocaine preference scores were calculated as in scpp . briefly , probes consisted of a reservoir ( polypropylene luer hub ) attached to a double cannula guide ( c235gs , 26ga , c / c distance 2.0 mm , 5 mm pedestal , cut 4 mm below pedestal , custom specified for mouse bilateral nac coordinates , plastics one inc ) . polyimide tubing ( 40 awg , 0.0031 " i d , 0.0046 " od , 0.00075 " wall , small parts ) was threaded through the stainless steel tubing of the cannula guide on one end , and out of a hole drilled into the luer hub to act as a flush outlet ( outflow tube ) on the other end . the dialysis membrane ( spectra / por , 13 kd molecular weight cut - off , spectrum labs ) was then threaded over the outflow tube and through the cannula guide ; ends were cut such that 500 m of dialysis membrane was exposed below the cannula guide and above the sealed end . junctions were sealed with bio - compatible epoxies ( epo - tek 730 , epo - tek 301 , epoxy technologies ) . in this design , a pharmacological agent could be intracranially delivered rapidly , continually , and concurrently to all members of the social group , without anesthesia . at postnatal day 35 - 40 , probes were implanted into the nac of male mice following bilateral craniotomy ( bregma 1.54 mm ; lateral 1.0 mm ) and attached to the skull using dental acrylic . previous reports indicate that for complete pharmacological effect , drug concentration in the reservoir must be 500 times the dose used for direct injections , thus otr and 5htr1b antagonists were applied at 10 mm ( l-368,899 ) and 85 mm ( nas-181 ) in a volume of 25 l saline . probe placement and competency was verified by post - hoc application of concentrated fluorescein sodium salt ( sigma - aldrich ) to reservoir prior to intracardial pfa perfusion and histology ( supplementary fig . 1f ) . rabies virus ( rbv ) was generated from a full length cdna plasmid containing all components of rbv ( sad l16 ; gift from dr . we replaced the rabies virus glycoprotein with egfp ( rbv - egfp ) , tdtomato ( rbv - tdtomato ) or cre - egfp to generate rbv expressing cre - egfp ( rbv - cre - egfp ) , egfp ( rbv - egfp ) or tdtomato ( rbv - tdtomato ) . to rescue rbv from this cdna we used a modified version of a published protocol . briefly , hek293 t cells were transfected with a total of 6 plasmids ; 4 plasmids expressing the rbv components ptit - n , ptit - p , ptit - g , and ptit - l ; one plasmid expressing t7 rna polymerase ( pcaggs - t7 ) , and the aforementioned glycoprotein - deleted rbv cdna plasmid expressing cre - egfp , egfp or tdtomato . for the amplification of rbv , the media bathing these hek293 t ( atcc ) cells was collected 3 - 4 days posttransfection and moved to baby hamster kidney ( bhk ) cells stably expressing rbv glycoprotein ( bhk - b19 g ) . after three days , the media from bhk - b19 g cells was collected , centrifuged for 5 min at 3,000 x g to remove cell debris , and concentrated by ultracentrifugation ( 55,000 x g for 2 hr ) . the adeno - associated viruses ( aavs ) used in this study were produced by the stanford neuroscience gene vector and virus core . briefly , aav - dj was produced by transfection of aav 293 cells ( agilent , inc ) with three plasmids : an aav vector expressing cre - egfp , aav helper plasmid ( phelper , agilent , inc ) , and aav rep - cap helper plasmid ( prc - dj , gift from mark kay , stanford ) . at 72 h after transfection , the iodixanol was diluted and the aav was concentrated using a 100 kda molecular weight cutoff ultrafiltration device . stereotaxic injection of viruses into nac was performed under general ketamine - medetomidine anesthesia using a stereotaxic instrument ( david kopf ) . a small volume ( 1 l ) of concentrated virus solution was injected bilaterally into nac core ( bregma 1.54 mm ; lateral 1.0 mm ; ventral 4.0 mm ) , unilaterally into the dorsal raphe ( drph ; bregma -3.3 mm ; lateral 0.0 mm ; ventral 3.35 mm ) , bilaterally into the ventral subiculum ( vsub ; bregma -2.95 mm ; lateral 3.1 mm ; ventral 4.35 mm ) , or bilaterally anterior cingulate ( acc ; bregma 1.0 mm ; lateral 0.3 mm ; ventral 1.25 mm ) at a slow rate ( 100 nl / min ) using a syringe pump ( harvard apparatus , ma ) . injection sites and viral infectivity were confirmed in all animals post - hoc by preparing sections ( 50 m ) containing the relevant brain region ( supplementary fig . briefly , after intracardial perfusion with 4% paraformaldehyde in pbs ( ph 7.4 ) , the brains were post fixed overnight in this same solution and the following day 50 m coronal , sagittal , or horizontal sections were prepared . primary antibodies were used at the following concentrations : mouse anti - oxytocin - neurophysin ( ot - np , 1:50 ; gift of harold gainer , nih bethesda , md ) ; rat anti - green fluorescent protein ( gfp , 1:1000 ; nacalai ) ; rabbit anti - parvalbumin ( parv , 1:750 ; swant ) ; rabbit anti - neuronal nitric oxide synthase ( nnos , 1:100 ; bd transduction laboratories ) ; rabbit anti - glial fibrillary protein ( gfap , 1:80 ; sigma - aldrich ) ; rabbit anti - choline acetyltransferase ( chat , 1:100 ; millipore ) ; rabbit anti - dopamine receptor protein ( darp , 1:100 , millipore ) ; sheep anti - tryptophan hydroxylase ( tryph , 1:100 millipore ) diluted in a solution containing 1% horse serum , 0.2% bsa , and 0.5% triton x-100 in pbs . after overnight incubation in primary antibody ( rt , shaker ) , slices were washed four times in pbs and then incubated with appropriate secondary antibody diluted at 1:750 for 2 h in pbs containing 0.5% triton x-100 . subsequently , slices were washed 5 times and mounted using vectashield mounting medium ( vector laboratories ) . to identify cells expressing gfp or tdtomato due to the injection of rbv - egfp or rbv - tdtomato into the nac , image acquisition was performed with a confocal microscope ( zeiss lsm510 ) using a 10x/0.30 plan neofluar and a 40x/1.3 oil dic plan apochromat objective . parasagittal slices ( 250 m ) containing the nac core were prepared from c57bl/6 and d1-tdtomato / d2-egfp bac transgenic mice on a c57bl/6 background using standard procedures . briefly , after mice were anesthetized with isoflurane and decapitated , brains were quickly removed and placed in ice - cold low sodium , high sucrose dissecting solution . slices were cut by adhering the two sagittal hemispheres brain containing the nac core to the stage of a leica vibroslicer . slices were allowed to recover for a minimum of 60 min in a submerged holding chamber ( 25c ) containing artificial cerebrospinal fluid ( acsf ) consisting of 119 mm nacl , 2.5 mm kcl , 2.5 mm cacl2 , 1.3 mm mgso4 , 1 mm nah2po4 , 11 mm glucose and 26.2 mm nahco3 . slices were then removed from the holding chamber and placed in the recording chamber where they were continuously perfused with oxygenated ( 95% o2 , 5% co2 ) acsf at a rate of 2 ml per min at 26 2c . for epsc recordings , bicuculline ( 20 m ) for ipsc recordings , dl-2-amino-5-phosphonovalerate ( dapv , 10 m ) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide ( nbqx , 5 m ) dissolved in dmso were added to block nmda and ampa receptors respectively . whole - cell voltage - clamp recordings from msns were obtained under visual control using a 40x objective . d1- and d2 msns in the nac core were identified by the presence of tdtomato and egfp , respectively , that were excited with uv light using bandpass filters ( hq545/30x ex for tdtomato ; hq470/40x ex for egfp ) . recordings were made with electrodes ( 3.56.5 m ) filled with 115 mm csmeso4 , 20 mm cscl , 10 mm hepes , 0.6 mm egta , 2.5 mm mgcl , 10 mm na - phosphocreatine , 4 mm na - atp , 0.3 mm na - gtp , and 1 mm qx-314 . excitatory and inhibitory afferents were stimulated with a bipolar nichrome wire electrode placed at the border between the nac core and cortex dorsal to the anterior commissure . recordings were performed using a multiclamp 700b ( molecular devices ) , filtered at 2 khz and digitized at 10 khz . epscs were evoked at a frequency of 0.1 hz while msns were voltage - clamped at 70 mv . input resistance and access resistance were monitored continuously throughout each experiment ; experiments were terminated if these changed by > 15% . summary ltd graphs were generated by averaging the peak amplitudes of individual epscs in 1 min bins ( 6 consecutive sweeps ) and normalizing these to the mean value of epscs collected during the 10 min baseline immediately before the ltd - induction protocol . oxytocin ( ot , tocris biosciences , 1 m , 10 minute ) was bath applied following the collection of baseline for induction of ot - ltd . for experiments examining the blockade of ot - ltd , slices were pre - incubated in antagonist ( otr - a , 1 m l-368,899 hydrochloride or 5htr1b - a , 20 m nas-181 ; tocris biosciences ) for at least 30 minutes prior to recording . for experiments examining the reversal of ot - ltd , 30 - 40 minutes following the collection of stable baseline epscs , 5ht1b - ltd was induced by 10 minute bath application of 2 m cp-93129 dihydrochloride ( tocris biosciences ) as described previously . for experiments examining the occlusion of ot - ltd , after stabilization of 5ht1b - ltd ( at 30 - 40 minutes post induction ) , miniature epscs were collected at a holding potential of 70 mv in the presence of ttx ( 0.5 m ) . 2 minutes after break - in ( sweep number 5 , 30s sweeps ) , thirty second blocks of events ( total of 200 events per cell ) were acquired and analyzed using mini - analysis software ( synaptosoft ) with threshold parameters set at 5 pa amplitude and <3 ms rise time . slices were incubated in the appropriate drug ( dissolved in acsf - bicuculline ) for 10 minutes prior to recording , and cross - cell comparisons were made . paired - pulse ratios ( ppr ) were acquired by applying a second afferent stimulus of equal intensity , 50 ms after the first stimulus , and then calculating the ratio of epsc2/epsc1 . coefficient of variance ( cv ) was calculated from the standard deviation divided by the average ( stdev / avg ) of 10-minute blocks ( minutes 0 - 10 , pre ; minutes 40 - 50 , post ) . comparisons between different experimental manipulations were made using a two- tailed , students t - test ( paired or unpaired , as appropriate ) with p < 0.05 considered significant . all statements in the text regarding differences between grouped data indicate that statistical significance was achieved , assuming normal distribution and equal variance . male young adult ( 4 - 6 weeks of age ) c57bl/6 ( charles river ) , drd1a tdtomato bac transgenic ( d1-tdtomato , gift of n. calakos , duke university , durham , nc ) , drd2egfp bac transgenic ( d2-egfp ) , oxtrtm1.1wsy homozygous ( cotr ko , jackson laboratory ) , or otr venus neo/+ ( otr - venus , gift of l.j . young , emory university , atlanta , ga ) mice backcrossed to c57bl/6 were used for all experiments . all procedures complied with the animal care standards set forth by the national institutes of health and were approved by stanford university s administrative panel on laboratory animal care . experimenters were blind to the treatment condition when subjective criteria were used as a component of data analysis , and control and test conditions were interleaved for all experiments . the protocol for social conditioned place preference ( scpp ) was shortened to 2 days of conditioning ( fig . animals were weaned ( or delivered from charles river ) at 3 weeks of age into home cages containing 3 - 5 cage - mates , and housed on corncob bedding ( bed - ocobs , 1/8 , pharmaserv ) . one to two weeks later , animals were subjected to experimental manipulations and returned to their home cage ( all cage - mates were of the same genotype and received the same experimental manipulation ) . animals were then placed in open field activity chamber ( env-510 , med associates ) equipped with infrared beams and a software interface ( activity monitor , med associates ) that monitors the position of the mouse . the apparatus was divided into two equally sized zones using a clear plastic wall , with a 5 cm diameter circular opening at the base ; each zone contained one type of novel bedding ( alpha - dri , pharmaserv , alpha chip , pharmaserv ; bed - ocobs , 1/4 , pharmaserv ; or kaytee soft granule , petco ) . the amount of time spent freely exploring each zone was recorded during 30-minute test sessions . after an initial test ( pre - conditioning trial ) to establish baseline preference for the two sets of bedding cues , mice were assigned to receive social conditioning ( with cage - mates ) for 24 hours on one type of bedding , followed by 24 hours on the isolate bedding cue ( without cage - mates ) on the other type of bedding . 24 hours later , animals received a 30-minute post - conditioning trial to establish preference for the two conditioned cues . animals were excluded ( pre - established criteria ) if they exhibited a pre - conditioning preference score > 1.5 or < 0.5 ( for an unbiased procedure ) ; pre - conditioning versus post - conditioning social preference scores were considered significant if paired student s t - test p values were < 0.05 . comparisons between experimental conditions were made using both normalized social preference scores ( time spent in social zone post over pre ) , and subtracted social preference scores ( time spent in social zone post minus pre ) ; these were considered significant if unpaired student s t - test ( two conditions ) , or anova ( three conditions , supplementary fig . 6 ) p values were < 0.05 . for cocaine - conditioned place preference ( ccpp ) , the apparatus was divided into two equally sized zones using plastic floor tiles with distinct visual and tactile cues ( grey & smooth or white & rough ) . after 5 days b.i.d . saline injections for habituation in the home cage , the amount of time spent freely exploring each zone was recorded during 30-minute test sessions . after an initial test to establish baseline preference for the two sets of cues , mice in each of the two treatment groups ( i.p . otr - a ) were randomly assigned in a counterbalanced fashion to receive cocaine ( 20 mg / kg ) or saline in the presence of one set of cues ( i.e. , an unbiased design ) . the second conditioning session was conducted 24 hours later in the presence of the other set of cues . the post - conditioning test session was conducted 24 hours after the second conditioning session to determine time spent in the presence of the cocaine versus saline associated cue . isolation and socially housed animals were not different in terms of ccpp so they were pooled for further analysis . pre - conditioning , post - conditioning , subtracted , and normalized cocaine preference scores were calculated as in scpp . briefly , probes consisted of a reservoir ( polypropylene luer hub ) attached to a double cannula guide ( c235gs , 26ga , c / c distance 2.0 mm , 5 mm pedestal , cut 4 mm below pedestal , custom specified for mouse bilateral nac coordinates , plastics one inc ) . polyimide tubing ( 40 awg , 0.0031 " i d , 0.0046 " od , 0.00075 " wall , small parts ) was threaded through the stainless steel tubing of the cannula guide on one end , and out of a hole drilled into the luer hub to act as a flush outlet ( outflow tube ) on the other end . the dialysis membrane ( spectra / por , 13 kd molecular weight cut - off , spectrum labs ) was then threaded over the outflow tube and through the cannula guide ; ends were cut such that 500 m of dialysis membrane was exposed below the cannula guide and above the sealed end . junctions were sealed with bio - compatible epoxies ( epo - tek 730 , epo - tek 301 , epoxy technologies ) . in this design , a pharmacological agent could be intracranially delivered rapidly , continually , and concurrently to all members of the social group , without anesthesia . at postnatal day 35 - 40 , probes were implanted into the nac of male mice following bilateral craniotomy ( bregma 1.54 mm ; lateral 1.0 mm ) and attached to the skull using dental acrylic . previous reports indicate that for complete pharmacological effect , drug concentration in the reservoir must be 500 times the dose used for direct injections , thus otr and 5htr1b antagonists were applied at 10 mm ( l-368,899 ) and 85 mm ( nas-181 ) in a volume of 25 l saline . probe placement and competency was verified by post - hoc application of concentrated fluorescein sodium salt ( sigma - aldrich ) to reservoir prior to intracardial pfa perfusion and histology ( supplementary fig . rabies virus ( rbv ) was generated from a full length cdna plasmid containing all components of rbv ( sad l16 ; gift from dr . we replaced the rabies virus glycoprotein with egfp ( rbv - egfp ) , tdtomato ( rbv - tdtomato ) or cre - egfp to generate rbv expressing cre - egfp ( rbv - cre - egfp ) , egfp ( rbv - egfp ) or tdtomato ( rbv - tdtomato ) . to rescue rbv from this cdna we used a modified version of a published protocol . briefly , hek293 t cells were transfected with a total of 6 plasmids ; 4 plasmids expressing the rbv components ptit - n , ptit - p , ptit - g , and ptit - l ; one plasmid expressing t7 rna polymerase ( pcaggs - t7 ) , and the aforementioned glycoprotein - deleted rbv cdna plasmid expressing cre - egfp , egfp or tdtomato . for the amplification of rbv , the media bathing these hek293 t ( atcc ) cells was collected 3 - 4 days posttransfection and moved to baby hamster kidney ( bhk ) cells stably expressing rbv glycoprotein ( bhk - b19 g ) . after three days , the media from bhk - b19 g cells was collected , centrifuged for 5 min at 3,000 x g to remove cell debris , and concentrated by ultracentrifugation ( 55,000 x g for 2 hr ) . the adeno - associated viruses ( aavs ) used in this study were produced by the stanford neuroscience gene vector and virus core . briefly , aav - dj was produced by transfection of aav 293 cells ( agilent , inc ) with three plasmids : an aav vector expressing cre - egfp , aav helper plasmid ( phelper , agilent , inc ) , and aav rep - cap helper plasmid ( prc - dj , gift from mark kay , stanford ) . at 72 h after transfection , the iodixanol was diluted and the aav was concentrated using a 100 kda molecular weight cutoff ultrafiltration device . stereotaxic injection of viruses into nac was performed under general ketamine - medetomidine anesthesia using a stereotaxic instrument ( david kopf ) . a small volume ( 1 l ) of concentrated virus solution was injected bilaterally into nac core ( bregma 1.54 mm ; lateral 1.0 mm ; ventral 4.0 mm ) , unilaterally into the dorsal raphe ( drph ; bregma -3.3 mm ; lateral 0.0 mm ; ventral 3.35 mm ) , bilaterally into the ventral subiculum ( vsub ; bregma -2.95 mm ; lateral 3.1 mm ; ventral 4.35 mm ) , or bilaterally anterior cingulate ( acc ; bregma 1.0 mm ; lateral 0.3 mm ; ventral 1.25 mm ) at a slow rate ( 100 nl / min ) using a syringe pump ( harvard apparatus , ma ) . injection sites and viral infectivity were confirmed in all animals post - hoc by preparing sections ( 50 m ) containing the relevant brain region ( supplementary fig . briefly , after intracardial perfusion with 4% paraformaldehyde in pbs ( ph 7.4 ) , the brains were post fixed overnight in this same solution and the following day 50 m coronal , sagittal , or horizontal sections were prepared . primary antibodies were used at the following concentrations : mouse anti - oxytocin - neurophysin ( ot - np , 1:50 ; gift of harold gainer , nih bethesda , md ) ; rat anti - green fluorescent protein ( gfp , 1:1000 ; nacalai ) ; rabbit anti - parvalbumin ( parv , 1:750 ; swant ) ; rabbit anti - neuronal nitric oxide synthase ( nnos , 1:100 ; bd transduction laboratories ) ; rabbit anti - glial fibrillary protein ( gfap , 1:80 ; sigma - aldrich ) ; rabbit anti - choline acetyltransferase ( chat , 1:100 ; millipore ) ; rabbit anti - dopamine receptor protein ( darp , 1:100 , millipore ) ; sheep anti - tryptophan hydroxylase ( tryph , 1:100 millipore ) diluted in a solution containing 1% horse serum , 0.2% bsa , and 0.5% triton x-100 in pbs . after overnight incubation in primary antibody ( rt , shaker ) , slices were washed four times in pbs and then incubated with appropriate secondary antibody diluted at 1:750 for 2 h in pbs containing 0.5% triton x-100 . subsequently , slices were washed 5 times and mounted using vectashield mounting medium ( vector laboratories ) . to identify cells expressing gfp or tdtomato due to the injection of rbv - egfp or rbv - tdtomato into the nac , image acquisition was performed with a confocal microscope ( zeiss lsm510 ) using a 10x/0.30 plan neofluar and a 40x/1.3 oil dic plan apochromat objective . parasagittal slices ( 250 m ) containing the nac core were prepared from c57bl/6 and d1-tdtomato / d2-egfp bac transgenic mice on a c57bl/6 background using standard procedures . briefly , after mice were anesthetized with isoflurane and decapitated , brains were quickly removed and placed in ice - cold low sodium , high sucrose dissecting solution . slices were cut by adhering the two sagittal hemispheres brain containing the nac core to the stage of a leica vibroslicer . slices were allowed to recover for a minimum of 60 min in a submerged holding chamber ( 25c ) containing artificial cerebrospinal fluid ( acsf ) consisting of 119 mm nacl , 2.5 mm kcl , 2.5 mm cacl2 , 1.3 mm mgso4 , 1 mm nah2po4 , 11 mm glucose and 26.2 mm nahco3 . slices were then removed from the holding chamber and placed in the recording chamber where they were continuously perfused with oxygenated ( 95% o2 , 5% co2 ) acsf at a rate of 2 ml per min at 26 2c . for epsc recordings , bicuculline ( 20 m ) was added to the acsf to block gabaa receptor mediated inhibitory synaptic currents . for ipsc recordings , dl-2-amino-5-phosphonovalerate ( dapv , 10 m ) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide ( nbqx , 5 m ) dissolved in dmso were added to block nmda and ampa receptors respectively . whole - cell voltage - clamp recordings from msns were obtained under visual control using a 40x objective . d1- and d2 msns in the nac core were identified by the presence of tdtomato and egfp , respectively , that were excited with uv light using bandpass filters ( hq545/30x ex for tdtomato ; hq470/40x ex for egfp ) . recordings were made with electrodes ( 3.56.5 m ) filled with 115 mm csmeso4 , 20 mm cscl , 10 mm hepes , 0.6 mm egta , 2.5 mm mgcl , 10 mm na - phosphocreatine , 4 mm na - atp , 0.3 mm na - gtp , and 1 mm qx-314 . excitatory and inhibitory afferents were stimulated with a bipolar nichrome wire electrode placed at the border between the nac core and cortex dorsal to the anterior commissure . recordings were performed using a multiclamp 700b ( molecular devices ) , filtered at 2 khz and digitized at 10 khz . epscs were evoked at a frequency of 0.1 hz while msns were voltage - clamped at 70 mv . input resistance and access resistance were monitored continuously throughout each experiment ; experiments were terminated if these changed by > 15% . summary ltd graphs were generated by averaging the peak amplitudes of individual epscs in 1 min bins ( 6 consecutive sweeps ) and normalizing these to the mean value of epscs collected during the 10 min baseline immediately before the ltd - induction protocol . oxytocin ( ot , tocris biosciences , 1 m , 10 minute ) was bath applied following the collection of baseline for induction of ot - ltd . for experiments examining the blockade of ot - ltd , slices were pre - incubated in antagonist ( otr - a , 1 m l-368,899 hydrochloride or 5htr1b - a , 20 m nas-181 ; tocris biosciences ) for at least 30 minutes prior to recording . for experiments examining the reversal of ot - ltd , 30 - 40 minutes post induction , otr - a was bath applied for 10 minutes . following the collection of stable baseline epscs , 5ht1b - ltd was induced by 10 minute bath application of 2 m cp-93129 dihydrochloride ( tocris biosciences ) as described previously . for experiments examining the occlusion of ot - ltd , after stabilization of 5ht1b - ltd ( at 30 - 40 minutes post induction ) , miniature epscs were collected at a holding potential of 70 mv in the presence of ttx ( 0.5 m ) . 2 minutes after break - in ( sweep number 5 , 30s sweeps ) , thirty second blocks of events ( total of 200 events per cell ) were acquired and analyzed using mini - analysis software ( synaptosoft ) with threshold parameters set at 5 pa amplitude and <3 ms rise time . slices were incubated in the appropriate drug ( dissolved in acsf - bicuculline ) for 10 minutes prior to recording , and cross - cell comparisons were made . paired - pulse ratios ( ppr ) were acquired by applying a second afferent stimulus of equal intensity , 50 ms after the first stimulus , and then calculating the ratio of epsc2/epsc1 . coefficient of variance ( cv ) was calculated from the standard deviation divided by the average ( stdev / avg ) of 10-minute blocks ( minutes 0 - 10 , pre ; minutes 40 - 50 , post ) . comparisons between different experimental manipulations were made using a two- tailed , students t - test ( paired or unpaired , as appropriate ) with p < 0.05 considered significant . all statements in the text regarding differences between grouped data indicate that statistical significance was achieved , assuming normal distribution and equal variance .

cone beam computed tomography ( cbct ) scanners for the oral and maxillofacial region were pioneered in the late 1990s independently by arai et al . in japan and mozzo et al . cbct has a lower dose of radiation , minimal metal artifacts , reduced costs , easier accessibility , and easier handling than multislice computed tomography ( msct ) ; however , the latter is still considered a better choice for the analysis of bone density using a hounsfield unit ( hu ) scale . oral implants require localized area of oral and maxillofacial area for radiation exposure ; so , cbct is an ideal choice . cbct scans help in the planning of oral implants ; they enable measurement of the distance between the alveolar crest and mandibular canal to avoid impingement of inferior alveolar nerve , avoid perforation of the mandibular posterior lingual undercut , and assess the density and quality of bone , and help in planning of the oral implant in the maxilla with special attention to the nasopalatine canal and maxillary sinus . hence , cbct reduces the overall exposure to radiation . social behaviors in species as diverse as honey bees and humans promote group survival but often come at some cost to the individual . although reinforcement of adaptive social interactions is ostensibly required for the evolutionary persistence of these behaviors , the neural mechanisms by which social reward is encoded by the brain are largely unknown . here we demonstrate that in mice oxytocin ( ot ) acts as a social reinforcement signal within the nucleus accumbens ( nac ) core , where it elicits a presynaptically expressed long - term depression of excitatory synaptic transmission in medium spiny neurons . although the nac receives ot receptor - containing inputs from several brain regions , genetic deletion of these receptors specifically from dorsal raphe nucleus , which provides serotonergic ( 5-ht ) innervation to the nac , abolishes the reinforcing properties of social interaction . furthermore , ot - induced synaptic plasticity requires activation of nac 5-ht1b receptors , the blockade of which prevents social reward . these results demonstrate that the rewarding properties of social interaction in mice require the coordinated activity of ot and 5-ht in the nac , a mechanistic insight with implications for understanding the pathogenesis of social dysfunction in neuropsychiatric disorders such as autism .

mutagenesis was performed on age - synchronized l4 animals using ethyl methanesulfonate ( ems ) according to standard protocols . briefly , when the animals reached the l4 stage , a large number of synchronized wild - type animals carrying the wyis85 transgene were suspended in a buffer solution and 20mmems ( sigma - aldrich ) for a period of four hours . following the incubation , these p0 animals were allowed to grow to adults , and once the f1 progeny from these animals became adults , the f2 offspring were age - synchronized . f2 embryos were obtained by bleaching f1 adults using a solution containing about 1% naocl and 0.1 mnaoh , washed in m9 buffer , and cultured for 24 hours . animals were then transferred and cultured on nematode growth medium ( ngm ) plates seeded with e. coli op50 until l4 stage . the strains used in this project included xa7810 : n2 , wyis85 ( pitr-1 pb::gfp::rab-3 ) ; xa7812 : lin-44(n1792)i , wyis85(pitr-1 pb::gfp::rab-3 ) . for imaging and screening , animals were washed and suspended in m9 solution containing 0.01 volume% triton x100 ( sigma - aldrich ) . after washing animals off of the plates , animals were allowed to settle , the supernatant removed and additional m9 added . animals were screened under a compound microscope using a 40 ( na=1.4 ) oil objective using the microfluidic chips . sorting decisions were made based on differences in the reporter expression pattern or intensity ; potential animals of interest were sorted into the mutant outlet and were collected directly from tubing connected to the mutant outlet with m9 solution containing 0.01% triton x100 . the equipment utilized during our experiments includes the following : peltier cooler ( pjt-5 30 mm square and pjt-6 40 mm square peltier coolers ) , copper heat exchanger ( custom designed and machined ) , peristaltic pump for coolant : 400 f / a ( watson marlow ) , digital i / o card ( pacdrive from ultimarc ) , solenoid valves and manifold ( 3-way 10 mm solenoid valve , 188 series , asco valve ) , microscope ( leica dm4500 ) with lens ( 63x oil , na=1.4 ) . and camera ( infinity 3 - 1 , lumenera ) . although manipulation and imaging of multicellular model organisms are an important part of modern biology , most methods are still labor intensive and time - consuming . recent work , largely through multiwell plate methods , and a modified flow cytometer ( copas ) , has made significant improvements in sample handling and low - resolution imaging of c. elegans , and d. rerio and d. melanogaster embryos . recently , microfluidic approaches have improved the throughput of high - resolution imaging of caenorhabditis elegans , but sorting has been based on user input or simplistic criteria , such as the local intensity . to date , all c. elegans screens , both automated and manual , have required human intervention to set the sorting boundaries , and the few automated screens have only been performed at very low resolution . thin glass capillary tubes have recently been used to image and then ablate axons in d. rerio , but image processing and ablation was performed manually using user input . similarly , microfluidic traps have been employed to align d. melanogaster embryos to enable highly controlled imaging conditions . automation of information extraction has largely been focused on post - processing of data acquired during an experiment . within multicellular model organisms , this has focused on understanding animal behavior and interactions , and with lineage tracking of cell fates . tracing of cell lineages from early to late embryogenesis has allowed tracking of c. elegans embryos through many divisions . a digital atlas of c. elegans cell locations , and methods to identify them , performing image processing to identify quantitative information in near real time during a screen , however , requires a framework that places a significantly higher value on speed and throughput . the framework provided in this work can be extended to tackling these problems for real - time screening of other model organisms . included on the website is software used during screening . because of differences in equipment this is primarily intended as a guide for development of customized code for the end user . the code contains functions used to operate the microfluidic device , as well as that to extract relevant information from the fluorescent images . the optimized feature extraction and phenotypical parameters can be modified to focus on identification of other small , fluorescent objects . the device is a standard two - layer pdms device , similarly to our previous work , fabricated using a rapid prototyping method and thermal bonding between layers ( sfig 4b ) . rather than using conventional fullclosure valves that rely on a curved cross - section of the flow - layer , although valves incapable of fully closing are typically considered drawbacks in microfluidic devices , in this case they could be used advantageously to simplify the design . because the valves always allow a small amount of flow ( supplementary fig . 2c ) , the device was designed using partial closure valves to position animals in the imaging channels , instead of suction channels . in addition to the development of a microfluidic device for screening , external systemic components were required to allow for automated sorting . this required a closed loop control system and the development of specific hardware to interface with and control the on - chip components . creating external components that would allow computerized control of on - chip components was necessary ( supplementary fig . because we needed to run the system for a very long period during the screen , a comprehensive framework was developed for handling errors robustly ( supplementary fig . this includes an external macroscale control components to be integrated with the microfluidic device to allow closed - loop control , an error handling routines to reduce the need for operator intervention for the closed loop control , and optimization of the operational sequence to minimize the amount of time spent per animal . external calls were to the machine learning libraries and a dll for valve control operation . due to potential stability issues with matlab operation , however , it could be advisable for future efforts to focus on the open microscopy environment and code in a more robust language such as java . the screening methodology was optimized to maximize the number of independent f2 animals screened , and to minimize the likelihood of screening clonal siblings . this was done by pooling f2 animals from a number of f1 mothers , and then screening a subset of this group . due to the screening methodology of the number of haploid genomes can not be directly calculated , but several assumptions have to be made regarding the sampling from f2 population . this results in approximately 1,500 f1 animals per plate , or 3,000 haploid genomes . given that f1 animals are a / b , we can assume that half of the animals are homozygous ( a / a or b / b ) , and the nave assumption is that for every 2 f2 animals screened , we have screened 1 haploid genome . on average , 500 f2 animals were screened from each of these populations , resulting in 250 haploid genomes . given that we were sampling from a larger population of animals , there is the possibility that an f2 animal could be homozygous for the same genome present in an animal that was previously screened . the probability of this occurring was calculated as 1(29993000)250=0.08 . given there were 3,000 haploid genomes present , we screened 0.08 * 3,000 = 240 . this yields a ratio of 240500=0.48 , implying that because we have screened a total of ~40,000 animals , that corresponds to nearly 20,000 haploid genomes . following screening , the animals identified were cloned and then verified by reimaging . once the identified mutants have been cloned and decontaminated , furthermore , because genetic validation by sequencing is such a slow and time consuming process , it is important to visually confirm that each of the animals to be mapped or sequenced indeed have an altered synaptic expression . validation was done using standard worm protocols using a worm - slide and sodium azide for immobilization . a small population ( ~1520 ) of animals of each genotype was imaged , and these images were used to determine whether the animals were actual mutants , and thus the accuracy of the screening . some of the mutants were phenotyped with larger numbers , and were characterized in greater detail to predict the potential pathway that was affected ( supplementary table 2 ) . each of the genotypes classified as mutants of interest were phenotyped . for the purpose of these results , animals that were sorted as mutants but failed to produce any offspring that could be used to verify whether the animals were correctly or incorrectly sorted , were removed from the results . to determine the accuracy of the screening protocol , images of all processed animals were collected and analyzed to determine whether animals were correctly or incorrectly sorted as wild - type animals . the false negative rate was determined by comparing the manual scores to the computer scores to determine the accuracy of the algorithm . the screen using the discriminative classifier resulted in a false positive rate of 69% , a false negative rate of 9% , and a theoretical enrichment of 6,000% . the screen using the outlier detection approach resulted in a false positive rate of 74% , a false negative rate of 7% , and an enrichment of 6,000% . the marginally higher false negative rate could be a result of the discriminative classifier not recognizing some mutant phenotypes , or merely the sample size . the false positive rates of these classifiers are similar to or better than the manual methods due to animal - to - animal variations and the manual method s subjectivity in scoring the phenotypes . these false positive animals also do not pose an issue in these rare - sort problems since it is a very small number of candidate mutants that need to be characterized in the following steps . false positives were calculated using the following equation where animals sterile animals were removed from the denominator : fp = wildtype sorted as mutantsanimals sorted as mutants . the false negative rate was calculated using the following equation : fn = mutants sorted as wildtypemutants sorted as mutants+mutants sorted as wildtype . the upper bound of the enrichment was calculated using the equation : enrichment = mutants correctly sorted / number of animals sorted as mutantsmutants in starting population / animals in starting population . the emerging field of optofluidics explores new possibilities to combine optical detection techniques with micro- and nanofluidics to develop miniaturized optical sensors with improved performance . the analytical techniques commonly used include surface plasmon resonance ( spr ) , surface - enhanced raman spectroscopy ( sers ) , and fluorescence imaging . sers , a powerful technique for biosensing and chemical identification , largely depends on the drastic enhancement of weak raman signal obtained from analyte molecules , upon their interaction with optical near - fields of metallic nanostructures . as an analytical technique , sers combines the benefits of label - free sensing , high sensitivity , and chemical specificity . an important aim is to further improve the capabilities of sers for diverse applications such as healthcare and environmental monitoring using substrates that can be fabricated reproducibly and have a uniform geometry . as the enhancement of electromagnetic fields occurs in nanoscale regions , termed as plasmonic hotspots , integration of microfluidics capable of directing the analyte flow and resultant concentration toward these hotspots can improve the sensitivity and reduce the detection time for sers . among various options , we explore metallic nanohole arrays fabricated in suspended silicon nitride ( si3n4 ) membranes because they provide a unique option to integrate sers with flow - through sensing , which has yet to be shown experimentally . nanohole arrays in thin metallic films can act as plasmonic substrates with multifarious applications . enhancement of the electromagnetic field and extraordinary optical transmission ( eot ) through the nanoholes has led to their application in plasmonic sensing . primarily , they have been used as a platform for spr refractive - index sensing . while groups have demonstrated nanohole - enhanced raman spectroscopy , the reported raman enhancement factors ( below 10 ) are weaker than that of other sers substrates . these previous experiments relied on low - throughput fabrication techniques such as focused ion beam ( fib ) or electron - beam lithography to pattern nanoholes in a metal film . suspended nanohole arrays have been utilized for spr sensing , but have not been utilized for sers . flow - through suspended nanoholes have been utilized for overcoming diffusion - limited transport in spr sensing . most of these methods , however , require external sources for generating pressure gradients , which can often damage the fragile membrane , and microfluidic tubing or an external power supply for creating an electric field . a simple and robust sample delivery mechanism is needed for flow - through sensing . in this work , nanoimprint lithography ( nil ) is used to fabricate periodic ag nanohole arrays over a millimeter - sized suspended silicon nitride ( si3n4 ) membrane with high throughput and reproducibility . to improve the raman enhancement factor ( ef ) on these large - area substrates , plasmon resonances of suspended nanohole arrays we report a high raman ef of up to 10 for silver - coated suspended nanoholes using this technique . furthermore , suspended nanohole arrays are capable of fast flow through the nanoholes driven by surface tension forces , which promote adsorption of the analytes toward the plasmonic hotspot and further boost the sers signal by 50 times compared to diffusion - limited transport . using this optofluidic sers substrate , we detect a 100 pm 4-mercaptopyridine ( 4-mp ) sample . suspended nanohole arrays were fabricated over a region of 1 mm in size using nanoimprint lithography ( nil ) , as shown in figure 1a c . low - stress silicon nitride ( 200 nm ) was deposited on both sides of single - sided polished ( 100 ) silicon wafers using low - pressure chemical vapor deposition ( lpcvd ) . these wafers were then patterned using photolithography and dry etching such that some regions on the back of the wafers had nitride removed , exposing the silicon . a nanoimprint mold was carefully placed on the resist so that it aligned well with patterns etched on the back of the wafer . the mold used for nanoimprinting had pillars with a 200 nm diameter , 500 nm periodicity , and 300 nm pillar height over a 1 cm 1 cm area . the sample was further processed to etch the nanohole arrays into the top nitride layer . the etching was done for a fixed time period such that the holes go about two - thirds of the way into the nitride . the nanoimprint resist was then cleaned off , and samples were placed in a koh bath for anisotropic etching . the samples were further dry - etched to remove the remaining nitride from the bottom of the nanoholes and obtain suspended open - ended nanoholes . silver ( 120 nm ) was then deposited on the top of the samples using an electron - beam evaporator . ( a ) a silicon chip after 200 nm low - stress nitride deposition and photolithography to expose desired regions on the backside . ( b ) nanoimprinting and dry etching was performed to transfer the nanohole array pattern to the top nitride membrane . ( c ) anisotropic koh etching of silicon was used to obtain the suspended nitride membrane . a final dry etch removed the remaining nitride from the bottom of the holes . the nanoimprinted region further has a 1 mm 1 mm suspended membrane in the center ( circled ) . for raman measurements of benzenethiol ( bzt ) and 4-mp solutions , a quartz cuvette with a 1 mm path length was used . a 785 nm diode laser with an incident power of 10 mw was loosely focused onto the sample using a 10 objective . the raman scattered light was collected in transmission mode using a 50 objective ( na 0.5 ) and passed onto a spectrometer ( ocean optics qe65000 ) through a multimode fiber after removing the excitation light with a notch filter . the acquisition time was 30 s. neat bzt ( 10 m ) and 0.6 m 4-mp solution were used to acquire the raman spectra . for formation of a self - assembled monolayer ( sam ) , chips were placed in 1 mm bzt ( eth . ) or 1 mm 4-mp ( aq . ) overnight . they were then rinsed thrice with ethanol ( bzt samples ) or deionized ( di ) water ( 4-mp samples ) over a period of 30 min . the parameters used for calculation of the enhancement factor are described in the supporting information . the chips were placed on a nikon eclipse lv 100 upright microscope stage with a 10 objective , and a fiber - optic spectrometer was used to record transmission spectra through the nanoholes . sucrose solutions of desired concentrations ( 01.5 m ) were used to tune the transmission spectra . data plots were prepared using matlab ( mathworks , inc . ) and graphpad prism version 5.04 ( graphpad software , inc . ) . three - dimensional ( 3d ) finite - difference time - domain ( fdtd ) simulations around the suspended nanoholes were performed using fullwave simulation software . a single hole was simulated with periodic boundary conditions on the faces intersected by the metal film to represent an infinite hole array and absorbing boundary conditions on the final two faces . a grid size of 3 nm was used around the hole in all dimensions . the index of refraction for the nitride was set at 2 , the optical constants for silver were measured via ellipsometry , and the refractive indices of materials surrounding the hole array were varied . a 30 nm thick sio2 film was deposited on the back of the samples using an electron - beam evaporator . using this directional deposition technique , the sidewalls of the nanoholes , which lie parallel to the direction of evaporation , were coated with a much thinner layer of silica . the desired volume of aqueous solution was then added to the top surface of the chips , as shown in figure 4 . fluorescent polystyrene beads were obtained from bangs laboratories ( poly(styrene/2%divinylbenzene / vinyl - cooh ) , mean diameter : 2.19 m ) and were used at a final concentration of 10 beads / ml in di water . a 10 l solution containing beads was added on the top of the chips for the three cases i , ii , and iii , as shown in figure 5 . the samples were left undisturbed until the solution was consumed through evaporation or flow . the top was then imaged using a fluorescence microscope to monitor accumulation of particles . for sers measurements , a 4-mp solution of desired concentration and volume was added to the top of the chips and allowed to completely flow through . sucrose solution was used to obtain the optimized sers signal from the chips , as discussed earlier . in the case of loss in the volume of sucrose from the top of the chip due to flow - through , suspended metallic nanohole arrays were fabricated on low - stress silicon nitride ( si3n4 ) membranes using nil ( figure 1a c ) . one such chip with a 1 cm 1 cm area of imprinted nanohole arrays is shown in figure 1d . the suspended membrane region , approximately 1 mm 1 mm defined by koh wet etching of si , consisting of open - ended nanoholes is also indicated . a scanning electron micrograph ( sem ) an sem image at higher magnification shows individual nanoholes in the silicon nitride membrane ( figure 1f ) . the holes are approximately 200 nm in diameter , and the array has a periodicity of 500 nm . silver ( thickness 120 nm ) was deposited over the chips using electron - beam evaporation . optimization of sers signal requires tuning of the plasmon resonances of the metallic substrate such that , for a given laser wavelength , there is maximum enhancement of electromagnetic field around nanoholes . generally , modification of the design of the plasmonic substrate such as changing the periodicity of nanohole arrays or altering the wavelength of the laser source is used for this tuning . here , we present a simple method to achieve desired plasmon resonance conditions on suspended nanohole arrays using refractive - index - based tuning . it has been established that surface - plasmon - mediated transmission can be modulated by the refractive index at the metal in fact , the shift in transmission peaks of nanohole arrays due to change in refractive index close to the sensor surface has been used for biosensing . we utilized this intrinsic property of metallic nanohole arrays to tune the resonance peaks . here , we used sucrose solutions of appropriate concentration on the nanohole array surface to shift the transmission spectra as desired and tested the evolution of the sers signal . sugars like glucose and sucrose have been reported to be very difficult to detect using sers owing to their small normal cross section and negligible adsorption to bare metal surfaces . this property makes them suitable to be used on a sers substrate without adding unwanted raman peaks . the schematic for sers measurement and the steps for signal optimization from the suspended nanohole array chips are illustrated in figure 2a . bzt and 4-mp were used to characterize the sers response of the chips as these molecules are known to have large scattering cross sections and can easily form self - assembled monolayers ( sams ) owing to thiol metal bonding on gold or silver . chips with 4-mp sam layers were used for the results shown in figure 2 . by increasing the concentration of the added sucrose solution , upon addition of approximately 1 m sucrose solution , the transmission peak at 600 nm corresponding to the ( 1,0 ) silver air interface was shifted to 785 nm ( figure 2b ) . ( a ) schematics showing the process of obtaining a sers signal from chips with air as dielectric media , resonance tuning using sucrose to improve the signal , and index matching to further optimize the signal . ( b ) figure showing normalized transmission maxima at around 600 nm corresponding to ( 1,0 ) metal air interface shifts to 785 nm after addition of the desired concentration of sucrose . ( c ) sers signal obtained from the same spot on the chip with the transmission peak tuned to different wavelengths . ( d ) sers signal obtained from the same spot before and after index matching . ( e ) sers signal obtained from 4-mp sam layers on suspended nanohole arrays as compared to fib milled dead - ended nanoholes . the signal from suspended nanoholes is about 50 times higher than that obtained from dead - ended samples . sers spectra were obtained from the sample with variation in the position of the transmission peak of the nanoholes ( figure 2c ) . these measurements were taken from the same spot on the sample to avoid any other sources of variation in signal . as expected , the maximum sers signal was obtained when the peak transmission wavelength of the nanoholes matched the laser wavelength at 785 nm . upon adding the same sucrose solution to the backside cavity of the suspended nanohole array membrane , a further increase ( up to 300% ) in the sers signal was observed ( figure 2d ) . it has been known that surface - plasmon - enhanced transmission through nanoholes can be enhanced up to an order of magnitude by matching the refractive index on either side of the metal film . three - dimensional ( 3d ) finite - difference time - domain ( fdtd ) simulations were performed to demonstrate the increase in the plasmonic field around the nanohole arrays with refractive - index - based tuning ( figure 3 ) . in the first case , air was used as the refractive index medium and weak plasmonic hotspots can be observed at the edges of the nanoholes ( figure 3a ) . with the addition of sucrose and filling of the nanoholes such that the transmission maxima is in resonance with the excitation wavelength ( 785 nm ) , more than an order of magnitude enhancement of the plasmonic field at the hotspots can be observed ( figure 3b , c ) . a further increase in electric field was obtained with sucrose present on both sides of the sample as well as inside the nanoholes ( figure 3d ) . the increase in electric field observed in these fdtd simulations correlates qualitatively with the measured increase in sers signal ( figure 2c , d ) . for further comparison , samples with dead - ended hole arrays were prepared using fib milling ; 120 nm thick silver was deposited on suspended nitride membranes and nanoholes with diameter 200 nm and periodicity 500 nm were milled through the silver but not through the nitride membrane . these samples were also placed in a solution of 4-mp overnight to form a sam layer . to measure the sers signal from the dead - ended hole array samples , a drop of sucrose solution was added to the substrate such that the transmission peak matches the laser wavelength . the sers signal obtained from an fib milled dead - ended hole array sample as compared to the signal obtained from the suspended nanohole array under optimized transmission conditions is shown in figure 2e . the signal obtained from suspended nanoholes was approximately 50 times higher as compared to dead - ended samples ( figure 2e ) . this difference could be attributed to a combination of a number of factors , including refractive index matching between the top and bottom sides of the suspended nanoholes , improved transmission , and nanoscale variations in geometry of the nanohole edges ( hotspots ) obtained via nanoimprinting as compared to ion milling . three - dimensional fdtd simulations for suspended nanohole arrays with an excitation wavelength of 785 nm . electric field intensity with ( a ) air as the dielectric medium around and inside the suspended nanoholes . ( c ) sucrose present on the top silver surface and filling top half of the nanoholes , whereas air is present elsewhere . concentration of sucrose is adjusted such that the transmission maxima through the nanoholes matches the excitation wavelength ( 785 nm ) . raman signals obtained from neat solutions of 4-mp and bzt were used to calculate enhancement factors for the chips . the vibration bands used for this calculation correspond to the in - plane ring breathing mode coupled to the c s stretching for the bzt and 4-mp samples . when bzt adsorbed to the silver surface , the vibration band was observed to shift from 1092 to 1072 cm . similarly for 4-mp , the vibration band shifted from 1115 cm in aqueous solution to 1099 cm upon adsorbing to the silver surface . enhancement factors of 10 were obtained for the suspended nanoholes for both 4-mp and benzenethiol samples . these numbers were obtained repeatedly over multiple spots on different samples using both bzt and 4-mp as analytes . in comparison , previous studies on nanohole array sers have reported efs in the range of 1010 . this improvement in the ef values ( 100 times ) obtained by our system confirms that suspended nanohole arrays can be used as excellent sers substrates . nanohole - based biosensors with integrated flow - through fluidics can provide one route to overcome diffusion - limited transport of analytes , improving the detection time and detection limit of the sensor . to facilitate the flow - through process , silica ( 30 nm ) , which is hydrophilic , as this deposition technique is highly directional , the sidewalls of the nanoholes , which lie parallel to the direction of evaporation , were coated with a much thinner layer of silica . the addition of the silica layer on the opposite side of sample injection drastically altered the flow properties of the chip . figure 4 shows the schematic of chips with silica on the backside , and a hydrophobic top surface . when an aqueous solution of analyte or particles was placed on the top of the chip , flow through the nanoholes was initiated as soon as the solution came into contact with the hydrophilic inner walls . figure 4c e shows snapshots of the flow in process , where a 2 l drop of water disappears through the nanohole membrane within 2 min . for membranes of size 1 mm , we obtained an approximate flow rate of 1 l / min . the top metal surface is hydrophobic , whereas the silica layer within the holes and on the backside of the chip is hydrophilic . ( b ) hydrophilic nanohole sidewalls suck in the solution , driving the flow toward the back of the chip ( shown as red arrows ) . ( c e ) bright - field images showing flow of a 2 l drop of water through the suspended nanohole array within 2 min . the water drop shrinks as it is sucked in through the hydrophilic nanohole sidewalls . the effect of flow - through concentration as compared to diffusion - limited adsorption or evaporation - driven concentration over suspended nanoholes was visualized using fluorescent 2 m polystyrene beads . in figure 5 , we compare the accumulation of polystyrene beads ( 10 beads / ml ) in three cases : ( i ) where the holes were dead - ended ; ( ii ) where the holes were open and concentration was driven by evaporation through the open - end of the nanoholes ; and ( iii ) holes were open and silica was deposited on the backside , leading to flow and concentration . schematics for the cross section of a single nanohole accompany the fluorescence images for each of the three cases in figure 5(a c ) . during the evaporation of a liquid drop on a surface , evaporation of solution from the pinned outer edges generates an outward flow and concentrates particles at the edges . this phenomenon , known as the coffee stain effect , is clearly visible in images ( a ) and ( b ) in figure 5 . the particles were randomly distributed within the area of the drop , as shown in figure 5a , as their position was determined by diffusion and the outer flow . in case ii , open - ended holes with no silica also promoted evaporation - based localized flow and accumulation of particles over the nanohole array was obtained . there was no significant difference in the time scales of evaporation for cases i and ii . for case iii ( open - ended holes with silica ) , the flow was based on surface tension and the time scale was an order of magnitude less than evaporation - based methods . this case demonstrates a significantly higher concentration as most of the solution was directed to flow through the nanoholes , improving particle aggregation over the holes ( figure 5c ) . the absence of a prominent outer ring for the drop indicates the dominance of nanohole directed flow and the relatively shorter time scale of the process . surface plots revealing the intensity of fluorescence for these three cases are shown in figure 5(d f ) . the average fluorescence from the accumulated beads over the suspended membrane area was calculated over three rounds of experiments and is shown in figure 5 g . bead concentration and time taken for consumption of solution over nanohole arrays for three cases . ( a ) case i : accumulation of 2 m polystyrene beads after evaporation of a 10 l drop over dead - ended nanohole arrays . the schematic shows cross section of a single dead - ended nanohole for this case . ( b ) case ii : the same experiment performed over suspended nanohole arrays with metal - coated ( hydrophobic ) sidewalls . ( c ) case iii : these samples had silica deposited on the back surface and sidewalls , resulting in surface - tension - directed flow . ( d f ) surface plots corresponding to ( a c ) representing the intensity of fluorescence over the suspended membrane . ( g ) average fluorescence intensity calculated from beads accumulated over the suspended membranes after 3 rounds of experiments . these results demonstrate that case iii surface - tension - induced flow - through promotes the most effective concentration of particles over the nanoholes and is an order of magnitude faster than purely evaporation - driven concentration . these results illustrate that surface - tension - induced flow through nanoholes promotes the most efficient concentration of particles and is at least an order of magnitude faster as compared to evaporation - based techniques . this principle can also be utilized for overcoming diffusion - limited interaction of analytes with the nanoholes for plasmonic sensing . for small analyte molecules , the passive flow generated by surface tension forces drives them to pass within less than 100 nm ( radius of the nanoholes ) of the hotspot , i.e. , edge of the nanoholes and the nanohole sidewalls . this short distance can be easily overcome by diffusion of analyte molecules increasing the probability of the analytes adsorbing on or close to the plasmonic hotspots , which can lead to enhancement of the raman signal of analytes . the 4-mp molecules , which have a thiol group , can be captured on the nanohole array substrate through formation of covalent thiol silver bonds . the molecules may also get physically adsorbed to silica layered regions inside the nanoholes through van der waals forces . to demonstrate the advantage of flow through the nanoholes over diffusion - based transport for sers sensing , we performed experiments where the nanohole substrates were prepared in two different ways . in the first case , 20 l of 100 nm 4-mp solution was allowed to completely flow through suspended nanoholes in about 20 min . for diffusion - based adsorption , substrates were submerged in a reservoir containing identical 4-mp solution for the same amount of time . samples with surface - tension - induced flow gave about 50 times higher signal as compared to diffusion - limited adsorption . the experiment was repeated over multiple sets of samples and similar results were observed ( figure s3 , supporting information ) . to demonstrate detection of low concentration samples , 50 l of 4-mp at a concentration of 100 pm ( i.e. , 5 fmol of 4-mp ) was allowed to flow through the sample for about an hour . the sers signal collected from this sample shown in figure 6b demonstrates the capability of the method to detect low concentrations of analyte molecules . the high sensitivity in this case results from the combination of two factors : large raman enhancement factor of the nanohole array substrate and flow - through - based concentration of molecules at the hot spot . ( a ) comparison of sers spectra obtained from flow - through of 100 nm 4-mp ( red spectrum ) as compared to diffusive transport ( blue ) . ( b ) sers spectra obtained after flow - through of 100 pm 4-mp for an hour . the limit of detection of the setup , in terms of 4-mp concentration , would depend on the duration of time the solution is allowed to flow through the nanoholes . however , it can be estimated in terms of number of molecules of 4-mp in solution . using the peak at 1099 cm in the sers signal shown in figure 6b , the signal - to - noise ( s / n ) ratio was calculated to be approximately 55 . assuming a linear concentration vs signal response ( figure s2 , supporting information ) , and calculating for a final s / n ratio of 3 , gave us a limit of detection of approximately 272 amol of 4-mp molecules in solution . we have demonstrated the fabrication of millimeter - sized suspended metallic nanohole arrays using nil and their potential for flow - through sers . refractive - index - based tuning with sucrose solution was implemented on this suspended nanohole array substrate for optimized sers . this tuning method was utilized to match the plasmon resonance peaks with the excitation laser wavelength . sucrose solution was used because it has a very weak intrinsic raman signal and , therefore , does not contribute detectable background to the measurements . this simple tuning method can also be utilized for plasmon - enhanced fluorescence where the plasmon resonance can be tuned based on the excitation or emission spectra of the fluorescent species . furthermore , this technique can easily be translated to other plasmonic substrates utilizing the principle that , by changing the refractive index of the dielectric medium around the metal , plasmon resonance conditions can be changed to optimize the detection of the optical signal from analyte molecules . the suspended ag nanohole arrays have a sers enhancement factor on the order of 10 , which is 2 orders of magnitude greater than those reported previously for nanohole arrays and compares well with other commonly used sers substrates . our demonstration of high sers enhancement factor from nanohole arrays can open up many possibilities , because the nanohole geometry enables trapping of biomolecules and small particles , formation of model membranes such as suspended lipid bilayers , as well as flow - through sensing to overcome the diffusion limit . to combine nanohole - enhanced sers with flow - through sensing , we have demonstrated surface - tension - induced passive flow and concentration of particles over the nanohole arrays . passive flow rates of around 1 l / min were obtained using this technique . this passive flow allowed us to overcome diffusion - limited transport of analytes and reduced the time required to consume the solution while bringing the analyte molecules closer to the plasmonic hotspots . our device design utilizing passive flow obviates the use of a bulky external pump and fluidic interconnects , thus improving the simplicity and portability of the process . thus , the simple and novel resonance tuning as well as passive flow - through technique discussed here on a suspended nanohole array platform can help alleviate the mass transport limitations and benefit wide applications in optofluidics .

morphometric studies in multicellular organisms are mostly performed manually because of the complexity of multidimensional features and lack of appropriate tools for handling these organisms . here we present an integrated system to autonomously ( i.e. without human supervision ) identify and sort mutants with altered subcellular traits in real - time . we performed self - directed screens of synapse formation 100 faster and found both novel genes and phenotypic classes previously unidentified in extensive manual screens . we present metallic nanohole arrays fabricated on suspended membranes as an optofluidic substrate . millimeter - sized suspended nanohole arrays were fabricated using nanoimprint lithography . we demonstrate refractive - index - based tuning of the optical spectra using a sucrose solution for the optimization of sers signal intensity , leading to a raman enhancement factor of 107 . furthermore , compared to dead - ended nanohole arrays , suspended nanohole arrays capable of flow - through detection increased the measured sers signal intensity by 50 times . for directed transport of analytes , we present a novel methodology utilizing surface tension to generate spontaneous flow through the nanoholes with flow rates of 1 l / min , obviating the need for external pumps or microfluidic interconnects . using this method for sers , we obtained a 50 times higher signal as compared to diffusion - limited transport and could detect 100 pm 4-mercaptopyridine . the suspended nanohole substrates presented herein possess a uniform and reproducible geometry and show the potential for improved analyte transport and sers detection .

dialysis - related amyloidosis ( dra ) diagnosed in patients with advanced renal insufficiency on maintenance hemodialysis often manifests as signs and symptoms of carpal tunnel syndrome ( cts ) , chronic arthropathy , presence of subchondral cysts and pathological fracture tendency . increased levels of beta-2-microglobulin ( bmg ) in the plasma of dialyzed patients plays an essential role in the pathogenesis of dra . cts is the most common complaint in dra , caused by pressure on the median nerve from complexes of amyloid , the main component of which is bmg . diagnosis of cts is based on signs and symptoms verified by nerve conduction [ 14 ] . the aim of this study was to evaluate the incidence of cts and identify factors influencing the development of cts in patients on maintenance hemodialysis , as well as results of its surgical treatment . the study included 386 patients ( 285 patients from the department of nephrology , university hospital , cracow , and 101 patients from the dialysis unit , st . patients were hemodialysed 3 times per week , for 45 hours each time , using cuprophane membranes ; while in the last 10 years cellulose or polysulphone , low - flux type dialyzers were used . diagnosis of cts was initially based on signs and physical symptoms verified by nerve conduction examination . clinical cts diagnosis was based on numbness , nocturnal pain in the median nerve distribution , and positive stimulating tests , particularly the tinel sign . a prolonged sensory and/or motor latency from the wrist to digits innervated by the median nerve the following parameters were evaluated : patient age , sex , duration of dialysis therapy , etiology of renal insufficiency , presence of anti - hcv antibodies , localization of av fistula , and presence of cysts and joint pain . concentrations of urea and creatinine before and after dialysis and potassium , calcium , and phosphorus were measured monthly . statistical analysis using the non - parametric mann - whitney test for unassociated variables compared age and duration of dialysis therapy for the groups of patients with cts and without cts . initial analysis of cts by sex , presence of anti - hcv antibodies and location of av fistula were verified using the chi - square test . carpal tunnel syndrome was diagnosed and verified using nerve conduction examination in 40 patients , who comprised 10.4% of the studied patient population on maintenance hemodialysis . causes of terminal renal insufficiency in cts patients were as follows : glomerulonephritis ( 45% ) , degenerative polycystic kidney disease ( 12.5% ) , chronic pyelonephritis ( 10% ) , diabetic nephropathy ( 5% ) , amyloidosis nephropathy ( 2.5% ) , lupus nephritis ( 2.5% ) , hypertensive nephropathy ( 2.5% ) , and renal cirrhosis of unknown origin ( 20% ) . patients with cts were aged between 36 and 83 years ( mean 54.5 years ) , while the asymptomatic patient group was aged 18 to 100 years ( mean 56.48 years ) ( figure 1 , table 1 ) . dialysis therapy in the patient group with cts ranged from 430 years ( mean 16.05 years ) ; while dialysis duration among patients without cts ranged from 0.216.4 years ( mean 4.51 years ) ( figure 2 , table 1 ) . statistical analysis showed that patients with cts were hemodialysed significantly longer ( p<0.00001 ) ( mann - whitney test ) . surgical release procedure was required in 5 patients ( 100% ) with cts on maintenance dialysis treated for a long period ( from 2530 years ) . in patients hemodialysed from 2024 years , cts developed in 5 patients ( 100% of the sub - group ) . in the group of patients less than half of the patients hemodialysed from 1014 years were operated on due to complaints in the course of cts ; they comprised 42.1% of this subpopulation ( 16 patients ) . four patients dialyzed for fewer than 10 years were found to have cts ( 1.6% ) ( table 2 ) . biopsies performed at the time of cts surgery confirmed dra . histological examination of biopsy specimens was performed in 21 out of 40 patients ( 52.5% ) , and all of these 21 biopsied patients presented with signs of amyloid deposits . among patients with cts , the majority were males ( m : f , 2:1 ) ( 27 males , 13 females ) , which did not reflect the ratio of both sexes in the studied population ( 229 males , 157 females ) . no statistically significant differences in incidence of cts by sex ( chi - square test , p = ns ) was noted . in the studied population , 43 patients were found to have positive anti - hcv antibodies . in the group of patients with cts , 19 patients were found to have positive anti - hcv antibodies , which equaled 47.5% of patients of this subgroup . in the group of 346 patients without cts , incidence of cts was statistically significantly higher in patients with positive anti - hcv antibodies ( chi - square test , p<0.00001 ) ( table 3 ) . all patients with cts complained of numbness , tingling and pain in the first 3 radial digits . the symptoms of paresthesia were more pronounced at night , causing insomnia and frequent waking . in 21 patients , these problems increased during dialysis . in some cases the diagnosis of cts was supported by positive tinel sign . in a number of patients muscle weakness , hypoesthesia and thenar hypotrophy nerve conduction studies showed prolonged distal sensory and motor latencies from the wrist to digits , innervated by the median nerve ( table 4 ) . regression of sensory and motor deficiencies was much slower and took 46 months . in 14 patients , cts developed in the wrist where the av fistula was located ; in 3 patients on the opposite side , and bilaterally in 23 patients ( 57.5% ) . no statistical differences were noted between the development of cts requiring surgical release procedure and location of the av fistula ( chi - square test , p = ns ) . a total of 72 surgical procedures of the median nerve release were performed in 40 patients with cts , in 9 patients due to recurrence of cts , and 23 on the opposite hand . the traditional surgical method was implemented where the transverse carpal ligament was severed , which later released the median nerve . during the surgical procedure , tumor - like changes were found in tendons and synovial membranes ; sometimes fibrotic adhesions , segmental tendon fragmentation of the finger flexors , or deep changes in the synovial membrane were also found . in 24 patients , such changes were found in the acetabulum of the hip joint , in the head of the femoral bone , radial and humeral bone , patella and the wrist bone . six patients had hip replacement performed ( endoprosthesis implantation ) . in patients with signs and symptoms of cts , nerve conduction and electromyography no statistically significant differences were noted in concentrations of the following biochemical parameters : urea , creatinine , potassium , calcium , phosphorus , sodium and liver parameters in patients with cts and the group of patients without cts . the frequency of clinical symptoms of cts in patients on chronic hemodialysis ranges between 2% and 31% . this is probably due to the number and characteristics of the evaluated patient group ; and to the degree to which diagnostic criteria is employed . most patients require surgical release procedure [ 58 ] . among the studied group of 386 patients on maintenance hemodialysis , the mean age of patients with cts was lower ( 54.5 ) than the mean age of patients without cts ( 56.48 years ) . others investigators noted statistically significant dependence between the age of hemodialysed patients and the occurrence of cts . the group of patients with cts was characterized by a 2-fold higher number of males ( 27 males , 13 females ) , which did not reflect the ratio of both sexes in the study population ( 229 males , 157 females ) . this data differs from the results of other authors ( ratio of m : f=1:1 ) or relates to other studies ( 67% of males in the group with cts ) . in the non - dialyzed patient population , cts is definitely more frequently diagnosed in females ( f : m=3:1 ) . the decreased percentage of females with cts in hemodialysed patients is supposedly due to common abnormalities that accompany terminal renal failure or to the fact that these patients do not perform as much movement of the wrist due to decreased physical activity . in the analyzed group of patients , no difference was noted between incidence of cts and patient sex . among the 40 patients with cts , , no connections have been found between the etiology of terminal renal failure and incidence of cts in dialyzed patients , except for schwarz et al , who found an increased incidence of cts in dialyzed patients after analgetic nephropathy . duration of dialysis therapy in the group of patients with cts was significantly longer ( mean 16.05 years ) in comparison with dialysis duration among patients without cts ( mean 4.51 years ) . this correlates with observations of other authors . all patients dialyzed for more than 20 years underwent surgical release procedure of the wrist ; whereas surgical procedures in cts patients dialyzed for less than 10 years were very rare . duration of dialysis therapy was the only statistically significant risk factor for cts . in the analyzed population of 386 hemodialysed patients , 43 were found to be anti - hcv positive ( 11.1% ) ; whereas in the subgroup of patients without cts only 24 patients were found to be hcv positive ( 6.9% ) . there were 19 patients with cts that had anti - hcv antibodies , which is 47.5% of this subgroup . hemodialysed patients with cts were found to have anti - hcv antibodies significantly more often . such a correlation has not been commonly noted by researchers , and is probably due to the fact that the percentage of patients with positive anti - hcv antibodies depends on the duration of dialysis therapy . one can speculate that this relationship may be due to stimulation of the liver by the inflammatory process to produce beta-2-microglobulin , and this in turn plays an essential role in the pathogenesis of dialysis - related amyloidosis . in the pre - erythropoietin era , hepatitis c infection was common in chronic dialysis patients when blood transfusions were routine . cts developed in 14 patients in the wrist with the av fistula , in 3 patients that had the av fistula on the opposite side , and in 23 patients bilaterally ( 57.5% ) . prevalence of cts in the wrist where the av fistula was located did not fulfill the criteria for statistical significance . a relationship between cts and the av fistula location has been postulated in many publications [ 6,13,1618 ] . vascular factors associated with the av fistula and mechanisms due to amyloid deposition must be considered , along with the substantial increase in venous pressure in the region of the av fistula , which , together with periodic patient water overload , generates pressure on the median nerve . clinical signs and symptoms such as paresthesia , increasing pain during the night and during dialysis , insomnia , and frequent waking during the night decrease patient quality of life . pharmacological therapy as symptomatic treatment only transiently decreases complaints . in 40 patients with cts , 72 surgical release procedures were performed ; 9 were due to recurrences after using the traditional surgical procedure ( surgically severing the transverse carpal ligament ) , and in 23 patients due to second hand localization . after the operative procedures , rapid relief of paresthesia and pain symptoms were observed ; return of sensory and motor deficits took 34 months longer . these results is in agreement with the findings of other authors , and indicate that recurrences of cts in dialyzed patients are more common in comparison to the non - dialyzed population . in the treatment of cts recurrences , excluding synovectomy , resection of the thickened tendinous sheaths of the finger flexor is recommended [ 12,17,1921 ] . duration of dialysis therapy is an independent and statistically significant risk factor in the development of cts . cts occurs significantly more often in anti - hcv - positive patients , which may be related to the longer period on dialysis . the prevalence of obesity is on the increase globally , both in developed and developing nations . in the united states , it is estimated that approximately 64.5% of adults can be classified as overweight or obese individuals . in addition to the morbidity associated with obesity , approximately 325,000 deaths in the united states each year among nonsmokers are attributable to obesity . bakari et al . evaluated obesity rates using body mass index ( bmi ) and waist - to - hip ratio ( whr ) among type 2 diabetic hausa - fulanis . among this group , 35% and 5% were overweight and obese , respectively , whereas 95% had central obesity when gender - specific whr was used . , in a study on type 2 diabetic patients in enugu , southeast nigeria , reported central obesity [ determined by waist circumference ( wc ) ] in 22.8% of the subjects studied . the use of different indices to determine or measure obesity clearly explains why the prevalence rates of obesity differ globally . among 6208 type 2 diabetic subjects seen in india , over 50% were found to be overweight or obese when bmi of > 25 kg / m was used , whereas 59.66% and 95.57% of men and women , respectively , had central obesity when gender - specific whr cut - off points of > 0.95 and > 0.85 , respectively , were used . insulin resistance with compensatory hyperinsulinemia has been suggested to underlie the clustering of cardiovascular risk factors , including glucose intolerance , hypertension , elevated serum triglycerides , low serum high density lipoprotein cholesterol and central obesity . central obesity has been shown to worsen the degree of insulin resistance . in the earlybird study , wc correlated significantly with homa - ir in both genders , while bmi correlated significantly with homa - ir in girls only . similarly , wc seems to be particularly associated with the risk for non - communicable diseases as shown by many other studies.[810 ] recently , the international diabetes federation ( idf ) proposed the use of ethnic - specific cut - off values for wc , having made it a compulsory criterion in its definition of the metabolic syndrome . several indices such as wc , whr , waist to height ratio ( whtr ) and sagittal abdominal diameter ( sad ) have been used as clinical measures of central obesity . magnetic resonance imaging ( mri ) and computed tomography ( ct ) are however considered the gold standard methods for determining the quantity of subcutaneous abdominal adipose tissue ( saat ) and intra - abdominal adipose tissue ( iaat ) . this gold standard , however , can not be routinely used in a clinic setting to measure these indices . wc is a simple and valid measure that may be used independently as an estimate of abdominal fat and it has been found to be more strongly associated with cardiovascular health risk . in this study , we compared the performances of two measures of central obesity , namely , wc and whr , in predicting the presence of cardiovascular risk markers in an apparently healthy nigerian population . this study was a cross - sectional descriptive survey of the inhabitants of enugu ( urban ) . it was formerly the capital of the former eastern nigeria and is currently the capital city of enugu state .. the state is one of the five states in the southeast geopolitical zone of nigeria . it is geographically located between longitude 726 e and 730 e and latitude 625 n and 628 n. enugu city is predominantly an urban christian community with a population of 722,664 people out of the total population of 3,257,298 for the whole state ( 2006 population census figure ) . its work force consists mainly of civil servants , business men , industrialists , farmers and students . the town is richly endowed with large deposits of coal which served as a major revenue source for nigeria before the era of oil boom . ethical clearance for the study was obtained from the ethics committee of the university of nigeria teaching hospital ( unth ) , and consent was obtained from all the participants . the subjects comprised apparently healthy individuals ( not known hypertensive or diabetic patients ) who were residents of enugu ( urban ) . a total of 1000 subjects aged 1870 years and who were of the igbo tribe were recruited through a multi - stage sampling procedure . subjects who were physically challenged ( either wheelchair bound or unable to stand ) and female subjects who were pregnant , based on their date of last menstrual period , were excluded . in stage one , five areas of the town , namely , emene , abakpa , trans - ekulu , asata and ogui layout , were selected by simple random sampling ( using the balloting technique ) . in the second stage , 200 participants were selected from among those who reported and got registered on the day of recruitment in each of the five areas . these subjects were invited for registration following health awareness campaigns executed in some of the churches within the five selected areas . those who were not selected were not interviewed but had their anthropometric indices measured and medical advice given based on the individual 's cardiovascular risk status . a total of 898 subjects ( 318 males and 580 females ) were used for analysis after cleaning up of the data . data collection and physical measurements were based on world health organization ( who ) 's steps instrument . it is a simple , standardized method for collecting and analyzing data for chronic disease risk factors in who member countries . it involves a sequential process which starts with gathering information on key behavioural risk factors ( step 1 ) and then moving to simple physical measurements ( step 2 ) . the physical measurements undertaken included height ( measured to the nearest 0.1 cm ) , weight ( recorded to the nearest 0.1 kg ) , wc and hip circumference ( recorded to the nearest 0.1 cm ) using non - stretching flexible linear tapes , and blood pressure ( recorded to the nearest whole number in mmhg ) using mercury sphygmomanometers ( accosons , essex , england ) . the landmark for the measurement of wc was the midpoint between the lowest rib and the iliac crest , as recommended by who . measurement was done at the end of expiration , with the arms by the side and patient standing with the feet together . both wc and hip circumference were measured in privacy with the subject in light clothing . the 1 and 5 korotkoff sounds were used to mark the systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) respectively . generalized obesity was defined as bmi 30 kg / m , while central obesity was defined according to the international diabetes federation ( idf ) ethnic - specific criteria as wc 94 cm and 80 cm for males and females , respectively . blood pressure was classified using the jnc 7 classification . risk category was defined using the hypertensive category ( sbp 140 mmhg and/or dbp 90 mmhg ) . comparison of means between two groups was done using the independent t - test while test of association / independence between categorical variables was performed using chi - square test of independence . the receiver operating characteristic ( roc ) analysis was used to compare the performance of wc and whr ( measures of central obesity ) as determined by the area under the curve ( auc ) . data analysis was conducted using statistical package for social sciences for windows ( spss ) version 10 ( chicago , il , usa ) . the subjects comprised apparently healthy individuals ( not known hypertensive or diabetic patients ) who were residents of enugu ( urban ) . a total of 1000 subjects aged 1870 years and who were of the igbo tribe were recruited through a multi - stage sampling procedure . subjects who were physically challenged ( either wheelchair bound or unable to stand ) and female subjects who were pregnant , based on their date of last menstrual period , were excluded . in stage one , five areas of the town , namely , emene , abakpa , trans - ekulu , asata and ogui layout , were selected by simple random sampling ( using the balloting technique ) . in the second stage , 200 participants were selected from among those who reported and got registered on the day of recruitment in each of the five areas . these subjects were invited for registration following health awareness campaigns executed in some of the churches within the five selected areas . those who were not selected were not interviewed but had their anthropometric indices measured and medical advice given based on the individual 's cardiovascular risk status . a total of 898 subjects ( 318 males and 580 females ) were used for analysis after cleaning up of the data . data collection and physical measurements were based on world health organization ( who ) 's steps instrument . it is a simple , standardized method for collecting and analyzing data for chronic disease risk factors in who member countries . it involves a sequential process which starts with gathering information on key behavioural risk factors ( step 1 ) and then moving to simple physical measurements ( step 2 ) . the physical measurements undertaken included height ( measured to the nearest 0.1 cm ) , weight ( recorded to the nearest 0.1 kg ) , wc and hip circumference ( recorded to the nearest 0.1 cm ) using non - stretching flexible linear tapes , and blood pressure ( recorded to the nearest whole number in mmhg ) using mercury sphygmomanometers ( accosons , essex , england ) . the landmark for the measurement of wc was the midpoint between the lowest rib and the iliac crest , as recommended by who . measurement was done at the end of expiration , with the arms by the side and patient standing with the feet together . both wc and hip circumference were measured in privacy with the subject in light clothing . the 1 and 5 korotkoff sounds were used to mark the systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) respectively . generalized obesity was defined as bmi 30 kg / m , while central obesity was defined according to the international diabetes federation ( idf ) ethnic - specific criteria as wc 94 cm and 80 cm for males and females , respectively . risk category was defined using the hypertensive category ( sbp 140 mmhg and/or dbp 90 mmhg ) . comparison of means between two groups was done using the independent t - test while test of association / independence between categorical variables was performed using chi - square test of independence . the receiver operating characteristic ( roc ) analysis was used to compare the performance of wc and whr ( measures of central obesity ) as determined by the area under the curve ( auc ) . data analysis was conducted using statistical package for social sciences for windows ( spss ) version 10 ( chicago , il , usa ) . males were older than females and had higher whr and dbp , whereas females had higher wc , bmi and sbp . the gender differences observed in all the variables except for sbp and dbp were significant [ table 1 ] . general characteristics of the subjects central obesity was more prevalent when whr ( 76.9% ) was used than when wc ( 66.5% ) was used . chi - square analysis showed that central obesity determined using both whr ( ( 1 ) = 5.15 ; p < 0.05 ) and wc ( ( 1 ) = 185.6 ; p < generalized obesity was found in 190 ( 21.2% ) subjects , while 339 ( 37.8% ) were overweight [ figure 1 ] . six ( 0.7% ) persons were undernourished ( bmi < 18.5 kg / m ) . weight categorization among the subjects according to body mass index using the jnc 7 classification , 430 ( 47.9% ) , 324 ( 36.1% ) and 144 ( 16% ) subjects were classified as having hypertension , pre - hypertension and normal blood pressure , respectively . greater proportion ( 42.7% ) of individuals had hypertension when sbp was used alone compared to 30.8% when dbp was used ( ( 1 ) = 9.2 ; p = 0.0024 ) . the auc showing the performances of wc and whr in predicting the presence of cardiovascular risk ( generalized obesity , hypertension and both obesity and hypertension ) is shown in figures 24 , respectively . receiver operating characteristic curve for waist circumference and waist - to - hip ratio for generalized obesity receiver operating characteristic curve for waist circumference and waist - to - hip ratio for hypertension receiver operating characteristic curve for waist circumference and waist - to - hip ratio for obesity and hypertension the values of the auc for each of the indices for generalized obesity , hypertension and hypertension / obesity are summarized in table 2 . based on its higher auc , areas under the curve for predicting the presence of obesity , hypertension , and obesity / hypertension the auc showing the performances of wc and whr in predicting the presence of cardiovascular risk ( generalized obesity , hypertension and both obesity and hypertension ) is shown in figures 24 , respectively . receiver operating characteristic curve for waist circumference and waist - to - hip ratio for generalized obesity receiver operating characteristic curve for waist circumference and waist - to - hip ratio for hypertension receiver operating characteristic curve for waist circumference and waist - to - hip ratio for obesity and hypertension the values of the auc for each of the indices for generalized obesity , hypertension and hypertension / obesity are summarized in table 2 . based on its higher auc , areas under the curve for predicting the presence of obesity , hypertension , and obesity / hypertension in this study , the performances of wc and whr were compared using roc analysis . roc curves are frequently used in several medical disciplines such as biomedical informatics , clinical chemistry and radiology . the roc curve plots sensitivity versus ( 1 specificity ) of a test as the threshold varies over its entire range . each data point on the plot represents a particular setting of the threshold , and each threshold setting defines a particular set of true - positive ( tp ) , false - positive ( fp ) , true - negative ( tn ) and false - negative ( fn ) frequencies , and consequently a particular pair of sensitivity and ( 1 specificity ) values . it was originally developed for radar applications in the 1940s , but roc analysis became widely used in medical diagnostics , where complex and weak signals needed to be distinguished from a noisy background . the area under an roc curve is equal to the probability that a randomly selected positive case will receive a higher score than a randomly selected negative case . the roc curve area is a good summary measure of test accuracy because it does not depend on the prevalence of disease or the cut points used to derive the curve . it is however suggested that once a test has been able to classify patients as either having a disease or not , the performance of the test for particular uses such as diagnosis or screening needs to be evaluated . as regards its use when comparing the accuracies of two tests as in this study , caution should be exercised as the roc curve area may be misleading if the curves cross each other . this study revealed that wc had higher aucs compared to whr in subjects who were classified as having generalized obesity alone , hypertension alone or both . the higher aucs therefore suggest that wc may be more useful and reliable than whr in predicting the presence of generalized obesity and hypertension or cardiovascular risk . in a similar study by pouliot et al . , sagittal abdominal diameter ( sad ) was identified to be better than other clinical measures or determinants of abdominal obesity wc among the various measures of central adiposity is very easy to measure and perhaps more time saving in a routine clinic setting . it is neither cumbersome at all compared to the gold standards ( ct , mri ) nor labourious requiring further measurements and calculations . the simplicity of its measurement and its relation to both body weight and fat distribution as a major advantage over bmi and waist - to - hip circumference ratio was highlighted by lean et al . studies have shown that anthropometric measures , such as bmi , whr , and wc cut - off levels , are not comparable across different racial populations . apart from the fact that wc is a good indicator of both the degree of obesity and the accumulation of visceral adipose tissue , the threshold values of wc corresponding to critical amounts of visceral adipose tissue do not appear to be influenced by sex or by the degree of obesity . taylor et al . , in a screening for regional fat distribution among adult women , found that wc significantly classified the subjects better than whr . al - sendi et al . also reported that wc is useful in identifying children ( 1217 years of age ) at risk of developing hypertension . among 768 middle - aged men from the olivetti heart study , wc was the strongest predictor of blood pressure , and also was related to heart rate , insulin concentrations , and insulin sensitivity . in this study , the aucs for both wc and whr for the subjects who had only hypertension were smaller when compared to those represented by obesity alone and by both obesity and hypertension . this can be explained by the fact that bmi is better related to body fat than hypertension though they can cluster in a single individual . this association is evidenced by the degree of relationships shown by the correlation coefficients ( not shown in the results ) . though the aucs were smaller in the hypertension group , wc still performed better than whr in this group . with respect to gender comparisons , females had a higher mean wc and bmi values , whereas the reverse was the case for whr . this pattern had earlier been observed among diabetic patients in the same ethnic region of nigeria and in cuban female scholars . hormonal differences particularly involving the adrenal and sex steroids have also been noted to influence body fat distribution.[3234 ] these gender patterns also appear to be established in childhood , especially with pubertal development . though the role of obesity as a health hazard in adults has been well recognized , its presence in adolescence has been associated with obesity in adulthood , thus emphasizing the importance of early detection and intervention directed at its treatment to avert the long - term consequences of obesity and development of cardiovascular diseases . adipose tissue is mostly distributed as subcutaneous fat ( 85% of total adipose tissue mass ) and then a smaller amount as intra - abdominal fat ( 15% ) in lean and obese persons . the relative contribution of intra - abdominal fat mass to total body fat is influenced by sex , age , race - ethnicity , physical activity , and total adiposity . the term visceral fat is commonly used to describe intra - abdominal fat , and intra - abdominal fat is made up of both intraperitoneal fat ( mesenteric and omental fat ) , directly draining into the portal circulation , and retroperitoneal fat , draining into the systemic circulation . currently , there is no universally accepted site for measuring intra - abdominal ( iaat ) and subcutaneous adipose ( saat ) tissue distributions . this remains a source of variation in data obtained from different studies and needs to be harmonized for effective comparison of data . abdominal circumference as measured by wc , actually measures the fat contributed by subcutaneous tissue which is under the skin of the abdomen and that deposited intra - abdominally . both saat and iaat have been found to correlate with insulin resistance . despite the obvious strengths of wc over whr and bmi , it is to be noted that adoption of different landmarks for its measurement may pose some limitations on the comparison of wc data generated from different studies . though some studies on wc involving subjects of african descent have been reported , characterization of wc among african populations still appears to be deficient , especially as it relates to diagnostic indices and threshold values . wc provides a unique indicator of body fat distribution , which can identify patients who are at increased risk of central obesity - related cardiometabolic disease , above and beyond the measurement of bmi . from this study , being a simpler index to measure , less time consuming and devoid of any calculations , we propose that clinicians will find wc a reliable means of assessing individuals cardiovascular risk status especially in a busy routine clinic setting .

summarybackgroundcarpal tunnel syndrome ( cts ) is the most common complication of dialysis - related amyloidosis ( dra ) developing in patients on long - term dialysis therapy . the aim of this study was to evaluate the incidence of cts and identify factors influencing the development of cts in patients on maintenance hemodialysis , as well as results of its surgical treatment.material/methodsthe study included 386 patients , among whom cts was diagnosed in 40 patients ( 10.4% ) on the basis of signs and physical symptoms , as well as by nerve conduction . the group of patients with cts and the group of patients without cts were compared according to age ( mean 54.50 vs. 56.48 years ) and duration of dialysis treatment . initial analysis of cts incidence by sex , presence of anti - hcv antibodies , and location of arterio - venous fistula ( av fistula ) was undertaken.resultsduration of dialysis treatment was the statistically significant risk factor for the development of cts ( 16.05 vs. 4.51 years ; p<0.0001 ) . among patients treated for a long period on hemodialysis ( 2030 years ) , 100% required surgical release procedures , while 66.66% of those treated for 1519 years , 42.1% of those treated for 1014 years , and 1.6% of those treated for less than 10 years . cts was diagnosed more often in anti - hcv - positive patients as compared with anti - hcv - negative patients ( 47.5 vs. 6.9% ; p<0.0001 ) . no significant differences were found when comparing cts incidence by sex or between the development of cts requiring surgical release intervention and location of the av fistula.conclusionssurgical release procedure of the carpal tunnel gave good treatment results in patients with cts . objective : to compare the performance of waist circumference ( wc ) and waist - to - hip ratio ( whr ) in predicting the presence of cardiovascular risk factors ( hypertension and generalized obesity ) in an apparently healthy population.materials and methods : we recruited 898 apparently healthy subjects ( 318 males and 580 females ) of the igbo ethnic group resident in enugu ( urban ) , southeast nigeria . data collection was done using the world health organization stepwise approach to surveillance of risk factors ( steps ) instrument . subjects had their weight , height , waist and hip circumferences , systolic and diastolic blood pressures measured according to the guidelines in the step 2 of steps instrument . generalized obesity and hypertension were defined using body mass index ( bmi ) and jnc 7 classifications , respectively . quantitative and qualitative variables were analyzed using t - test and chi - square analysis , respectively , while the performance of wc and whr was compared using the receiver operating characteristic ( roc ) analysis . p value was set at < 0.05.results:the mean age of the subjects was 48.7 ( 12.9 ) years . central obesity was found in 76.9% and 66.5% of subjects using whr and wc , respectively . wc had a significantly higher area under the curve ( auc ) than whr in all the cardiovascular risk groups , namely , generalized obesity ( auc = 0.88 vs. 0.62 ) , hypertension alone ( auc = 0.60 vs. 0.53 ) , and both generalized obesity and hypertension ( auc = 0.86 vs. 0.57).conclusion : wc performed better than whr in predicting the presence of cardiovascular risk factors . being a simple index , it can easily be measured in routine clinic settings without the need for calculations or use of cumbersome techniques .

with over 100 different molecular targets , curcumin is the archetypal pleiotropic dietary agent,1 and is currently at the forefront of biomedical research , as cogently testified by the over 5150 entries for curcumin in pubmed , ie , about 10% of those for aspirin , the best known drug . the redundancy of targets makes it difficult to decipher the clinical translation of the biochemical signature of curcumin , but there is general agreement that the most important clinical targets of curcumin are transcription factors ( nf - kb , stat3 , nrf2 ) , and that curcumin can exert beneficial effects by modulating the genomic and cell - signaling pathways involved in the inflammatory response.1,2 for this reason , the effects of curcumin are not expected to be immediately measurable but rather to develop gradually.1 the dismally low oral bioavailability of curcumin has long hampered the clinical translation of its medicinal potential,3 but this issue has now been substantially improved by various formulation strategies , with dispersion in lecithin , as in meriva ( indena spa , milan , italy ) being the best documented in terms of comparative pharmacokinetics4 and clinical efficacy.59 during a series of recent clinical studies of meriva for various chronic diseases,59 a rapid analgesic effect was observed within 12 hours of ingestion by some patients . similar anecdotal observations were reported by other users , raising the issue of the significance of these findings . these reports , and the recent discovery that curcumin can desensitize or inhibit a series of transient receptor potential ion channels involved in the generation of painful stimuli , ie , transient receptor potential cation channel 1 ( trpa1 ) and transient receptor potential cation channel subfamily v member 1 ( trpv1),1012 provided a rationale to investigate the activity of meriva in the mitigation of acute pain . here we report a pilot comparative study of the analgesic properties of meriva and two popular analgesic drugs , acetaminophen and nimesulide . the mechanism of action of acetaminophen has long been elusive , but has recently been related to the desensitization , by its quinone metabolite , of spinal trpa1,13 an ion channel that is also sensitive to the desensitizing activity of curcumin.10,11 as a powerful and rapidly acting cyclo - oxygenase 2 inhibitor,14 nimesulide outperforms curcumin as a direct inhibitor of this enzyme but is nevertheless largely devoid of the genomic capacity to downregulate inflammatory cytokines , as shown by curcumin.15 patients were excluded if they had a history of heart , renal , hepatic , or autoimmune disease , cancer , or gastroduodenal pathology . the lower and upper age limits for eligibility were 18 and 70 years , respectively . all patients signed a written informed consent before entering into the study , in accordance with the declaration of helsinki . the subjects received four tablet containers , containing nimesulide 100 mg in the first container , acetaminophen 500 mg in the second container , and meriva 500 mg in the third and fourth containers . the composition of meriva is curcumin ( 20% ) , phosphatidylcholine ( 40% ) , and micro - crystalline cellulose ( 40% ) . participants were blinded to the product contained in the pills , that were simply named as a ( nimesulide ) , b ( acetaminophen ) , and c and d ( meriva ) . meriva was supplied by indena spa , and nimesulide and acetaminophen were from commercial sources . the sequence of treatment was selected accordingly to the number of tablets to be taken , ie , one for nimesulide , two for acetaminophen , and three ( first cycle ) or four ( second cycle ) for meriva . the subjects were instructed to take one tablet of a on the first day of pain after enrollment , followed , after 48 hours of discontinuance , by two tablets of b , and , after further 24 hours of discontinuance , by three tablets of c. this protocol comprised the first cycle . in the protocol for the second cycle , subjects took one tablet of a on day 4 , followed by two tablets of b and four tablets of d , always with the same 2448-hour discontinuance cycle between treatments . the discontinuance between treatments was included in order to allow washout of the parent compound and/or active metabolites before intake of further treatment . this was of particular importance for nimesulide , which has an half - life of 25 hours but generates an active metabolite , ie , 4-hydroxynimesulide . therefore , a 48-hour washout was always observed between treatments a and b and of 24 hours between treatments b and c. fourteen subjects participated in the first cycle and 15 participated in the second cycle ; the difference in patient numbers was due to the fact that one patient , on antibiotic therapy for dental pain caused by pulpitis , was enrolled when the first cycle of the study had already been completed . all participants were allowed to take a second dose of the same product if pain perception was still significant 12 hours after the first dose . this opportunity was not utilized in practice , except for meriva , which has a shorter duration of action than the two other agents . after each intake of medication , the subjects completed a questionnaire , with the assistance of a clinician , canvassing the following items : pain perception , estimated according to the five - point visual analog scale devised by scott - huskisson ( 0 , nil ; 1 , slightly perceptible ; 2 , mild ; 3 , severe ; 4 , intolerable ) ; tolerability on the day of treatment ( poor , fair , good , excellent ) ; compliance ( poor , fair , good , very good ) ; and adverse events on the day of treatment and during the two following days . pain perception scores were analyzed according to a randomized block factorial design , and comparison between mean values were done using the tukey - kramer test . correspondence analysis and the pearson chi - square test were used to assess tolerability , compliance , and presence of adverse events . the subjects received four tablet containers , containing nimesulide 100 mg in the first container , acetaminophen 500 mg in the second container , and meriva 500 mg in the third and fourth containers . the composition of meriva is curcumin ( 20% ) , phosphatidylcholine ( 40% ) , and micro - crystalline cellulose ( 40% ) . participants were blinded to the product contained in the pills , that were simply named as a ( nimesulide ) , b ( acetaminophen ) , and c and d ( meriva ) . meriva was supplied by indena spa , and nimesulide and acetaminophen were from commercial sources . the sequence of treatment was selected accordingly to the number of tablets to be taken , ie , one for nimesulide , two for acetaminophen , and three ( first cycle ) or four ( second cycle ) for meriva . the subjects were instructed to take one tablet of a on the first day of pain after enrollment , followed , after 48 hours of discontinuance , by two tablets of b , and , after further 24 hours of discontinuance , by three tablets of c. this protocol comprised the first cycle . in the protocol for the second cycle , subjects took one tablet of a on day 4 , followed by two tablets of b and four tablets of d , always with the same 2448-hour discontinuance cycle between treatments . the discontinuance between treatments was included in order to allow washout of the parent compound and/or active metabolites before intake of further treatment . this was of particular importance for nimesulide , which has an half - life of 25 hours but generates an active metabolite , ie , 4-hydroxynimesulide . therefore , a 48-hour washout was always observed between treatments a and b and of 24 hours between treatments b and c. fourteen subjects participated in the first cycle and 15 participated in the second cycle ; the difference in patient numbers was due to the fact that one patient , on antibiotic therapy for dental pain caused by pulpitis , was enrolled when the first cycle of the study had already been completed . all participants were allowed to take a second dose of the same product if pain perception was still significant 12 hours after the first dose . this opportunity was not utilized in practice , except for meriva , which has a shorter duration of action than the two other agents . after each intake of medication , the subjects completed a questionnaire , with the assistance of a clinician , canvassing the following items : pain perception , estimated according to the five - point visual analog scale devised by scott - huskisson ( 0 , nil ; 1 , slightly perceptible ; 2 , mild ; 3 , severe ; 4 , intolerable ) ; tolerability on the day of treatment ( poor , fair , good , excellent ) ; compliance ( poor , fair , good , very good ) ; and adverse events on the day of treatment and during the two following days . pain perception scores were analyzed according to a randomized block factorial design , and comparison between mean values were done using the tukey - kramer test . correspondence analysis and the pearson chi - square test were used to assess tolerability , compliance , and presence of adverse events . compliance with treatment was very good for nimesulide and acetaminophen , good for meriva 1.5 g ( corresponding to 300 mg of curcumin ) and fair to good for meriva 2 g ( corresponding to 400 mg of curcumin , table 2 ) . the latter observation can be explained by the fact that intake of four pills was uncomfortable for some subjects . in general , all treatments were well tolerated ( table 3 ) , with tolerability being excellent for acetaminophen and meriva 1.5 g , poor to fair for nimesulide , and fair to good for meriva 2 g. no side effects were noticed for acetaminophen and meriva 1.5 g , but a few subjects reported gastrointestinal discomfort ( strong heartburn and gastroesophageal reflux , requiring antacid therapy ) with nimesulide . meriva 2 g also induced stomach heaviness , nausea , and heartburn in some participants , mostly those who had previously reported these side effects with nimesulide ( table 4 ) . however , unlike the gastric discomfort associated with nimesulide , that induced by meriva had an early onset , so was possibly nonspecific , being related , apart from an increased sensitivity to gastric injury , to the sheer challenge of ingestion of four pills . to study the time course of analgesic activity , pain intensity was evaluated by the visual analog scale before and 0.5 , 2 , 3 , 4 , 6 , 8 , 12 , and 24 hours after administration . the efficacy and safety results obtained for nimesulide and acetaminophen are consistent with those reported in the literature . a significant reduction in pain perception was observed 13 hours after taking nimesulide and 24 hours after taking acetaminophen . conversely , meriva 1.5 g had a statistically significant effect only after 3 hours , with an overall analgesic effect significantly lower than that of the other two drugs . in this cycle , nimesulide showed the highest and long - lasting analgesic effect , followed by acetaminophen . while the results for nimesulide and acetaminophen were not markedly different from those observed in the first cycle , the higher dose of meriva ( 2 g ) reduced pain perception after 2 hours . the analgesic effect of meriva 2 g lasted 4 hours , and a second dose was then necessary in some cases ( after 68 hours for headache , 8 hours for neuropathic pain , and 12 hours for neuralgia and pain from osteoarthritis ) . the analgesic effect of this meriva dose was lower than that of nimesulide , but higher than that associated with acetaminophen . curcumin can attenuate thermal hyperalgesia associated with diabetic neuropathic pain by inhibition of tumor necrosis alpha and nitric oxide release,16 have an antihyperalgesic effect in a formalin - induced orofacial pain model in rats,17 and decrease trpv1-mediated pain hypersensitivity.12 further , intrathecal administration of curcumin significantly decreased the sensitivity of rats in the formalin test.18 the relatively quick onset of these activities is at odds with the mainly genomic anti - inflammatory mechanism(s ) of curcumin , suggesting a direct activity on the mechanism of translation of inflammatory stress into a painful sensation , a process where thermal transient receptor potential play a critical role.19 curcumin behaves as a combined trpv1 inhibitor12 and trpa1 desensitizer,10,11 and this direct action is complemented by that mediated via inflammatory mediators , a major class of thermal transient receptor potential modulators.19 however , the clinical relevance of these observations has not yet been evaluated . to fill this gap in our knowledge , we carried out a pilot comparative study of curcumin ( formulated as meriva to improve its poor oral absorption)4 and two popular analgesic drugs ( acetaminophen and nimesulide ) . at a dose of 2 g ( corresponding to 400 mg of curcumin ) , meriva had a clear analgesic effect in subjects affected by acute pain , confirming anecdotal reports of the pain - relieving properties of this curcumin formulation . at this dose , the activity was higher than that associated with 500 mg of acetaminophen , while a lower dose of meriva ( 1.5 g , corresponding to 300 mg of curcumin ) gave only transient and often unsatisfactory relief of pain , indicative of suboptimal therapeutic plasma concentrations . the analgesic effect of meriva achieved significance only 2 hours after administration , as observed for acetaminophen . conversely , nimesulide was more rapidly acting , with strongest pain - relieving properties being reported one hour after administration . at both doses used , the tolerability of meriva was substantially better than that of nimesulide , which often requires concomitant administration of antacids to alleviate the gastric irritation associated with its use . the similarity of action of curcumin and acetaminophen supports the view that these compounds share the same mechanism of action , while nimesulide is a powerful inhibitor of cyclo - oxygenases,14 a class of enzymes only modestly inhibited by curcumin in a direct way . taken together , our results show that preclinical data on the analgesic properties of curcumin have a clinical translation , at least at a dose of 2 g as in the meriva formulation . while this dose of meriva is significantly higher than that used to relief chronic inflammatory conditions ( 11.2 g / day),8 its pain - relieving activity could benefit from general down - regulation of the inflammatory response induced by curcumin , given that transient receptor potential - mediated mechanisms of analgesia are magnified by attenuation of inflammation . overall , this would translate into better control of chronic pain , providing a rationale for the inclusion of meriva in protocols of treatment for acute pain of various origins . the posttraumatic immune response is characterized by a complex set of pro- and anti - inflammatory reactions in order to restore homeostasis and was shown to be organ and cell specific [ 2 , 3 ] . splenic immune response and cellular immunity are known to be depressed after trauma - hemorrhage leading to an increased susceptibility to infectious complications [ 4 , 5 ] . several studies have demonstrated that splenocyte functions , such as proliferative and cytokine production capacity as well as macrophage antigen presentation function are suppressed following trauma hemorrhage [ 69 ] . in regards to t - cell function , a posttraumatic shift from splenic th1 cytokine ( il-2 , ifn- , and tnf- ) to th2 cytokine ( il-4 , il-5 , il-10 ) production has been reported [ 1015 ] . these splenic immune alterations are well described in rodent trauma models consisting of laparotomy and hemorrhagic shock [ 3 , 1619 ] and appear already 2 hours after insult induction [ 12 , 15 , 2023 ] . simple hemorrhage and laparotomy alone were demonstrated to result in a marked depression of splenic immune function , and no differences were seen in the extent of depression in the early stage , if these two insults were combined . however , a prolonged splenic immunodepression of 7 days was seen after combined trauma hemorrhage , whereas splenic immune suppression only persists up to 3 days following isolated hemorrhage or laparotomy . with a later onset at day 7 , splenic immune alterations following femoral fracture and hemorrhage seem to differ from the aforementioned trauma models . however , results are inconsistent due to different study designs ( e.g. , animal gender , genetic strain , and protocol of fracture induction ) . data regarding the role of isolated femur fracture in early stage splenic immune alterations are lacking , but are of special clinical interest as femoral fracture itself as well as the stabilization by femoral nailing seems to have a significant impact on the inflammatory response and the susceptibility to infectious complications . furthermore , it is still controversially discussed whether interleukin ( il)-6 , produced by many different cell types ( e.g. , macrophages , endothelial , and t cells ) , exerts pro- or anti - inflammatory effects in the posttraumatic immune response . il-6 seems to have both , pro- and inflammatory effects , depending on whether it is acting in a paracrine or endocrine manner . additionally , the effects of il-6 seem to be influenced by the stimulus and the model of inflammation used in experimental studies . the aim of the present pilot study was to investigate whether a femoral fracture with or without hemorrhagic shock has an impact on the splenic cytokine production capacity at an early posttraumatic stage ( 4 hours ) and to observe the effects of il-6 under these conditions . prior to initiation , the study was approved by the animal welfare committee of the state of lower saxony . the experiments were performed in 18 male c57bl/6 il-6 knockout ( il-6 ) mice aged 810 weeks and weighing 22.0 3.0 g. as a control 18 male c57bl/6 mice of similar weight ( wild type ; wt ) were used . the animals were bred and raised under specific pathogen - free conditions in the central animal facility of our institution . throughout the study period , wt and il-6 animals were randomly assigned into sham groups ( s ) ( each of 6 animals , only anaesthesia ) and 2 experimental groups , in which either an isolated femur fracture or a combination of hemorrhage and femoral fracture were induced . all procedures were performed after deeply anesthetizing the animals with 2.2 0.3 mg of ketamine ( ketanest 100 mg / kgbw ) and 0.33 0.045 mg of xylazine ( rompun 15 mg / kgbw ) . a standardized femur fracture was induced in experimental groups using a blunt guillotine device with a weight of 500 g as previously described . this resulted in an a - type femoral fracture combined with a moderate soft - tissue injury . a hemorrhagic shock was induced by withdrawing 60% of the total blood volume ( body weight ( in g ) x 0.04 ml ) via puncturing the orbital plexus . resuscitation using sterile ringer 's lactate was performed with four times the shed blood volume in the tail vein after 1 hour . experimental animals were sacrificed four hours after induction of hemorrhage and fracture , whereas sham mice were four hours after the first anaesthesia . splenocytes were isolated as previously described . in brief , spleens were gently ground between frosted microscope slides to produce a single cell suspension . the erythrocytes were lysed with lysis buffer and the remaining cells were washed with pbs by centrifugation ( 300 g , 15 min , 4c ) . after centrifugation , cells were resuspended in rpmi 1640 ( gibco ) containing 10% heat inactivated fbs and antibiotics ( 50 u / ml penicillin , 50 g / ml streptomycin and 5 g / ml gentamycin , all from gibco ) to get a final concentration of 110 cells / ml . the splenocytes were then cultured in the presence of 10 g / ml lps ( lps from e.coli 0111:b4 , sigma - aldrich inc . , steinheim , germany ) at 37c , 95% humidity , and 5% co2 for 24 hours . after incubation , the cell free suspension cytokine concentrations in cell - free supernatants were determined with cytokine bead array inflammatory kits using flow cytometry according to the manufacturer 's instructions ( bender medsystems , vienna , austria ) . statistical analysis was performed using spss computer software ( spss 11.5 , chicago , il ) . . comparisons between groups were performed using one - way analysis of variances ( anova ) , student 's t - test or the rank - sum test ( mann - whitney u - test ) . the tnf- secretion of stimulated splenocytes was significantly reduced following isolated femoral fracture in wt and in il-6 mice compared to sham animals ( figure 1 ) . in il-6 mice , the decrease of the tnf- release was significantly attenuated in comparison to wt animals ( p < 0.05 ) . in sham groups , no significant differences concerning the tnf- secretion were observed between wt and il-6 mice ( p > 0.05 ) . combined hemorrhage and femoral fracture resulted in a further significant decrease of the tnf- release in wt as well as in il-6 mice compared to animals with an isolated femoral fracture ( p < 0.05 ) . again , a significantly attenuated depression of the tnf- production was demonstrated in il-6 mice compared to wt animals ( p < 0.05 ) as shown in figure 1 . the il-6 productive capacity of stimulated splenocytes was significantly decreased after femoral fracture compared to sham animals ( p < 0.05 ) . the combination of hemorrhagic insult and femoral fracture resulted in an additional significant suppression of the il-6 release ( p < 0.05 ) as shown in figure 2 . in il-6 mice , the mean il-6 secretion was < 6 pg / ml in all study groups due to the lacking il-6 gene . therefore , no significant differences in il-6 release between different study groups were observed in il-6 mice ( p > 0.05 ) . in wt mice , femoral fracture with or without hemorrhagic shock did not result in a significant increase of the il-10 production of splenocytes compared to sham animals ( p > 0.05 ) . in il-6 mice , the il-10 release of stimulated splenocytes was significantly decreased in all study groups compared to wt animals ( figure 3 ) . thereby , the il-10 production was significantly decreased following femoral fracture with or without hemorrhagic shock compared to the sham group ( p < 0.05 ) . no significant differences concerning the il-10 release were demonstrated between il-6 mice with an isolated femoral fracture and animals with an additional hemorrhagic shock ( p > 0.05 ) . trauma results in a significant impairment of splenic immune functions which has been supposed to increase the susceptibility to septic complications as a result of depressed cellular immunity [ 3337 ] . operative stabilization of major fractures ( e.g. , femoral fractures ) is supposed to additively enhance the trauma - induced inflammatory changes [ 38 , 39 ] , which has resulted in an ongoing discussion about the optimal timing of definitive fracture stabilization . despite the important role of the spleen in the immune response after trauma , the effects of a femoral fracture as an isolated injury or in combination with hemorrhagic shock on the early - stage splenic cytokine production has yet not been investigated . furthermore , the role of il-6 within these splenic immune alterations has not been elucidated . in this context , it is well described that systemic cytokines are mainly released by kupffer cells as the largest population of tissue macrophages after trauma hemorrhage and infectious stimulus [ 2 , 40 ] . as the pathophysiological role of splenic il-6 has been discussed controversially , we focused on the importance of splenocytes and locally generated il-6 in the posttraumatic inflammatory response . an isolated femoral fracture resulted in a significantly depressed cytokine production capacity of splenocytes at an early stage of the posttraumatic course . the depression of splenic cytokine release was enhanced in case of an additional hemorrhagic shock . the splenic immunodepression in terms of reduced tnf- secretion of splenocytes was significantly attenuated in the absence of il-6 . the production capacity of splenocytes for il-10 was suppressed in the absence of il-6 in all study groups . in contrast to wt animals , splenic il-10 release was significantly decreased following femoral fracture with and without hemorrhagic shock . in the present study , we were able to show that femoral fracture with and without hemorrhage results in a marked alteration of splenic cytokine production capacity at an early posttraumatic stage ( 4 h ) . in accordance , several studies reported a splenic immunodepression 2 h and 4 h following laparotomy and hemorrhagic shock [ 12 , 15 , 2023 , 41 , 42 ] . in contrast , splenic immune depression after femoral fracture and hemorrhage seems to appear later in the posttraumatic course . described that the typical splenic th1/th2 shift was evident at day 7 , but not at day 1 after trauma hemorrhage . further studies reported comparable results with marked alterations of splenic cytokine release in response to femoral fracture and hemorrhage at day 7 after insult induction [ 4346 ] . on the other hand , monroy et al . as well as stapleton et al . found no significant alterations in splenic cytokine secretion seven days after combined femoral fracture and hemorrhage . several studies used female animals in different estrus cycle status , which probably have a marked impact as splenic immune response exhibits a gender dimorphic pattern . additionally , we induced a closed femoral shaft fracture , which was shown have an greater impact in terms of splenic immune suppression compared to open femoral fracture used in other studies . as previous studies described an association between the extent of splenic immune depression and the posttraumatic susceptibility to infectious complications [ 12 , 13 , 15 , 49 , 50 ] , the enhanced depression of splenic cytokine production capacity following combined femoral fracture and hemorrhagic shock might have clinical implications in the setting of multiple trauma in terms of priority and timing of definite fracture stabilization . however , the transfer of our results to the clinical setting is limited as measuring the cytokine production capacity of splenocytes only reflects a part of the t cell ( namely , cd4 + t - cells ) and splenic immune function . based on our data , we could not make any statement concerning cd8 + t - cell function and proliferation capacity of splenocytes . furthermore , it is not possible to differentiate whether depressed cytokine release is caused by a decreased production capacity of cd4 + t - cells , splenic macrophages , and/or dendritic cells as we measured cytokine secretion in the overall splenocyte culture . as the splenic immune response after trauma hemorrhage has been reported to result in a shift from th1 to th2 cytokines , we would have expected an increased production capacity of splenocytes for il-10 . in contrast , we only observed a nonsignificant increase of splenic il-10 release following femoral fracture and hemorrhage in wt animals . this might be explained by the fact that we used c57bl6 mice , which were shown to be hyporesponsive to trauma hemorrhage compared to animals of the c3h / hen strain used in other studies . moreover , it might be argued that femoral fracture and hemorrhage does not result in a th1/th2 shift because of the missing effect on the th2 cytokine release in our study . in this context , it might be suggested that the depression of splenic tnf- and il-6 release results from a decreased production capacity of splenic macrophages rather than cd4 + t cells . in order to clarify this point separate isolation of splenic macrophages and/or assessment of more cd4 + t - cell - specific mediators ( il-2 , inf- , il-4 , and il-5 ) furthermore , significant differences in the splenic il-10 release may not become apparent already 4 hours after trauma hemorrhage . also for answering the question when the splenic immune function is restored after femoral fracture and hemorrhage , further studies with additional time points ( e.g. , 24 hours , day , 3 days , and 7 days ) based on our results , it could be assumed that il-6 might exert anti - inflammatory effects on the splenic immune function after trauma - hemorrhage . in the present study , we observed the principle depressive effect of femoral fracture and hemorrhagic shock on the splenic tnf- release in wt and il-6animals . however , the depression of cytokine release was significantly attenuated in the absence of il-6 . the increased tnf- levels might be explained by a potentially anti - inflammatory role of il-6 in order to restore tnf- production ( negative feedback mechanism ) , which itself has been described as a potent inductor of il-6 synthesis [ 51 , 52 ] . the significance of this observation on immune competent cells in other tissue compartments as well as on the incidence of infectious complications and outcome after trauma has to be clarified in further studies . in the present study , the secretion of il-10 was significantly lower in il-6 mice in all study groups , again pointing towards an anti - inflammatory effect of il-6 on splenocytes , possibly by inducing il-10 synthesis . for mucosal cells , where only a little or no increase of il-10 mrna was observed in il-6 mice after hemorrhage , whereas a marked increase in wt mice was found . in the same study , in contrast , after a septic insult significantly increased il-10 plasma levels were observed in il-6 mice compared to wt animals . due to the results of the present studies and the observations made in the literature it might be assumed that the kind of the initiating inflammatory stimulus as well as the observed body compartment might be essential for the net effect of il-6 . it has been postulated that the net effect of il-6 on the host inflammatory response reflects a balance of two opposing effects , a paracrine effect of il-6 that promotes inflammation , and an endocrine effect of il-6 that diminishes inflammation . as il-6 mice were used in the present study , the results have to be interpreted carefully . in il-6 mice , there might be a functional redundancy in the regulation of the immune response following a traumatic insult [ 1 , 55 , 56 ] . in il-6-deficient mice , other proinflammatory mediators may take over effects of il-6 resulting in an only partially impaired inflammatory response compared to wt animals . furthermore , the present study is limited by the fact that facs analyses of cell free supernatants were done only once . this may lead to a significant variation of data as shown by the standard deviations in the present study . the depression of splenic cytokine production capacity after femoral fracture with and without hemorrhagic shock at an early posttraumatic stage underlines the importance of adequate treatment strategies and identification of high risk patients in order to prevent posttraumatic complications . the absence of il-6 results in significant changes of posttraumatic splenic cytokine release indicating that the posttraumatic modulation of il-6 synthesis might be a potential target for therapeutic interventions . however , further studies especially with an additional infectious stimulus are needed to identify the relevance of such therapies .

in addition to its anti - inflammatory activity , meriva , a proprietary lecithin formulation of curcumin , has been anecdotally reported to decrease acute pain in patients with various chronic diseases . given that curcumin can desensitize transient receptor potential a1 , a nociceptor seemingly also mediating the analgesic effect of acetaminophen , as well as inhibiting and downregulating the expression of cyclo - oxygenase 2 , the selective target of nimesulide , a nonsteroidal anti - inflammatory agent , we carried out a pilot comparative study of the acute pain - relieving properties of these three agents . at a dose of 2 g ( corresponding to 400 mg of curcumin ) , meriva showed clear analgesic activity , comparable with that of a standard dose ( 1 g ) of acetaminophen , but lower than that of a therapeutic ( 100 mg ) dose of nimesulide . the analgesic activity of lower ( 1.5 g ) doses of meriva was less satisfactory , and the onset of activity was longer than that of nimesulide for both doses . on the other hand , gastric tolerability was significantly better than that of nimesulide and comparable with that of acetaminophen . taken together , our results show that the preclinical analgesic properties of curcumin have clinical relevance , at least at a dose of 2 g as the meriva formulation . while this dose is significantly higher than that used to relieve chronic inflammatory conditions ( 11.2 g / day ) , its pain - relieving activity could benefit from the general downregulation of the inflammatory response induced by curcumin , considering that the transient receptor potential channel - mediated mechanisms of analgesia are magnified by attenuation of inflammation . in patients on treatment with meriva , this would also translate into better control of acute pain , providing a rationale for the analgesic properties associated with this curcumin formulation . splenic immune function is known to be depressed following hemorrhage . the present study investigates the effects of femoral shaft fracture , isolated or in combination with hemorrhage , on early stage cytokine production capacity of splenocytes and observes the role of il-6 under these conditions . male il-6 knockout ( il-6/ ) and wild - type mice ( wt ) were randomly divided into three groups : sham ( s ) , isolated femoral fracture ( fx ) , and femoral fracture + volume controlled hemorrhage ( th - fx ) ( n = 6 per group ) . animals were sacrificed four hours after induction of hemorrhage and fracture . cytokine release ( tnf- , il-6 , and il-10 ) of isolated and lps - stimulated splenocytes was determined by cytometric bead array . femoral fracture with or without hemorrhage caused a suppression of in vitro cytokine production capacity of splenocytes at an early posttraumatic stage in wt and il-6/. in the absence of il-6 , the profile of splenic cytokine secretion is significantly altered , identifying this cytokine as a potential therapeutic target to modulate the posttraumatic immune response .

lipoma is the most common soft tissue mesenchymal neoplasm in adults , which is located in any part of the body . the majority occurs in the upper - half of the body , particularly the trunk , head , and neck region , and it is rarely described in the oral cavity . conventional lipoma has been divided into three forms according to the origin of the localization : superficial lipoma ( arise within subcutaneous tissue ) , deep lipoma ( arise within deep soft tissue ) and parosteal lipoma ( arise within the surfaces of bone ) . the histological characteristics and types vary , which include angiolipoma , angiomyolipoma , myolipoma , osteolipoma and chondrolipoma . both osteolipomas and chondrolipomas are rare entities.[24 ] however , occurrence of osteolipomas or in other term lipomas with oseous and cartilagenous metaplasia are less common than other subtypes of lipomas . the term of osteochondrolipoma has been used for the lipomas containing both cartilage and bone which , are very rarely seen and generally associated with a paraosteal localization . to our knowledge , a 64 year old male patient with painless mass on his mandibulary symphysis region , was referred to the department of oral and maxillofacial surgery , faculty of dentistry on september , 2007 . the lesion had been first noticed 2 months prior of the presentation to the clinic with no significant enlargement . clinical examination of the oral cavity revealed a well demarcated , firm , round and exophitic lesion of approximately 2 cm in diameter at the symphysis region of the left mandible . the lesion was firmly attached to the bone in the clinical examination and the overlying mucosa was intact and of normal color . radiographically , at the site of the lesion some degree of radioopacity was observed [ figure 1 ] . under local anesthesia , the patient was taken to the operating room and the lesion was completely excised with an intraoral approach . during the surgical intervention , the encapsulated lesion was not adherent to the underlying periosteum and overlying mucosa , thus the lesion was dissected and removed easily [ figure 2 ] . the surgical specimen was histopathologically evaluated for final diagnosis at department of oral pathology , faculty of dentistry , gazi universitys . the 6-month postoperative follow - up showed healing of the oral mucosa without any recurrency . preoperative panoromic radigoraph exhibiting the dense radioopacity ( arrow ) in the symphysis region of the mandible intraoperative view of the lesion with encapsulation macroscopically , the gross specimen was a 2.7 1.8 1.4 cm encapsulated mass mostly comprising of adipose tissue with a few number of encapsulated nodular structures . on section , focal areas of hyaline chondroid structures and lamellar bone with fatty marrow were seen throughout the lesion . both islands of bone and cartilage were surrounded with well defined fibrous tissue [ figure 3 ] . there was no histological evidence of malignancy , such as mitosis , cellular atypia , necrosis , and hemorrhage . histochemistry with alcian blue periodic acid - schiff stain showed acid mucopolysaccaride presence in chondroid tissue ; hence , this was hyaline chondroid . on immunohistochemistry , adipocytes were positively stained with vimentin antibody , while both adipocytes and chondrocytes were positive for s-100 protein [ figure 4 ] . histopathological features of osteochondrolipoma showing mature adipose tissue containing cartilage islands and lamellar bone trabecules ( h and e , 40 ) chondrocytes and adipocytes demonstrating positivity for s 100 protein ( immunoperoxidase , 40 ) however , they are relatively uncommon in the oral cavity with incidence of 14% . variants of lipoma especially osteolipoma and chondrolipoma are very rare lesions which are located in the oral cavity . furthermore , intaoral lipoma with both osteoid and cartilaginous metaplasia has been reported just in two cases . we presented an osteochondrolipoma case that differed from the typical arrangement by two rare alterations . first , the lesion showed well - defined cartilaginous and osseous areas within lipoma that is also considered as osseous and cartilaginous metaplasia . in the a study of fregnani et al . , fibrolipoma was reported as the dominant subtype in 46 lipoma cases , in which neither osteolipoma nor chondrolipoma was found . two lipomas with cartilaginous or osseous metaplasia was reported out of 125 lipoma cases in another study , whereas none of the cases represented osteochondrolipoma . although , any case in the name of oral osteochondrolipoma has not been reported , lipoma with osteoid and cartilaginous metaplasia has been represented by two cases in the oral region . the osteochondrolipoma is the specific term of the lipoma with mature cartilaginous and osseous differentiation ; thus , both terms represents the same entity . some authors consider such type of lipoma as a benign mesenchymoma due to consisting of two or more mesenchymal elements such as lipocytes , chondrocytes , osteocytes , and fibroblasts . jones et al . stated that the term of benign mesenchymoma should be used strictly to describe an unencapsulated neoplasm composed of two or more mature mesenchymal tissues not ordinarily associated with each other , excluding fibrous connective tissue . in the present case , as the tumor is well demarcated and partially encapsulated and moreover the prominent component is mature adipose tissue , the tumor is considered as osteochondrolipoma . second alteration that took part in this case , was the parosteal localization of the lesion . the parosteal lipoma which is defined as lipoma exhibits a conguous relationship with the periosteum , usually demonstrating some form of attachment to periosteum with an underlying osseous reaction , is rarely seen in the oral cavity . various types of osseous cortical responses from a reactive overproduction of bone to cortical erosion can be seen underlying bone . the radiopacity in our case may reflect either the reactive bone production of underlying bone or the density of cartilagenous and ossous component of the tumor . multipotent cells of mesenchyme , different cell lines and metaplastic differentiation of the adipose tissue are three hypothesis regarding the origin of the tumor . however , considering its parosteal location , histological findings of well - formed cartilage and bone tissue within the adipose tissue neoplasm , it is decided to be a true neoplasm of mesenchymal tissue stem cells showing characteristics of three different tissues originating from the mesenchyme . electrostatic interactions comprise one of the principle interatomic forces , along with exchange - repulsion , dispersion , and polarization or induction . the importance of electrostatic interactions is paramount at long range and for polar molecules . much development effort has been focused on computational treatment of long - range electrostatics , e.g. , the development of particle - meshed ewald ( pme ) methods . electrostatic interactions at short range have received less consideration until recently . at close distances , a spherical approximation of atomic charge distributions is insufficiently accurate and use of atomic multipole expansions provides much greater flexibility in modeling complex electrostatic potentials near a molecular surface , an insight which inspired the development of the amoeba force field . nonetheless , at very close interatomic distances , when electron clouds overlap , a point multipole approximation becomes inadequate . the electrostatic potential within a spherical electron cloud no longer behaves as a simple 1/r interaction potential at small separation distances . such deviation from a simple coulomb potential is referred to as a penetration effect . while the charge penetration effect leads to a negative correction to energy at typical molecular interaction distances , where the electron electron penetration is dominant , it can be repulsive at very short range . a recent study by lewis and co - workers reported the counterintuitive result that any ring substitutions of the benzene dimer ( parallel ) with electron - withdrawing or electron - donating groups yield more favorable electrostatic contributions than the unsubstituted benzene benzene dimer itself . this result is contrary to the conventional thought that such interactions are correlated with the ability to withdraw or donate electrons to the cloud as described by the hunter sanders rules . sherrill and co - workers suggested this is because the electrostatic interactions in such systems at the stacking distance exhibit a significant charge penetration effect . the multipole model , which the hunter sanders rules are based upon , can not correctly account for such effects . moreover , in a recent study of aromatic crystals , a charge penetration corrected amoeba - like model predicted better crystal properties than the uncorrected model . it was shown that point atomic multipoles consistently predict positive ( repulsive ) electrostatic interactions between stacked or t - shaped benzene dimers while symmetry - adapted perturbation theory ( sapt ) suggests an opposite trend toward attractive interactions . the current amoeba force field seemingly compensates for penetration with a less repulsive van der waals interaction , so the total interaction energy is reasonable at certain dimer configurations . however , explicit incorporation of the penetration effect provides much better anisotropy in crystal packing and makes the overall force field more transferable . in another study of organochlorine compounds using the amoeba model , it was found that the transferability of chlorine van der waals parameters was unsatisfactory , likely due to lack of an explicit penetration correction . there have been previous attempts to incorporate the charge penetration effect into implicit solvent models , multipole - based electrostatic models , charge - density - based ( including gaussian multipole ) models , and combined quantum and molecular mechanics ( qm / mm ) models . generally , the charge penetration correction involves breaking the atom - centered point charge into an effective core and a valence electron density , as suggested by gordon et al . and piquemal et al . in this way , the electrostatic interaction between two atoms is described as a sum of interactions between core and valence charge densities , which can be modeled with empirical exponential functions . alternatively , rigorous integration over the two charge densities can be used to model short - range electrostatic interactions , with a significantly greater expenditure of computational effort . others have explored incorporating charge penetration effects into the qm / mm framework , using either screened molecular mechanics ( mm ) charges or simple empirical damping corrections . such screened mm charges are typically parametrized for qm / mm applications and may be not directly applicable in full mm calculations , e.g. , to reproduce the attractive sapt electrostatic energy in a stacked benzene benzene conformation , unless an explicit term for interactions of the valence charge densities is included , as in the model recently proposed by wang and truhlar . in this study , the charge is revisited , implemented , and extensively tested in the context of the amoeba force field and using a new parametrization strategy . the charge penetration corrected amoeba point multipole model ( multipoles + cp ) is developed using a comprehensive set of small molecule complexes , and the parameters are determined for h , c , n , o , p , s , f , cl , and br to cover the elements commonly found in organic and biological molecules . to facilitate model development in this and future studies , a new database of sapt2 + decomposed quantum mechanical energies constructed for 101 small molecule pairs , each at 7 different intermolecular distances ( the s1017 database ) , is presented . in order to systematically examine the electrostatic and other components of intermolecular forces , the s101 and s1017 databases of homo- and heterodimers of common organic molecules have been constructed . the first 66 pairs , which cover the majority of the typical organic interactions of h , c , n , and o atoms , are taken from the s66 database from hobza et al . in addition , 15 complexes containing halogen atoms ( f , cl , and br ) , six complexes containing sulfur , and four complexes containing phosphorus have been added . furthermore , 10 monomer water complexes , which encompass amino acid side chain analogs ( including the charged ones ) missing in the s66 data set , have also been added , yielding a total of 101 pairs . to construct the s1017 database , definitions of the intermolecular distance vectors from the s668 database of hobza et al . were used . unlike s668 , each of the 101 model complexes were placed at seven separation distances , corresponding to 0.70 , 0.80 , 0.90 , 0.95 , 1.00 , 1.05 , and 1.10 times the equilibrium intermolecular distances . compared against the s668 database , the s1017 set includes more dimer configurations at very short separations , which have been rarely investigated but are essential to the study of penetration effects and exchange repulsion interactions . we have selected 0.7 times the equilibrium distance as the lower bound because the sapt calculations below show that at this close distance the electrostatic energy is about 50% of the exchange repulsion energy or higher ; i.e. , both electrostatic and van der waals ( vdw ) components are important in the total interaction energy . as these short distances are being sampled in molecular dynamics simulations at room temperature and even more so at higher temperatures , their contributions to the simulated bulk properties are nonnegligible . thus , it is essential to ensure the charge penetration model behaves correctly at these short distances . the newly added structures among the 101 complexes were optimized at the mp2/cc - pvtz level of theory with counterpoise correction using the gaussian09 program . for each of the resulting 707 dimer configurations , the interaction energy has been decomposed using sapt2 + analysis provided by the psi4 program . the sapt2 + calculation returns electrostatic , exchange repulsion , induction , and dispersion energies , all to second order with respect to intramolecular electron correlation . exact definitions of each component can be found in figure 1 of sherrill et al . it should be noted that dispersion energy can only be separated from other effects in long range when two molecules do not overlap . thus , at van der waals distances , it may be more appropriately to refer to this as dispersion - like or damped dispersion energy . this should be kept in mind even though for simplicity the term dispersion is used throughout the discussion . all sapt calculations were carried out using dunning s correlation consistent basis sets at both aug - cc - pvdz and aug - cc - pvtz levels . the complete basis set ( cbs ) limits of the sapt2 + energies were also estimated . ( data can be found in the supporting information ) schematic view of monomers and dimers in the s101 data set . different configurations of the same dimers , e.g. , menh2water , phenol water , benzene benzene , and mecl mecl , are included in the data set to take into account the orientational effect . a two - point extrapolation strategy has been used to estimate the complete basis set limit of the exchange repulsion and dispersion energy at the sapt2 + level of theory . this is similar to helgaker s scheme but with an optimized p value ( eq 1 ) . such a protocol has previously been applied to extrapolate the dispersion energy of dft - sapt calculation in earlier study.1 different p values , 3.0 for exchange repulsion and 4.3 for dispersion energy , were obtained using the small pairs and subsequently applied for extrapolation over the full s1017 database . since the truncated terms in the sapt2 + dispersion energy make a considerable contribution to the total interaction energy , dispersion energies obtained at the sapt2+/cbs level are scaled by a factor f in order to match the sapt total interaction energy to those obtained at the ccsd(t)/cbs level of theory ( eq 2).2 by minimizing eq 2 using the 66 pairs in the s66 data set , a scale factor of f = 0.89 has been determined and used to construct the s1017 database . in order to model the charge penetration effect , the method of piquemal et al . is revisited . their original model corrects the charge charge and charge dipole interactions . here , we propose to retain the charge , each atomic point charge is divided into an effective core and a damped valence electron distribution . thus , the electrostatic energy between two atomic charges can be written as3where r is the interatomic distance ; z is the positive effective core charge , which is set to be equal to the number of valence electrons of each atom ; q is the net charge of the atom , thus ( z q ) can be considered as the magnitude of the ( negatively charged ) electron cloud ; and and are two parameters controlling the magnitude of the damping of the electron cloud when the atom is interacting with the core and with electrons from other atoms , respectively . thus , the total electrostatic energy between two atoms now involves three components , the core the first method involves fitting the damped potential to the qm electrostatic potential at short range , near or within the molecular surface . by considering a probe charge of + 1e as a particle with an effective core charge of + 1e and having no valence electrons , ( z2 thus , the electrostatic potential can be written as4 once z and q are determined , can be obtained easily by fitting eq 4 to the qm electrostatic potential . in the second method , is intuitively set to be the same as the number of valence electrons ( except the hydrogen atom):5 when z and are fixed in eq 3 , the electrostatic energy is more attractive when is greater . this is in accordance with the intuition that atoms having a larger electron cloud may exhibit a stronger penetration effect . although the final parameters for h , c , n , o , p , s , f , cl , and br were derived based on the second method , the performance of both methods is examined for h , c , n , and o containing molecules in later sections . as the distance between two atoms increases , thus , the electrostatic interaction at medium and long distances can still be accurately modeled via a multipole expansion , as the penetration correction diminishes rapidly with distance . as the data will show , the penetration correction is only significant when atomic separation is shorter than the sum of atomic van der waals radii and thus does not affect the reciprocal space portion of an ewald summation approach such as particle mesh ewald ( pme ) . in addition , to ensure the continuity between the real and reciprocal space , a switching function is used near the real space ewald cutoff distance ( typically 7 for atomic multipole pme ) to ensure the penetration correction completely disappears:6where r is the interatomic distance and rl and ru are the lower and upper bounds of the switching function . quadrupole interactions . following previously detailed procedures , an initial set of atomic multipole moments for each molecule was obtained from distributed multipole analysis ( dma ) at the mp2/6 - 311 g * * level of theory . then the dipole and quadrupole moments were further optimized by fitting to the electrostatic potential calculated at the mp2/aug - cc - pvtz level . the same strategy has been used in developing the amoeba force field for small molecules , proteins , and organochloroine compounds previously . with the addition of the penetration correction described here , monopole monopole ( charge charge ) interactions are calculated using eq 3 while all other terms in the amoeba retain their original form . the sapt and amoeba multipole based intermolecular interaction energies were compared on exactly the same dimer structures . since h , c , n , and o are the most common elements in organics and biomolecules , their parameters were determined first within the new charge penetration formalism . a training set of 357 molecule pairs was used , consisting of 157 pairs of hydrogen - bonded complexes , 127 pairs of dispersion - dominant complexes , and 87 pairs with mixed features of both . for example , an initial set of parameters for sp or sp carbon , nonpolar hydrogen , and hydrogen attached to sp c were obtained by selecting a smaller number of pairs ( e.g. , 87 ) from the dispersion - dominant and mixed complexes . similarly , other parameters such as those for sp oxygen and polar hydrogen were obtained initially from water dimers . then , one or more sets of initial parameters for all h , c , n , and o atom types were optimized together using the entire training set . once the parameters for these four elements were finalized , further parametrization for p , s , f , cl , and br was carried out using the subsets in s1017 . in each of these subsets , an 80/20 ratio for the training and testing complexes was maintained to ensure a sufficient amount of data points for each atom type . an optimization program written in python , using the quasi - newton and nelder mead simplex methods from the scipy library , was applied to all of the parametrization work . the first derivative of the sum of unsigned errors with respect to each parameter was calculated numerically . in order to examine the convergence of the sapt2 + energy toward the basis set limit , five small molecule pairs were selected and calculated using the aug - cc - pvxz ( x = d , t , q , or 5 , abbreviated as axz in the following paragraphs ) basis sets . ethyne ( t - shaped ) , and ethyne water ( cho ) . the energy difference between different axz ( x = d , t , q , and 5 ) basis sets for the total interaction energy and each energy component , including electrostatic , induction , exchange repulsion , and dispersion energy , are compared . in general , a steady decrease in the energy gaps between adz the difference between aqz and a5z basis sets of all of the energy components as well as the total interaction energy are already well below 0.05 kcal / mol . in particular , the differences in electrostatic and induction energies are even smaller , at 0.003 and 0.005 kcal / mol , respectively . this implies that the difference between a5z and a bigger basis set , e.g. , a6z , should be even smaller and negligible . therefore , the results obtained using the a5z basis set were used to approximate the complete basis set limit . since sapt2 + calculations are computationally expensive , the practical size of the basis set has been limited to aug - cc - pvtz for most molecule pairs in the s101 database . to obtain an estimate of the sapt2 + energy at the cbs limit , extrapolation as shown in figure 2 , the electrostatic and induction energy components converge quickly to the cbs limit ( approximated by a5z results ) . the mean unsigned errors between atz and a5z of five pairs are 0.018 and 0.010 kcal / mol for the two components , respectively . therefore , for electrostatic and induction energies , the results obtained with the atz basis set are considered a reasonable approximation of the cbs limit . for exchange repulsion and dispersion energy , a two - point scheme was applied to extrapolate the energy calculated at adz and atz to the cbs limit . difference between the sapt2 + energy components calculated using aug - cc - pvxz ( x = d , t , and q ) basis set with the value obtained using aug - cc - pv5z . as mentioned in the previous section , different energy components converge at different rates with respect to the basis set size . electrostatic and induction energies calculated using the atz basis set are sufficiently converged , while the exchange repulsion and dispersion energy terms are not . thus , a two - point extrapolation scheme is used to extrapolate the exchange repulsion and dispersion energy at atz basis set to the complete basis set limit . the extrapolated dispersion energy is further scaled by a factor of 0.89 to compensate for higher order dispersion terms missing in the sapt2 + approach ( refer to methods for details of the extrapolation and scale factor determination ) . then the total sapt2 + interaction energy is obtained by summing up the individual energy components . finally , the quality of the sapt2 + interaction energy at different basis set levels was examined by comparing with the ccsd(t)/cbs interaction energy of the s66 data set ( figure 3 ) . in general , the sapt2+/cbs estimates with scaled dispersion energy ( which will be referred to as sapt2+/cbs / scaled to distinguish from the sapt2+/cbs values without scaling ) have the smallest mean unsigned error ( mue ) among tested combinations , given mue values of 0.16 , 0.68 , 0.47 , and 0.17 kcal / mol ( and rmse of 0.25 , 0.80 , 0.56 , and 0.21 kcal / mol ) for sapt2+/cbs / scaled , sapt2+/cbs , sapt2+/atz , and sapt2+/adz , respectively . it is not surprising that the sapt2+/adz combination yields very reasonable total interaction energy as this is consistent with a previous report . however , the small error in the total interaction energy of sapt2+/adz is due to the error cancellation of individual energy components . to best estimate individual energy components , sapt2+/cbs / scaled results remain the most accurate choice in this study . however , for the larger systems where aug - cc - pvtz calculations are not practical , sapt2+/adz may be considered as an alternative . for all 707 pairs in the s1017 database reported in this study , the same strategy ( sapt2+/cbs / scaled ) is applied to calculate the individual energy components . errors of sapt2 + interaction energy compared to ccsd(t)/cbs estimation for dimers in the s66 data set . sapt2+/aug - cc - pvdz energy is shown in solid circles , sapt2+/cbs is shown in triangles , and sapt2+/cbs / scaled is shown in hollow circles . the electrostatic interaction energy due to amoeba point multipoles as well as the charge penetration correction ( multipoles + cp ) are calculated and compared with sapt2 + data for the s1017 data set ( excluding 77 complexes containing an ethyne molecule due to a lack of amoeba parameters ) ( supporting information table s3 ) . in our current model , the parameters z and for each atom are uniquely determined by the element type . is set equal to z , or if z is less than 2 , then is set to 2 . the only parameter to be determined for the penetration correction is in eq 3 . for each of the h , c , n , and o elements , carbon , nitrogen , and oxygen all have three values representing sp , sp , and aromatic cases . for sulfur , distinct values are used for sulfide and sulfur iv , while p , f , cl , and br have only a single value per element in current parametrization . in general , after fitting of parameters , the new electrostatic model with charge penetration correction shows excellent agreement with the sapt2 + results ( figure 4 ) . taking the valence- parameter set as an example , for dimers near the equilibrium distances ( rmin ) , i.e. , 0.90 , 0.95 , 1.00 , 1.05 , and 1.10 of rmin , the mean unsigned error ( mue ) of the original point multipoles is 3.16 kcal / mol , which is reduced about 5-fold to 0.57 kcal / mol after inclusion of the charge penetration correction ( table 3 ) . for the dimers at very short separation , i.e. , 0.70 and 0.80 of rmin , the mues for the corrected and uncorrected electrostatic energy are 3.28 and 19.16 kcal / mol , respectively . as shown in figure 4 , it is striking that point - multipole - based electrostatic energy alone yields very large errors for dimers in close contact , and the simple charge penetration correction applied here is able to systematically improve agreement with sapt - derived electrostatics . based upon the mean unsigned errors , the charge penetration corrected model results in a percentage error of 13.6% and 13.4% at near - equilibrium and very short separations , respectively . in contrast , the uncorrected model has errors of 53% and 69% for these same two distance ranges . it is clear the charge penetration corrected model not only reduces the magnitude of absolute and relative errors compared to sapt but also provides consistent performance over a range of distances . in the uncorrected model , the percentage of error at very short distances is larger than at near - equilibrium distances , due to the increased effect of short - ranged charge penetration . plots of multipole electrostatic energy ( kcal / mol ) against the reference sapt2+/aug - cc - pvtz calculation for ( a ) near - equilibrium ( 0.90 , 0.95 , 1.00 , 1.05 , and 1.10 ) complexes taken from the s1017 data set , ( b ) expanded plot of the boxed region in a , and ( c ) short - range ( 0.70 and 0.80 ) complexes in the s1017 data set . the uncorrected amoeba point multipole energy ( multipoles only ) is shown in red circles , and the charge penetration corrected point multipole energies using the valence- parameter set ( multipoles + cp ) are denoted by blue crosses . charge penetration corrected model using the fitted- parameter set . for s66 dimers at near - equilibrium separations and using uncorrected amoeba multipoles , the hydrogen - bonded complexes exhibit the largest mean unsigned error of 4.41 kcal / mol , compared to mues of 3.08 and 2.08 kcal / mol for dispersion - dominant and mixed complexes ( table 4 ) . this is not surprising since the hydrogen - bonded complexes generally have the strongest electrostatic interactions . however , in terms of relative errors , the dispersion - dominant complexes carry the largest error at 105% , while the hydrogen - bonded and mixed complexes have the mean percentage of errors of 30% and 58% , respectively . it is somewhat surprising the dispersion - dominant complexes have such absolute and relative errors , as they are normally considered to have the weakest electrostatic interaction among the three types . charge penetration corrected model using the fitted- parameter set . to help understand why dispersion - dominant complexes have such large relative errors , the electrostatic energies of benzene dimers and stacked and t - shaped complexes , as well as hydrogen - bonded water dimers , are shown in figure 5 . for the uncorrected amoeba model , the calculated electrostatic energy is positive for the benzene pairs yet qm calculations suggest the interaction is attractive with a negative electrostatic energy . taking the electrostatic energy for this pair at the equilibrium distance as an example , the sapt2+/cbs / scaled calculation yields a value of 2.6 kcal / mol , while the uncorrected amoeba multipoles give + 1.0 kcal / mol , an error of 3.6 kcal / mol or 138% . for the t - shaped benzene dimer , the sapt2+/cbs / scaled and the uncorrected amoeba multipoles have values of 2.2 and 0.4 kcal / mol , respectively . the unsigned error is 1.8 kcal / mol or 82% of the sapt values , both somewhat less than for the complex . in contrast , although the hydrogen - bonded water dimer has the larger electrostatic energy of 7.2 kcal / mol , the uncorrected model has an unsigned error of 1.6 kcal / mol and a relative error of only 22% . this trend is in accordance with the averaged errors reported in table 4 and suggests the electrostatic interaction in dispersion - dominant complexes is the most charge penetration dependent . first , in the nonpolar molecules , the electron distribution is more balanced ; i.e. , there is more electron density on the hydrogen atoms , hence a stronger penetration effect for hydrogens . second , in the stacked benzene dimer , interactions between heavier atoms , carbon carbon for example , suffer stronger charge penetration effect , thus weight more in electrostatic energy . for hydrogen - bonded pairs , although the percentage of error is relatively low for the uncorrected atomic multipoles , the absolute error remains significant . therefore , a correction is still necessary in order to achieve better accuracy in the force field . it is notable that , after the charge penetration correction , the mean unsigned errors of all three types of complexes are reduced to 0.50.6 kcal / mol near the equilibrium distances , which is approaching the possible error of the qm calculation itself . plots of the electrostatic energy profiles of water water and benzene benzene dimer complexes . the valence- parameter set was used in calculations of the charge penetration corrected model ( multipoles + cp ) . the vertical line indicates the equilibrium distance . as mentioned in methods , an alternative way to derive the parameter is to fit the penetration - damped electrostatic potential ( eq 4 ) to the target qm values . an attempt to use this fitting strategy has been also made , and the resulting parameters have been compared . the parametrization of is restricted to a single unique value for each element type , as before . a brute force scanning of the parameter using a grid size of 0.1 was used to search for the global minimum since the parameter is less sensitive than . all 13 monomers ( excluding ethyne ) in the s66 data set were used in fitting of the for h , c , n , and o elements . then parameters were determined as before with values fixed to their potential - fitted values . the penetration parameter set obtained this way will be referred to as the fitted- set , while the parameter set with based on eq 5 will be referred as the valence- set . with the fitted- parameters , the rmse of the electrostatic potential of the 13 monomers calculated using eq 4 is greatly reduced to 0.07 kcal / mol , compared to an rmse of 0.95 kcal / mol for the valence- ( see supporting information table s2 ) . the performance of the two sets of parameters has been compared using the s101 data set . the overall performance of the two parameter sets is very similar to mean unsigned errors of 1.35 and 1.33 kcal / mol for the valence- and fitted- sets , respectively ( table 3 ) . for near - equilibrium pairs , the valence- set has a marginally better mue of 0.57 kcal / mol against 0.72 kcal / mol for the fitted- set . for short - ranged pairs , the fitted- set with a mue of 2.84 kcal / mol yet is slightly better than a mue of 3.28 kcal / mol of the valence- set . similar trends in rmses of the two sets of parameters are also observed . however , the valence- parameter set tends to have more balanced performances for hydrogen - bonded , dispersion - dominant , and mixed complexes , giving the mues of 0.50 , 0.53 , and 0.61 kcal / mol for the three groups , respectively ( table 4 ) . in contrast , the fitted- set , with a mue of 0.47 kcal / mol for the hydrogen - bonded complexes , shows subtly better agreement with sapt results yet has slightly worse performances for the aromatic compounds . the mues of the dispersion - dominant and mixed complexes are 0.69 and 0.74 kcal / mol , respectively ( electrostatic energy of individual pairs can be found in table s3 in the supporting information ) . nonetheless , the two sets of parameters all have excellent agreement with the sapt results for the whole s1017 database , while the fitted- set yields better electrostatic potential than the valence- set . the charge penetration model also exhibited good transferability during the fitting of parameters . although three atom types are used for h , c , n , and o in the current parametrization , restriction to a single for each element also results in reasonable accuracy . simply applying the arithmetic mean of the three parameters in valence- parameter set for each element ( supporting information table s1 ) increases the mue by only 0.1 to 0.65 kcal / mol for the near - equilibrium pairs in the s66 set ( table 5 ) . for pairs with shorter distances , the mue increases by 0.8 to 3.52 kcal / mol in the same set . only marginal improvements in mues were found after optimizing the parameters for each element starting from the averaged value . we believe this demonstrates the robustness and transferability of the charge penetration correction and the parametrization strategy . for the purpose of retaining flexibility , we recommend the use of three atom types for each of the h , c , n , and o elements in our final model . an additional set of parameters which has a unique for each element is also presented . the charge penetration effect is usually overlooked in molecular mechanical models and traditional force fields . our results show that dma - derived point multipoles systematically underestimate the sapt electrostatic interaction energy at typical molecular interaction distances based on the 1017 dimers studied here ( see the supporting information ) . an exponential damping function providing a simple charge charge penetration model suitable for force field incorporation has been revisited , along with a new parametrization strategy . the s1017 sapt - decomposed quantum mechanical energy database is developed as a reference for parameter training and for use in future force field comparison . the database is an extension of the s66 and s668 data set previously developed by hobza and co - workers , with additional prototype molecular complexes . the decomposed energies are calculated at the sapt2+/aug - cc - pvtz level of theory , with exchange the dispersion energy is further scaled to compensate for missing higher order terms in the sapt2 + method . the total sapt interaction energy is in excellent agreement with ccsd(t)/cbs results , which are currently considered to be the gold standard for estimation of intermolecular interactions , with a mean unsigned error of 0.16 kcal / mol for the s66 data set . thus , the sapt results should provide a reliable reference for force field development . by replacing the idealized charge charge ( coulomb ) interaction with the charge penetration corrected model ( eq 3 ) in the amoeba framework , the accuracy of calculated electrostatic energies for the s1017 database is improved by 5-fold . for the five distance pairs near the equilibrium distances ( i.e. , 0.901.10 times the equilibrium distance ) , the mean unsigned error of the charge penetration corrected and uncorrected point multipole models are 0.57 and 3.16 kcal / mol , respectively ; for the extremely close distance separations ( i.e. , 0.70 and 0.80 times the equilibrium distance ) , the mean unsigned errors of the two models are 3.28 and 19.16 kcal / mol , respectively . the improvement for the corrected model is significant and shows a consistent agreement with the quantum mechanics data at both long and short distances . the robustness and transferability of this model is also reflected in the use of very limited ( element - based ) parameters . the charge penetration correction is short - ranged and rapidly converges to the classical coulomb interaction beyond 67 . thus , it can be completely incorporated into the real space of ewald summation without any additional computational cost in reciprocal space . because simulations including penetration correction are clearly feasible , there is ongoing work dedicated to the optimization of parallel scaling the coupled penetration / smooth particle mesh ewald approach . quadrupole penetration ) are also possible and have been implemented in models such as sibfa . nonetheless , the simple empirical charge penetration model presented in this work provides us with an efficient approach to achieve accurate electrostatic energy that is systematically modeled after sapt quantum mechanical energy decomposition . the change in the electrostatic component requires re - examination of the van der waals interaction to arrive at a balanced representation of the total energy . overall we expect this improvement in the electrostatic component will alleviate the need for error compensation via other components and lead to more balanced and transferable potential energy functions in general . the comprehensive sapt database we developed in this work will also be useful for many others who are interested in understanding intermolecular forces or evaluating different empirical models .

osteochondrolipoma is a rare benign soft tissue neoplasm . it is occasionally considered to be a variant of adipose tissue neoplasm lipoma showing multiple differentiation pathways of pluripotent stem cells . as with the lipomas they can be seen at any location and show cartilagenous and osteoid differentiation when located parosteally . we present a case of osteochondrolipoma located at the symphysis of the mandible . to our knowledge , this is the first reported case of an oral osteochondrolipoma associated with parosteal localization . classical molecular mechanics force fields typically model interatomic electrostatic interactions with point charges or multipole expansions , which can fail for atoms in close contact due to the lack of a description of penetration effects between their electron clouds . these short - range penetration effects can be significant and are essential for accurate modeling of intermolecular interactions . in this work we report parametrization of an empirical charge charge function previously reported ( piquemalj .- p . ; j. phys . chem . a2003 , 107 , 1035326313624 ) to correct for the missing penetration term in standard molecular mechanics force fields . for this purpose , we have developed a database ( s1017 ) of 101 unique molecular dimers , each at 7 different intermolecular distances . electrostatic , induction / polarization , repulsion , and dispersion energies , as well as the total interaction energy for each complex in the database are calculated using the sapt2 + method ( parkert . m. ; j. chem . phys.2014 , 140 , 09410624606352 ) . this empirical penetration model significantly improves agreement between point multipole and quantum mechanical electrostatic energies across the set of dimers and distances , while using only a limited set of parameters for each chemical element . given the simplicity and effectiveness of the model , we expect the electrostatic penetration correction will become a standard component of future molecular mechanics force fields .

anterior cervical spine fusion and stabilization is a well established procedure for cervical myelopathy , radiculopathy , neoplasms , and cervical trauma2 ) . although injuries to the pharynx and esophagus are known complications of anterior cervical spine surgery , delayed pharyngeal or esophageal perforation is rare7,9,10 ) . here , we describe a rare but potentially life - threatening complication after anterior cervical spine fusion and plating . the authors highlight this issue by presenting this case , which had no associated morbidity , and include a review of the relevant literature . a 43-year - old man was admitted to our institute with a 3-month history of dysphagia and neck pain with swelling . he was paraplegic due to a c6 - 7 fracture and dislocation and has been operated on 8 years previously . initial surgical treatment included anterior corpectomy of c7 and anterior iliac crest graft placement using a plate and screws . hematological studies including erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) were normal . a simple lateral radiograph and a computed tomography scan showed partial anterior migration of the lower screw . a hydro - soluble contrast swallow image confirmed esophageal perforation ( fig . the loose screw was removed and esophageal perforation was found during surgery and repaired directly by a cardiovascular team(fig . the patient was fed using a nasogastric tube for 3 weeks and subsequently oral feeding was gradually resumed . further progress was favorable , and a contrast study performed at 3 weeks postoperatively showed no evidence of fistula . anterior cervical fusion and plate fixation is an effective procedure for the treatment of cervical myelopathy or radiculopathy and cervical spine trauma . plating has been reported to achieve a fusion rate of up 98% , and to result in early mobilization , reduced graft - related complications ( especially for multilevel fusion ) , and to avoid late deterioration of the cervical spine alignment obtained at surgery1,4 ) . the complication rate after anterior cervical plating is generally low and decreases with surgeon 's experience . according to zeidmann14 ) , the overall complication rate associated with anterior cervical spinal fusion is approximately 5% , and pharyngo - esophageal perforation is uncommon , but nevertheless of the utmost importance because of the possibility of graft infection leading to osteomyelitis , mediastinitis , sepsis , and death6 ) . acute injury can be caused iatrogenically during surgical approach due to inappropriate placement or dislodgement of sharp - toothed retractor blades in the esophagus . retraction is particularly dangerous when a nasogastric tube is positioned because the wall of the hypopharynx or esophagus may be " trapped " by a high - pressure claw between the retractor and the tube , causing ischemic injury and secondary perforation8 ) . delayed esophageal injuries are due to chronic compression or contact and subsequent necrosis , abscess formation , and perforation due to graft dislodgement or screw migration with or without plate failure4,7 ) . screw dislodgement often follows a benign course and is completely asymptomatic , due to the small diameters of the screws used and slow migration from the external to the internal mucosa , which permits spontaneous tissue repair of the defect caused repetitive friction between the retropharyngo - esophageal wall and the plating system(normally positioned with adhesion ) , traction - type pseudodiverticulum , and perforation are other causes of delayed injury11 ) . the complications of esophageal perforation range from asymptomatic with local infection to mediastinitis and death . the clinical course depends on the etiology , location , and timing of the perforation . asymptomatic perforation has as well been reported as incidental oral extrusion of screw even years after anterior cervical spine stabilization5 ) . patients generally present with swallowing difficulty , regional swelling , neck pain , dysphagia , weight loss , dysphonia , subcutaneous emphysema , and fever ; our patient presented with dysphagia and neck pain with regional swelling12,13 ) . conservative treatment may be preferred for small , contained defects of less than 1 cm , and consists of the elimination of oral feeding , tube feeding to restore fluid and nutritional balance , and intravenous antibiotics . some cases need surgical repair , such as , perforation closure with a primary suture or sternocleidomastoid or pectoralis major flap repair13 ) . we operated on our patient to remove the offending screw due to evident fistula confirmed by esophagography and esophagoscopy . direct repair was effective in achieving a successful perforation repair of the esophageal perforation with an early return to oral feeding . we report a rare case of delayed esophageal perforation caused by screw displacement after anterior cervical spine plating . careful periodic follow - up is necessary , and when encountered , early surgical closure following removal of the offending screw is mandatory . acute ophthalmia neonatorum ( on ) continues to play an important role in causing severe ocular problems of newborns in countries where primary health care coverage is insufficient , as in the case of angola . unfortunately , in most clinical obstetric wards of luanda , the nation 's capital , there is no regular plan of on prophylaxis , nor is there any capacity for determining the microorganisms involved in the etiology of this entity . therefore , most of the acute conjunctivitis cases in the newborns are empirically diagnosed as on and treated without having information on the causative agent . although prenatal screening and treatment of pregnant women are very effective for the prevention of on , this approach can be difficult to implement in developing countries . in some african countries , at the time of delivery , a large percentage of expectant mothers have had little or no prenatal care . in industrialized countries , on prophylaxis was performed by silver nitrate eyedrops and currently by topical erythromycin , tetracycline ointment , and others . in other developed countries , emphasis is placed on maternal surveillance , and there is no systematic prophylaxis of on . the use of povidone - iodine ( p - i ) has been advocated for the less developed countries , but few well - designed prospective , randomized , and controlled clinical studies have been performed with this agent . even while doubts remain regarding the real efficacy of p - i for prevention of on caused by either chlamydia trachomatis ( ct ) or neisseria gonorrhoeae ( ng ) [ 4 , 5 ] , this substance is well tolerated , has a broader spectrum than other anti - infectious agents , does not induce resistance , and is more effective than some antibiotics that are more expensive for prophylaxis [ 6 , 7 ] . after the civil war in angola ( 19742002 ) , , we conducted a pilot study structured as a maternity ward prospective series and found that 12% of the newborns had signs of acute bilateral conjunctivitis . an additional problem was the inability to determine the microorganisms involved [ 911 ] because microbiological studies were not routinely performed at either maternity wards or eye centers . based upon these existing conditions , we decided to evaluate different tests that could provide necessary microbiological information to the ophthalmologists and then select the best one for implementation in a nationwide on prophylaxis program . our initial attempt was to use the simplest available diagnostic tool , standard staining of conjunctival swabs by giemsa and gram stains . however , this provided little useful information regarding the active on agent(s ) . following this , we attempted to use multiplex polymerase chain reaction ( pcr ) of maternal endocervical and neonatal conjunctival specimens for microorganism identification . the pcr assay has been previously validated with conjunctival and endocervical samples collected from this study ( manuscript accepted ) . in brief , we demonstrated that this technique could be used to identify the three major microorganisms , for example , ct , ng , and mycoplasma genitalium ( mg ) , involved in sexually transmitted infections ( stis ) . this was successful not only in endocervical samples , but also in conjunctival smears , where it never had been used . therefore , our recommendation for angola 's health authorities was to provide this kind of microbiological identification technique . the primary purpose of this work was to analyze the efficacy , limitations , and obstacles of 2.5% p - i eyedrops for the prophylaxis of on in angola . these data are intended to provide the local health authorities with enough information to develop a national prophylactic campaign against on based on the routine use of p - i eyedrops for every newborn . a secondary objective was to provide additional data on the etiological agents both in conjunctival smears from newborns and in endocervical samples from their mothers and the possible relationship of these agents with clinical risk factors . an interventional , randomized , and prospective study with a blinded , randomized control group was designed . the study was conducted at the general augusto n'gangula specialized hospital ( hgeag ) and the health center of samba ( css ) , both in luanda , angola . approval was provided by the ethical commission of the faculty of medicine of the agostinho neto university ( luanda , angola ) . after the explanation of the objectives and methodology of the investigation the study was performed in accordance with the ethical standards from the 1964 declaration of helsinki and its later amendments . the target population for this study consisted of 317 mothers and their newborns from the hgeag ( 173 ) and the css ( 144 ) , recruited from 7 december 2011 to 22 november 2012 . the inclusion criteria consisted of healthy children weighing at least 2.3 kg and a gestation period of at least 37 weeks . newborns not meeting the inclusion criteria and those with respiratory distress at birth were excluded . additionally , because of the possible deleterious effect of iodine , newborns were excluded if their mothers were diagnosed with thyroid disease according to clinical data contained in her medical record . maternal data were collected through a questionnaire that included age , race , education , parity , number of prenatal visits , pathology during pregnancy , and duration of rupture of the amniotic sac . neonates were randomly distributed into two groups , a and b , by blocked randomization with a fixed block size of 4 . newborns in group b received instillation of a drop of p - i 2.5% in the bottom of the lower sac of both eyes immediately after a basic eye examination and the collection of conjunctival smears within 3 hours of birth . custom 2.5% p - i eyedrops were prepared by a certified spanish pharmacy ( carreras , barcelona , spain ) following the standards of good manufacturing practices . ocular samples were obtained from both eyes of the newborns by vigorous swabbing across the inferior tarsal conjunctiva . all samples were taken by ophthalmologists and medical personnel previously trained in these procedures by an expert microbiologist from spain ( pm ) . samples were collected with flocked swabs in universal transport medium ( copan italia s.p.a . , brescia , italy ) , stored at 70c , and shipped to the department of microbiology and immunology at the hospital clnico universitario of valladolid , valladolid , spain . dna extraction was performed according to routine laboratory standards with the gxt dna / rna reagents in a genoxtract extractor ( hain lifescience , nehren , germany ) . a multiplex pcr assay that coamplified dna sequences of ct , ng , mg , and an internal control was performed using the bio - rad dx ct / ng / mg kit ( bio - rad , hercules , ca , usa ) , according to manufacturer 's instructions . the ophthalmologist responsible for the study ( ia ) administered the p - i eyedrops . the basic eye examination given to all newborns included pupil light responses , eyelid position and movement , appearance of the conjunctiva , corneal size and transparency , iris appearance and symmetry , lens transparency , quality and symmetry of retinal red reflex ( brckner test ) , and the size , position , and shape of the pupil . an information sheet with explanations about the signs of ophthalmia / acute conjunctivitis ( red eye and ocular secretions and/or eyelid edema ) was given to all participating mothers . they were also given instructions to return with their children for observation after discharge from the maternity ward in any case , even if their baby did not show these signs . the mobile phone number of each mother was noted ( only two mothers gave no indication of telephone contact ) . between the 5th day and 7th day postpartum , phone calls were made to the mothers to bring their infants for observation , especially if they presented ocular signs of on or any other ocular alterations . the minimum sample size was calculated taking in consideration the suspected prevalence of on detected in our previous study and with an expected reduction of on in at least 30% in the prophylaxis group ( group b ) . to detect a 8% difference between treatment groups with a significance level of 5% and power of 80% , infection rates of the different on bacteria in the endocervical samples of the mothers and in the conjunctival samples of their newborns and the presence of clinical risk factors were compared by test . the mean age of the 317 participating mothers was 25 years ( range : 1452 years ) , with the majority between 14 and 24 years . some of the mothers , 28% , had a basic level of education and 2.2% were illiterate and the remainders have good reading skill . parity varied from 0 to 9 births , with 82% of the mothers having had 3 previous deliveries . the number of prenatal consultations of the mothers ranged from 0 to 9 , with an average of 4 consultations . thirty - eight cases ( 1.2% ) did not have any prenatal consultation , and 112 cases ( 35% ) had less than four . a total of 96 mothers ( 30.4% ) referred some pathology during pregnancy , predominately urinary infection in 81 mothers ( 25.6% ) and vulvovaginitis in 13 others ( 4.1% ) . a total of 70 mothers ( 22% ) presented with premature rupture of membrane ( prom ) . thirty - one of them had more than 6 hours before delivery , and 39 had less than 6 hours . data were collected from 317 children , but a total of 72 newborns were excluded ( 22.6% ) from the study for low weight , respiratory distress , death , or transfer of the mother to a more specialized center for dystocic delivery ( table 1 ) . newborns , 123 females and 118 males , had a gestational age of 36 to 40 weeks , and an average weight of 3.260 kg . a total of 42 ( 17.1% ) had ocular pathology at the time of delivery , including 31 ( 12.6% ) with conjunctival hyperemia and 11 ( 4.4% ) with on signs , including conjunctival hyperemia plus eyelid edema and/or purulent secretion . three out of the 11 suspected on cases were born from mothers with urinary infections during pregnancy . for five of the newborns with signs of on , the amniotic sac did not rupture prematurely . for the other six due to technical difficulties in the preparation of the samples for transport to spain for pcr analysis , there was no information available regarding the presence or absence of ct , mg , and ng for 6 maternal endocervical samples and 13 newborn conjunctival smears . a total of 543 valid samples were analyzed , 232 from conjunctival smears and 311 from endocervical samples ( table 2 ) . the most common etiologic agent in newborns was ct ( n = 4 ) , followed by mg ( n = 2 ) and then ng ( n = 1 ) . pcr gave positive results in 28 mothers , with a predominance of mg ( n = 19 , 6.1% ) , followed by ct ( n = 8 , 2.1% ) and ng ( n = 2 , 0.5% ) . eleven of the 28 mothers ( 39.3% ) who were infected with ct , mg , or ng presented risk factors for mother - to - child transmission . the factors included prom at delivery time ( n = 6 , all positive for mg ) , vulvovaginitis ( n = 3 ) , urinary tract infection ( n = 1 ) , and urinary tract infection plus vaginitis ( n = 1 ) . of the cases with prom with > 6 hours of labor , one had ng and 3 had mg . the mother - to - child transmission rates were 50% for both ct and ng and 10.5% for mg ( manuscript accepted ) . chi - square analysis showed no significant correlation between cases with external signs of acute conjunctivitis and the presence of urinary or vaginal infections in the mother at the time of delivery . the newborns were randomly distributed into group a , in which the newborns received no ocular prophylactic treatment ( n = 130 ) , and group b , in which the newborns received p - i prophylaxis in both eyes ( n = 115 ) . despite the efforts made with every mother to perform a follow - up visit within 7 to 10 days after delivery , only 16 children were evaluated , nine from group a and seven from group b. ten children , 7 from group a ( controls ) and 3 from group b ( p - i treated ) , did not show any ocular pathology . two ( one from each group ) had acute bilateral conjunctivitis after the third day postpartum . one from group a had a small conjunctival hemorrhage in one eye , and another , from group b , had jaundice of both conjunctivas . the nearly complete disappearance of on in developed countries has been the result of a combination of factors , including prophylactic measures and , above all , better prenatal care [ 13 , 11 , 13 ] . however , currently in angola and other west african nations , no prophylactic measures are used , and it is routinely very difficult to identify the pathogenic agents . thus , diagnosis of on is based on clinical signs , and systematic follow - up of children after birth is almost impossible . ng and ct are currently the most common on etiologic agents , accounting for 60% of all cases in countries without neonatal prophylaxis [ 1416 ] . although gonococcal infection is less common in developed countries , it continues to be a problem in developing countries such as kenya where on has an incidence as high as 4% of live births for ng and 8% for ct can infect the fetus by ascending from the vagina to the uterus and can be present in the newborn at birth as an acute conjunctivitis ; however , other agents usually have an incubation period of 414 days before clinical signs . the aim of this study was to evaluate the efficacy of intervening at birth by providing p - i as a prophylactic agent for on . the weak response of mothers returning for a second ophthalmic examination for their newborns prevented the achievement of this goal . maternal ignorance about the implications of on and cultural factors resulted in poor cooperation of mothers in the follow - up evaluations . even after careful oral and written explanations were given to mothers about the signs and symptoms of on and the ocular and systemic risk for the affected children , and even after most of the mothers were personally contacted by mobile phone , only a very small number of children , 5.7% of the global sample , returned for a second examination . these kinds of problems have been recognized by other authors [ 6 , 7 , 14 ] . since 1990 , p - i has been considered as a potential prophylactic agent of on . several studies have established that p - i has broad spectrum of action and is effective against most agents of on , unlike other prophylactic agents previously used [ 1621 ] . p - i does not induce bacterial resistance , has low toxicity , is of very low cost , and is stable for several months after opening . even more , the transient brownish staining of the ocular surface after instillation could be useful as an indicator of its effective application . in 2002 , isenberg reported that 2.5% p - i was not irritating to the eye . overall , the studies showed that when trained personnel applied p - i after hygienic and general care of the newborn , the results are superior to the other prophylactic agents [ 16 , 19 ] . nevertheless , a number of arguments have been raised against its use as a prophylactic agent , including the possibility that it could be confused with a detergent , the lack of effect of p - i against viruses , and lack of studies proving that it is safe in newborns [ 15 , 19 ] . our study also shows for the first time the frequency of mg infection and some associated clinical findings in a cohort of angolan mothers and their newborns ( manuscript accepted ) . mg is an emerging cause of stis and has been implicated in urogenital infections of men and women worldwide . mg has a prevalence of 7.3% and 2% in high- and low - risk populations , respectively . there is also evidence that this microorganism has the potential to cause ascending infection and may play an important role in the on [ 22 , 23 ] . besides the fact that on is a potential cause of blindness , it can also result in serious systemic complications when nasopharyngeal colonization during vaginal delivery evolves to otitis , pneumonitis arthritis , sepsis , and meningitis [ 13 , 15 , 24 ] . risk factors associated with on are genitourinary infection and prom [ 10 , 13 , 15 ] . in this study , a total of 108 mothers ( 34.1% ) presented some of these risk factors . according to world health organization recommendations , prenatal care of mothers should include identification and management of infections including hiv , syphilis , and other stis . regular screening for stis is not routine in prenatal care in angola although the majority of mothers included in this study did have the prenatal appointments recommended by who . most of them had 4 or more prenatal visits , and the percentage of mothers without prenatal care was considerably lower than the one obtained by our group in the same maternity ward three years before ( 1.2% versus 14.7% ) , showing an improvement of the health system of the country . prenatal studies in mothers of similar age as in this study are very important because the age group between 15 and 25 years of age is considered to be at risk of stis . actually , genital ct ( serotype d - k ) and ng infections are especially common in this age group of african women ; therefore , their neonates are also a risk group for on and systemic complications [ 10 , 13 , 15 ] . our data show that the prevalence of ct in our population was relatively low when compared to previous studies in angola and other african populations . other studies of pregnant women have shown prevalence rates from about 6% in tanzania to 13% in cape verde . the prevalence in our study was much lower , which confirms the relatively low presence of ct in angola . ng and ct infections have common clinical features in women . both produce silent clinical infections , are transmitted efficiently to newborns , and can lead to sterility or chronic infection . both generate silent carriers , causing severe clinical consequences over time . according to historical data , around 3% of newborns with ng ophthalmia will develop complete blindness if untreated , and 20% will have some degree of corneal damage . in africa , the prevalence rate of ng among pregnant women ranges from 0.02% in gabon to 7.8% in south africa . our results showed a lower prevalence of ng , 0.5% , among mothers , confirming our previous results ( manuscript accepted ) . the results of the present study also allow us to estimate the rates of mother - to - child transmission which were 50% for ct and ng and 10.5% for mg . to our knowledge , this is the first study that estimates the rate of mg transmission in angola . in our cohort , a total of 12 newborns presented signs of acute conjunctivitis at the first ophthalmic evaluation , although laboratory tests were positive for mg only in one case . on the other hand , there were six cases that did not show signs of acute conjunctivitis at the first observation but were nevertheless positive for ct , mg , or ng . neonatal infections are of great epidemiological importance , so focused screening efforts should be made to reduce the number of infected pregnant women and thereby the rate of vertical transmission . to a similar extent as seen for other major stis in africa , our work clearly shows a mirror effect when considering matches between infected newborns and their mothers . this result also indicates the need to locally improve public information about primary health care particularly that oriented to eye care . without this knowledge , the participation of community members in studies like ours will not be effective . in summary , we determined the prevalence and incidence of ct , ng , and mg , which are all implicated in angolan cases of newborn on . we also determined that the mother - to - child transmission rates of ct and ng were 50% and 10.5% for mg , which was identified for the first time in angolan newborns . unfortunately , due to low compliance in follow - up clinical assessments , we were unable to achieve the original goal of testing the efficacy and safety of p - i systematic prophylaxis for preventing on .

although anterior approaches to the cervical spine are popular and safe , they cause some of complications . esophageal perforation after anterior spinal fusion is a rare but potentially life - threatening complication . we present a rare case of delayed esophageal perforation caused by a cervical screw placed via the anterior approach . a 43-year - old man , who had undergone surgery for complete cord injury at another orthopedic department 8 years previously , was admitted to our institute due to painful neck swelling and dysphagia . radiological studies revealed a protruding screw and esophageal perforation . the perforation was found during surgery and was successfully repaired . this case emphasizes the need for careful long - term follow - up to check for delayed esophageal perforation in patients that have undergone anterior cervical spine plating . purpose . to determine the efficacy of povidone - iodine ( p - i ) prophylaxis for ophthalmia neonatorum ( on ) in angola and to document maternal prevalence and mother - to - child transmission rates . methods . endocervical samples from mothers ( n = 317 ) and newborn conjunctival smears ( n = 245 ) were analysed by multiplex polymerase chain reaction ( pcr ) for chlamydia trachomatis ( ct ) , neisseria gonorrhoeae ( ng ) , and mycoplasma genitalium ( mg ) . newborns were randomized into a noninterventional group and an interventional group that received a drop of p - i 2.5% bilaterally after conjunctival smear collection . mothers were trained to identify signs of on and attend a follow - up visit . results . forty - two newborns had ocular pathology , and 11 ( 4.4% ) had clinical signs of on at the time of delivery . maternal pcr was positive for mg ( n = 19 ) , ct ( n = 8) , and ng ( n = 2 ) . six newborns were positive for ct ( n = 4 ) , mg ( n = 2 ) , and ng ( n = 1 ) . mother - to - child transmission rates were 50% for ct and ng and 10.5% for mg . only 16 newborns returned for follow - up . conclusions . lack of maternal compliance prevented successful testing of prophylactic p - i efficacy in on prevention . nevertheless , we documented the prevalence and mother - to - child transmission rates for ct , ng , and mg . these results emphasize the need to develop an effective angolan educational and prophylactic on program .

this work was supported , in whole or in part , by the one hundred person project of sun yat - sen university ( xz ) , national natural science foundation 81302262 ( xz ) , the basser research center for brca ( lz ) , the national institutes of health r01ca142776 , r01ca190415 , p50ca083638 ( lz ) , the ovarian cancer research fund ( lz and xh ) , the breast cancer alliance ( lz ) , department of defense ( lz ) , and the marsha rivkin center for ovarian cancer research ( lz ) . the indications for our approach were as follows : ( 1 ) isolated pcl avulsion fracture ; and ( 2 ) posterior tibial sag present at 90 of knee flexion . intra - articular lesions except pcl avulsion fracture were excluded using preoperative magnetic resonance image because of posteromedial approach and the consequential prone position . a c - arm device was used to avoid the penetration of growth plate by the anchors in pediatric or adolescent patients . patients were placed in the prone position , with the knee flexed 20 to 30. an oblique incision of < 10 cm was made between semitendinosus tendon and medial head of the gastrocnemius ( fig . the medial head of the gastrocnemius was held laterally to expose the posterior capsule of the knee joint . capsulotomy with an l - shaped incision was made longitudinally and then extended laterally to expose the fracture site ( fig . after debridement of fracture site , 2 suture based anchors ( 3.5 mm ; biomet sports medicine , warsaw , in , usa ) were inserted into the superomedial and superolateral region of the fracture site . each suture limb from the proximal row was placed through the hole at the end of the push - lock device ( 3.5 mm ; bio - pushlock , arthrex inc . , naples , fl , usa ) . pilot holes for the push - lock device were created using a punch just inferior to the distal edge of the fracture site . after the device was fully engaged in the pilot hole , the sutures were cut . the use of a knotless anchor provides a compressive force and consequently fixes the reduced avulsion fragment strongly to the fracture bed ( fig . if the patients were children , the anchors were inserted so as not to penetrate a physeal plate . a well - padded plaster splint was applied to the knee at 5 to 10 of flexion . on the first day , after surgery , the drain was removed and patients were encouraged to start quadriceps muscle strengthening exercise and begin non - weight bearing walking using crutches . after 2 weeks , protected range of motion exercise was started and partial weight bearing ambulation using crutches was permitted with locked brace in full extension . six weeks later , the brace was unlocked and the patients began full weight bearing ambulation . at 8 weeks , the brace was removed and patients were encouraged to increase activity gradually . a 20-year - old man presented with a right knee injury resulting from a motor vehicle accident . clinical examination revealed a swollen knee , limited range of motion and posterior tibial sag at 90 on knee flexion . the knee range of motion was 135 with a flexion 135 and flexion contracture 0 after 2 weeks postoperatively . the patient was able to return to his usual daily activities after postoperative 6 months ( fig . 2 ) . a 13-year - old boy presented with left knee injury resulting from a fall . clinical examination revealed hemarthrosis of the knee , limited range of motion , and posterior tibial sag . the knee range of motion was 140 with flexion 140 and flexion contracture 0 after 4 weeks postoperative . a well - padded plaster splint was applied to the knee at 5 to 10 of flexion . on the first day , after surgery , the drain was removed and patients were encouraged to start quadriceps muscle strengthening exercise and begin non - weight bearing walking using crutches . after 2 weeks , protected range of motion exercise was started and partial weight bearing ambulation using crutches was permitted with locked brace in full extension . six weeks later , the brace was unlocked and the patients began full weight bearing ambulation . at 8 weeks , the brace was removed and patients were encouraged to increase activity gradually . a 20-year - old man presented with a right knee injury resulting from a motor vehicle accident . clinical examination revealed a swollen knee , limited range of motion and posterior tibial sag at 90 on knee flexion . the knee range of motion was 135 with a flexion 135 and flexion contracture 0 after 2 weeks postoperatively . the patient was able to return to his usual daily activities after postoperative 6 months ( fig . a 13-year - old boy presented with left knee injury resulting from a fall . clinical examination revealed hemarthrosis of the knee , limited range of motion , and posterior tibial sag . the knee range of motion was 140 with flexion 140 and flexion contracture 0 after 4 weeks postoperative . currently , open reduction or arthroscopic fixation is more commonly used in treatment of displaced pcl tibial bony avulsion.3456 ) the latter has both advantages and disadvantages . since the site of attachment of the pcl to the tibia is located deep within the posterior tibial plateau , multiple arthroscopic sutures and tunnels are required , making the procedure more challenging and difficult . furthermore , some authors reported that arthroscopic fixation using suspensory device needs a sizeable drill hole that may break thinner bone fragments.9 ) open reduction can be performed with a traditional s - shaped approach , but this incision is associated with injury to adjacent neurovascular structures . in contrast , we used a less invasive posteromedial approach , which had several advantages , including exposure of the posterior capsule through the gap between the medial head of the gastrocnemius and the semitendinosus muscle . with the approach in this study , it is possible to minimize risks of damage to vessels and nerves , as well as providing satisfactory exposure of the fracture site . similar methods using posteromedial approach with suture anchors are previously reported.6 ) these studies used 2 suture anchors and fixed bony fragment by knotting with the sutures of 2 suture anchors . the most important advantage of the present suture bridge fixation technique with posteromedial incision , is its indication for use regardless of the thickness , size , and comminution of bony fragment . this technique is similar to the double - row , trans - osseous equivalent or suture bridge techniques for rotator cuff repair or internal reduction and fixation of greater tubercle fractures of the humerus . it allows for a simple reproducible anatomic reduction and compression of the fracture fragment , even though the fragment is small or comminuted . furthermore , suture anchor is relatively small , as compared to other fixation devices , such as cannulated screws , wires , sutures , and absorbable screws . the anchor also offers strong purchase , and the attached suture can resist strong tension , resulting in reliable fixation . previous study on anterior cruciate ligament avulsion fracture , reported that suture bridge construct provides superior fixation with regard to ultimate load , as compared with standard screw fixation and suture fixation in biomechanical analysis of suture bridge fixation for tibial eminence fractures.10 ) similarly , the suture bridge mattress fixation in this study may provide enough ultimate load and compressive force to the avulsed fragment and thus maximize the surface area for bony healing . furthermore , there is no risk of causing comminution . in conclusion , the results of the study indicated that the posteromedial approach , combined with suture anchors , is a suitable treatment for isolated tibial avulsion fractures of the pcl .

long non - coding rnas ( lncrnas ) are defined as rna transcripts larger than 200 nucleotides that do not appear to have protein - coding potential . accumulating evidence indicates that lncrnas are involved in tumorigenesis . our work reveals that lncrna fal1 ( focally amplified lncrna on chromosome 1 ) is frequently and focally amplified in human cancers and mediates oncogenic functions . we presented a surgical technique including a suture bridge technique with relatively small incision for the reduction and fixation of posterior ligament avulsion fractures . a suture anchor was used to hold the avulsed fragment and a knotless anchor was used to continuously compress the bony fragment into the fracture site , thereby maintaining reduction during healing .

cichlid fishes have undergone spectacular radiations in different parts of the world . in particular , the species flocks of the east african great lakes are well - known examples for rapid evolution and speciation [ 15 ] . tanganyika , malawi , and victoria is inhabited by hundreds of mostly endemic cichlid species [ 6 , 7 ] . notably , most of the diversity is found in the littoral habitat , whereas reduced species richness in the deep benthal and pelagial seems to be a common phenomenon in all east african great lakes [ 710 ] . at least three factors may have contributed to this pattern : ( i ) reduced niche diversity in the pelagic and in deepwater benthic zones , ( ii ) a narrow ambient light spectrum consisting only of short - wavelength blue light and hence less promotive of diversification mechanisms contingent on color perception than the shallow clear - water habitats [ 1114 ] , and ( iii ) the absence of strong barriers to gene flow . indeed , deepwater cichlid species often have lake - wide distributions with very low , if any , population genetic structure over large geographic distances [ 10 , 15 , 16 ] ( see also the lake tanganyika clupeid limnothrissa miodon and the centropomid lates stappersii ) . on the other hand , high levels of genetic differentiation , sometimes accompanied by phenotypic divergence on small geographic scales , are characteristic for the species - rich guild of stenotopic rock - dwelling cichlid species [ 1931 ] . however , allopatric diversification in the fragmented littoral zone was not necessarily accompanied by the evolution of pre- or postzygotic isolation , so that secondary contact imposed by lake level fluctuations has often led to hybridization or introgression between previously allopatric taxa [ 3235 ] . even today , substrate breeders of the tribe lamprologini can be found in mixed - species pairs , and interspecific fertilizations occur in communally nesting , shell - breeding lamprologines . indeed , phylogenetic analyses of predominantly littoral cichlid lineages revealed that interspecific hybridization has played , and still plays , an important role in the evolution of these fish [ 32 , 34 , 3641 ] . thus , in the majority of recent molecular studies on species relationships within littoral tribes , especially when comparing mitochondrial and nuclear phylogenies , the explanation for the tree topologies involved the claim of introgression and hybridization between established lineages in addition to incomplete lineage sorting ( reviewed in ) . we hypothesize that this is not the case in tribes composed of deepwater species . lacking the geographic structure introduced by littoral habitat heterogeneity , deepwater species may still be spatially separated by distance , by segregation of breeding grounds , by variable hydrological conditions [ 16 , 43 , 44 ] , or by large - scale fragmentation of the lake basin during major droughts [ 45 , 46 ] . generally , however , the potential barriers to gene flow for deepwater species are less insurmountable than those met by stenotopic littoral cichlids . specifically in lake tanganyika , the evolution of stenotopy regarding depth , bottom type , or light intensity may have been prohibited by the seasonal upwelling of anoxic waters . we postulate that given the high potential for gene flow , diversification of lineages will either be curtailed ( as suggested by the relative species paucity ) or be attended by strong reproductive isolation right from the start . this would imply that introgression following cladogenesis occurred at much lower rates , if at all , in the deepwater species than in littoral cichlids . we test this hypothesis in the deepwater cichlid tribe bathybatini by comparing phylogenetic trees based on mitochondrial sequence data with trees obtained with nuclear multilocus aflp data , an approach which has previously revealed hybridization in littoral cichlids and other contexts [ 34 , 36 , 37 , 39 , 40 , 4854 ] . moreover , in several studies , phylogenetic inference on species relationships has benefited from the use of multilocus genetic data , and we expect that the aflp data collected in the present study will also contribute to the resolution of intergeneric relationships within the bathybatini , which are still debated due to conflicting or ambiguous molecular and morphological evidence [ 46 , 5559 ] . the tribe bathybatini ( sensu takahashi ) comprises 17 species in three genera : ( 1 ) the genus bathybates comprises six large ( 3040 cm ) , piscivorous species preying mainly on pelagic freshwater clupeids ( b. fasciatus and b. leo ) , benthic cichlids ( b. graueri , b. vittatus , and b. ferox ) , or undefined prey ( the rare , elusive b. horni ) , in addition to the small ( 20 cm ) b. minor , which is a specialized clupeid hunter . in accordance with their trophic niches , coulter distinguished three morphotypes among the bathybates species , the fast - swimming fusiform predators b. fasciatus , b. leo , and b. horni , the generalized shape of the benthic feeders b. graueri , b. vittatus , and b. ferox , and the small clupeid - mimicking b. minor , which mingles with its prey and accompanies the diurnal clupeid migrations . based on trawl net and gill net catches , b. minor were classified as pelagic , b. fasciatus and b. leo as chiefly bathypelagic and the remaining four species ( b. graueri , b. vittatus , b. ferox , and b. horni ) as chiefly benthic . except for b. minor , which was never found below 70 m , bathybates species descend to depths of 150200 m. ( 2 ) the member of the monotypic genus hemibates , h. stenosoma , is an abundant benthic species on the muddy bottom of southern lake tanganyika feeding on fish and shrimps mainly at depths between 100 and 200 m . ( 3 ) the small - bodied ( < 15 cm ) species of the genus trematocara ( formerly assigned to the genera trematocara and telotrematocara , ) comprise nine benthic and bathypelagic species feeding on a variety on invertebrate prey , fish larvae , and phytoplankton . they are found at maximum depths of 75 to 200 m . following the upward movement of zooplankton , some species release their fry in shallow areas , but overall , data on bathybatine breeding behaviour is anecdotal or lacking [ 43 , 60 ] . the species are sexually dimorphic , with males of bathybates and hemibates exhibiting species - specific patterns of dark stripes , bars and dots on a silver background and egg - spots on the anal fins , and males of the silvery trematocara with dark dorsal fin markings . all bathybatini have large eyes , which promote not only the detection of prey and predators but possibly also mate - recognition in the dark depths . in line with the latter , the monochromatic patterning of males may be viewed as adaptation to the short - wavelength dominated visual environment , in contrast to the colourful patterns of the mouthbrooding cichlids in the shallow , light - flooded littoral . a recent phylogenetic study based on three mitochondrial genes supported the monophyly of bathybates as well as of the species therein and indicated a polytomy of three equidistant lineages representing bathybates , hemibates , and trematocara . within bathybates , b. minor appeared ancestral to a radiation of the six large species , which showed a basal split of b. graueri and low statistical support for the branching order of the remaining species ( figure 1 ) . the short internal branches among the large bathybates species supported a rapid radiation at approximately 2.32.7 mya , coinciding with the rapid diversification of other lake tanganyika cichlids [ 28 , 45 , 62 ] . competition and resource partitioning as well as potential geographic isolation during an extreme low - stand of the lake were proposed as promoters of bathybates speciation . this study is based on a total of 38 specimens , representing all seven bathybates species , hemibates stenosoma as well as trematocara unimaculata and t. macrostoma ( table 1 ) . all specimens were obtained between 1999 and 2011 from local fishermen at lake tanganyika and identified to species by s. koblmller . unfortunately , it was not possible to obtain a comprehensive taxon sampling for the genus trematocara as , because of their small size and hence low market value , these fish ( except for the largest species t. unimaculatum ) are not caught by local fishermen . fin clips or white muscle tissue were preserved in ethanol and dna was isolated using a proteinase k digestion / high salt precipitation method . the ten primer combinations used for selective amplification were ecori - aca / msei - caa , ecori - aca / msei - cag , ecori - aca / msei - cac , ecori - aca / msei - cat , ecori - act / msei - cat , ecori - act / msei - caa , ecori - act / msei - cag , ecori - act / msei - cac , ecori - acc / msei - caa , and ecori - acc / msei - cac . selective amplification products were visualized using an abi 3130xl automated sequencer ( applied biosystems ) along with an internal size standard ( genescan-500 rox ; applied biosystems ) . polymorphic positions were initially identified using genemapper 3.7 software ( applied biosystems ) in a range of 50500 bp . in order to adjust misaligned bins and avoid size homoplasy , bins containing ambiguous low intensity peaks in a large proportion of the samples and entire profiles with short read - lengths or very low peak heights were deleted . these preprocessed , un - normalized peak - height data were analyzed with aflpscore 1.4a , which optimizes thresholds for locus retention and phenotype calling based on estimated error rates . phenotype calling thresholds were set as absolute or relative depending on the number of retained loci and the achieved error rates obtained with each option . 20 replicate samples were included to calculate the mismatch error rate for all unique loci . a neighbour joining ( nj ) tree based on nei and li 's distances was constructed in paup 4.0b5 . bootstrap values from 1000 pseudoreplicates were used as standard measure of confidence in the inferred tree topology . although accurate models for bayesian tree construction using aflp datasets do exist ) , the high demands for processing power make them unfeasible to use [ 67 , 68 ] . thus , bayesian phylogenetic inference ( bi ) was conducted in mrbayes 3.1.2 , employing the restriction site model with the noabsencesites coding bias correction [ 68 , 70 ] . the dirichlet prior for the state frequencies was set to ( 2.44 , 1.00 ) matching the actual 0/1 frequencies in the dataset . posterior probabilities were obtained from metropolis - coupled markov chain monte carlo simulations ( 2 independent runs ; 10 chains with 8,000,000 generations each ; chain temperature : 0.2 ; sample frequency : 1,000 ; burn - in : 4,000,000 generations ) . chain stationarity and run parameter convergence were checked in tracer 1.5 . to test for homoplasy excess introduced by hybridization , we conducted a tree - based method as outlined by seehausen by removing single species from the dataset and observing the change in bootstrap values in the nj tree ( see also [ 34 , 40 ] ) . in theory , the inclusion of a hybrid taxon in a multilocus phylogeny introduces homoplasy with clades that contain its parental taxa . hybrid taxa should be intermediate to the parental taxa since they carry a mosaic of parental characteristics . thus , decreasing the amount of homoplasy in the dataset by removing the hybrid taxon should increase the bootstrap support for the clades that include the parental taxa or their descendants , whereas removing nonhybrid taxa should have no effect on the statistical support of other nodes ( figure 2 ) . testing for consistency between mtdna- and aflp - based tree topologies employed two different strategies . in a first test we evaluated whether our aflp - nj - topology can be explained by the mtdna data of koblmller et al . . using the mitochondrial sequences , we inferred the log likelihood of the aflp - nj - topology data by constraining maximum likelihood ( ml ) tree search to a topology identical to the species tree suggested by the nj analyses of our aflp data , applying the substitution model used in this previous study ( hky+i+g ) . to test for significant differences between the unconstrained and constrained mtdna topology we performed an ml - based shimodaira - hasegawa ( sh ) test ( full optimization , 1,000 bootstrap replicates ) in paup . in a second test we evaluated by means of a bayes factors approach whether the mtdna tree can be explained by our aflp data , and whether the aflp - nj and bi trees differ significantly from each other . we performed bi searches constraining the topology to that of the mtdna - topology and the interspecific relationships implied by the aflp - nj - tree in mrbayes 3.1.2 employing the same settings as above . bayes factor comparison using the harmonic means of the likelihood throughout different runs [ 77 , 78]among the three alternative phylogenatic hypotheses was performed in tracer 1.5 . values of 2 ln bf ( two times the difference between the harmonic means of the two models ) > 10 are considered strong evidence for support of one model over another . the final aflp dataset consisted of 659 unique loci with a mismatch error rate of roughly 3% , which falls within the acceptable limit for mismatch error rates as defined by . both the nj and bi analysis yielded largely congruent and well - supported topologies with only minor differences between them ( figures 3(a ) and 3(b ) ) . whereas all species were monophyletic in the nj tree , bathybates graueri was not resolved as a monophylum in the bi tree , but as paraphylum including the well - supported clade of the other large bathybates species ( figure 3(b ) ) . despite this minor topological difference , bayes factor comparison strongly supports the bi tree over the nj tree ( 2 ln bf = 26.828 ; figure 3(c ) ) . we note , however , that the two - state model implemented in mrbayes does not fully cover the complex genetic process of aflp evolution and thus provides accurate phylogenetic inference less likely than distance methods [ 67 , 79 , 80 ] . hence , the observed differences between aflp and nj tree topologies might be attributed to this problem . both the nj and bi analyses support the monophyly of all three genera with the genus trematocara representing the most ancestral branch ( figures 3(a ) and 3(b ) ) . within the genus bathybates , the small and morphologically most distinct member of the genus , b. minor , was sister taxon to the remaining large bathybates species . branch lengths among the large bathybates species are rather short and some received rather low statistical support , indicating a period of rapid cladogenesis . nevertheless , both nj and bi analyses revealed a largely consistent phylogenetic pattern within the large bathybates species . both sh - test and bayes factor comparison revealed significant differences between mtdna and aflp phylogenies ( sh - test : ln l of 9505.385 versus 9585.142 for mtdna versus aflp - nj - topology - constraint , p < 0.001 ; bayes factors : 2 ln bf of 88.576 between mtdna and aflp - bi - topology and 61.748 between mtdna and aflp - nj - topology , figure 3(c ) ) . the homoplasy excess test provided no evidence for introgressive hybridization ( data not shown ) . in the original classification of lake tanganyika cichlid tribes by poll , bathybates and hemibates were included in a tribe bathybatini as sister group to the trematocarini ( equivalent to the genus trematocara ) , a hypothesis supported both by lepidological as well as allozyme data . in contrast , based on morphological characteristics , stiassny and takahashi proposed a sister group relationship of bathybates and trematocara . currently , all three genera , bathybates , hemibates , and trematocara , are united in the tribe bathybatini . a previous mtdna phylogenetic study remained equivocal with regard to the two competing morphological classifications and suggested that bathybates , hemibates , and trematocara diverged rapidly from their common ancestor . in the present aflp phylogeny , the length differences between the most ancestral branches favour poll 's original classification with trematocara as sister group to hemibates + bathybates [ 55 , 57 , 58 ] . consistent with the mitochondrial phylogeny , the aflp data confirm the split between bathybates minor and the larger members of the genus bathybates with b. graueri as their most basal representative and identifies a period of rapid cladogenesis at the onset of the diversification of the large bathybates species . however , mtdna and aflp phylogenies differ significantly with respect to the branching pattern among the remaining large bathybates species . introgressive hybridization ( including the possibility of complete mtdna replacement ) and ancient incomplete lineage sorting are two alternative sources of topological disagreement between nuclear and mitochondrial trees [ 40 , 41 , 8183 ] , resulting in similar phylogenetic patterns that are difficult to resolve by strict hypothesis testing . circumstantial inference can be based on the fact that lineage sorting is expected to lag behind rapid cladogenetic events , such that the rapid radiation of the large bathybates species predisposes this clade to mitonuclear phylogenetic incompatibilities without implying postcladogenetic introgression [ 72 , 73 ] . likewise , monophyly of species in both mitochondrial and nuclear trees ( excepting the paraphyly of b. graueri in bayesian aflp tree ) and negative tests for homoplasy excess in the aflp data do not indicate the presence of hybrid taxa in the genus bathybates . these findings support our hypothesis that deepwater species are less prone to introgressive hybridization and hybrid speciation than littoral species , but reservations arise on the one hand from the possibility that events of introgression were not detected in our samples and data and given the power of our analyses , and on the other hand from the small number of species in the phylogeny . principally , rates of interspecific introgression may not differ between littoral and deepwater cichlids , but will nonetheless lead to higher incidences of introgression in the species - rich groups than in a less speciose clade . if this was the case , the lack of a signal of introgression in bathybates and hemibates would be fully explained by the low diversification rate of the lineage and hence limited opportunity for interspecific hybridization , without implying the evolution of complete reproductive isolation early on during diversification . the branching order of hemibates and the basal bathybates species , which is reconstructed congruently by mtdna and aflp markers , suggests repeated transitions between benthic and bathypelagic feeding mode ( ecological data from ) . the basal h. stenosoma represents a benthic generalist feeding on shrimps and various species of fish . the next split led to the specialized pelagic clupeid hunter b. minor , which mimics its prey in size and coloration and stages surprise attacks from within the sardine shoals . then , b. graueri took a step back to the benthic habitat , specialized on cichlid prey and evolved a large body size . the chronology of the following radiation of the large bathypelagic clupeid hunters and benthic cichlid hunters remains unresolved , but involved at least one transition from benthic to bathypelagic habitat preferences . depth preferences may vary between these species , such that speciation may have involved both niche and spatial segregation . the apparent ecological differentiation among lineages may have reduced the fitness of hybrids [ 8587 ] and may have promoted the evolution of a mate recognition system , perhaps based on the species - specific melanic patterns of male hemibates and bathybates . the efficacy of monochromatic black , silvery , and white body and fin patterns in mediating assortative mating in the dark , short - wavelength dominated environment has recently been demonstrated for deepwater cichlid species of lake malawi . these sympatric and morphologically similar species differ primarily in male nuptial patterns and their reproductive isolation is corroborated by genetic differentiation estimates . however , there is increasing evidence that color pattern is not the only cue for mate recognition in cichlids fish and it is possible and likely that auditory and olfactory cues play a role in mediating assortative mating in deepwater species , too . in concert with previous mitochondrial data , the present study provides an informative phylogeny of the species in the deepwater genera hemibates and bathybates . as far as the branching pattern can be resolved the rapid radiation within bathybates mirrors a burst of speciation observed in several other cichlid tribes of lake tanganyika and reveals a congruent cladogenetic pattern across vastly different habitats , which suggests some kind of synchronization by environmental factors [ 27 , 45 , 46 , 62 ] . consistent with the hypothesis that lineages evolving in the weakly structured deepwater habitat would develop stronger reproductive isolation than the allopatric lineages of the fragmented littoral , our data provided no evidence for the presence of hybrid taxa in the deepwater dwelling genus bathybates . in further support of the hypothesis , introgressive hybridization is also not indicated by the mitochondrial and aflp phylogenies of the lake tanganyika cichlid genus xenotilapia , which includes several deepwater - dwelling species comprising the prey of the benthic bathybates and hemibates . an increased sample size to evaluate this pattern will be attained by the analyses of additional open - water and deepwater species , for example , the tribe limnochromini and the genus trematocara . huge economic losses have been seen in livestock production causing problems in animals and humans . the use of chemotherapeutic drugs to control internal and external parasites is a widespread practice in livestock production and several broad - spectrum anthelmintics are available in the market for the control of helminthosis . currently , failure of anthelmintics and reduced efficacy due to resistance of nematodes in sheep and goats are becoming a threat in some countries and are of increasing importance in certain african countries [ 2 , 3 ] . according to waller , most of the nematodes of domestic animals have shown resistance to common anthelmintics especially in warm and humid parts of the world and this has been suspected to be due to frequent dosing and poor therapeutic strategies [ 58 ] . drug resistance was first reported in the late 1950 's [ 9 , 10 ] and early 1960 's [ 1114 ] . by the nineties , recently , lots of works have been done on this within and outside of africa [ 1522 ] . this has led to the search for a more eco - friendly anthelmintic that can tackle the resistance issue . v. amygdalina has been shown to have potentials as an anthelmintic in some animal species and humans [ 2325 ] . adediran et al . worked on v. amygdalina as an anthelmintic on goats though it was the stalk and leaves that were fed raw to the goats . a comparison to establish the efficacy of this plant relative to well known and established synthetic anthelmintics to which resistance has been reported is therefore needed . we investigated the response of anthelmintic - naive goats that have acquired helminth infections naturally to treatment with three commonly used anthelmintics and v. amygdalina in southwest nigeria . faecal egg count reduction test ( fecrt ) , the commonest nematicide evaluation means [ 26 , 27 ] , has some shortcomings primarily due to the fact that egg count does not correspond to worm burdens . the fecrt can however be standardized by randomization of animals into treatment groups for even dispersion of egg counts and drug effectiveness , accurate dosing to make sure no administered drug is lost , and proper tagging of animals in each treatment group to ensure correct sampling . our study was conducted with strict adherence to the standardisation method to ensure accuracy of our data on the drugs we evaluated . the objective of this study was to evaluate resistance in west african dwarf goats to levamisole , albendazole , and ivermectin and v. amygdalina . the goat owners were interviewed before purchase to establish the treatment history of the herd . they were allowed to stabilize by giving feed , water , and mineral lick supplement . after two weeks , the animals were divided into four treatment groups ( group a , ivermectin treatment , group b , levamisole treatment , group c , albendazole treatment , and group d , v. amygdalina treatment and group e was untreated reference group ) . the drugs ( ivermectin , levamisole , and albendazole ) were acquired from a veterinary drug store located in the study area while the plant , v. amygdalina , was collected from the environment and taken for authentication at the department of botany in the institution . the v. amygdalina leaves after identification were taken to the laboratory where the aqueous extract was prepared . two kilograms of v. amygdalina leaves was hand - washed in two litres of distilled water and passed through a 2 mm sieve and the filtrate was used within one hour . each animal in group a was given a dose of ivermectin injection of 0.2 mg / kg body weight subcutaneously individually . each animal in group b was given levamisole injection at a dose rate of 8.0 mg / kg body weight subcutaneously ; albendazole bolus was administered orally to each animal in group c at a dose rate of 10 mg / kg while the animals in group d were drenched with 5 mls per kg body weight of the plant aqueous extract . faecal samples were collected fresh from the rectum of the animals pretreatment and posttreatment in air - tight bags and taken to the laboratory for analysis . posttreatment samples were collected on days 1 , 2 , 4 , 7 , and 14 . the samples were then subjected to the faecal egg counts using the mcmaster method described by . the faecal culture , floatation , and sedimentation techniques of faecal examination faecal egg counts , helminth ova , and larvae identified were recorded . the percentage reduction in faecal egg count was calculated for each treatment group using the arithmetic mean of the egg count for each group at day 7 with the fecr version 4 software . resistance to an anthelmintic was considered to be present if the percentage reduction in egg count was less than 95% , and the lower 95% confidence limit is less than 90 . the helminth ova identified during pretreatment faecal examination were haemonchus contortus , trichostrongylus spp . , ostertagia ostertagi , oesophagostomum spp . , chabertia sp . , , ostertagia ostertagi , oesophagostomum spp . , charbetia sp . , and strongyloides were identified at larva culture . h. contortus was identified in 70% of the goats , trichostrongylus in 61% , oesophagostomum in 56% , chabertia in 15% , and strongyloides in 10% . posttreatment , h. contortus larvae were the most common followed by trichostrongylus spp . and oesophagostomum spp . the percentage reduction in faecal egg count was 96% for each of ivermectin , levamisole , and albendazole and 100% for v. amygdalina . the statistical analysis showed a lower 95% confidence limit ( cl ) of 89 for ivermectin and levamisole and 91 for albendazole ( table 1 ) . this result implies that there is low resistance in nematodes of goats to ivermectin and levamisole while there is susceptibility to albendazole and v. amygdalina . the arithmetic means of the faecal egg count for the different groups are presented in figure 1 . anthelmintic drugs such as albendazole , ivermectin , and levamisole are used to treat parasitic infection in ruminants . the regular use of these drugs and sometimes improper use make the issue of resistance inevitable . according to , it has been observed that frequent use of the same group of anthelmintic , use of anthelmintics in suboptimal doses , prophylactic mass treatment of domestic animals , and frequent and continuous use of a single drug have contributed to the widespread development of anthelmintic resistance in helminths . research reports of anthelmintic resistance in goats in africa have been relatively few although this is more likely to be as a result of low number of studies carried out in this area and not because cases of resistance were not observed . this present study showed that there is a measure of resistance to ivermectin and levamisole in nigerian wad goats while albendazole is still an effective drug . also v. amygdalina has been shown to be an alternative source of treating helminths in goats . it is recommended to keep animals on dry lot for 12 to 24 hours after deworming to ensure that eggs and larvae that survive the anthelmintics are not deposited on safe pasture . this presupposes that 24 hours after treatment there should not be viable helminth eggs ; therefore , the hatching of viable eggs seven days after treatment in our study is an indication that the worms survived . ivermectin , being the most widely used because of its effect against ectoparasites , has been grossly overused ; hence it is highly susceptible to resistance development . this is corroborated by the works of who worked in howell et al . in the united states . our results however differ from the works of who worked on ivermectin in sheep nematodes in oyo state , nigeria , but did not record any resistance . this is possibly as a result of the different assays used and the species of small ruminant involved . ademola used sheep and the larval development assay ( lda ) while goats were used in this study and the fecrt was used as the assay . although goats and sheep have similar genera of nematodes , it has been reported that nematodes in goats herd usually develop anthelmintic resistance more rapidly . also , the fecrt we used is an in vivo assay while lda is an in vitro assay which according to may be useful as a supplement to fecrt . in ethiopia , the reports [ 3439 ] that albendazole , ivermectin , and levamisole were effective in goats though resistance to albendazole was suspected are also at variance with the results in this study . difference in location , brand of drug available , and breed of goat used for experiments and level of resistance already developed by helminths are likely reasons for the disparity . levamisole in goats has been known to produce adverse reactions such as muscle tremors and hyperesthesia with irritability even at recommended doses ; subsequently the subcutaneous administration inducing a lower blood peak of the drug is often preferred in goats . based on the adverse reactions in goats , levamisole is therefore often administered with caution using the lower dose range which may at times result in suboptimal dosing when given through the subcutaneous route , resulting in the gradual development of resistance . the 100% efficacy recorded for v. amygdalina is also corroborated by an earlier report by . the implication of this is that the plant could be a good alternative to the commonly used synthetic anthelmintics . the plant is also readily available and it is found as a shrub in most rural backyard gardens in developing countries to which nigeria belongs . the study has shown that the common anthelmintics used in west african dwarf ( wad ) goats in nigeria are still effective although low level of resistance was recorded for ivermectin and levamisole . however , the presence of any degree of resistance is of concern to small ruminant production . also , we conclude that vernonia amygdalina presents a ready alternative that would likely take care of anthelmintic resistance in common goat nematodes . strategies recommended to control helminths include a better use of existing drugs , use of vaccines for helminths , growth regulators , and biological control . however , maintaining parasites in refugia and not exposing them to anthelmintics have been identified as key in controlling and delaying the development of resistance because susceptible genes are preserved . according to chiejina and behnke , the wad goats should be explored for their unique resilience and genetic improvement and looked into reducing the use of chemical anthelmintics . in line with these recommendations , better drug - use and control as well as targeted and planned treatment routines should be carried out to keep resistance low and ensure adequate control of helminth parasites in wad in southwest nigeria . use of ethnoveterinary plants like v. amygdalina as an alternative source for effective anthelmintic treatment is also highly recommended . the plant presents an environmentally friendly alternative that will essentially reduce reliance on use of chemicals .

hybridization among littoral cichlid species in lake tanganyika was inferred in several molecular phylogenetic studies . the phenomenon is generally attributed to the lake level - induced shoreline and habitat changes . these allow for allopatric divergence of geographically fragmented populations alternating with locally restricted secondary contact and introgression between incompletely isolated taxa . in contrast , the deepwater habitat is characterized by weak geographic structure and a high potential for gene flow , which may explain the lower species richness of deepwater than littoral lineages . for the same reason , divergent deepwater lineages should have evolved strong intrinsic reproductive isolation already in the incipient stages of diversification , and , consequently , hybridization among established lineages should have been less frequent than in littoral lineages . we test this hypothesis in the endemic lake tanganyika deepwater cichlid tribe bathybatini by comparing phylogenetic trees of hemibates and bathybates species obtained with nuclear multilocus aflp data with a phylogeny based on mitochondrial sequences . consistent with our hypothesis , largely congruent tree topologies and negative tests for introgression provided no evidence for introgressive hybridization between the deepwater taxa . together , the nuclear and mitochondrial data established a well - supported phylogeny and suggested ecological segregation during speciation . anthelmintic drug resistance has led to the search for alternatives in controlling helminth infections . fifty west african dwarf goats without history of anthelmintic treatment were divided equally into five groups . group a was treated with ivermectin injection subcutaneously , group b with levamisole subcutaneously , group c with albendazole orally , and group d with aqueous extract of vernonia amygdalina and group e was untreated control . faecal samples were collected before treatment from each animal and larval culture was carried out . faecal egg count reduction ( fecr ) test was carried out for each group and the data analysed using fecr version 4 to calculate percent reduction in faecal egg count . predominant helminth infections from larval culture were haemonchus contortus ( 70% ) , trichostrongylus spp . ( 61% ) , and oesophagostomum spp . ( 56% ) . mixed infection was present in all the animals . from the fecr test vernonia amygdalina extract was more effective against helminths ( 100% ) , compared to ivermectin 96% , levamisole 96% , and albendazole 99% . the lower 95% confidence limit was 89 for ivermectin and levamisole and 91 for albendazole . there is low resistance to ivermectin and levamisole and susceptibility to albendazole while v. amygdalina has great potentials that could be explored for the treatment of helminth diseases in goats .

maca ( lepidium meyenii walp . ) is an endemic highland crop of the central andes which is grown from central peru to bolivia and northwestern argentina [ 1 , 2 ] . this plant has great potential as an adaptogen and appears to be promising as a nutraceutical in the prevention of several diseases . maca roots have been traditionally used to increase the rate of fertility in both humans and livestock . over the past 20 years , commercial maca products have gained popularity as dietary supplements for aphrodisiac purposes and for increasing fertility and stamina . recently , maca industry develops fast in yunnan province , china . in yunnan , until the end of 2010 , maca growing and promotion areas reached 175 hm and maca yield achieved 780 t , taking up to 90% and 93% , nationwide . mineral elements in food are very important because the quality of many functional foods and medicines depends on the content and type of minerals . in the us , national surveys show that micronutrient inadequacies are widespread and mineral supplement helps fulfill micronutrient requirements in adults and children . up to now , only limited studies on selected elements in maca from different origins have been carried out [ 10 , 11 ] . moreover , in some studies , no reference material had been certified for elemental analysis , so there has been doubt about accuracy of the determination . in the present study , inductively coupled plasma optical emission spectroscopy ( icp - oes ) was used to determine the contents of eight elements ( b , co , cr , cu , li , na , ni , and zn ) in maca samples , and a comparison was made between the samples from china and peru . standard sample solutions of b , co , cr , cu , li , na , ni , and zn obtained from standard material center of china were used to make a mixed calibration curve in the range of 0400 g ml . ten maca samples were collected from four places of yunnan , china , and four samples were from peru . these samples were washed with deionized water thoroughly , dried to a constant weight , grounded into powder using an agate mortar , passed through a 60-mesh sieve , and stored in the plastic bags . 500 mg of each maca sample was weighed into an acid washed teflon digestion tube . 8 ml of hno3 , 2 ml of h2o2 , and 1 ml ultrapure water were added to the vessel . samples were digested in a microwave dissolver ( ethos one , milestone , italy ) equipped with ptfe vessels . the extract was transferred into a cuvette and made up to 25 ml with ultrapure water . simultaneous multielement detection of b , co , cr , cu , li , na , ni , and zn was performed with icp - oes ( icpe-9000 , shimazu , japan ) . the optimal instrumental conditions for icp - oes are shown in table 2 . blank experiments were prepared in the same way . to ensure the precision of the experiment , the certified reference material gbw10028 ( dried herb powder astragalus membranaceus ) was used for validation of the method ( table 4 ) . the relative standard deviation ( rsd ) was found to be below 8% , and the recoveries range from 92% to 109% , which proved that this method was accurate and precise . contents of b , co , cr , cu , li , na , ni , and zn in maca samples are shown in table 5 . boron affects fat and lipid metabolism , minerals and mineral metabolism , vitamin d , and bone development . our b values in maca from china and peru were 8.121 mg kg dw and 6.612 mg kg dw , respectively , which were in agreement with that reported in the literature ( 8.8 mg kg dw ) . cobalt is an essential micronutrient in the form of vitamin b12 , but cobalt is toxic in larger doses or long - term exposure at a low level . the adverse effects of cobalt relate to various organs and tissues and may include a possible carcinogenic potential . the contents ( mg kg dw ) of co , cr , and li were , respectively , < 0.023 , < 1.13.5 , and 0.0200.17 for the samples from china and < 0.023 , < 1.12.3 , and 0.0350.063 from peru . up to now , there was no other report on the contents of co , cr , and li in maca . its compounds show vast array of biological actions , such as anti - inflammatory , antiproliferative , and biocidal activities . cu contents in the samples from china ( 2.531 mg kg dw ) were higher than the samples from peru ( < 2.1 mg kg dw ) . published data available on cu in maca from china were 4.032 mg kg dw [ 10 , 11 , 13 , 18 ] , while those from peru were 1.562 mg kg dw [ 10 , 11 , 1922 ] . 302600 mg kg dw which is similar to the value ( 672400 mg kg dw ) in maca from china in literatures [ 11 , 13 , 18 ] . however , our data on na in the samples from peru ( < 3041 mg kg dw ) are lower than the values reported ( 110190 mg kg dw ) in the literatures [ 11 , 1922 ] . the contents of ni in the samples were 0.0854.5 mg kg dw from china and 0.681.7 mg kg dw from peru . however , higher value ( 11.3 mg kg dw ) was found in the literature on the maca from china . zinc is essential for a number of physiological functions and plays a significant role in many enzyme actions in the living systems . zn contents in samples were found to be 1939 mg kg dw from china and 2739 mg kg dw from peru , which were in accordance with the values for maca in literature available ( 2589 mg kg dw from china and 1658 mg kg dw from peru ) [ 10 , 11 , 13 , 1822 ] . cu contents in all of the maca samples from china , as well as na contents in two samples from china , were remarkably higher than those values in other samples . myrtle ( myrtus communis l. ) is an evergreen shrub belonging to the family of mirtaceae that grows spontaneously throughout the mediterranean area . in italy it grows along the coast and in the inner hills , and it is spread especially in the islands , where it is one of the most characteristic species . myrtus communis had history in the popular and traditional medicine : the essential oil obtained from leaves and , sometimes , flowers and berries has been used for its tonic and balsamic properties , and it is used in flavour and fragrance industries . in sardinia natural formations are still the main source for the production of a traditional liqueur , that every year reaches 3 million bottles . essential oils are gaining remarkable interest for their potential multipurpose use as antioxidant , antibacterial , and antiseptic agent [ 14 ] ; the essential oil obtained from the leaves was used in the past for the treatment of lung disorders . the isolation of essential oils from myrtus communis leaves is usually obtained by hydrodistillation method with a clevenger - type apparatus , according to the italian official pharmacopoeia . the chemical composition of the essential oils , analysed by gas / cromatography ( g / c ) , generally exhibits -pinene , 11% ; 1,8-cineole , 16% ; linalool , 12% ; -terpineol , 7% ; and limonene , 5% . the sardinian myrtle oil is characterized by the lack of myrtenyl acetate and by a higher content of limonene . . showed the biological activities of tannins , including anticancer and antioxidant . in our previous studies ( data not published ) the antimicrobial properties of myrtus essential oil against several clinical strains and in particular against helicobacter pylori were studied , and we obtained encouraging results . considering these results , in this study , we have used this essential oil towards strains of m. tuberculosis and strains of mycobacterium avium subsp . in this study we investigated the antimicrobial properties of the essential oil of myrtus communis against two reference strains : m. tuberculosis h37rv ( virulent strain ) and m. tuberculosis h37ra ( avirulent strain ) and 8 clinical isolates of m. tuberculosis resistant to one or more drugs , collected in the department of biomedical sciences , microbiology of the university of sassari , italy . we also studied the antimicrobial properties of the essential oil towards two human strains of mycobacterium avium subsp . the antibacterial activity of the oil was assessed by the proportional method used for mycobacterium tuberculosis as described in nccls - national committee for clinical laboratory standards . briefly , isolate suspensions of m. tuberculosis in 7h9 broth were adjusted to an optical density of 1 mcfarland , and two dilutions , 10 and 10 , were plated onto 7h10 agar with a different concentration of essential oil ( 16% , 14% , 12% , 10% , 8% , 4% , 2% , 1% , and 0.17% v / v ) . we also investigated the antimicrobial properties of some components of m. communis and , in particular , limonene , 1 - 8 cineole and -pinene , using the proportional method . in table 1 we report the data obtained with the essential oil in toto . towards all the strains of m. tuberculosis , including the extensively drug - resistant ( xdr ) , the oil of m. communis tested showed an mic of 0.17% ( v / v ) , whereas against the two m. paratuberculosis strains showed an mic of 2% ( v / v ) . subsequently we tested each different compound ( limonene , 1 - 8 cineole , -pinene ) to see if it had different antimicrobial properties . as far as limonene concerned , it showed towards all the strains an mic of 2% ( v / v ) ; about 1 - 8 cineole , for 4 strains , including h37rv , the mic was of 2% ( v / v ) , while the mic shown for other 4 strains was of 16% ( v / v ) ; -pinene showed an mic of 1% ( v / v ) for 3 strains , and for one strain an mic of 2% ( v / v ) , for 3 others an mic of 8% ( v / v ) , and for only one mic was of 16% ( v / v ) ( table 2 ) . limonene and -pinene are monoterpenic that , according to the literature , are used as expectorant , antalgic , revulsive , antitussive , mucolitic , and decongestant ; about 1 - 8 cineole it is an oxide used as expectorant , mucolitic , and decongestant . we compared the activity of our essential oil with four standard antitubercular drugs : streptomycin , isoniazid , rifampin , and ethambutol , performing according to international protocols . two clinical strains were rifampicin resistant , three strains were resistant to 2 drugs ( one streptomicin and rifampicin resistant , one streptomicin and isoniazid resistant , and one isoniazid and rifampicin resistant ) , one was resistant to 3 drugs ( streptomicin , isoniazid , and ethambutol ) . the essential oils screened in toto have a better antimicrobial activity than each single compound against all mycobacteria tested . the results presented here may contribute to the knowledge of the antimicrobial properties of myrtle and our aim is to carry on further studies . the chemical composition of the essential oil of m. communis exhibited qualitative differences that depended on different geographical areas and from the season in which the leaves were picked up . the essential oil that we used for antimicrobical in vitro assay contained a high quantity of monoterpenic and oxide that , according to literature , do not have antimicrobical activity . from our studies emerged that the essential oil in toto might have a good activity towards m. tuberculosis , although the individual compounds ( except -pinene ) showed in all the strains a higher mic . the results from the myrtle oil in toto showed a good activity towards m. tuberculosis but not toward m. paratuberculosis . the mic registered against m. tuberculosis was 0.17% ( v / v ) in comparison with an mic of 2% ( v / v ) observed toward m. paratuberculosis . a limit of our study is the small amount of the essential oil that did not allow to perform the mic towards all the strains used . testing and large clinical studies are necessary to verify the potential use of the essential oils of myrtle as antitubercular drug . given the excellent results that we obtained in this study , we would expand the research with further studies , to value the possible cytotoxic effects , and eventually to perform tests using in vivo mouse model .

contents of eight mineral elements in maca ( lepidium meyenii walp . ) from china and peru were determined by inductively coupled plasma optical emission spectroscopy . cu contents in maca samples from china ( 2.531 mg kg1 dry weight , dw ) were higher than the samples from peru ( < 2.1 mg kg1 dw ) . na in two samples from china was found to be significantly of high content ( 2400 and 2600 mg kg1 dw ) . the contents ( mg kg1 dw ) of b , co , cr , li , ni , and zn were , respectively , 8.121 , < 0.023 , < 1.1~3.5 , 0.0200.17 , 0.0854.5 , and 1039 for the samples from china , while being 6.612 , < 0.023 , < 1.1~2.3 , 0.0350.063 , 0.681.7 , and 2739 for the samples from peru . mycobacterium tuberculosis is the etiological agent of tuberculosis . the world health organization has estimated that 8 million of people develop active tb every year and the situation is complicated by an increase of mycobacterium tuberculosis strains resistant to drugs used in antitubercular therapy : mdr and xdr - tb . myrtle leaf extracts , used as an antiseptic in sardinian traditional medicine , have strong antibacterial activity as several investigations showed . in this study we investigated the antimicrobial properties of the essential oil of myrtus communis against clinical strains of m. tuberculosis and m. paratuberculosis .

additional supporting information may be found in the online version of this article at the publisher 's web site . the mesenchymal tumors of the gastrointestinal ( gi ) tract have been called as various names including stromal tumor of unknown malignant potential ( stump ) , gastrointestinal stromal tumors ( gist ) , gastrointestinal autonomic nervous tumors ( gant ) and gastrointestinal pacemaker cell tumors ( gipact ) ( 1 , 2 ) . after stem cell kinase receptor cd117 ( c - kit ) was introduced , the gists presumed to derive from stem cells differentiating to the gi pacemaker cells , known as interstitial cells of cajal ( icc ) , because nearly all gists are implicated with c - kit gene abnormality and cd117 ( c - kit ) overexpression ( 1 ) . even though the new entity of gist were initially introduced from the ultrastructural observation , the number of ultrastructural studies on gists ( 3 - 5 ) have been limited , compared to the quite a few ultrastructural reports of gants ( 6 - 8 ) . gists can occur primarily in every intraabdominal site including omentum , mesentery or peritoneum in addition to gi tract . cd117 is being demonstrated increasingly in a wide variety of tumors including inflammatory myofibroblastic tumors , intraabdominal desmoids ( 9 ) , angiomyolipoma and perivascular epithelioid cell tumors ( 10 ) , and malignant peripheral nerve sheath tumor ( 11 ) , which give rise to quite confusion . we investigated the gist and gant ultrastructurally to identify the specific ultrastructure of these tumors , any clue of their histogenesis and the comparison of the gists and gants . fifteen cases of gists including 2 cases of gants were surgically resected at the ilsan paik hospital , inje university from february 2000 to may 2003 . among them , thirteen cases had been carried out the electron microscopic studies . the preservation and quality of processing of all cases were excellent or enough for evaluation . the pathologic diagnoses and grading of gists were made by 3 pathologists , based on morphology and immunohistochemistry with miettinen et al . 's grading system of gists , which recommended the grading by tumor size and mitotic rate along with the location ( 12 ) . we excluded c - kit negative pure smooth muscle tumors , schwannomas and other gastrointestinal mesenchymal tumors . age range was 24 to 76 yr old ( mean : 58.4 yr old ) . the size range was 0.3 cm to 39 cm in greatest diameter ( mean : 9.1 cm ) . eight cases were located in the stomach , 5 cases were in the small intestine , one in the mesocolon and one in the liver . the last one was late recurrent gist 10 yr after resection of primary gastric gist . after sampling for electron microscopic ( em ) study , tumor tissue was fixed in 10% neutral formalin solution . for em study the tissue fixed in 3.0% glutaraldehyde was washed in cold 0.1 m phosphate buffer ( ph 7.4 ) and postfixed in 1% osmium tetroxide , buffered with 0.1 m phosphate . sections were cut with an ultramicrotome , stained with uranyl acetate and lead citrate , and examined by em ( jeol , japan ) . for immunohistochemical study , all representative formalin ( cold 10% buffered ) fixed and paraffin - embedded tumor tissues were cut to 2 - 3 m thickness . all antigens used here including c - kit , cd34 , vimentin , smooth muscle actin ( sma ) , desmin , s100 protein , synaptophysin , cd56 and ki67 ( mib-1 ) were summarized in table 2 . after deparaffinization and rehydration , the sections were subjected to high temperature antigen unmasking in a citrate buffer in the autoclave ( 121 ) for 20 min . after blocking in 2% skimmed milk , the sections were incubated with primary antibodies with ideal dilution for 60 min at room temperature . the sections were incubated with biotinylated secondary antibody , which is polyvalent and universal ( prediluted , immunotech , marseille , france ) , and the expression was detected using the peroxidase labeled streptavidin biotin complex technique according to the manufacturer 's recommendations . normal small intestine and resident mast cells were served as the positive tissue control for all used antibodies . for antibody control , most tumors were large and ranged from 0.3 cm to 17.5 cm ( mean : 9.1 cm ) . the cut surfaces of the tumors showed pinkish gray fish flesh to granular appearance with some hemorrhage and necrosis . the tumors were mainly located in the muscular wall of the intestine with or without involvement of the mucosa and serosa . one case of the small intestinal gists showed overt multiple mesenteric metastatic nodules . among thirteen cases of gists , focal cytoplasmic vacuolization was often seen . no signet ring - like or oncocytic cells or cells with a clear cytoplasm generally mitotic counts were low but various in each case from 0/50 to 40/50 hpf . eleven cases were pure gist , 2 cases were gist with smooth muscle differentiation and 2 cases were gant . according to the miettinen et al . report ( 2002 ) , two cases were malignant having metastasis , six cases were probably malignant , three cases were low malignant potential and two cases were categorized as probably benign . all of the fifteen gists including two gants were robustly positive for vimentin ( 100% ) and cd117 ( c - kit ) ( 100% ) ( fig . 1 - 3 ) . 1 ) , strongly but focally . gants revealed strong but focally positive for s-100 protein and neurofilament ( nf ) ( fig . synaptophysin was only focally and less intensely immunoreactive in one case of gants ( fig . p53 was positive in 61.5% ( 8/13 cases ) of gists and 100% ( 2/2 cases ) of gants . in normal adults stomach and intestine , well - developed c - kit immunoreactive icc were present around the myenteric plexus and muscle layers ( fig . icc had well developed cytoplasmic organelles such as endoplasmic reticulum , mitochondria , golgi apparatus and caveolae . the polygonal cells usually had rich interdigitating cytoplasmic processes and most of them were villous ( filopodia - like ) ( fig . the nuclei were oval to irregularly elongated and generally heterochromatic , but some nuclei were exceptionally euchromatic ( fig . 5 , 6 ) . there were also abundant wavy intermediate filaments , which were generally dispersed in the cytoplasm . the cytoplasmic organelles were abundant , smooth and rough endoplasmic reticulum , mitochondria , lysosomes and golgi apparatus . there were desmosome or hemidesmosome - like junctions as well as a few gap junctions ( fig . a discrete external lamina was usually absent but incontinuous external lamina material was present in half of the gists ( fig . caveolae or pinocytotic vesicles were rarely seen . a case with focal immunohistochemical smooth muscle differentiation showed ultrastructual differentiation toward smooth muscle , such as cytoplasmic myofilaments ( fig . small round dense core neurosecretory granules were observed in gants , measuring 40 - 150 nm in diameter ( fig . 7 ) , but gists also had electron dense granules , favor lysosomes , which could be confused with neurosecretory granules . no definite synaptic junction , synaptic vesicles or microtubles most tumors were large and ranged from 0.3 cm to 17.5 cm ( mean : 9.1 cm ) . the cut surfaces of the tumors showed pinkish gray fish flesh to granular appearance with some hemorrhage and necrosis . the tumors were mainly located in the muscular wall of the intestine with or without involvement of the mucosa and serosa . focal cytoplasmic vacuolization was often seen . no signet ring - like or oncocytic cells or cells with a clear cytoplasm generally mitotic counts were low but various in each case from 0/50 to 40/50 hpf . eleven cases were pure gist , 2 cases were gist with smooth muscle differentiation and 2 cases were gant . according to the miettinen et al . report ( 2002 ) , two cases were malignant having metastasis , six cases were probably malignant , three cases were low malignant potential and two cases were categorized as probably benign . all gants were probably malignant . all of the fifteen gists including two gants were robustly positive for vimentin ( 100% ) and cd117 ( c - kit ) ( 100% ) ( fig . 1 - 3 ) . 1 ) , strongly but focally . gants revealed strong but focally positive for s-100 protein and neurofilament ( nf ) ( fig . synaptophysin was only focally and less intensely immunoreactive in one case of gants ( fig . p53 was positive in 61.5% ( 8/13 cases ) of gists and 100% ( 2/2 cases ) of gants . in normal adults stomach and intestine , well - developed c - kit immunoreactive icc were present around the myenteric plexus and muscle layers ( fig . in normal controls , the myenteric icc had heterochromatic and lobulated nuclei with slender cytoplasmic processes ( fig . icc had well developed cytoplasmic organelles such as endoplasmic reticulum , mitochondria , golgi apparatus and caveolae . the polygonal cells usually had rich interdigitating cytoplasmic processes and most of them were villous ( filopodia - like ) ( fig . the nuclei were oval to irregularly elongated and generally heterochromatic , but some nuclei were exceptionally euchromatic ( fig . 5 , 6 ) . there were also abundant wavy intermediate filaments , which were generally dispersed in the cytoplasm . the cytoplasmic organelles were abundant , smooth and rough endoplasmic reticulum , mitochondria , lysosomes and golgi apparatus . there were desmosome or hemidesmosome - like junctions as well as a few gap junctions ( fig . a discrete external lamina was usually absent but incontinuous external lamina material was present in half of the gists ( fig . immunohistochemical smooth muscle differentiation showed ultrastructual differentiation toward smooth muscle , such as cytoplasmic myofilaments ( fig . small round dense core neurosecretory granules were observed in gants , measuring 40 - 150 nm in diameter ( fig . 7 ) , but gists also had electron dense granules , favor lysosomes , which could be confused with neurosecretory granules . no definite synaptic junction , synaptic vesicles or microtubles were found in gants . in all two cases of gants , in 1983 , mazur and clark first introduced the vague term ' gastrointestinal stromal tumor ' because many mesenchymal tumors of the gastrointestinal tract did not express the known markers of the smooth muscle or schwann cell ( 13 ) . perineurial or mesenchymal nerve sheath cells of myenteric plexus were thought as a possible origin of these stromal tumors ( 13 ) . after that , owing to lots of efforts to clarify these tumors , a new entity of gists and its pathogenesis have been established . gists are most commonly occurred in the stomach ( 60 - 70% ) and the small intestine ( 20 - 30% ) . in addition , the tumors arose in the esophagus , colon , rectum , omentum and mesentery have been reported less than 10% . for predicting prognosis , ma et al . ( 14 ) graded the gists into benign ( < 5 cm in size and < 5 mitosis/50 hpf ) , malignant ( > 5 mitosis/50 hpf ) , and borderline lesions ( > 5 cm in size but < 5 mitosis/50 hpf ) regardless of site . however , several reports suggested that stromal tumors should be analysed as a site - specific fashion because tumors from different location brought out different growth patterns and prognosis . according to suggestion of miettinen et al . in the grading ( 12 ) , 2 of our gists were malignant having metastasis , 6 were probably malignant , 3 were low malignant potential and 2 were probably benign . all 2 gants were probably malignant . for making a diagnosis of gists , immunohistochemical stainings of the cd117 and ultrastructural examination are required , because morphological heterogeneity of the gists gives rise to quite confusion . except rare cases , the diagnosis of the gist should apply to the neoplasm displaying c - kit immunoreactivity ( 15 ) . the exceptions were very clearly described in the paper of christopher et al . ; including fixation artifact , technical error of immunostaining , sampling error , cessation of kit expression perhaps following sti-571 therapy and very rare cases ( 2% ) with real lack of kit mutation and/or kit expression ( 15 ) . gists can show focal differentiation to autonomic nerve or smooth muscle , but pure smooth muscle or schwann cell derived tumors , such as leiomyoma , leiomyosarcoma and schwannoma are not gists . morphological heterogeneity of gists such as extreme epithelioid or spindle shape of the tumor cells or marked myxoid or cystic changes are well known . tumors of our cases also showed variable histology , but immunohistochemically , our cases were compatible with gists or gants based on expression of vimentin ( 100% ) , cd117 ( 100% ) , and cd34 ( 66.7% ) . two cases with focal immunoreactivity for sma , were diagnosed as gists with smooth muscle differentiation . gants showed variable immunoreactivity for s-100 protein , nf , synaptophysin in addition to cd117 . besides a tumor size over 5 cm , mitotic rate still seems to be the best predictor for prognosis of gist . also mib-1 proliferation index above 4% and p53 positivity indicate a more aggressive course ( 17 , 18 ) . however , cunningham et al . found no relationship between p53 immunoreactivity and survival ( 19 ) . in our series , p53 was positive in high percentage ( 69.2% , 9/13 cases ) of gists and 100% ( 2/2 cases ) of gants , reflecting the high percentage of high - grade tumors . until now , the ultrastructural reports of gists ( 3 - 5 ) were rare than those of gants ( 6 - 8 ) . gists may originate from the pluripotent precursor cells of the icc ( 5 ) . according to the electron microscopic study on kitw - lacz/+ transgenic mice , kit - expressing cells in the outer parts of the musculature had scattered caveolae , inconspicuous basal lamina and numerous mitochondria , whereas those in the submuscular region they had more pronounced myoid features . the iccs are located in most parts of the digestive system , especially in myenteric plexus , submucosal meissner 's plexus and third plexus . iccs demonstrate species- , region- , and location - dependent variations in the ultrastructures ( 20 ) . they are communicated each other or with smooth muscle by peg and socket or gap junctions . its cytoplasm characteristically possesses cisternae of smooth endoplasmic reticulum and intermediate filaments ( 21 ) . in present study , normal iccs present in the myenteric plexus had the similar ultrastructures as the previously reported ones . in the gists , the polygonal shaped neoplastic cells usually had rich interdigitating or villous cytoplasmic processes , but spindle shaped cells rarely showed villous processes . the tumor cells were closely apposed each other in all cases but spindle cells had more interstitium , which was fibrous . in our cases , small round neurosecretory granules were definitely found in gants , measuring 40 - 150 nm in diameter . interestingly , gists also had many small electron dense granules , which were compatible with lysosomes . the prominent cytoplasmic organelles were quite a few smooth and rough endoplasmic reticulum , and some mitochondria , lysosomes and golgi apparatus . abundant intermediate filaments were dispersed in the cytoplasm and cytoplasmic processes . in 2 cases , desmosome - like or hemidesmosome - like junctions as well as one of our cases was a recurrent gist occurred in the liver 10 yr lag period after resection of gastric gist , which was previously diagnosed as leiomyosarcoma . in all 15 cases , the caveolae were rarely seen except one case , which showed smooth muscle differentiation . all of these features were comparable with ultrastructure of icc cells except reduced caveolae and gap junctions ( 20 - 22 ) . described that ultrastructural site - specific similarities and differences of the gists ( 4 ) , however , our cases showed different ultrastructures according to the cell shape , i.e. , whether it is epithelioid or spindle , irrespective of location . lee et al . described that the clinicopathologic , histologic , immunohistologic , and molecular features of gants were similar to gists , indicating that gants merely represents a phenotypic variant of gists ( 23 ) . in the ultrastructural level , although gists and gants have overlapping features , the ultrastructural characteristics of gants were dense core neurosecretory granules and skenoid fibers . neurosecretory granules and skenoid fibers are not specific for gant because the latter can be seen in smooth muscle tumors and the former are present in various neuroendocrine tumors and small cell carcinomas . however , when we differentiate gants from the gists , identification of these two features may be very helpful . in our case from exclusion diagnosis of smooth muscle or schwannian tumors on the base of absence of hallmarks of these tumors and inclusion diagnosis of gists and gants can be easily made from electron microscopic examination . the most common and important ultrastructural features of gists were rich villous cytoplasmic processes and dispersed intermediate filaments and those of gants were neurosecretory granules and skenoid fibers . ultrastructural analysis gives much benefit for identification of the lineage differentiation of neoplastic cells and differential diagnosis from other mesenchymal tumors and between gist and gant .

this report describes that a regular positive electrospray ionization mass spectrometry ( ms ) analysis of terpendoles often causes unexpected oxygen additions to form [ m + h + o]+ and [ m + h + 2o]+ , which might be a troublesome in the characterization of new natural analogues . the intensities of [ m + h + o]+ and [ m + h + 2o]+ among terpendoles were unpredictable and fluctuated largely . simple electrochemical oxidation in electrospray ionization was insufficient to explain the phenomenon . so we studied factors to form [ m + h + o]+ and [ m + h + 2o]+ using terpendole e and natural terpendoles together with some model indole alkaloids . similar oxygen addition was observed for 1,2,3,4-tetrahydrocyclopent[b]indole , which is corresponding to the substructure of terpendole e. in tandem ms experiments , a major fragment ion at m / z 130 from protonated terpendole e was assigned to the substructure containing indole . when the [ m + h + o]+ was selected as a precursor ion , the ion shifted to m / z 146 . the same 16 da shift of fragments was also observed for 1,2,3,4-tetrahydrocyclopent[b]indole , indicating that the oxygen addition of terpendole e took place at the indole portion . however , the oxygen addition was absent for some terpendoles , even whose structure resembles terpendole e. the breakdown curves characterized the tandem ms features of terpendoles . preferential dissociation into m / z 130 suggested the protonation tendency at the indole site . terpendoles that are preferentially protonated at indole tend to form oxygen addition peaks , suggesting that the protonation feature contributes to the oxygen additions in some degrees . 2014 the authors . journal of mass spectrometry published by john wiley & sons , ltd . gastrointestinal stromal tumors ( gists ) are the most common mesenchymal tumors in the gastrointestinal tract ( git ) . although interstitial cells of cajal has been suggested as origin of this tumor , the cytological and ultrastructural features of gists are heterogeneous and unclear . a total 10 cases of normal gastrointestinal tissue ( control ) , 13 gists of the stomach ( 8) , small intestine ( 3 ) , mesocolon ( 1 ) and liver ( 1 ) , and 2 gastrointestinal autonomic nervous tumor ( gant ) of small intestine were ultrastructurally studied . normal interstitial cells of cajal ( icc ) were abundantly present around the myenteric plexuses or individually scattered through the wall of git . icc was characterized by slender cytoplasmic processes , well - developed endoplasmic reticulum ( er ) , mitochondria , golgi apparatus , caveolae and intermediate filaments . the gists and gants had overlapping ultrastructures . the most common and important ultrastructural features of gists were rich villous cytoplasmic processes , dispersed intermediate filaments and abundant ser , and those of gants were neurosecretory granules and skenoid fibers . compared with icc , the gists and gants had remarkably reduced caveolae and gap junctions . our study suggested that ultrastructural analysis gives much information to investigate lineage differentiation of neoplastic cells and make a differential diagnosis of these tumors from other mesenchymal tumors and between gists and gants .

the asian tropopause aerosol layer is composed of sulfate , primary organics , and secondary organics the north american tropospheric aerosol layer is mostly composed of sulfate and secondary organics aerosol optical depth of asian tropopause aerosol layer increases by 0.002 from 2000 to 2010 the asian tropopause aerosol layer is composed of sulfate , primary organics , and secondary organics the north american tropospheric aerosol layer is mostly composed of sulfate and secondary organics aerosol optical depth of asian tropopause aerosol layer increases by 0.002 from 2000 to 2010 recently , a layer of enhanced aerosol scattering associated with the asian summer monsoon was observed by stratospheric aerosol and gas experiment ii ( sage ii ) [ thomason and vernier , 2013 ] and calipso [ vernier et al . , 2011a ] . the observed enhancement , referred to as the asian tropopause aerosol layer ( atal ) , occurs during june - july - august in the broad region bounded by 0160e , 1545n . calipso backscatter observations show that the atal has been present since calipso observations began in 2006 . show that the atal existed since 1999 in sageii data but was not detected prior to 1998 . the asian monsoon circulation transports asian pollution from the boundary layer to the stratosphere , where satellites observe strong enhancements of carbon monoxide , hcn , and h2o [ park et al . , 2009 ; randel et al . , however , in the first modeling study of the atal , neely et al . show that the atal may originate from emissions over a wide region , including both europe and asia . the results of neely et al . also suggest that the atal was likely present before 1998 , but observations were most likely obscured by volcanic aerosol ( i.e. , from the pinatubo eruption in 1991 ) . one caveat of the neely et al . both sage ii [ thomason and vernier , 2013 ] and calipso [ vernier et al . , 2011a ] observe a similar aerosol layer over north america , the north american tropospheric aerosol layer ( natal ) , which is weaker than the atal . the natal is associated with the north american monsoon located in the southwestern united states and mexico [ adams and comrie , 1997 ] . the sources , composition , and physical properties of the natal are likely to differ from the atal since the underlying lower tropospheric chemistry is greatly different . for example , the natal does not exhibit enhanced stratospheric co [ park et al . the transport mechanisms in the natal are also likely somewhat different than in the atal . show that both the atal and natal are regions in which water vapor enters the stratosphere in june - july - august ( jja ) ; however , the isotopic composition of the water differs . aerosol that is pumped into the stratosphere may affect climate significantly by scattering sunlight back to space and absorbing radiation locally [ stocker et al . therefore , aerosol layers in upper troposphere and lower stratosphere ( utls ) may be important if they persist or their particles are transported into the stratosphere . the properties of the atal and the natal remain unclear due to limited in situ measurements . the composition and size distribution of the particles are important properties to determine since they control the impact of the aerosol on the radiation field . this study will address the composition , source , and optical properties of the atal and natal using a sectional aerosol model . we analyze the properties of the atal and natal using a general circulation model ( community earth system model version 1 ( cesm1 ) ) coupled with a sectional aerosol microphysics model ( community aerosol and radiation model for atmospheres ( carma ) ) [ toon et al . , 1988 ; bardeen et al . , 2008 ] . cesm1/carma includes primary emitted organics , secondary organics , dust , sea salt , black carbon , and sulfate . in carma , we track two types of aerosol in two sets of radius bins . one type consists of sulfuric acid particles formed through nucleation and condensation of water and sulfur acid vapor , which primarily occurs locally . the other set of bins includes particles containing mixtures of organics , black carbon , sea salt , dust , and condensed sulfate . gas phase sulfur chemistry is modeled using the algorithms described in english et al . . carma includes formation of cloud - borne sulfate through aqueous chemistry as well as so2 formation from dimethyl sulfide as simulated by model for ozone and related chemical tracers ( mozart ) [ emmons et al . , mozart [ emmons et al . , 2010 ] tracks five species of volatile organic compounds as precursors of secondary organic aerosol ( soa ) . to better quantify the formation of soa , carma applies a volatility - basis - set approach that was introduced in pye et al . . the aerosol scavenging schemes are from rasch et al . and barth et al . the scavenging coefficient below cloud is dependent on aerosol size and precipitation rate , similar to henzing et al . . the core is composed of black carbon and dust , while the shell is composed of a combination of all other materials that are possibly in a liquid state including sulfate , organics , salt , and condensed water . this radiative information is then coupled to the dynamics of cesm1 through its radiation code . the simulations are carried out with 2 horizontal resolution and 56 vertical levels . in the region from 13 to 18 km altitude the simulations use about seven vertical levels . a 5 year spin - up run was first carried out so that the lower stratospheric aerosol layer reached a quasi - equilibrium state . then two sets of ensembles are carried out for the years 2000 and 2010 with prescribed sea surface temperature , respectively . organic emission rates , including primary particles and gases that form secondary particles , are taken from amann et al . and the global fire emission database ( gfed version 3 ) [ van der werf et al . : anthropogenic so2 emission rates come from edgar - ft2000 ( http://www.mnp.nl/edgar/ ) , with the exception that asian emission inventories are taken from the regional emission inventory for asia [ ohara et al . , 2007 ] . so2 emissions from biomass burning are estimated by gfed ( version 2 ) [ van der werf et al . , 2006 ] and biogenic emissions of volatile organic compounds ( vocs , including isoprene and monoterpenes ) are estimated by guenther et al . . model results are compared to the limited in situ aerosol measurements in the asian utls . caribic ( civil aircraft for the regular investigation of the atmosphere based on a instrument container ) provides observations of trace gases and aerosols since 1997 through intercontinental flights at 912 km altitude , which is at the lower edge of the atal [ brenninkmeijer et al . , 2007 ] . 2014 ] from 2005 to 2014 collected during 330 flights from frankfurt in germany to east asia ( guangzhou , china ) , southeast asia ( bangkok , thailand ) , and the indian subcontinent ( chennai , india ) . detailed sampling and elemental analysis methods are described in nguyen et al . and martinsson et al . . figure1 shows the simulated aerosol extinction ratio , defined as the total extinction ( i.e. , aerosol extinction plus molecular extinction ) divided by the molecular extinction , at a wavelength of 1000 nm averaged from 15 to 45 n , over the period june , july , and august 2000 . a layer of enhanced aerosol scattering , box 1 , extends from 14 km to 18 km vertically , and from 0 to 120e . the location and shape of the simulated layer of enhanced scattering is consistent with sage ii observations from thomason and vernier . however , our model simulation also predicts an aerosol enhancement mainly composed of mineral dust extending from the surface up to 13 km above africa , box 2 , which is not seen in the calipso [ vernier et al . , 2011a ] or sage ii [ thomason and vernier , 2013 ] observations . thomason and vernier used a filter for eliminating the effects of clouds on their observations , which depends on the aerosol absolute extinction and the extinction ratio at two wavelengths ( 1000 nm and 525 nm ) . when we apply the same filter , the layer over africa disappears , which suggests that the cloud filter used in thomason and vernier may mask a fraction of aerosol that has relatively big particles with high extinction coefficients . aerosol extinction ratio at 1000 nm averaged between 15 and 45n during jja seasons using emissions from 2000 . in addition to simulating the atal , our model also predicts a weaker , less extended aerosol layer near 15 km over north america centered at 100w longitude , box 3 , consistent with the location of the north american monsoon [ adams and comrie , 1997 ] . [ vernier et al . , 2011a ; thomason and vernier , 2013 ] and modeled by neely et al . . the simulated natal peak extinction ratio is only 48% of that of the simulated atal . below the natal , another aerosol enhancement region extending up to 13 km is also predicted by the model , box 4 . this lower layer is mainly composed of soa over north america where copious biogenic vocs are known to be emitted . similarly , the atal is adjacent to a lower layer extending downward and eastward centered around 120e , box 5 . both layers associated with asia are composed of materials transported from the surface . however , those in the atal are pumped up by the asian summer monsoon and confined into the anticyclone . the simulated peak value of the atal extinction ratio is about 3 times that observed by sage ii [ thomason and vernier , 2013 ] . relative humidity is high and extinction increases strongly with high humidity . in reality , however , particles may not grow due to the low temperatures in the utls . sensitivity tests with no hygroscopic growth in the upper troposphere and above shows up to 30% decrease in the extinction of the atal , which still leaves the model a factor of 2 higher than observations . it is also conceivable that sage ii - derived products preferentially exclude aerosol with high extinctions that are colocated with cloud due to filtering that attempts to remove cirrus cloud [ thomason and vernier , 2013 ] . in situ data if , in fact , particles do not grow when relative humidity increases at low temperatures , it would be important for theories of cirrus cloud formation [ koop et al . , 2000 ] . in addition , the model may overestimate the aerosol loading in the atal region possibly due to an underestimation of wet scavenging . the ratio of the extinction at 525 nm to 1020 nm in the model is centered at 33.5 , while from sage ii it is 44.5 [ thomason and vernier , 2013 ] , which is consistent with the model overestimating size , or the sage ii retrieval algorithm rejecting larger aerosols . the modeled particle effective radius is between 0.1 and 0.2 m in the atal region . unfortunately , there are no in situ data to compare the model results to in this region . show the tropical tropopause layer ( ttl ) contains sulfate and organic particles in roughly equal proportions based on two campaigns over tropical central america . in addition , while english et al . and neely et al . show that sulfates alone can not explain the extinction in the ttl observed by sage ii below about 20 km , brhl et al . show that in addition to sulfates , organics contribute to the aerosol extinction in the ttl observed by sage ii . caribic provides data on the sulfur - to - carbon mass ratio in the upper troposphere over europe and asia , as shown in figure2 . simulated values are averaged from 200 to 300 mb , pressures which are below the atal . because the caribic data ( circles ) are from samples take during individual flights over a period of time , actual meteorological conditions produce some variability . both simulations and observations show a strong decline of the sulfur - carbon ratio from higher latitudes ( e.g. , europe , 5070% sulfur - to - carbon ( s / c ) ) to lower latitudes ( e.g. , chennai , bangkok , and guangzhou , 1020% s / c ) . part of the reason for the decline in the s / c ratio as latitude decreases is that the tropopause is lower at higher latitudes so that more stratospheric sulfate is present along planes of constant pressure as shown in figure2 . in addition , more organics are emitted over india and china than over europe , which are transported upward in the asian summer monsoon . cesm1/carma simulations ( not shown in this paper ) suggest that the s / c ratio does not vary by more than 10% from 14 to 16 km in the middle to upper troposphere but increases rapidly in the lower stratosphere . sulfur - to - carbon ( s / c ) mass ratio of jja shown in the panel averaged from 200 to 300 mb using 2010 emissions . the simulation results shown in figure3 suggest the atal to be mostly composed of organics ( roughly 60% by mass ) and sulfate ( roughly 40% ) given 2010 emissions . the simulated atal and natal is mostly composed of sulfate and organics , which is consistent with froyd et al . who showed that the aerosol composition over central america is also a mixture of organics and sulfate . simulations also suggest that the effective radius of mixed particles ( mostly composed of organics at 100 mb ) is between 0.2 and 0.3 m in both atal and natal , while the effective radius of pure sulfate particles ( 33% of total sulfate mass is in pure sulfates , leaving 67% of sulfate in the mixed particles ) is below 0.1 m , indicating pure sulfates were been formed locally to the layers . ( a ) simulated organic mass ( g / m ) between 100 mb to 230 mb ; ( b ) simulated sulfate . according to the cesm1/carma results shown here , primary organic aerosol ( poa ) and secondary organic aerosol ( soa ) make comparable contributions to the atal ( figure4 ) . ( top ) poa and ( bottom ) soa mass mixing ratios ( ppb ) simulated by cesm1/carma in the atal during the jja season . in addition to the atal and natal ( see figure1 ) , this study finds other aerosol enhancement features in the upper troposphere that are not currently recognized in observations . these aerosol features extend downward and eastward from both the atal and the natal ( an additional enhancement due to dust is found over africa ) . the layer downward and eastward of the natal is also composed of soa associated with strong biogenic emission from the surface . , 2011 ; vernier et al . , 2011b ] . using a conservative method to remove ice clouds in sageii and calipso data , vernier et al . show the aerosol optical depth ( aod ) at 525 nm of the atal has increased from 0.003 in year 2000 to roughly 0.005 in year 2010 . suggest recent anthropogenic increases in so2 from asia can not explain a global stratospheric aerosol increase since 2000 , which is largely due to small volcanic eruptions . here we explore the contribution of asian emissions to the aerosol in the utls by completing two sets of simulations with ensembles of three members each . one set of ensembles uses emissions for the year 2000 and the other emissions for the year 2010 . the emissions in midlatitudes , especially anthropogenic so2 and vocs vary between 2000 and 2010 over asia , europe , and north america . figure5 shows column aod ( from 13 to 18 km ) at 532 nm averaged from 15n to 45n in jja of 2010 ( red curve ) and 2000 ( blue curve ) , respectively . error bars denote data variability ( standard deviation ) inside ensemble runs ( three members with slightly different initial conditions ) . the modeled aod of the atal is up to a factor of 2 higher than reported in vernier et al . as discussed in the context of figure1 . this test suggests that the aod was generally higher in 2010 for latitudes from 15 to 45n at all longitudes . aod changes at other longitudes including the natal ( 70w120w ) are within data variability inside ensembles . this study suggests the emission increase in asia between 2000 and 2010 has lead to an increase in the optical depth of the atal region by about 0.002 on average , which is similar to that the estimate by vernier et al . . the sulfate mass fraction in the atal is similar between 2000 and 2010 ( roughly 40% ) ; the growth in optical depth comes from increased emissions of so2 ( increasing by 13% ) , primary organics ( 7.3% ) , and anthropogenic soa ( 30% ) in the region of atal ( 1545n , 0120e ) . column aod ( from 13 to 18 km , at 532 nm ) averaged from 15 to 45n in jja . a three - dimensional climate model coupled with a sectional aerosol model ( cesm1/carma ) is used to analyze the composition and optical properties of the atal and the natal . the modeled aerosol extinction ratio peaks in the utls over asia in a layer extending from 14 to 18 km , which is consistent with sage ii observations [ thomason and vernier , 2013 ] and calipso observations [ vernier et al . , 2011a ] . the simulations reproduce the s / c ratio established by in situ measurements of the caribic observatory [ martinsson et al . , 2014 ] from 200 mb to 300 mb over europe and asia . the s / c ratio decreases from higher latitudes to lower latitudes , which possibly results in part from more organics emitted over india and china than over europe . according to the simulations , the atal is mostly composed of mixed organics and sulfates with an effective radius of 0.20.3 m , and in situ generated sulfate particles with an effective radius below 0.1 m . due to the high occurrence of cirrus clouds over india and china , aerosol measurements from satellite - based remote sensing instruments are difficult . we predict larger extinctions in the atal than observed , which could be due to satellites failing to detect the aerosol in high humidity , cloudy regions . alternatively , the hygroscopic growth of the aerosols in our model may be too great , which may be of importance to theories of cirrus cloud nucleation . or , our model may overpredict aerosol mass or size in the atal . our simulations reproduce the natal with weaker intensity compared with the atal during the jja season as observed . while the atal has a strong poa contribution , the simulated natal is mostly composed of biogenic soa . simulations also suggest recent increased anthropogenic emission of so2 as well as organic aerosols and gases in asia lead to an increase in optical depth of the atal region by 0.002 on average between 2000 and 2010 . californium-252 is an artificial element with a half - life of 2.645 years , and it decays via either alpha emission ( 96.9% ) or spontaneous fission ( 3.1% ) . cf emits both photons and neutrons ( 2.3110 n / s/g ) of varied energy with potential for both clinical brachytherapy and neutron capture therapy ( nct ) applications . the relatively high neutron yield and long half - life , when compared to other spontaneous fissioning isotopes , clinical successes with cf sources are undoubtedly due in part to the theoretical advantages inherent in treating tumors with fast neutrons in general and with cf in particular . the effectiveness of cf might further be improved by augmenting the cf dose to tumor with an additional dose by neutron capturer loading to the tumor itself . fast neutrons emitted by the cf source scatter in tumor tissue and lose their energy by multiple scattering to eventually become thermal . increasing the probability of occurrence thermal neutron capture by neutron capturer cases dose rate enhancement in tumors loaded with these materials . materials such as b , gd and s have been proposed as agents for neutron capture . indeed , the combination of cf brachytherapy and neutron captures may improve tumor dose noticeably . following the capture by b ( bnct ) , high linear energy transfer ( let ) alpha particles and li nuclei are released . these heavy particles deposit their energy in the range of 5 - 9 mm ( tumor cell limit ) and therefore , the destructive effects of the resulted particles are limited to boron loaded cells . the method gadolinium neutron capture therapy ( gdnct ) is a recently proposed therapy modality , mainly based on the action of auger and internal conversion electrons generated by gd after neutron capture . the capture reaction in gd has the form of gd ( n , )gd and the emitted gamma rays make dose enhancement . the potential effect of enhancing nct near the surface of the target volume by addition of s has been proposed as well . the neutron capture reaction for s has the form of s(n , )si and has its most important resonance at 13.5 kev . in a study by porras , an enhancement of the neutron absorbed dose by s was observed in a high concentration of s ( between 1 and 10 mg / g ) , for a monoenergetic neutron source of 13.5 kev and for tumors at small depths . the purpose of this study is to evaluate the dose distribution in the presence of uniform distribution of neutron capturer materials and to determine the effect of these materials on dose rate enhancement in brachytherapy with cf source . therefore , careful analysis of different components of the radiation field and a detailed characterization of dose distributions in the absence and presence of neutron capturer materials must be carried out . in this study , neutron - ray flux and energy spectra , neutron and gamma dose rates and dose enhancement factor are determined in the vicinity of a cf source in water phantom with and without same concentration ( 200 ppm ) of b , gd and s using monte carlo mcnp5 code in the present study , a cf applicator tube ( at ) source available from oak ridge national laboratory ( ornl ) was modelled . the geometry of cf source is shown in figure 1 . the cylindrical active core is made of californium oxide , cf2o3 with 12 g / cm density . the length and radius of the active cylinder is 1.5 and 0.615 cm , respectively , which is located in a primary capsule of pt / ir-10 percentage mass , with inner and outer diameters of 1.35 and 1.75 mm , respectively , and inner and outer lengths of 15.50 and 17.78 mm , respectively . the secondary capsule has inner and outer diameters of 1.80 and 2.80 mm , respectively , and inner and outer lengths of 17.82 and 23.14 mm , respectively . further , the 0.635 mm diameter bodkin eyelet through the secondary capsule is also included in the source geometry of cf at source the monte carlo simulation of radiation therapy allows accurate prediction of radiation dose distribution delivered to a patient . in the present work , a complete dosimetric data set for the cf at source in water , in the absence and presence of neutron capture materials was obtained using monte carlo mcnp5 code . the source was positioned in the center of a 15 cm radius spherical phantom filled with water of 0.998 g / cm3 mass density , or capture materials - water mixture for uniform distribution of b , gd and s capture materials throughout the water phantom . the dose rate was determined in a cylindrical annulus 0.2 cm thick0.2 cm deep positioned along the transverse axis at distances ranging from 0.25 to 10 cm from the source center . assuming kerma equality with absorbed dose at different distances , f6 tally was used to calculate the particle dose of all components including thermal neutrons , epithermal neutrons , fast neutrons , induced gamma rays and source gamma rays . the neutron dose , source gamma ray and induced gamma ray doses were calculated separately . to calculate particle flux , particle fluence was calculated with f4 tally and then was multiplied by 2.3110 , since the calculations were performed assuming one microgram of cf source . the capture product dose ( absorbed dose by capture materials ) resulted from the capture of thermal neutrons by b , gd and s was calculated using the fluence - to - kerma conversion factors . the neutron dose is the sum of source fast neutron dose resulted from elastic scattering of fast neutrons in water and the capture product dose which is resulted from thermal neutron capture by b , gd and s. the neutron energy spectrum for cf source was assumed to be maxwellian spectrum with an average energy of 2.1 mev and the most probable energy of ~0.7 mev . photon spectrum of the cf source was taken from the study by fortune , and has photon energies in the range of 0.019.79 mev . the thermal neutron region was defined to be below 0.5ev , the epithermal neutron region is from 0.5ev to 10 kev and the fast neutron region is over 10 kev . the s(, ) thermal neutron scattering library ( lwtr.01 t ) was used in order to calculate the transport of low energy neutrons . in the present study , a cf applicator tube ( at ) source available from oak ridge national laboratory ( ornl ) was modelled . the geometry of cf source is shown in figure 1 . the cylindrical active core is made of californium oxide , cf2o3 with 12 g / cm density . the length and radius of the active cylinder is 1.5 and 0.615 cm , respectively , which is located in a primary capsule of pt / ir-10 percentage mass , with inner and outer diameters of 1.35 and 1.75 mm , respectively , and inner and outer lengths of 15.50 and 17.78 mm , respectively . the secondary capsule has inner and outer diameters of 1.80 and 2.80 mm , respectively , and inner and outer lengths of 17.82 and 23.14 mm , respectively . further , the 0.635 mm diameter bodkin eyelet through the secondary capsule is also included in the source geometry of cf at source the monte carlo simulation of radiation therapy allows accurate prediction of radiation dose distribution delivered to a patient . in the present work , a complete dosimetric data set for the cf at source in water , in the absence and presence of neutron capture materials was obtained using monte carlo mcnp5 code . the source was positioned in the center of a 15 cm radius spherical phantom filled with water of 0.998 g / cm3 mass density , or capture materials - water mixture for uniform distribution of b , gd and s capture materials throughout the water phantom . the dose rate was determined in a cylindrical annulus 0.2 cm thick0.2 cm deep positioned along the transverse axis at distances ranging from 0.25 to 10 cm from the source center . assuming kerma equality with absorbed dose at different distances , f6 tally was used to calculate the particle dose of all components including thermal neutrons , epithermal neutrons , fast neutrons , induced gamma rays and source gamma rays . the neutron dose , source gamma ray and induced gamma ray doses were calculated separately . to calculate particle flux , particle fluence was calculated with f4 tally and then was multiplied by 2.3110 , since the calculations were performed assuming one microgram of cf source . the capture product dose ( absorbed dose by capture materials ) resulted from the capture of thermal neutrons by b , gd and s was calculated using the fluence - to - kerma conversion factors . the neutron dose is the sum of source fast neutron dose resulted from elastic scattering of fast neutrons in water and the capture product dose which is resulted from thermal neutron capture by b , gd and s. the neutron energy spectrum for cf source was assumed to be maxwellian spectrum with an average energy of 2.1 mev and the most probable energy of ~0.7 mev . photon spectrum of the cf source was taken from the study by fortune , and has photon energies in the range of 0.019.79 mev . the thermal neutron region was defined to be below 0.5ev , the epithermal neutron region is from 0.5ev to 10 kev and the fast neutron region is over 10 kev . the s(, ) thermal neutron scattering library ( lwtr.01 t ) was used in order to calculate the transport of low energy neutrons . to validate our monte carlo simulation , the computed dose rates were compared with experimental and simulated values published in the literature . figure 2 and 3 show a comparison between our simulated neutron and total gamma ray dose rates ( total gamma ray dose is the sum of source gamma - ray dose and induced gamma ray dose ) with the experimental measurements of colvett and the simulated calculations of krishnaswamy . there is a good agreement between values with small discrepancies at distances close to the source . these discrepancies might be explained by different modelled energy spectra for neutron and gamma rays emitted from cf source in simulation studies . also , in the regions close to the source , the dose gradient is extremely steep , and experimental measurement values depend on the accuracy and sensitivity of the measurement device to rapidly changing radiations dose . simulated and experimental neutron dose rates for the water phantom comparison of total gamma - ray dose rates for the water phantom after validation , the validated computer code was applied to evaluate the effect of neutron capturers on dose rate distribution . figure 4 shows the behavior of the cf neutron energy spectra calculated at the same distance along the transverse direction of the source in water phantom in the absence and presence of capturer materials . as it is seen in this figure , in the presence of gd and b capturer materials , neutron flux has decreased in the thermal energy region while it is not seen at the epithermal and fast energy regions . the reduction of thermal neutron flux in the media containing gd and b is the direct result of thermal neutron capture process by these materials and hydrogen in water . difference in the rate of this reduction depends on the magnitude of thermal neutron capture cross - section of these materials . the no - change in the neutron energy spectrum in the presence of s may be resulted from both neutron spectrum of cf source with varied energy and low concentration of s in this study . neutron energy spectrum at 3 cm distance from the source , in water phantom with and without the presence of capture materials figure 5 and 6 show the flux of fast , epithermal and thermal neutrons at different distances from the source in water phantom with and without the presence of capture materials . obtained result shows that the effect of capture materials on the epithermal and fast neutron fluxes is impalpable . in figure 6 , the thermal neutron flux increases as afterwards , there is a dramatic decrease due to the absorption of thermal neutrons by capture materials and hydrogen . there is a neutron flux ( neutron flux is the sum of thermal , epithermal and fast neutron flaxes ) depression of about 57% in b , 80% in gd and 0.0005% in s loadings . it can be concluded that this depression emanates from the thermal neutron flux depression due to thermal neutron capture by the capture materials . epithermal and fast neutron fluxes in water phantom in the absence and presence of capture materials thermal neutron flux in water phantom in the absence and presence of capture materials figure 7 shows neutron dose rate as a function of distance in water phantom with and without the presence of capture materials . there is significant enhancement of neutron dose in the presence of gd and b. it can be concluded that the difference in the amount of this enhancement given that the neutron dose is the sum of source fast neutron ray dose and capture product dose which is resulted from difference in capture product dose rate . in other words , enhancement rate of neutron dose in the presence of capture materials depends on the type of capture products . neutron dose rate distributions in the absence and presence of capture materials at different distances from the source figure 8 provides the comparison between capture product doses in terms of distance from the source . we notice that there is a resemblance and relation between the increase of capture product dose in figure 8 and depression of thermal neutron flux in figure 6 , with increasing distance from the source . the increase rate of capture product dose resulting from depression of thermal neutron flux is maximum for gd and is minimum for s. in other words , the difference in the amount of capture product dose is a direct result of difference in magnitude of the thermalization process of neutrons by these capture materials . capture material dose rate distribution for 200-ppm concentrations at different distances from the source figure 9 shows the source and the induced gamma ray doses calculated in water phantom with and without the presence of capture materials at different distances from the source . we notice that the existence of capture materials does not alter source gamma dose rate but does reduce the induced gamma dose rate . induced gamma rays are produced by thermal neutron capture reactions of 1h(n,)2h in water . the reduction of induced gamma dose in the capture material loading is a result of hydrogen proportion reduction in capture material loaded media compared to only water medium which results in occurrence reduction of thermal neutron capture reactions of 1h(n,)2h and , consequently , to reduction of induced gamma dose rate . contrary to neutron dose , the induced gamma dose in media containing gd is lower than b because of higher ability of gd toward b in thermal neutron capture which results in fewer thermal neutrons existing to be captured by hydrogen and , induced gamma dose increases in a lower trend in media containing gd . in other words , contribution of induced gamma dose in enhancing total dose rate is further in media containing b toward gd . source and induced gamma ray dose as a function of distance figure 10 shows the total dose rate as a function of distance in water phantom in the absence and presence of capturer materials . the enhancement rate of total dose in media containing gd is more than that of b and s. the reason for it will be due to higher neutron dose and lower induced gamma dose in media containing gd compared to b and s as higher amount of gd product dose than b and s. total dose rate versus distance away from the source to determine the effect of capture materials on dose enhancement rate , dose enhancement factor ( def ) is used which is defined as the ratio of total dose in a tumor containing the capture material to total dose in the same tumor without the presence of capture material . dose enhancement factor values for different capture materials are presented in table 1 . according to data of this table , the value of def increases with increasing distance from the source and reaches its highest value equal to 3.258 and 1.476 for gd and b , respectively at the distance of roughly 8 cm from the source center , and after that decreases . in other words , the effectiveness rate of gd and b capture materials in enhancing dose rate depends on the tumor distance from the source . increase in the value of def with increasing distance from the source despite the decline in ray intensity is due to both decrease neutron average energy in the effect of attenuation , and increasing the less energetic scattered rays arrived to depth that makes increase the occurrence probability of thermal neutron capture by capture materials and subsequently dose rate enhancement . enhancement rate of total dose in the presence of s is not significant since its def is equivalent to one . dose enhancement factor at different distance from the source for b , gd , and s in this study , a detailed characterization of dose distribution in the absence and presence of b , gd and s neutron capturers has been carried out for cf brachytherapy source using monte carlo simulation . obtained result shows that tumor loading with gd and b neutron capturers in neutron brachytherapy with cf source makes significant dose enhancement due to the increase in occurrence probability of thermal neutron capture by these materials . the results also show that the magnitude of dose augmentation with this therapy design will depend not only on the capture product dose , but also on the tumor distance from the source . this dependence is resulted from both difference in the magnitude of thermalization process of neutrons by these materials and the decrease of neutron average energy due to attenuation that make increase the occurrence probability of thermal neutron capture . s is not a suitable agent for dose increase by neutron capture in brachytherapy with cf source . in other words , s makes dose enhancement under specific conditions in which these conditions depend on neutron energy spectra of source , the s concentration in tumor and tumor distance from the source .

recent studies revealed layers of enhanced aerosol scattering in the upper troposphere and lower stratosphere over asia ( asian tropopause aerosol layer ( atal ) ) and north america ( north american tropospheric aerosol layer ( natal ) ) . we use a sectional aerosol model ( community aerosol and radiation model for atmospheres ( carma ) ) coupled with the community earth system model version 1 ( cesm1 ) to explore the composition and optical properties of these aerosol layers . the observed aerosol extinction enhancement is reproduced by cesm1/carma . both model and observations indicate a strong gradient of the sulfur - to - carbon ratio from europe to the asia on constant pressure surfaces . we found that the atal is mostly composed of sulfates , surface - emitted organics , and secondary organics ; the natal is mostly composed of sulfates and secondary organics . the model also suggests that emission increases in asia between 2000 and 2010 led to an increase of aerosol optical depth of the atal by 0.002 on average which is consistent with observations.key points the asian tropopause aerosol layer is composed of sulfate , primary organics , and secondary organics the north american tropospheric aerosol layer is mostly composed of sulfate and secondary organics aerosol optical depth of asian tropopause aerosol layer increases by 0.002 from 2000 to 2010 background : in neutron interaction with matter and reduction of neutron energy due to multiple scatterings to the thermal energy range , increasing the probability of thermal neutron capture by neutron captures makes dose enhancement in the tumors loaded with these materials . objective : the purpose of this study is to evaluate dose distribution in the presence of 10b , 157gd and 33s neutron capturers and to determine the effect of these materials on dose enhancement rate for 252cf brachytherapy source . methods : neutron - ray flux and energy spectra , neutron and gamma dose rates and dose enhancement factor ( def ) are determined in the absence and presence of 10b , 157gd and 33s using monte carlo simulation . results : the difference in the thermal neutron flux rate in the presence of 10b and 157gd is significant , while the flux changes in the fast and epithermal energy ranges are insensible . the dose enhancement factor has increased with increasing distance from the source and reached its maximum amount equal to 258.3 and 476.1 cgy / h/g for 157gd and 10b , respectively at about 8 cm distance from the source center . def for 33s is equal to one . conclusion : results show that the magnitude of dose augmentation in tumors containing 10b and 157gd in brachytherapy with 252cf source will depend not only on the capture product dose level , but also on the tumor distance from the source . 33s makes dose enhancement under specific conditions that these conditions depend on the neutron energy spectra of source , the 33s concentration in tumor and tumor distance from the source .

the transposable element ( te ) was first described by mcclintock as a controlling element that jumps from one position to another in the maize chromosome in the mid-1940s , long before the discovery by watson and crick of the double helix structure of dna as a genetic element . it was a time when , after the rediscovery of mendel 's law of heredity in 1900 , the cytogenetic study of chromosomes was at the forefront of genetics and when genes were thought to be beads on a string located on the chromosome . it was in the late 1970s and early 1980s when insertion sequence ( is ) elements and transposons ( tn ) were discovered and found to be similar to mcclintock 's controlling elements , and her ac / ds elements were confirmed at the dna sequence level as a te . salient features of dna transposable elements ( dtes ) , including terminal inverted repeats ( tirs ) , target site duplication ( tsd ) , the transposase gene , and simple and replicative transposition mechanisms have been well established by extensive molecular biology and biochemistry studies . then , retrotransposable elements ( rtes ) , including long terminal repeat ( ltr)-retrotransposons , non - ltr - retrotransposons , and other retro - elements , were being added to the repertoire since the 1980s to make the picture diverse and complicated . many new families of tes are being added , especially from eukaryotes , even by computational screening in the post - genome era , which has necessitated a new classification based on their structures and mechanisms of transposition . the presence of foldback intercoil ( fbi ) dna was first reported by kim in 1985 and shown by a space filling model to mediate intra - molecular homologous recombination of inversions and deletions . it was further shown in 1987 that fbi dna can mediate inter - molecular dna rearrangements , such as site - specific insertions , at the foldback tip and dna transpositional integration at the intercoil end of the detached dte . it will be examined in this review how fbi dna mediation of transposition can be extended to different classes and families of rtes . readers are suggested to refer to many good reviews available on the classification , structures , genetic contents , and function of the gene products and on the mechanisms of transposition with excellent diagrammatic illustrations [ 5 , 9 , 10 , 11 , 12 ] . this review will minimize duplicate descriptions and focus on the mechanistic features that are relevant to the application of fbi dna to the mechanisms of dna transposition . to build the basis for comparison and understand transposition mechanisms , it is necessary to consider te structures based on the presence or absence of the following factors : 1 ) rna intermediates before transposition , 2 ) tirs or ltrs , 3 ) transposase or reverse transcriptase ( rt ) and other proteins related to autonomous or non - autonomous transposition , 4 ) tsd , and 5 ) other features , like dna replication modes , hairpin structure , and enhancers . transposition of dtes involves direct movement of the dte intermediate either by a simple cut - and - paste mechanism , as in tn5 , tn7 , and tn10 , or by replicative transposition , in which case one copy stays in the original site and a daughter dte copy appears in a distant location [ 16 , 17 , 18 ] . step - wise description of the transposition events includes the requirement and pairing of both dte ends [ 13 , 19 ] , binding of the transposase proteins to the terminal sequences , double - strand breaks ( dsbs ) of the dte terminals and target ends , ligation of the 3'-oh donor end and the 5'-p target end , and gap repair of the single - strand gaps on the target that were generated by dsb . in the case of the mu bacteriophage , the gap can be repaired while the entire transposon gets replicated in a cointegrate mechanism [ 21 , 22 ] . transposition of rtes involves an rna intermediate that is reverse - transcribed by rt into a cdna . this double - stranded linear dna functions as the direct precursor for integration by an integrase protein into a new location in the host chromosome in a ' copy and paste ' manner , as in the ' cut - and - paste ' transposition reaction in dtes [ 20 , 23 ] . tirs are a hallmark of dtes , as ltrs have been that of rtes until they were joined by non - ltr retrotransposons , which have a 5'-untranslated region ( 5 ' utr ) and 3 ' utr , as typified by line-1 ( l1 ) , or which has neither of the terminal elements , as typified by the alu element . it becomes clear that the presence of terminal repeats in the tes is no longer a common requirement for the transposition reaction . the description of the protein factors above will suffice for this review , except for the long interspersed nuclear element ( line ) open reading frames ( orfs ) . the retrotransposition and integration of line have been viewed as a coupled process , called target - primed reverse - transcription [ 5 , 24 ] . however , this model has recently been enhanced by the finding that l1 reverse - transcription does not require base pairing between the primer and template . therefore , the cdna that was reverse - transcribed from the mrna expressed from the genomic copy of line is inserted into the host genome . tsds are generated in almost all classes and families of tes , except for the helitron dte superfamily [ 6 , 9 , 10 ] . the size of the tsd varies from 2 to 11 , but the sequences are not conserved . the ubiquitous presence of tsds , which are generated by gap fillings ( gfs ) after te integration on the target , reflects the unity in transposition reactions , regardless of dte or rte , and the presence or absence of terminal repeats . yet , the meaning of the small oligomer size of the duplicated target is seldom discussed in terms of dna structure . helitron , a eukaryotic dte , is considered , together with polintons , a third class of tes for many unique aspects . they contain 16 - 20-bp hairpins 10 - 12 nucleotides inside of the 3'-end and transpose precisely between the 5'-a and t-3 ' with no tsd generation . helitrons indiscriminately capture and mobilize gene sequences and may impact hybrid vigor or heterosis in maize . since the hairpin sequence corresponds to the fbi dna motif , it will be interesting to consider whether it can not only serve as a terminator or rolling circle replication but also serve as a replication origin for its own replication and for frequent gene capture activities , which the helitron is capable of . polinton is 15 - 20 kb long , with a 6-bp tsd and 100 - 1,000-bp tir at both ends . polintons code up to 10 proteins , including a family of b dna polymerases ( polbs ) , a retroviral - like integrase , an a transposase , and an adenoviral - like cysteine protease . polintons , however , with their structural characteristics and presence of tsd , should follow non - homologous end - joining ( nhej ) with the gf transposition mechanism , as ordinary dtes . what makes it unique is the presence of a self - encoded polb and a short 1 - 3-bp terminal tandem repeat that supports a protein - primed self - synthesis mechanism for polinton propagation . this mechanism calls for an extrachromosomal single - stranded polinton intermediate that forms a racketlike structure and follows polb - mediated replication to become double - stranded before being integrated into the host genome . it has been proposed that eukaryotic dtes follow three types of transposition mechanisms according to different replication modes : cut - and - paste for most dtes , rolling circle replication for helitrons , and synthetic replication for polintons . in fact , there seems to be many different cases of transposon - related replication that need to be clarified in the future . as mentioned earlier , continuation of dna replication of an inserted te molecule , like in mu co - integration , may also fill the gap to generate tsds . it should be emphasized that dna transposition has been grouped in two : simple , conservative , or ' cut - and - paste ' mechanism and replicative transposition [ 16 , 17 , 29 ] . in tn5 , at the outer ends , ori - c - like 9-bp sequences are found , which may serve to direct host replication functions to the ends of the element during transposition . a synthetic mechanism is proposed for polinton propagation to explain protein - primed replication of a racket - like structure of a single - stranded polinton . if it gets to be understood that the heteroduplex structure that is derived from fbi dna may serve as the template of dna replication , many of the diverse cases of dna replication may be clarified ( see below ) . fbi dna is formed by the folding back at one point of a non - helical parallel track of double - stranded dna at as sharp as 180 and the intertwining of the bent helixes within each other 's major groove to form an intercoil with a diameter of 2.2 nm ( fig . fbi dna could be considered a double - stranded version of a hairpin or stem - and - loop structure of rna and single - stranded dna . but , such sharp bending and formation of a four - stranded intercoil are not considered possible with a double helical b - dna with a diameter of 2.0 nm . in an extended definition , fbi dna may contain a loop and a much longer stem under biological conditions . intercoil dna is designated to distinguish itself from supercoil dna , which is thicker than 2.2 nm . intercoil dna is also termed to avoid the use of four - stranded dna , which has the connotation of four - stranded base pairing among homologous repeats . it needs to be emphasized that intercoil dna is formed by two intertwining duplexes of any sequence , but when homology is met at a certain region , homologous recombination may proceed via heteroduplex formation with the help of the proper enzymes involved . fbi dna was first formulated based upon a rare but unique electron microscopy ( em ) configuration of plant mitochondrial dna ( mtdna ) molecules and space - filling models [ 7 , 8 ] . once the fbi dna structure was realized with the space filling model , it was possible to perform homologous recombination with direct contact of the repeat sequences . the intra - molecular homologous recombination of a deletion and inversion occurs via 1 ) synapsis of the repeats in the intercoil , 2 ) heteroduplex formation by 90 base flipping , and 3 ) nick - and - close resolution of the sugar phosphate backbone crossing over . inter - molecular homologous recombination , known as site - specific insertion , on the other hand , is mediated by direct contact between the attp site at the tip of the fbi dna and the attb site on the target dna . perpendicular approach of the foldback tip onto the target site effectively resolves the year - long topological puzzle encountered in four - stranded base pair winding of two circular dna molecules and explains that 6- or 7-bp attp and attb sites correspond to the diameter of the approaching fbi dna tip and the target width . dna transposition is explainable by the perpendicular approach of the detached transposon ends in intercoil form to the host target , as described in detail below . transposition of a dte molecule in fbi dna configuration is tested by a space filling model , as presented in fig . 1 and as a detailed diagram in fig . 2 . the te molecule , detached from the host by a dsb in a blunt end cut , approaches , as shown in the diagram , at a perpendicular angle to the target site . both 3'-oh ends of the te ligate with the 5'-p ends of the staggered - cut target , leaving the overhang target ends single - stranded , which would be repaired by gap filling . as shown in the diagram ( fig . 2 ) , when the te ends approach the target on the minor groove side , the target size would be 5 bp on the minor groove side and 7 bp on the major groove side . this explains the meaning of the average size of the 5 - 7-bp target duplication , as it matches the diameter of the incoming intercoil dna , 2.2 nm . in reality , the target sizes vary between 2 to even over 10 bp in a te superfamily - specific manner . this tsd size variation could be explained by several factors - namely , local squeeze and stretch of the dna double helix ; unequal cutting positions of the te ends , as exemplified in tn7 by the 3-nt overhang by staggered dsb ; the characteristics of the endonucleases involved ; and even temporary loosening of the intercoil end during a very elaborate and concerted coordination of the transpososome structure . in earlier days , when is and tn , and even the ltr retrotransposons , were the major players of transposition , it was the presence of terminal repeats that attracted attention for explaining the transpositional mechanism , as in homologous recombination . therefore , it was somewhat puzzling to explain the cases of non - ltr retrotransposons with asymmetric termini sequences . but , it is now tsd that attracts attention as a common factor for the transposition mechanism of all tes , except for helitrons . it is the structural nature of the intercoil , which is formed by intertwining duplexes , regardless of the homology between the two termini . when both ends of a te synapse in the form of an intercoil , are cut by a dsb off the host duplex strand , and approach the target in perpendicular direction , their 3'-oh ends ligate to the staggered 5'-p ends of the broken target . the resulting overhang single - strand dna gaps on both flanking ends of the target are repaired to generate the accompanying tsd . the significance of the terminal repeats is then the homologous recombination that is required for further transactions of recombination , replication , and transcription . the enhancer is a cis element of dna that affects , as a third factor , dna transactions , such as transcription , homologous recombination , and transposition [ 31 , 32 , 33 , 34 ] . it has remained somewhat of an enigma , because the presence of an enhancer element and its binding proteins at a distance in a sequence and orientation non - specific manner enhances the efficiency of the reaction by 1,000 times . rather unique configurations of rigid rod - like dna with a big protein complex at one end [ 35 , 36 fig . 4 - 2 ] may shed light on the enhancer concept and give us an idea of what actually happens under cellular conditions ( fig . 3 ) . folding back of a dna duplex at a distant point would bring an enhancer site into juxtaposition with the base of a te , which has also branched out as fbi dna ( figs . 4 and 5 ) . proteins binding to the enhancer site and to both ends of the te and any other additional factors together would form a tight dna - protein complex , called a transpososome . this transpososome would provide a tight and stable surgical platform for a series of highly concerted precision cut - and- paste reactions : namely , dsb of the te termini , sealing of the host duplexes by nhej , staggered cuts on the target site , nhej of the te 3'-oh ends and target 5'-p ends , and finally , gap - filling of the 5 - 7-nucleotide single - strand portions to flank the transposed te . in replicative transposition ( fig . first , the gap - filling reaction during tsd may continue on to the te molecule by displacement or polarized replication , even though the direction of replication may be reversible . second , as shown in the diagram , tirs in the fbi dna configuration may transform into heteroduplexes and serve as primers for bidirectional replication of the internal te , thus keeping one copy at the original site and depositing the other copy to the target site in juxtaposition . inversion of the replicated te may result from homologous recombination , as predicted [ 7 , 16 ] . tsd serves as a common landmark of unity on transposition mechanisms for both dtes and rtes . the only exception so far is the helitron superfamily , which lacks tsd and employs a rolling circle replication model . as long as a tsd is generated during te integration into the host chromosome , it indicates that the immediate intermediate of integration is in a double - stranded dna form , regardless of rna or single - stranded dna being the initial intermediate . from the em pictures of fbi and rigid rod configurations of native dna ( fig . it becomes apparent that vertical insertion of the dsb ends of the te in an intercoil alignment is probably the best - fitting mechanism of dna transposition . small sizes of the target , around 6 - 7 bp , encountered in tsd as well as in attp and attb in site - specific insertion all point to the fact that they correspond to the diameter of fbi dna 2.2 nm . the transpososome , made of fbi dna configurations of the te and the enhancer element , may effectively abolish lingering mechanistic puzzles about keeping four broken strands of the te and the host from flying apart and answer how a te molecule jumps onto a new target site at a distance . replicative transposition is explained likewise , even utilizing the terminal homology as a possible origin of replication . it would be , however , difficult , if not impossible , to reproduce such a transpososome complex in vitro by fbi dna configuration , which is critically needed for em observations and crystallography analysis . there are four main modes of transactions by which the fbi dna motif may be utilized in dna functions in the cell : alpha deletion , omega inversion , needle point site - specific insertion , and nhej - gf integration of te . with such an abundant presence in strategically important positions in the genome and very dynamic functional roles , it is hoped that fbi dna receives the proper recognition and interpretation for a better understanding of the life process . smoking is a major risk factor associated with the development and progression of a variety of cancers . smoking is estimated to account for approximately 4 - 5 million deaths worldwide and approximately 443,000 deaths each year in the united states alone [ 2 , 3 ] . sufficient evidence has accumulated to conclude that tobacco smoking caused cancers not only of the lung , but also of the lower urinary tract including the renal pelvis and bladder , upper aero - digestive tract including oral cavity , pharynx , larynx , and esophagus , and pancreas [ 2 , 4 ] . recent lines of evidence have showed that smoking tobacco can also cause cancers of the nasal cavity , paranasal sinus , nasopharynx , stomach , liver , kidney , cervix , uterus , breast , adenocarcinoma of the esophagus , and myeloid leukemia . of the thousands of chemicals in tobacco smoke , polycyclic aromatic hydrocarbons and nicotine - derived nitrosamines have been identified as the major and potent carcinogens [ 5 , 6 ] . the metabolites of these agents form dna adducts and cause mutations in vital genes like rb , p53 , and k - ras in smokers [ 79 ] . while the induction of these cancers is mediated by tobacco - specific nitrosamines as well as other carcinogens present in the tobacco smoke , it is becoming clear that signaling through the nicotinic acetylcholine receptors contribute to the growth , progression , and metastasis of a variety of cancers . nicotine , which is the major addictive component of tobacco smoke , acts through nicotinic acetylcholine receptors ( nachr ) [ 911 ] , but is not thought to be carcinogenic . the expression of nachrs in central and peripheral nervous system is associated with smoking dependence and addiction . it was generally believed that nachrs are only expressed in nervous system and at neuromuscular junctions ( muscle type nachrs ) . however , the discovery of widespread expression of nachrs in mammalian cells , including cancers , suggested its direct role in cancer progression [ 1315 ] . this paper deals with certain aspects of nicotinic receptor signaling in nonneuronal cells that lead to increased cell proliferation and survival , angiogenesis , tumor growth , and metastasis . nachrs are a complex of five subunits forming hetero- or homopentamers to form a central ion channel [ 16 , 17 ] . the neuronal nachrs can be homomeric composed of 7 , 8 , or 9 subunits or with the combinations of 26 or 10 subunits with 24 subunits ( heteromeric nachrs ) . the muscle type nachrs may be comprised of combinations of 1 subunits with 1 , , , or subunits . both neuronal as well as muscle nachr higher binding to 42-nachrs results in desensitization of the receptor , which could be the reason that 7-nachr is the major stimulator of cancer development and progression in vivo . in addition to nicotine , tobacco - specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) can also bind to 7-nachr , and n - nitrosonornicotine ( nnn ) binds to heteromeric -nachrs . the affinity of nnk for the 7-nachr was found to be 1,300 times higher than nicotine , whereas the affinity of nnn for heteromeric -nachrs was 5,000 times higher than that of nicotine [ 21 , 22 ] . since the discovery of ubiquitous presence of nachrs in mammalian cells , studies from many laboratories have linked nachrs with various pathological conditions including tumor growth and angiogenesis [ 13 , 23 ] . in earlier studies , nicotine was found to stimulate endothelial - cell proliferation via nachr at concentrations lower than those obtained in blood after smoking . as described in the later part of this paper , many studies have correlated the exposure of nicotine or other tobacco smoke components with induction of pathological neovascularization through the activation of nachr [ 23 , 25 ] . studies from our laboratory have suggested that nicotine can enhance the growth and metastasis of pre - established lung tumors . altogether , these studies proposed the involvement of tobacco smoke components in various aspects of tumorigenesis and vascular dysfunctions in smokers . extensive research by many groups has successfully associated the physiological effect of nicotine and its derivatives with the direct activation of nachrs . small cell lung carcinoma ( sclc ) pulmonary neuroendocrine cells ( pnecs ) and sclc cells express high levels of the 7-nachr , whereas heteromeric nachrs were undetectable [ 27 , 28 ] . at the same time , both hetero- and homomeric nachrs are found to be expressed in nonsmall cell lung carcinoma cells of different histologic subtypes [ 19 , 29 ] . recently , differential expression pattern of achr subunit gene was studied in nsclc patients who were smokers or never smokers . higher expression of chrna6 and chrnb3 combination was correlated with nsclcs in nonsmokers , whereas lower expression was correlated with nsclcs in smokers . additionally , increased expression of chrna1 , chrna5 , and chrna7 subunit genes was correlated with short - term exposure to nicotine . human biopsies of mesothelioma as well as of normal pleural mesothelial cells were found to express functional 7-nachr [ 31 , 32 ] . studies from the russo laboratory have shown that inhibition of nachrs by -cobratoxin ( -cbt ) can inhibit the growth of a549 tumors in immunocompromised mice . these findings strengthen the hypothesis that modulation of nachrs upon chronic exposure to tobacco may contribute to the development and progression of cancer . in the following sections , we will summarize the findings to support the hypothesis . attempts have been made to elucidate the molecular events that mediate nicotine - induced cell proliferation . activation of nachr through nicotine or nnk has been found to activate protein kinase c ( pkc ) , the serine / threonine kinase raf-1 , the mitogen - activated kinases erk1 and erk2 , and the transcription factors fos , jun , and myc through the selective activation of 7-nachr in sclc . it has been reported that ( )-nicotine stimulated tumor cell proliferation via secretion of the neurotransmitter serotonin , and the growth stimulatory effect of nicotine or nnk could be blocked by selective serotonergic receptor antagonists [ 27 , 35 , 36 ] . in a recent report , the effects of acute and repetitive exposure to nicotine was shown to induce a neuronal - like appearance in n417 sclc cell line , which produced bigger and more vascularized tumors in mice through activation of cxcr4/cxcl12 axis . a prominent increase in the expression of cxcr4 nsclc cell lines from large - cell carcinoma , squamous - cell carcinoma , and adenocarcinoma , all showed the activation of pi3k - akt pathway and nf-b activation in response to nicotine or nnk treatment [ 38 , 39 ] . in addition , frequent loss of the tumor suppressor gene foxo3a was reported in carcinogen - induced lung adenocarcinoma . in nnk - treated lung cancer cells , restoration of foxo3a in foxo3a - deficient cells increases sensitivity to apoptosis caused by a dna - damaging intermediate of nnk . this study proposed that foxo3a might play a role in lung adenocarcinoma suppression by providing a protective response to carcinogenic stress . experiments from our laboratory have shown that nicotine stimulation affects various components of cell cycle regulatory machinery [ 26 , 29 , 41 ] . exposure to nicotine resulted in activation of raf-1 , induction of cyclin d and cyclin e - associated kinase activity as well as rb phosphorylation , which led to the dissociation of e2f1 from rb . further , it was observed that stimulation with nicotine caused the dissociation of rb from e2f - responsive proliferative promoters ( cdc6 and cdc25a ) , while there were increased amounts of e2f1 bound to them . these molecular events were correlated with increased proliferative effects of nicotine in nsclc cell lines a549 ( human bronchioalveolar carcinoma ) , nci - h23 , nci - h441 ( lung adenocarcinoma ) , and nci - h226 ( pleural effusion squamous cell carcinoma ) as well as on primary normal human bronchial epithelial cells ( nhbes ) , small airway epithelial cells ( saecs ) , human aortic endothelial cells ( haecs ) , and human microvascular endothelial cells from lung ( hmec - ls ) . the mitogenic effects of nicotine were abrogated by 7 subunit antagonists , -bungarotoxin , and methylallyl aconitine ( maa ) , whereas it was unaffected by -lobeline ( 42 subunit inhibitor ) or dihydro -erythoidine ( dhe ; 32 and 42 subunit inhibitor ) , suggesting that 7 subunits primarily mediated the mitogenic effects of nicotine in nsclc cells . we have further illustrated that upon nicotine stimulation , the scaffolding protein -arrestin-1 forms a complex with nonreceptor tyrosine kinase - src and gets recruited to the nachrs . these results suggested that 7-nachr - mediated stimulation of cell proliferation is through a -arrestin-1-src signaling axis in nsclc ; ( see also figure 1 ) . other than lung cancer , activation of 7-nachr and heteromeric nachrs expressing 3 and 5 subunits have been reported in oral and esophageal keratinocytes . similar to lung cancer cells , nnk was found to bind with high affinity to 7-nachr , whereas nnn was found to bind to heteromeric nachrs with higher affinity . esophageal cancer - het-1a cells stimulated with nnk or nnn showed increased mrna transcripts and expression of pcna and bcl-2 , and transcription factors gata3 , nf-b , and stat1 . however , induction of ras - raf - erk1-erk2 cascade , the jak2-stat3 pathway and nf-b activation was associated with enhanced cell proliferation through these nitrosamines in immortalized oral epithelial cells . in addition , chronic exposure of nicotine or environmental tobacco smoke on oral keratinocytes selectively upregulated 5- and 7-nachr subunits , resulting in intensified signaling responses to nicotine . the secreted mammalian ly-6/urokinase plasminogen activator receptor - related protein-1 ( slurp-1 ) is recently identified as an endogenous ligand for the 7 subunit of the nicotinic acetylcholine receptor ( nachr ) . the expression levels of slurp1 and slurp2 ( secreted mammalian ly-6/urokinase plasminogen activator receptor - related protein-2 ) transfection of the cells with slurp1 or slurp2 cdna reduced the nitrosamine - induced colony formation in soft agar while inhibiting the growth of nnk - transformed keratinocytes in mouse xenografts . slurp1 bound to 7-nachr and slurp2 bound to nachrs expressing the 3 subunit [ 22 , 43 ] . similar results were demonstrated recently where ht-29 human colon cancer cells treated with nicotine resulted in increased cell proliferation and a marked reduction in the protein expression of slurp1 via 7-nachrs activation . recently , nicotine mediated upregulation of foxm1 expression was found in primary oral keratinocytes which was associated with induction of genomic instability . a centrosomal protein cep55 as well as a dna helicase and putative stem cell marker hells , were found to be novel targets of nicotine - induced foxm1 expression and correlated with oral cancer progression . experiments with human mammary epithelial - like mcf10a or cancerous mcf7 cells revealed that treatment of these cells with nicotine enhances the activity of protein kinase c ( pkc ) alpha with cdc42 as a downstream target for nicotine - induced proliferation and migration . it has also been suggested that nicotine - induced proliferation of human breast cancer cell is dependent on 9-nachr and cyclin d3 expression . the effects of nicotine on a population of cancer stem cells in mcf-7 human breast cancer cells were examined , using aldehyde dehydrogenase ( aldh ) as a stem cell marker . this study found that nicotine increases the stem cell population via 7-nachr and the pkc - notch dependent pathway . apart from direct responses through nachrs , indirect signaling events may also contribute to nicotine - induced tumor growth and survival . since nachrs are cation channels , it can stimulate signaling cascades by the influx of ca2 + through the opened 7-nachr . ca2 + channel blockers are shown to significantly reduce dna synthesis in response to nicotine or nnk in sclcs . also , nachr - mediated systemic increase in stress neurotransmitters , adrenaline , and noradrenaline , which are -adrenergic agonists , are also shown to stimulate -adrenergic receptor - initiated camp signaling and transactivation of egfr cascade through egf secretion in nnk - treated small airway epithelial cells [ 50 , 51 ] . nicotine is found to induce systemic or cellular increase in noradrenaline and significantly enhance the growth and angiogenesis of pancreatic , gastric , and colon cancer - xenografts with increased expression of erk1-erk2 , cox2 , prostaglandin e2 , vegf , and transactivation of -adrenergic as well as egfr signaling in colon cancer cells [ 5255 ] . activation of erk1-erk2 and stat3 in response to nicotine has also been reported in bladder cancer cells downstream of nachrs and -adrenergic receptors . importantly , apart from nachrs , direct interaction of nnk with -adrenergic receptor has been proposed as a novel mechanism , which may significantly enhance the high cancer - causing potential of these nitrosamines [ 50 , 57 ] . similar to the activation via neurotransmitters , nnk binding to -adrenergic receptor was also found to activate adenylyl cyclase - camp - pka - creb cascade and transactivation of efgr . additionally , an additive effect of estrogen receptors and nachrs was also demonstrated in promoting the growth of a549 tumors in athymic nude mice . cotreatment of nicotine and estradiol resulted in increased cell proliferation as well as vegf secretion from cancer cells , leading to increased tumor growth as well as microvascular density within the tumor . recently , the chronic exposure to estrogen and nnk was shown to have synergistic effects on cell proliferation and production of noradrenaline and adrenaline , by upregulating 7-nachrs in immortalized small airway epithelial cells . in addition to the effect on tumor growth , epidemiological and clinical data implicate that in patients with cancer , continued smoking causes resistance to therapy by blocking the induction of apoptosis . various studies have linked the activation of nachr resulting in inhibition of apoptotic pathways . in sclc cells , nnk was shown to phosphorylate bcl-2 at ser70 which promoted its interaction with c - myc that significantly enhanced the half - life of the c - myc protein . this functional cooperation of bcl2 and c - myc resulted in promoting cell survival and proliferation . this effect could be blocked by the pkc inhibitor staurosporin , the erk1-erk2 inhibitor pd98059 or silencing of myc [ 61 , 62 ] . additionally , mesothelioma cells also showed nicotine - stimulated proliferation through 7-nachr - mediated ca - dependent activation of the erk1-erk2 cascade and inhibited apoptosis by induction of nf-b and phosphorylation of bad at ser112 ( bcl-2 antagonist of cell death ) . in nsclcs , constitutive activation of akt similarly , nicotine or nnk exposure displayed akt - mediated growth and nf-b - mediated resistance to apoptosis in human airway epithelial cells as well as lung cancer cells [ 38 , 39 ] . treatment of cells with the phosphatidylinositol 3-kinase ( pi3k ) inhibitor ly294002 or specific depletion of akt was shown to block both nicotine - induced bax phosphorylation and cisplatin resistance in nsclc cells . in addition to these signaling events , results from our laboratory revealed a significant role for the iap proteins xiap and survivin in nicotine - mediated chemoresistance in nsclcs in vitro . chromatin immunoprecipitation assays demonstrated that nicotine stimulation caused an increased recruitment of e2f1 and concomitant dissociation of retinoblastoma tumor suppressor protein ( rb ) from survivin promoter in nsclc cells . moreover , ablation of e2f1 levels caused abrogation of survivin expression and protective effects of nicotine against cisplatin - induced apoptosis in a549 cells . in the above study , chemoprotective effect of nicotine was found to be mediated through 3/4-nachr activation and could be abrogated by agonists of these subunits . it was also found that nicotine stimulation enhanced the levels of xiap at the protein level . nicotine induces the activation of akt , which is known to phosphorylate xiap and prevent its proteasome - mediated degradation . in agreement with this , an akt inhibitor could abrogate the antiapoptotic effects of nicotine in a549 cells . in other studies , the cooperative effect of nicotine and nnk was investigated for their transforming ability in various lung epithelial or cancer cells . exposure to nicotine or the combination of nicotine and nnk for one week augmented bcl-2 expression , accompanied by an increased resistance to cisplatin - induced apoptosis . this study also showed that the combination treatment promoted cell proliferation and anchorage - independent growth as compared to nnk exposure alone . in another study , nicotine - induced mcl-1 phosphorylation significantly enhanced the half - life of mcl-1 , which conferred long - term survival potential . specific depletion of mcl-1 by rna interference blocked nicotine - stimulated survival and enhanced apoptotic cell death . nicotine - mediated activation of 7-nachr has also been linked with the expression of ppar/ protein by inhibiting ap-2 protein expression and dna binding activity to the ppar/ gene promoter . antagonist and short interfering rna against 7-nachr as well as inhibitors of phosphatidylinositol 3-kinase ( pi3k ; wortmannin and ly294002 ) and mammalian target of rapamycin ( mtor ; rapamycin ) blocked the expression of ppar/ protein demonstrating a novel mechanism by which nicotine could promote human lung carcinoma cell growth . these studies show that signaling through the nachrs could promote cell proliferation and survival , utilizing multiple signaling cascades . angiogenesis , the formation of new blood vessels from pre - existing vasculature , is a complex multistep process involved in a number of physiological processes such as wound healing , embryogenesis and reproduction . in addition , angiogenesis is necessary for the sustained growth of the primary tumor as well as metastatic dissemination . the proangiogenic activity of nicotine is mediated by nicotinic acetylcholine receptors , particularly 7 subunit . the pioneering study by villablanca ( 1998 ) demonstrated the ability of nicotine to induce endothelial cell proliferation . this observation was followed by the elegant studies from the john cooke 's laboratory suggesting a cholinergic pathway for nicotine - induced angiogenesis where they demonstrated complete inhibition of endothelial network formation using nonselective nachr antagonist mecamylamine in an in vitro angiogenesis model . although several nachr isoforms are expressed on endothelial cells , a similar inhibition was obtained only with the selective 7-nachr antagonist -bungarotoxin , confirming the specific involvement of 7-nachr . further , in vivo pharmacological inhibition of nachr and a genetic disruption of 7-nachr expression significantly inhibited inflammatory angiogenesis and reduced ischemia - induced angiogenesis and tumor growth . they also provided anatomic and functional evidence for nicotine - induced angiogenesis and arteriogenesis when they observed that nicotine accelerated the growth of tumor and atheroma in association with increased neovascularization . nicotine increased endothelial - cell growth and tube formation in vitro , and accelerated fibrovascular growth in vivo . in a mouse model of hind - limb ischemia , nicotine increased capillary and collateral growth , and enhanced tissue perfusion . these effects of nicotine were mediated through nicotinic acetylcholine receptors at nicotine concentrations that are pathophysiologically relevant and suggested a possible role for the endothelial production of nitric oxide , prostacyclin , and vascular endothelial growth factor [ 7074 ] . nicotine has been demonstrated to stimulate postnatal angiogenesis , having an antiapoptotic effect on endothelial cells . it was observed that nicotine stimulated postnatal vasculogenesis on endothelial progenitor cells ( epcs ) . furthermore , the antiapoptotic effect of nicotine was blocked completely by nicotinic acetylcholine receptor ( nachr ) antagonist hexamethonium bromide . recent studies have shown that apart from cigarette smoking , exposure to secondhand smoke also could induce angiogenesis . a positive correlation between secondhand smoke exposure and concentrations of nicotine in the body further , it was found that the levels of nicotine exposure from secondhand smoke were comparable to those of active smokers . in a mouse model where lewis lung cancer cells were implanted subcutaneously into mice , which were then exposed to sidestream smoke ( shs ) or clean room air and administered vehicle or mecamylamine ( an inhibitor of nachr ) ; shs significantly increased tumor size , weight , capillary density , vegf , and mcp-1 levels , and circulating endothelial progenitor cells ( epc ) . mecamylamine partially inhibited the effects of shs on these angiogenic processes and nearly abolished the effects of shs on tumor capillary density suggesting that nicotine mediated the effects of shs on tumor angiogenesis and growth . several recent studies have implicated that nicotine - induced angiogenesis could be mediated by growth stabilization and transmigration of endothelial progenitor cells ( epc ) [ 75 , 78 , 79 ] . nicotine accelerated the growth of syngenic colon cancer cmt93 cells when grown subcutaneously in mice by inducing angiogenesis via bone marrow derived epcs . to determine if the angiogenic effects of nicotine is mediated by epc mobilization , heeschen et al . used a model of mouse parabiosis and found that nicotine enhances epc mobilization into the vasculature of the ischemic tissue . this effect may be due to the direct actions of nicotine on epc proliferation , migration and/or mobilization as suggested by in vitro models and plasma markers used in the investigation . they also noticed that in the absence of acute ischemia , nicotine did not stimulate epc mobilization . the activation of nachrs in response to ischemia induced the release of proangiogenic factors like vegf and stem cell derived factor-1 , both of which are regulated by hypoxia , which in turn facilitates epc mobilization . evidence from another study also demonstrated that nicotine promotes angiogenesis via stimulation of nachr - dependent endothelial cell migration . nachr antagonism not only abolished nicotine - induced human microvascular endothelial cells ( hmvec ) migration but also abolished migration induced by bfgf and attenuated migration induced by vegf . transcriptional profiling identified gene expression programs which were concordantly regulated by all 3 angiogens ( nicotine , vegf , and bfgf ) , a notable feature of which includes corepression of thioredoxin - interacting protein ( txnip ) , endogenous inhibitor of the redox regulator thioredoxin . furthermore , interestingly , nachr antagonism abrogates growth factor ( vegf- and bfgf- ) mediated induction of thioredoxin activity suggesting the requirement of nachr activation in endothelial cell migration , a key angiogenesis event . the proangiogenic effects of nicotine have been found to be mediated by 7-nachr on endothelial cells by activating erk / map kinase pathway , pi3 kinase / akt pathway , and nf-b [ 23 , 25 , 83 , 84 ] . further , nicotine has been shown to induce the proangiogenic factors like vegf and hif-1 in nsclc cell lines . pharmacologically blocking nachr - mediated signaling cascades , including the ca2+/calmodulin , src , protein kinase c , pi3k / akt , mapk / erk1/2 , mtor pathways , significantly attenuated nicotine - induced upregulation of hif-1. these proangiogenic and invasive effects of nicotine were partially abrogated by depleting hif-1 using sirna techniques . nicotine also enhanced the activity of matrix metalloproteinase 2 and 9 and expression of plasminogen activators in a cox2 and vegfr2-dependent manner . the proangiogenic effect of nicotine has been shown to be dependent on src activity by our laboratory . the inhibition of src , using chemical inhibitors or sirna has been shown to inhibit endothelial cell proliferation , migration , and angiogenic tubule formation on matrigel . as mentioned earlier , studies from our laboratory suggest that the scaffolding protein -arrestin-1 causes the activation of src . oligomeric complex comprising of nachr , -arrestin-1 , and src is vital for nachr signaling . in addition , depletion of -arrestin-1 caused abrogation of endothelial cell proliferation and angiogenic tubule formation [ 29 , 41 ] . these data suggest that nicotine behaves in a manner analogous to growth factors and induces cell cycle progression in endothelial cells . epithelial to mesenchymal transition ( emt ) is a biological process that allows a polarized epithelial cell , which normally interacts with the basement membrane through its basal surface , to undergo multiple biochemical changes with a signature of more advanced and less differentiated cancer that allow it to assume a mesenchymal phenotype . this enhanced migratory capacity , invasiveness , resistance to apoptosis , and greatly increased production of ecm components [ 8789 ] . this process results in degradation of basement membrane and the formation of a mesenchymal like cell , which can migrate away from the epithelial layer in which it originated . epithelial to mesenchymal transition ( emt ) is involved in tumor progression from noninvasive tumor cells into metastatic carcinomas . recent studies from our laboratory demonstrated that nicotine can induce invasion and migration in cell lines derived from lung cancer , breast cancer , and pancreatic cancer via 7-nachr - mediated signal transduction pathways . the proinvasive effects of nicotine were mediated by 7-nachr in lung cancer cells while 7-nachr and dhe sensitive nachrs mediated invasion of breast cancer cells . long - term treatment of lung cancer and breast cancer cells with nicotine was found to diminish levels of epithelial markers namely -catenin and e - cadherin and upregulate mesenchymal proteins like fibronectin and vimentin , indicative of disruption of cell - cell contacts and increased motility . in addition to facilitating emt , nicotine and nnk have been shown to affect various aspects of tumor cell invasion and migration . for example , both nicotine and nnk are shown to promote the invasion of nsclc by phosphorylation of and m - calpains . several lines of evidence show that calpain - mediated proteolysis mediates various aspects of cell physiology including cell migration and invasion . nicotine was found to induce phosphorylation of both and m - calpains via 7-nachr ; the binding of nicotine to 7-nachr in turn was found to activate src and pkc - iota , leading to enhanced invasion and migration of nsclc cell line h1299 . similarly , nnk also could promote invasion and migration through phosphorylation of and m - calpains in a 7-nachr - dependent fashion . several observations in patients suggest that those exposed to tobacco carcinogens are more likely to develop larger , more vascularized tumors with a high propensity for metastatic spread and resistance to chemotherapy . about 30% of lung cancer patients who are smokers continue to smoke after they have been diagnosed , which might result in increased adverse medical consequences such as increased tumor progression , development of a second cancer , greater recurrence , greater cancer - related mortality and reduced quality of life [ 92 , 93 ] . while these studies demonstrate a role for tobacco carcinogens in the initiation , growth , and progression of cancers , the relative contribution of nicotine by itself to these processes is not well explored . a recent study from our laboratory demonstrated that nicotine by itself can induce the growth and metastasis of tumors in immunocompetent mice , independent of other tobacco carcinogens . nicotine administered either intraperitonially or by commercially available transdermal patches could substantially promote tumor growth . similar effects were observed on implanted tumors as well as tumors induced by tobacco carcinogen , nnk . furthermore , mice exposed to nicotine showed significantly enhanced lung metastasis as well as tumor recurrence after surgical removal of the primary tumor , indicating that nicotine can enhance the growth and metastasis of pre - established lung tumors . as mentioned earlier , repetitive exposure to nicotine on sclc - n417 cells resulted in neuronal - like appearance along with increased adhesion to the extracellular matrix . these changes were accompanied by enhanced migration through collagen matrices and adhesion to and transmigration across lymphatic endothelial cell monolayers . accumulating evidence from epidemiological studies suggest a strong association between smoking and pulmonary metastatic disease in women with breast cancer . in a murine model of metastatic mammary cell cancer , cigarette smoke exposure was associated with an increase in the total pulmonary metastatic burden providing experimental support for an adverse effect of smoking on the metastatic process and suggesting a possible mechanism for smokers ' increased breast cancer mortality . in addition , it was observed that cigarette smoking was correlated with increased lymph node metastases at mastectomy in women older than 50 years of age suggesting that tobacco usage may potentiate the early spread of malignant disease . although numerous studies have indicated the role of nicotine exposure in tumor promotion , little is known about the molecular mechanisms by which nicotine promoted breast tumor development , especially on the metastatic process of breast cancer . at least four different subunits of nachrs including 5 , 7 , 9 , and 4 are shown to be expressed in breast cancer cells . it has been demonstrated that in addition to proliferative effect , nicotine promoted migration of breast cell lines ( mammary epithelial cell line mcf10a and breast cancer cell line mcf7 ) through a signaling cascade involving pkc activation and its downstream effector cdc42 . exposure to nicotine has shown to increase the expression of 9-nachr in breast cancer cells [ 47 , 97 ] . studies using a soft agar transforming assay and a mouse xenograft model demonstrated that noncancerous human breast epithelial cell line , mcf10a , could be neoplastically transformed by exposure to either a cigarette smoke condensate or the tobacco specific carcinogen , nnk [ 98 , 99 ] . in a recent study , 9-nachr expression was silenced in mda - mb-231 breast cancer cells which resulted in reduced proliferation and tumorigenic potential in both in vitro and in vivo assays , indicating the role of 9-nachr in breast carcinogenesis . cigarette smoking has recently been recognized as a risk factor for gastric cancer and long - term exposure of nicotine - induced emt like changes in gastric cancer cell lines by activating erk/5-lox signaling pathway . a study on the association between cigarette smoking and pancreatic cancer showed that smokers had a significantly higher risk ( 70% ) of developing pancreatic cancer compared to nonsmokers [ 103105 ] . accumulating evidence suggests that nicotine induces expression of osteopontin , a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival , proliferation , invasion , and metastasis . rats exposed to cigarette smoke showed a dose - dependent increase in pancreatic osteopontin expression . in addition , analysis of cancer tissues from invasive pancreatic ductal adenocarcinoma ( pda ) patients , the majority of whom were smokers , showed the presence of significant amounts of osteopontin in malignant ducts and the surrounding pancreatic acini . further studies suggested that nicotine contributes to pda metastasis by inducing mmp9 and vegf expression and osteopontin mediated these effects . an osteopontin isoform , opnc , is selectively inducible by nicotine and is highly expressed in pda tissues from smokers which induced the expression of monocyle chemoattractant protein ( mcp-1 ) indicating a proinflammatory role of nicotine . altogether , these results suggest that nicotine plays a key role in the regulation of the complex cellular cascades that modulate cell adhesion , invasion , and migration leading to metastasis . as summarized in figure 1 , nicotine , the principal addictive component of tobacco smoke , as well as other nitrosamines have been found to act through nachrs on nonneuronal cells to facilitate tumor growth , angiogenesis , metastasis , survival , and chemoresistance by regulating diverse signaling pathways . binding of agonist to nachr facilitates the complex formation between the receptor , scaffolding protein -arrestin and tyrosine kinase src . activation of src was found to be important for cancer as well as endothelial cell proliferation and angiogenic tube formation in vitro . proliferative effect of nachr - activation was also supported by indirect stimulation of -adrenergic receptor ( -ar ) signaling . further , chemotherapy - induced apoptosis was found to be blocked by nicotine - induced survivin expression as well as nf-b activation . activation of nachr is also correlated with emt - like changes and metastatic dissemination of primary tumor cells . given the ability of nicotine to affect various aspects of tumor growth and metastasis , antagonists of nachr signaling might be beneficial in controlling the growth and progression of tumors . recently , alpha cobratoxin ( -cbt ) has been shown to block the growth of a variety of nsclc and mesothelioma cell lines both in vitro and in vivo [ 109 , 110 ] . the most striking effect of -cbt was its ability to effectively inhibit the metastatic potential of lung cancer cells transplanted into nude mice , indicating the possibility of using nachr antagonists as adjuvant therapy in preventing metastatic spread . at the same time , the potential side effects of nachr antagonists on the brain and central nervous system need to be investigated before using them as a viable drug for combating lung cancer . moreover , the direct role of nicotine alone on several aspects of tumorigenesis raises the need to revisit the potential tumor promoting effects of nicotine - replacement therapy . also , the modulation effects of secondhand smoke on nachrs require detailed investigation in the future .

foldback intercoil ( fbi ) dna is formed by the folding back at one point of a non - helical parallel track of double - stranded dna at as sharp as 180 and the intertwining of two double helixes within each other 's major groove to form an intercoil with a diameter of 2.2 nm . fbi dna has been suggested to mediate intra - molecular homologous recombination of a deletion and inversion . inter - molecular homologous recombination , known as site - specific insertion , on the other hand , is mediated by the direct perpendicular approach of the fbi dna tip , as the attp site , onto the target dna , as the attb site . transposition of dna transposons involves the pairing of terminal inverted repeats and 5 - 7-bp tandem target duplication . fbi dna configuration effectively explains simple as well as replicative transposition , along with the involvement of an enhancer element . the majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the fbi dna - mediated perpendicular insertion of the paired intercoil ends by non - homologous end - joining , together with gap filling . a genome - wide perspective of transposable elements in light of fbi dna is discussed . cigarette smoking is highly correlated with the onset of a variety of human cancers , and continued smoking is known to abrogate the beneficial effects of cancer therapy . while tobacco smoke contains hundreds of molecules that are known carcinogens , nicotine , the main addictive component of tobacco smoke , is not carcinogenic . at the same time , nicotine has been shown to promote cell proliferation , angiogenesis , and epithelial - mesenchymal transition , leading to enhanced tumor growth and metastasis . these effects of nicotine are mediated through the nicotinic acetylcholine receptors that are expressed on a variety of neuronal and nonneuronal cells . specific signal transduction cascades that emanate from different nachr subunits or subunit combinations facilitate the proliferative and prosurvival functions of nicotine . nicotinic acetylcholine receptors appear to stimulate many downstream signaling cascades induced by growth factors and mitogens . it has been suggested that antagonists of nachr signaling might have antitumor effects and might open new avenues for combating tobacco - related cancer . this paper examines the historical data connecting nicotine tumor progression and the recent efforts to target the nicotinic acetylcholine receptors to combat cancer .

ischemic mitral regurgitation can be defined as moderate to severe mitral leak precipitated by acute myocardial infarction . valve repair is now the procedure of choice , but some cases can pose difficult anatomy . most patients with ischemic mitral regurgitation have predominant annular dilatation at the posterior commissure and require only ring annuloplasty . if papillary muscle elongation or rupture occurs , gore - tex artificial chordal replacement performs well . with ischemic mitral regurgitation accompanying posterior ventricular aneurysms , standard trans - atrial mitral repair provides the best results , with associated aneurysms being repaired concurrently . surgical approaches and technical outcomes of mitral repair in ischemic mitral regurgitation are illustrated in 5 patients using operative images and echocardiograms . each method is illustrated , including ring annuloplasty , pericardial leaflet augmentation , artificial chordal replacement , and ventricular aneurysm repair . using these techniques , virtually all ischemic mitral regurgitation can be repaired , with consequential patient benefits , even in the most complex anatomy . ischemic mitral regurgitation has been shown to have better outcomes when managed with valve repair . using combinations of annular , leaflet , and chordal procedures , even complex ischemic mitral regurgitation can undergo autologous reconstruction with excellent long - term results . several divergent concepts currently exist about the prognosis and management of ischemic mitral regurgitation ( imr ) . they range from little can be done to modify the dismal prognosis - to mitral repair can cure the disorder ; from mitral repair should be uniform - to prosthetic replacement achieves more certain results . one problem is that conclusions have been based on multiple different surgical approaches between centers , and also , statistical techniques often have been suboptimal . thus , it is likely that outcome benefits did vary between institutions , with differences in surgery and other factors , accounting for the divergent conclusions . the goal of this paper is to review repair techniques for imr used over the past 25 years at duke university medical center and to document observed outcome characteristics . imr can be defined as mitral regurgitation precipitated by myocardial infarction , with essentially normal leaflet and chordal support . imr usually is associated with myocardial necrosis , rather than reversible ischemia , and the infarction is usually inferior . fact , mitral valve competence requires the integrated function of all five aspects of the valve - annulus , leaflets , chordae , papillary muscles , and ventricular wall . as defined in george burch s classic 1966 papillary muscle dysfunction paper , most cases involve predominant posterior annular dilatation , but others experience leaflet tethering due to infarct expansion . rarely , papillary muscles can elongate or rupture , producing leaflet prolapse . the common type of imr involves the posterior wall , usually is caused by right and/or circumflex coronary occlusion , produces moderate posterior - lateral left ventricular ( lv ) dysfunction , and usually will not reverse with revascularization alone . as described in the 1980 s , the decision to repair the valve should be based on the appearance of moderate or severe imr on the intra - operative transesophageal echo ( tee ) , sometimes with provocative volume loading from the pump before bypass . surgical techniques . posterior annular dilatation is an important mechanism of imr , and has been almost uniformly corrected in our practice with a true - sized full rigid or semi - rigid ring . it has been shown that fixed reduction in anterior - posterior annular geometry is extremely important in this entity . these patients often have minor leaflet abnormalities , such as fetal commissural cusps or clefts , predisposing them to opening up the valve with ischemic posterior annular dilatation . occasionally , leaflets can be tethered by infarct expansion , contributing to lack of valve closure . as langer showed , a posterior leaflet autologous pericardial patch can be used to compensate for the tethering , and this approach provides a large margin of safetylong - term , especially if further geometric changes occur . for leaflet prolapse due to elongated or ruptured papillary muscles , gore - tex artificial chords can be placed to a viable papillary muscle , adjusting chordal lengths after ring placement , as we have described . the first patient is a 77 year old man , experiencing an acute circumflex infarct that produced acute moderate to severe mitral regurgitation ( mr ) and congestive heart failure . on tee ( figures 1 a , b ) , there is loss of valve coaptation at the posterior commissure ( red arrow ) , due to annular dilatation from a large posterior lateral infarct ( green arrow ) . this produced the characteristic loss of coaptation area and a central mr jet , but both leaflets are at the annular plane and not tethered or prolapsed ( figure 1c ) . after simple insertion of a full rigid ring , the valve is entirely competent ( figure 1d ) . panel a is a short axis view of the left ventricle , showing an expanded posterior - lateral wall infarct ( green arrow ) . panel b shows a gap in mitral coaptation at the posterior commissure during systole ( red arrow ) , producing a central regurgitant jet ( panel c ) , but the leaflets are at the annular plane . this is the most common echocardiographic appearance of ischemic mitral regurgitation . in panel d , the second patient is a 55 year old obese diabetic who experienced a right coronary infarct and imr 6 months earlier that were treated with right coronary artery ( rca ) stenting . despite revascularization , imr and she then developed an in - stent stenosis in the rca and an additional left anterior descending coronary artery ( lad ) lesion , and her ventricle showed residuals of the inferior infarct , with generalized dilatation . there was loss of central mitral coaptation , due to annular dilatation , a hint of tenting and leaflet tethering ( figure 2a ) , and moderate to severe mr with a central jet ( figure 2 b).when the valve was inspected , however , the posterior leaflet seemed fine with good closure and minimal tethering on valve testing by ventricular distension with cold saline ( figure 2c ) . therefore , the valve is repaired by simple placement of a carpentier ring , with good recovery of competence , and no residual leak ( figure 2d ) . so , most ischemic mitral regurgitation ( mr ) can be repaired with full ring annuloplasty alone , which achieves excellent results , with a documented 2% late reoperation rate , 9% late moderate mr recurrence rate , and quite satisfactory echocardiographic results . clinical images in a patient with chronic ischemic mitral regurgitation . panel a shows a hint of leaflet tethering ( green arrow ) , with both leaflets slightly below the annular plane . in panel c , the posterior leaflet seems to coapt well ( red arrow ) with no evidence of tethering during valve testing with cold saline injection . therefore , a simple ring annuloplasty was performed , and in panel d , the valve is completely competent . the third patient is a frail elderly female presenting with an acute circumflex infarct and severe imr , treated initially with percutaneous coronary intervention ( pci ) . unlike the first 2 patients , she had prominent posterior leaflet tethering by echocardiography ( figure 3a ) , and severe lv dysfunction , mainly in the lateral wall due to the infarct . on emergency angiography , she had an occluded circumflex artery , but also lad and rca stenoses . the circumflex was stented , but the imr persisted , the heart failure remained severe , and the ejection fraction continued at a 0.20 level . a magnetic resonance viability study was performed . in imr patients with low ejection fractions , we will operate if 2 of the 3 regions appear viable . in fact , this patient seemed to have good residual myocardium everywhere . upon inspecting the valve , the right aspect of the posterior leaflet was tethered , and the posterior leaflet would nt coapt , even with saline distension . in figure 3b , one can see the posterior leaflet pulled down into the ventricle with tethering from infarct expansion . a gluteraldehyde - fixed autologous pericardial patch was inserted into the posterior leaflet to compensate for the tethering , and the valve became nicely competent . most of what is seen in figure 3c is patch , with the posterior leaflet moving down into the ventricle to become the entire coaptation area , and the valve is entirely competent ( figure 3d ) panel a shows both leaflets tented into the ventricle and not closing properly due to tethering ( green arrow ) . direct inspection in panel b reveals taught posterior leaflet chords at the posterior commissure , inhibiting valve closure ( red arrow ) . after insertion of an autologous pericardial patch into the posterior leaflet and placement of a carpentier ring ( panel c ) , the valve is entirely competent with a large coaptation area ( panel d ) . the margin of safety provided by the patch may reduce later imr recurrence with further infarct expansion , or other problems . a transthoracic echocardiogram , obtained 6 months postoperatively , showed good valve opening , and only trivial residual leak . the fourth patient is a 57 year old man who had a major right coronary infarct , producing severe mr , a posterior lv aneurysm , and severe heart failure . he also had lad disease , and - there was a large posterior wall aneurysm ( figure 4a ) . the aneurysm was resected and closed primarily , approximating viable aneurysm edge to viable edge and removing all non - contractile scar . upon inspecting the valve , he had a chronically ruptured posterior papillary muscle at the posterior commissure , with severe prolapse that was not appreciated on the echo ( figure 4b ) . a 2 - 0 gore - tex artificial chord was placed to a viable papillary muscle and stuffed into the ventricle for later retrieval . after ring placement , after repair , the valve was nicely competent ( figure 4c ) , with no residual leak ( figure 4d ) . panel a is the left ventriculogram from a patient with severe ischemic mitral regurgitation and a large posterior wall aneurysm ( green arrow ) . in panel b is shown an associated chronically ruptured papillary muscle producing prolapse of a posterior commissural cusp ( red arrow ) . in panel c , an artificial chord has been placed to the commissural cusp , along with a carpentier ring , and the valve is completely competent ( panel d ) . combined chords and patches . the last patient is a 61 year old man with a remote coronary bypass for an inferior infarction , and severe persistent imr due to simultaneous leaflet tethering and prolapse - a rare combination . in figure 5a , one can see 2 separate jets of imr , with the posterior leaflet tethered down into the ventricle ( figure 5b ) and a segment of anterior leaflet prolapsing ( figure 5c ) . thus , the patient had simultaneous posterior leaflet tethering from infarct expansion , and anterior leaflet prolapse due to papillary muscle elongation . with a patch to the posterior leaflet and an artificial chord to the anterior leaflet , the valve was completely competent with good leaflet position and no residual leak ( figure 5d ) . this case illustrates that some varieties of imr can be quite complex , but with various combinations of full rings , pericardial patches , and artificial chords , the vast majority can be effectively repaired . intraoperative transesophageal echo frames from an ischemic mitral regurgitation patient with both posterior leaflet tethering and anterior leaflet prolapse . after repair of both lesions with a posterior leaflet pericardial patch and a chord to the anterior leaflet , the valve is fully competent ( panel d ) . outcome characteristics of mitral repair.from several sources , it is now clear that the generally poor prognosis after mitral valve repair for imr ( a 56% 5-year unadjusted survival in our series ) is due primarily to the adverse patient profiles that are characteristic of this population , such as advanced age , numerous co - morbidities , and left ventricular dysfunction . if these baseline risk factors are taken into account with a cox model ( figure 6 ) , valve repair seems to restore these patients to an adjusted survival that is similar to standard coronary bypass . also of note in this analysis , is the average 14% better risk - adjusted survival with valve repair , as compared to prosthetic valve replacement , which is consistent with most recent analyses . survival curves over 15 years of follow - up adjusted with a cox proportional hazards model for differences in all important baseline patient characteristics . the blue curve represents adjusted survival characteristics for 16,209 patients having coronary bypass alone without intraoperative transesophageal ( group 1 ) . the red curve represents adjusted survival for 3,181 patients with mild - to - moderate intraoperative transesophageal treated with coronary bypass alone ( group 2a ) . the green curve is adjusted survival for 416 patients with moderate - to - severe intraoperative transesophageal managed with mitral valve repair ( group 2b ) . and finally , the brown curve represents adjusted survival of 106 patients with moderate - to - severe intraoperative transesophageal receiving mitral valve replacement ( group 2c ) . mitral repair for moderate - to - severe intraoperative transesophageal restored adjusted survival to levels equivalent to standard coronary bypass patients with similar risk profiles . mitral valve replacement achieved an average 14% lower risk - adjusted survival over 15 years , as compared to valve repair . in summary , the pathophysiology of ischemic mr can be complex , with aspects involving the annulus , leaflets , chords , papillary muscles , and ventricular wall . based on our clinical experience , mitral valve repair should be the procedure of choice for moderate or worse imr , as encountered in the coronary disease population . finally , effective mitral repair restores imr patients to a prognosis that is similar to standard coronary bypass , for a given patient s age and co - morbidities , and repair seems superior to valve replacement in all subsets . a dna sequence contains six potential open reading frames ( orfs ) , three on one strand and three on the reverse strand . however , typically only one of the six is actually expressed because it is associated with appropriate genetic signals that specify the dna strand and the reading frame to be transcribed and translate . exceptions occur in which more than one open reading frame is translated into a protein , as has long been observed in the case of viral genes , where it was suggested that this property permitted a high packing density of information ( 1 ) . however , analysis of the coding potential of 481 prokaryotic genomes revealed the surprisingly high frequency of alternate orfs of annotated genes especially in high g + c rich genomes , where almost every annotated orf exhibits an alternative orf that could potentially encode a protein of 100 amino acids or more ( 2 ) . the frequency of alternate open reading frames in high g + c genomes gives rise to the possibility that this property could be exploited to evolve novel genetic information and it is important to be able to detect this potential . however , this high frequency also provokes serious problems of gene annotation , where the incorrect orf may inadvertently be mis - annotated as the coding sequence . this potential for error is especially problematic when automatic gene prediction programs are used to annotate genomes , but errors can also slip by human annotators . the problem is exacerbated if an alternative orf is mis - annotated and the error is propagated in subsequent genome annotations . alterorf provides a searchable database of all possible alternative orfs in sequenced prokaryotic genomes that are potentially capable of encoding proteins of 100 amino acids or more . the objectives are 2-fold : to improve genome annotation by indicating possible errors in orf identification and , perhaps more important in the long term , to predict instances of genes that potentially could give rise to more than one protein . annotated protein coding genes were extracted from completely sequenced prokaryotic genomes in the genome database of ncbi . all alternative orfs , potentially encoding 100 amino acids or more , were extracted from each gene sequence using perl scripts and the bioperl application programming interface ( api ) ( 3 ) . using the standard genetic code , the in silico translated amino acid sequence of each alternative orf was searched for similarity in completely sequenced prokaryotic genomes ( 4 ) and for conserved domains and motifs using cdd ( 5 ) , pfam ( 6 ) , cog ( 7 ) , kog ( 8) , smart ( 9 ) and uniprot . ( 10 ) . hierarchical clustering using the software hcluster_sg developed as part of the treefam project ( 11 ) was used to build sequence families with the alternate orfs . blast e - values were normalized from 0 to 100 ( with 100 corresponding to e - value 0.0 ) . the resulting information was stored in a relational database built with microsoft sql server 2005 . release 1.0 ( september 2007 ) contains approximately 1.5 million annotated genes from 481 organisms and about 3 million alternate orfs . of these 942 856 ( 33% ) occur in frame 1 , 621 306 ( 21% ) in frame 2 , 322 284 ( 11% ) in frame 3 , 350 805 ( 12% ) in frame + 2 and 675 525 ( 23% ) in frame + 3 . the following are provided for each alternate orf sequence : ( i ) conserved domains and motifs including cdd ( 5 ) , pfam ( 6 ) , cog ( 7 ) , kog ( 8) , smart ( 9 ) and uniprot . ( 10 ) and ( ii ) blast results with annotated sequences in completely sequenced prokaryotic genomes and alternate orfs identified in alterorf . the cross genera conservation of some alternate orfs suggests that they might represent new protein families or domains and hierarchical clustering ( 11 ) was used to build sequence families from conserved alternate orfs . the alterorf database can be accessed through a simple and easy to use web interface at www.alterorf.cl . the database can be searched by protein i d ( derived from ncbi ) , by organism and by sequence using a sequence search service . in addition , an option is provided to analyze complete genome sequences not present in the database . searching by protein i d : a protein i d can be used to recover the original annotated gene that appeared in the database ( e.g. genbank ) , and also any alternate orf(s ) associated with that gene . if alternate orfs are detected , tables providing information regarding domains , motifs and protein family are displayed with links to further information . searching by organism : the user can select an organism from a pulldown menu or index for a pre - analyzed list of annotated protein coding genes with alternate orfs . searching by protein sequence : a search using a protein sequence can be carried out against all sequences stored in alterorf using wu - blast ( blast.wustl.edu/ ) . downloading data : all data in the alterorf database can be freely downloaded by ftp . additional information on the use of alterorf can be found in the faqs and tutorial sections .

introductionischemic mitral regurgitation can be defined as moderate to severe mitral leak precipitated by acute myocardial infarction . valve repair is now the procedure of choice , but some cases can pose difficult anatomy . this review will illustrate current techniques for repairing complex ischemic mitral regurgitation.methodsmost patients with ischemic mitral regurgitation have predominant annular dilatation at the posterior commissure and require only ring annuloplasty . full rigid rings are used preferentially . with leaflet tethering , adjunctive autologous pericardial patches are effective in restoring leaflet coaptation . if papillary muscle elongation or rupture occurs , gore - tex artificial chordal replacement performs well . with ischemic mitral regurgitation accompanying posterior ventricular aneurysms , standard trans - atrial mitral repair provides the best results , with associated aneurysms being repaired concurrently.resultssurgical approaches and technical outcomes of mitral repair in ischemic mitral regurgitation are illustrated in 5 patients using operative images and echocardiograms . each method is illustrated , including ring annuloplasty , pericardial leaflet augmentation , artificial chordal replacement , and ventricular aneurysm repair . using these techniques , virtually all ischemic mitral regurgitation can be repaired , with consequential patient benefits , even in the most complex anatomy.conclusionsischemic mitral regurgitation has been shown to have better outcomes when managed with valve repair . using combinations of annular , leaflet , and chordal procedures , even complex ischemic mitral regurgitation can undergo autologous reconstruction with excellent long - term results . alterorf is a searchable database that contains information regarding alternate open reading frames ( orfs ) for over 1.5 million genes in 481 prokaryotic genomes . the objective of the database is to provide a platform for improving genome annotation and to serve as an aid for the identification of prokaryotic genes that potentially encode proteins in more than one reading frame . the alterorf database can be accessed through a web interface at www.alterorf.cl

cardiovascular disease is the leading cause of death in end - stage renal disease ( esrd ) patients receiving long - term peritoneal dialysis ( pd ) therapy . data from the canada and united states ( canusa ) peritoneal dialysis study showed that nearly half of the mortality in pd patients was due to cardiovascular disease . according to data from the united state renal data system ( usrds ) , this trend has remained more or less the same in the recent years . vascular / valvular calcifications are important and highly prevalent complications in esrd patients including those receiving pd therapy and very much contributed to the exceedingly high cardiovascular mortality in this population . numerous observational cohort studies demonstrated the prognostic importance of vascular / valvular calcification in esrd patients . using plain radiographs to estimate number of arterial sites with calcification including carotid artery , abdominal aorta , and iliofemoral axis , both the presence and extent of vascular calcifications are strong predictors of cardiovascular and all - cause mortality in the esrd patients . abdominal aortic calcification detected nonquantitatively using plain lateral abdominal radiographs has also been shown to be an independent predictor of all - cause mortality and cardiovascular death in hemodialysis patients . using multislice computed tomography ( msct ) that enables quantification of calcification , block et al . demonstrated a significant mortality effect of the severity of coronary artery calcium score in incident hemodialysis patients . cardiac valvular calcification , detected using echocardiography , also predicts all - cause mortality and cardiovascular death in chronic pd patients . notably , patients with both aortic and mitral valvular calcification showed the highest risk of mortality and cardiovascular death compared to those with either heart valve calcification or no valve calcification . these data suggest that the presence of vascular or valvular calcification , irrespective of the sites involved , is indicative of a poor prognosis in the dialysis population including patients on pd . in this paper , we reviewed the prevalence , significance , contributing factors , and emerging mechanisms for this important complication in the pd population . furthermore , we discussed therapeutic strategies that may be useful in retarding calcification burden in the pd patients . the reported prevalence of coronary artery calcification in the esrd patients ranged from 40% to nearly 100% [ 714 ] . there are very little data in pd patients but the available evidence suggest that vascular and valvular calcification is an equally highly prevalent complication in the pd population . in two small surveys , at least 60% to 80% of the pd patients , were complicated with coronary artery calcification [ 15 , 16 ] . the prevalence of cardiac valvular calcification ranges from 32% to 47% in pd patients [ 17 , 18 ] , in contrast to 19% to 84% in hemodialysis patients [ 1924 ] . in the general population , coronary artery calcium score provides a quantitative estimate of total atherosclerotic plaque burden and correlates with obstructive coronary artery disease . in nondialysis chronic kidney disease ( ckd ) patients , similar association was observed between the severity of coronary artery calcium score and obstructive coronary artery disease . study in hemodialysis patients demonstrated a close relation between the severity of coronary artery calcification and prevalence of atherosclerotic vascular disease . this is in keeping with recent similar analysis in pd patients showing that the severity of coronary artery calcium score was associated with the prevalence of atherosclerotic vascular disease ( unpublished observation ) . on the other hand , the severity of coronary artery calcium score is predictive of arterial stiffening in patients with ckd and is associated with increased risk of left ventricular hypertrophy . unlike in the general population , where calcification occurs mainly in the intimal layer and reflects atherosclerotic disease burden , vascular calcification in esrd patients typically occurs both in the intimal and medial layer . the medial calcification , known as monckeberg 's medial calcinosis is also prominent in patients with diabetes . medical calcification promotes arterial stiffening and is associated with increased risk of left ventricular hypertrophy and impaired coronary perfusion . while both medial and intimal type of calcifications predict mortality and cardiovascular death in esrd patients , intimal calcification was associated with worse survival compared to medial calcification . on the other hand , cardiac valvular calcification is a marker reflecting generalized atherosclerosis and calcification in the pd patients . . showed in a cohort of young hemodialysis adults of rapid progression in coronary artery calcification over a mean follow - up period of 20 months . in the subsequent study by block et al . , patients who had baseline coronary artery calcifications showed significant progression in coronary calcification while those without baseline calcification remained free from calcification during follow - up . these data suggest that preexisting coronary artery calcification is one of the key factors predicting further progression of vascular calcification in esrd patients . our recent analysis also reported similar finding in pd patients ( unpublished observation ) in that pd patients without significant baseline vascular calcification remained relatively free of calcification with time on dialysis . these data raised the possibility of some protective mechanisms or genetic factors in play that prevent patients from developing vascular calcification . the high incidence of vascular / valvular calcification in pd patients is contributed by both traditional and so - called nontraditional or kidney disease - related risk factors . age is one of the major clinical factors associated with vascular / valvular calcification in dialysis patients [ 7 , 10 , 17 , 33 ] . some studies showed that the degree of coronary or valvular calcification increased with increasing duration of dialysis [ 7 , 8 , 17 ] . disturbed mineral metabolism with resulting hyperphosphatemia has been suggested to play a major contributing role for vascular / valvular calcification in esrd patients [ 8 , 10 , 12 , 17 ] . according to a retrospective analysis from the usrds , an elevated serum phosphorus or an elevated calcium phosphorus product predicts all - cause mortality as well as cardiovascular mortality in hemodialysis patients . notably , patients with a high serum phosphorus 6.5 mg / dl was associated with an increased risk of death from coronary artery disease , sudden death , infection or other causes , compared with the low phosphorus group . the netherlands cooperative study on the adequacy of dialysis ( necosad ) study compared hemodialysis and pd patients and showed that hyperphosphatemia with resulting high calcium phosphorus product had similar predictive value for mortality in pd and hemodialysis patients . having a serum phosphorus level > 1.78 mmol / l was associated with a time - dependent adjusted hazard ratios of 1.6 and 1.4 for all - cause mortality in pd and hemodialysis patients , respectively . these data clearly indicated that optimizing phosphorus control is equally important in pd as in hemodialysis patients . in vitro study showed that inorganic phosphate induced an osteoblastic phenotypic change in vascular smooth muscle cells which led to the deposition of calcium and phosphate - containing apatite crystals . this is in keeping with cross - sectional studies demonstrating an association between hyperphosphatemia and vascular calcification in esrd patients [ 8 , 10 , 12 , 33 ] . hyperphosphatemia is also associated with an increased risk of valvular calcification in patients on pd . previous longitudinal study showed a significant association between serum phosphorus and calcium phosphorus product with changes in coronary artery calcification over 1 year in pd patients , providing further evidence to support the involvement of hyperphosphatemia in vascular / valvular calcification in pd patients . as in hemodialysis patients , pd patients have a high prevalence of hyperphosphatemia . according to the necosad study , around 40% of the long - term pd patients had serum phosphorus level above the kidney disease outcome quality initiative ( k / doqi ) target of 1.78 this is very similar to our survey showing that around 40% of chinese pd patients had serum phosphorus level above the k / doqi target . one of the major clinical factors associated with hyperphosphatemia in pd patients was dietary protein intake . residual renal function is the other significant factor associated with serum phosphorus control in pd patients . on the other hand , among anuric pd patients , serum phosphorus showed the strongest correlation with pd clearance followed by normalized dietary protein intake . additionally , a total urea clearance of at least 2.0 and creatinine clearance of at least 60 l / wk per 1.73 m appeared to be optimal clearance targets that maintain serum phosphorus below 5 mg / dl in continuous ambulatory pd patients . given that it is extremely difficult to achieve a total weekly creatinine clearance of over 60 l with pd alone , these data suggest potential limitation of pd alone to achieve adequate phosphorus control in anuric pd patients . in keeping with these observations , our recent study demonstrated an increased prevalence of valvular calcification among anuric pd patients and was partly mediated via an increased calcium phosphorus product . apart from alterations in phosphorus metabolism , some of the therapeutic strategies for abnormal mineral metabolism and renal bone disease have been suggested to have the potential of worsening vascular calcification in esrd patients [ 8 , 40 ] . the use of very high doses of calcium - based binders for phosphorus control as well as the administration of large doses of vitamin d analogs for control of secondary hyperparathyroidism may contribute to episodes of hypercalcemia and/or hyperphosphatemia and may thus aggravate vascular calcification . chertow and coworkers have shown in a longitudinal study that calcium - based phosphate binders are especially associated with progressive coronary artery and aortic calcification in hemodialysis patients when mineral metabolism is poorly controlled . patients with low bone activity appeared to be particularly susceptible to worsening of vascular calcification and stiffening when exposed to an excess calcium load . this may be due to the inability of adynamic bone to incorporate extra calcium load and thus increases the risk of extraskeletal calcification . vascular / valvular calcification is now recognized to be an active , cellular - mediated process involving an osteogenic phenotypic change of vascular smooth muscle cells along with the dynamic interactions between calcification inducers and inhibitors . this is first evident by the accumulation of macrophages and t - lymphocytes other than low - density lipoprotein and lp(a ) lipoprotein in early aortic valve calcification [ 43 , 44 ] . subsequent study from our group showed an important link between inflammation and valvular calcification in pd patients . among patients with high calcium phosphorus product , the presence of both inflammation and malnutrition increased the prevalence of valvular calcification as compared to those with no evidence of inflammation and malnutrition . notably , even among patients with normal calcium phosphorus product , the presence of inflammation and malnutrition was associated with at least twofold increase in the prevalence of valvular calcification as compared to those with no inflammation or malnutrition at all . these data suggest that inflammation is predictive of an increased risk of valvular calcification independent of calcium phosphorus product . the presence of inflammation also predicts a more adverse clinical outcome among pd patients with valvular calcification . histopathological study showed markedly increased expression of c - reactive protein messenger ribonucleic acid ( mrna ) in the coronary vessels of renal patients compared to nonrenal patients in both calcified and noncalcified parts of arteries , suggesting that uremia itself was associated with increased vascular inflammation and that may mediate the development of vascular calcification . this is further supported by in vitro data showing that pooled uremic serum can induce accelerated calcification of vascular smooth muscle cells , regardless of the level of phosphorus . however , the exact uremic toxins that increase vascular calcification are currently not clear . in hemodialysis patients , c - reactive protein showed similar association with annualized change in coronary artery calcium score and has been associated with progression of abdominal calcification . inflammation has also been inversely related to circulating fetuin - a or alpha - heremans - schmid glycoprotein ( ahsg ) in both hemodialysis and pd patients [ 48 , 49 ] . these data add circumstantial evidence to support possible causal link between inflammation and calcification and warrant further investigation . circulating calcification inhibitors are increasingly recognized to play an important role in modifying the calcification risk of esrd patients . the presence of extensive calcification in various organs including the kidneys , lungs , myocardium , skin , and blood vessels in ahsg or fetuin - a deficient mice being fed a mineral and vitamin d rich diet provides novel evidence to support a crucial role of fetuin - a in inhibiting ectopic calcification . even though some studies suggested higher fetuin - a levels in pd than hemodialysis patients , lower serum fetuin - a was linked to an increased risk of all - cause mortality and cardiovascular death in both hemodialysis and pd patients . lower fetuin - a was associated with more inflammation and valvular calcification in pd patients and was inversely related to more aortic calcification in pediatric dialysis patients . all these data lent supporting evidence that fetuin - a may be involved in inhibiting vascular and valvular calcification . other circulating inhibitor such as pyrophosphate has recently been shown to be inversely related to the severity of coronary artery calcification and its circulating level did not seem to be affected by the severity of kidney disease , the modality of dialysis or inflammatory activity . matrix gla - protein ( mgp ) belongs to a family of the n - terminal -carboxylated ( gla ) proteins that require a vitamin k - dependent -carboxylation for their biological activation . the development of spontaneous vascular calcification in mgp - deficient mice was novel evidence to support a key role of mgp in inhibiting vascular calcification . a study in the general population indicated an inverse correlation between blood mgp levels and coronary artery calcification . however , the assay measured total blood levels of mgp without differentiating between undercarboxylated ( uc ) and carboxylated ( cmgp ) and without taking into account of tissue deposition . more recently , study showed that serum total uc - mgp was significantly reduced in hemodialysis patients compared to nonrenal failure population and markedly reduced in patients with calciphylaxis . serum total uc - mpg level was inversely correlated with serum phosphate but positively associated with serum fetuin - a . in a very recent study , lower circulating levels of dephosphorylated - carboxylated mgp ( de - cmgp ) patients with lower dp - cmgp level were also associated with more calcifications as denoted by a composite semiquantitative calcification score . however , it remains further elucidation whether circulating mgp directly reflects the biologically active mgp in the vasculature and may play a causative role in vascular calcification . osteoprotegerin ( opg ) belongs to the tumor necrosis factor - receptor gene superfamily and serves as a soluble decoy receptor for the receptor activator of nf--ligand ( rankl ) . the demonstration of medial calcification of the aorta and renal arteries in opg knockout animal suggests opg and its signaling pathway may play a role in medial arterial calcification . in the general population , serum opg was positively related to the extent and severity of coronary artery disease . serum opg was higher in dialysis patients than healthy controls and was independently associated with the severity of abdominal aortic calcification in hemodialysis patients . furthermore , both serum c - reactive protein and opg were higher among rapid progressors of abdominal calcification compared to slow progressors . further study will be required to investigate the exact role of opg in vascular calcification in esrd patients . other potential mechanisms of vascular calcification include vascular smooth muscle cells apoptosis and fgf-23 and klotho activity . there is in vitro evidence that chronic mineral dysregulation is capable of triggering vascular smooth muscle adaptation and release of matrix vesicles and that ultimately culminates in vascular smooth muscle cell apoptosis and vascular calcification [ 63 , 64 ] . the fibroblast growth factor-23 ( fgf-23)klotho activity plays an important role in the systemic regulation of phosphate homeostasis [ 65 , 66 ] . studies have shown that both fgf-23 and klotho knockout mice develop extensive vascular and soft tissue calcification [ 67 , 68 ] . serum calcium and 1,25 hydroxyvitamin d levels increased sodium phosphate cotransporter activity in both fgf23 and klotho ablated mice increases renal phosphate reabsorption which in turn can facilitate calcification . very recent study showed that klotho may exert direct effects on vascular smooth muscle cells and its deficiency may directly induce vascular calcification in uremia , suggesting that klotho may act as a novel circulating inhibitor of vascular calcification . current therapeutic strategies for vascular / valvular calcification in the esrd population are largely linked to management of mineral bone disorder associated with ckd such as control of hyperphosphatemia , avoiding , hypercalcemia and control of secondary hyperparathyroidism . a recent prospective analysis comparing 3555 patients who began treatment with phosphorus binders during the first 90 days after initiating hemodialysis versus 5055 patients who remained untreated clearly showed that the early use of phosphorus binders was independently associated with better survival . so far , there are no data as to whether the type of phosphorus binders may affect progression of vascular calcification in pd patients . study in pd patients showed that sevelamer hydrochloride provides a similar reduction in serum phosphorus compared to calcium - based binders as in hemodialysis patients . a number of prospective open - label randomized controlled trials in hemodialysis patients including the treat to goal study , and the renagel in new dialysis patients ( rind ) study , comparing sevelamer versus calcium - based binders suggested a slower progression of coronary artery calcification with sevelamer hydrochloride as compared to calcium - based binders . notably , in both studies [ 11 , 32 ] , sevelamer and calcium - based binders - treated patients achieved comparable serum phosphorus control , but serum calcium was significantly lower in calcium - based binder - treated patients versus sevelamer - treated patients . the reduced calcium load with resulting lower serum calcium may partly contribute to the slower progression of vascular calcification in sevelamer - treated patients . in the extended followup of the rind study , a significant survival benefit was observed in the sevelamer - treated patients as compared to patients treated with calcium - based binder . on the other hand , the calcium acetate renagel evaluation-2 ( care 2 ) study reported similar progression of coronary artery calcification with sevelamer and calcium acetate when statins were used to achieve a similar serum ldl - cholesterol in hemodialysis patients . in the dialysis clinical outcomes revisited ( dcor ) study , no significant difference was observed in the overall all - cause mortality between sevelamer and calcium - based binder treated hemodialysis patients . however , there is some suggestion that sevelamer treatment may be associated with lower overall mortality but not cardiovascular - related mortality among elderly patients . there is no data on the effect of lanthanum carbonate or other newer phosphorus binders on the progression of vascular calcification as compared to calcium - based binders in dialysis patients . given the current available evidence , the kidney disease improving global outcome ( kdigo ) guideline suggests restricting the dose of calcium - based binders in patients with stage 5d ckd including those on pd , in the presence of persistent or recurrent hypercalcemia , arterial calcification , adynamic bone disease , and persistently low parathyroid hormone . vitamin d receptor activator ( vdra ) has been the standard treatment for secondary hyperparathyroidism in patients with ckd . there is animal data to suggest that different vdras have differential effects on vascular calcification in uremia . in the study by mizobuchi et al . , paricalcitol , a selective vdra , showed no effects on serum calcium , phosphorus and aortic calcium content in uremic animals in contrast to other vdra such as calcitriol or doxercalciferol which increased serum calcium , phosphorus , and aortic calcium content . in addition , doxercalciferol treatment increased mrna and protein expression of bone - related markers runx2 and osteocalcin in the aorta , while paricalcitol did not . similar data suggest differential effects of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized apo - e knockout animals . there are observational data to suggest that use of paricalcitol was associated with survival advantage over calcitriol in hemodialysis patients . randomized controlled trials will be required to determine whether paricalcitol may be associated with less progression of vascular calcification compared to other vdra in the dialysis population . cinacalcet hydrochloride is a calcimimetic agent that inhibits parathyroid hormone secretion from the parathyroid cells by activation of the ca receptor and emerges as a novel therapeutic agent for the treatment of secondary hyperparathyroidism in patients with ckd . previous double - blind randomized placebo - controlled trial showed that cinacalcet had comparable efficacy in both hemodialysis and pd patients and effectively reduced calcium , phosphorus , and parathyroid hormone . in the more recent 33-week achieve study that randomized hemodialysis patients to receive either cinacalcet and low - dose vitamin d analog or flexible vitamin d analog , a more significant reduction in parathyroid hormone ( pth ) ( median percent change , 47% versus 11% ) , calcium , and phosphorus was observed in the cinacalcet and low - dose vitamin d analog treatment group as compared to flexible vitamin d analog group . studies in uremic animal models showed that calcimimetic is capable of inhibiting vascular calcification in contrast to calcitriol which induces vascular calcification [ 81 , 82 ] . in the advance trial , a prospective , randomized , controlled trial that evaluated the progression of vascular and cardiac valve calcification in 360 prevalent adult hemodialysis patients with secondary hyperparathyroidism after receiving treatment with cinacalcet plus low - dose vitamin d sterols versus flexible doses of vitamin d sterols alone , a trend towards less progression of coronary artery agatston calcium score was observed in the cinacalcet - treated group after 52 weeks versus the flexible vitamin d sterol group but did not reach statistical significance . the difference in the corresponding changes in volume scores between the two groups were , however , of statistical significance . furthermore , increases in calcium scores were consistently less in the aorta , aortic valve and mitral valve among subjects treated with cinacalcet plus low - dose vitamin d sterols versus the flexible vitamin d sterol group , and the differences were significant at the aortic valve . these data suggest that the use of cinacalcet may be associated with slower progression of vascular / valvular calcification as compared to flexible vitamin d sterol - treated patients . we await the results of another randomized trial , namely , the evolve ( evaluation of cinacalcet hcl therapy to lower cardiovascular events ) study to investigate whether cinacalcet treatment may confer survival benefit in hemodialysis patients with secondary hyperparathyroidism . there is so far no randomized controlled study in pd population that addresses the issue of vascular / valvular calcification and clinical outcomes in relation to vdra or cinacalcet . studies in in vitro cell culture model or ex vivo model suggested that aortic calcification requires the elevation of both calcium and phosphorus and that calcium rather than phosphate appears to have a more profound effect on aortic calcification [ 63 , 84 ] . however , in pd patients , a low calcium pd fluid may be associated with exacerbation of secondary hyperparathyroidism . thus , to help reduce calcium loads yet without exacerbating secondary hyperparathyroidism , the previous k / doqi guideline suggested that the dialysate calcium concentration in hemodialysis or pd should be maintained at 2.5 meq / l ( 1.25 mmol / l ) . the more recent 2009 kdigo guideline suggested that the dialysate calcium concentration in hemodialysis or pd patients should be between 2.5 ( 1.25 mmol / l ) and 3 meq / l ( 1.5 mmol / l ) . a dialysate calcium concentration above 3 meq / l ( 1.5 mmol / l ) is not recommended in dialysis patients as it results in positive calcium balance and increased risk of hypercalcemia . therefore , maintaining a neutral calcium balance appeared a reasonable goal in managing pd patients . there is so far very little data on the effect of dialysate calcium concentration on vascular calcification in pd patients . a small nonrandomized study suggested that patients receiving pd fluid of high calcium concentration , namely 1.75 mmol / l showed an increase in arterial stiffening as compared to those using low calcium concentration pd fluid which showed no change . these preliminary data suggest that high calcium exposure through pd solution may play a role in progressive arterial stiffening through an increase in vascular calcification and warrant further investigation . vascular and valvular calcifications are equally highly prevalent in esrd patients receiving pd treatment as in hemodialysis patients and are predictive of an increased mortality and adverse cardiovascular outcomes . current therapeutic strategies that may be of use in limiting progressive vascular / valvular calcification in pd patients are largely linked to management of mineral bone disorder associated with ckd including control of hyperphosphatemia , avoiding hypercalcemia , and control of secondary hyperparathyroidism . randomized controlled trials will be required to determine whether these strategies may reduce calcification burden and clinical outcomes in the pd population . with the development of chemotherapy , control of metastatic colorectal cancer has increased in recent years . aggressive resection of distant metastases , local recurrence , or peritoneal dissemination has been performed , and good results have been reported [ 1 , 2 ] . particularly , aggressive resection has been recommended for hematogenous metastasis ; however , in pulmonary resection , the presence of mediastinal lymph node recurrence has been reported as a poor prognostic factor . recommendations for the resection of distant lymph node metastasis are lacking , but it is thought that solitary metastatic lesions should be resected . the development of various diagnostic imaging such as pet / ct has contributed enormously to performing preoperative staging and detecting postoperative recurrence . here , we present the case of a patient who had a solitary swollen mediastinal lymph node with high accumulation on pet / ct after primary resection of rectal cancer . however , histopathological results showed no metastasis , and he was diagnosed with reactive lymphadenitis . we report 1 case study of false - positive diagnosis on pet / ct and compare our findings with previous literature . a 60-year - old patient presented with positive fecal occult blood and was subsequently diagnosed with rectal cancer ( rs ) . histopathological examination revealed moderately differentiated adenocarcinoma , and the pathological stage was pt3n1m0 , pstage iiia ( fig . the oral fluoropyrimidine agent s-1 ( 80 mg / m per day , administered for 4 weeks with a 2-week resting period ) was administered for adjuvant chemotherapy . on postoperative day 239 , a ct scan revealed a solitary swollen mediastinal lymph node ( fig . 2a ) . the patient reported no symptoms of common cold , no palpable lymph nodes , and no murmur in the chest . laboratory data , including blood count , biochemical findings , and c - reactive protein , were within normal range . the serum cea level was also normal ( 1.8 ng / ml ; normal value , < 5.0 ng / ml ) ; before primary surgical treatment , it was 5.8 ng / ml . t2-weighted mri showed iso - high intensity , and diffusion - weighted mri showed a high signal at the tracheal bifurcation ( fig . pet / ct was performed , and abnormal f - fdg uptake appeared in the tracheal bifurcation . thoracoscopic mediastinal lymph node resection was performed in the prone position on postoperative day 281 . the surgical procedure was as follows : ( 1 ) a 12-mm port was inserted into the fifth intercostal space on the posterior axillary line with an 8-mm hg pneumothorax ; ( 2 ) a 12-mm camera port was inserted into the ninth intercostal space on the lower scapula line ; ( 3 ) a 5-mm port was inserted into the seventh intercostal space on the posterior axillary line . on histopathological examination , the resected specimen measuring 33 22 mm in diameter showed enlarged germinal centers without an epithelial component . immunostaining was performed to detect follicular lymphoma ; bcl-2 was negative , and the patient was diagnosed with reactive lymphadenitis ( fig . oral capecitabine ( xeloda , 3,600 mg per day , administered for 2 weeks with a 1-week resting period ) was administered for 6 months . imaging follow - up was performed 2 months after thoracoscopic lymph node resection by ct scan . we confirmed that the suspicious lymph node was resected and other recurrent lesions did not appear . the patient is alive at more than 15 months after his second surgery and is being followed up closely . since the publication of colorectal cancer treatment guidelines in japan , providing appropriate treatment for these patients has become mandatory . therefore , accurate preoperative diagnosis is important , and postoperative recurrence must be evaluated accurately by various methods of diagnostic imaging . given the advent of fdg- pet and pet / ct in recent years , pet imaging has become essential for improving the detection rate . studies have reported that pet imaging is more useful than mri or multidetector - row ct ( mdct ) [ 4 , 5 ] . however , in colorectal cancer , the diagnostic capability for lymph node metastasis ( reported as 2951% sensitivity , 85100% specificity , and 6775% diagnostic accuracy ) is insufficient compared to that for the primary tumor [ 6 , 7 , 9 ] . especially for sensitivity , diffusion - weighted mri is more useful than fdg - pet . in our hospital , the diagnosis rate of lymph node metastases using preoperative pet / ct for 42 patients with colorectal cancer tended to be lower in specificity ( 77% sensitivity , 71% specificity , and 74% accuracy ) . generally , local inflammation , abscess formation , inflammatory diseases , and lymphomas may be considered as the causes of false - positive lymph nodes on pet . although false - positive para - aortic lymph nodes are occasionally identified on pet , to our knowledge , pet false - positive cases associated with colorectal cancer in mediastinal lymph nodes have not been found . the use of pet / ct for identification of mediastinal lymph nodes has been reported . physiological accumulation is often observed in hilar lymph nodes and has the following features : ( 1 ) nearly equal accumulation intensity on both sides , and ( 2 ) no swollen lymph nodes in the mediastinum on ct . on the other hand , solitary mediastinal lymph node metastases of cases of colorectal cancer the present case did not meet the features of physiological accumulation in hilar lymph nodes and showed a solitary enlarged lymph node , high suvs , and high intensity on diffusion - weighted mri . therefore , we diagnosed the patient with mediastinal lymph node metastasis of colorectal cancer and performed surgical treatment . on the other hand , less invasive surgery is desirable for surgical treatment , and advances in video - assisted surgery have been remarkable in recent years . thoracoscopic esophagectomy has become widespread because it provides a magnified view and an easier approach to the mediastinum . the biggest advantage of the prone position is a good view of the surgical field without displacing the heart and right lung because of gravity and artificial pneumothorax . our case maximized the benefits of the prone position , and the patient had a good postoperative course . even though histopathological findings showed lymphadenitis , the patient had no distinct physical findings and inflammatory response . the possibility of viral infection can not be ruled out , although it was difficult to diagnose correctly before surgical treatment . in cancer treatment , it is necessary to decide the appropriate strategy on a case - by - case basis with due consideration of the limits of various methods of diagnostic imaging . moreover , evaluations of pre- and postoperative colorectal cancer recurrence and metastasis using integrated fdg pet / contrast - enhanced ct have been reported . kitajima et al . reported 170 postoperative cases , with 93.2% sensitivity , 95.8% specificity , and 94.7% accuracy . there is no consistent strategy for appropriate treatment of solitary lymph node metastasis in colorectal cancer patients . especially for pet - positive lesions , both sensitivity and specificity may be insufficient . mediastinoscopy and needle biopsy with bronchoscopy are useful methods ; however , the possibility of disseminated cancer cells remains . in conclusion , our case report suggests that it is necessary to understand the limits of the diagnostic accuracy of pet / ct in determining the indication for surgical treatment . additionally , the thoracoscopic approach in the prone position is one of the easy - to - use approaches to the mediastinum .

cardiovascular disease accounts over half of the total mortality in peritoneal dialysis ( pd ) patients . in addition , there is an increasing recognition of a high prevalence of vascular and valvular calcification that may contribute to the increased all - cause and cardiovascular mortality in the pd patients . disturbed mineral metabolism in association with chronic kidney disease has been suggested as one of the major contributing factors to the increased vascular / valvular calcification in this population . in this paper , we provide an overview of the prevalence and importance of this complication in the pd patients . in addition , we review the contributing factors and some emerging mechanisms for this complication . furthermore , we discuss some therapeutic strategies that may be useful in limiting the progression of vascular / valvular calcification in the pd population . 18f - fluorodeoxyglucose ( fdg ) positron emission tomography and computed tomography ( integrated fdg pet / ct ) has been used to diagnose recurrence and differentiate postoperative changes from lymph node metastasis in colorectal cancer , although its accuracy is questionable . we report a prone thoracoscopic surgery for a rectal cancer patient in which false - positive mediastinal lymph nodes were found on fdg - pet / ct . a 60-year - old man underwent a laparoscopic high anterior resection and d3 lymph node dissection for rectal cancer . the histopathological diagnosis was moderately differentiated adenocarcinoma of the rectum , stage iiib ( pt3n1m0 ) , necessitating oral fluoropyrimidine agent s-1 . after the primary surgery , a solitary mediastinal lymph node measuring 30 mm in diameter was detected , and abnormal accumulation was confirmed by fdg - pet / ct ( suvmax , 11.7 ) . thoracoscopic resection was performed in the prone position , but histopathological results showed no metastasis . he was subsequently diagnosed with reactive lymphadenitis . the patient was discharged on postoperative day 4 in good condition and is alive without recurrence 12 months after surgery . pet / ct is useful for the detection of colorectal cancer recurrence ; however , it does have a high false - positive rate for mediastinal lymph nodes . there is a limit to its diagnostic accuracy , and one must determine the indication for surgical treatment carefully . surgery in the prone position is a useful and minimally invasive approach to the mediastinum and allows aggressive resection to be performed .

colistin belongs to the polymyxin class of antibiotics which is a group of cationic polypeptides . however , the popularity rapidly faded in 1970s because of significant renal and neurological toxicity and was replaced with less toxic antibiotics with a comparable or broader antibacterial spectrum such as aminoglycosides . with the recent emergence of multi drug - resistant ( mdr ) gram - negative organisms , in particular pseudomonas aeruginosa , acinetobacter baumannii , and klebsiella pneumonia , colistin has been reconsidered as a potential therapeutic agent for the past 10 - 15 years . this has not only increased the clinical usage of this ancient drug , but has also increased the prevalence of all the toxicities related to colistin . neurotoxicity is the second most common toxicity after nephrotoxicity following colistin therapy . among the various manifestations of its neurotoxicity , several cases of respiratory failure due to colistin were reported in the previous era . however , a 31-year - old white female with history of spina bifida , paraplegia , hydrocephalus status post ventriculo - peritoneal shunt , osteomyelitis of the left hip status post left leg amputation , and chronic sacral decubitus ulcer presented with foul smelling discharge from her sacral wound for 2 weeks . the culture from the wound grew two biotypes of pseudomonas aeruginosa : one was resistant to all antibiotics except for colistin while the other was resistant to all antibiotics except for colistin and imipenem . in addition , methicillin - resistant staphylococcus aureus ( mrsa ) grew from the intraoperative bone culture . she was started on colistimethate sodium ( cms ) 200 mg intravenously every 12 hours for her mdr p. aeruginosa . she was then discharged to a sub - acute rehabilitation center on cms and vancomycin . after three days , she was noted to have sudden respiratory distress in the sub - acute rehabilitation center for which emergency medical service ( ems ) was called . on arrival she was immediately started on bag - valve mask ventilation ( bvm ) and was taken to the emergency room ( er ) . in the er , she was noted to have oxygen saturation at 80s on bvm and was emergently intubated . shortly after resuscitation , her arterial blood gas analysis revealed ph of 7.45 , paco2 of 25 , and pao2 of 280 mmhg ( on fio2 of 100% ) . her labs were significant for leukocytosis with white cell count of 20,000/mm and an acutely elevated creatinine of 2.2 mg / dl ( baseline creatinine was 0.9 mg / dl at the time of discharge ) with metabolic acidosis with bicarbonate of 18 mmol / l secondary to her renal failure . on further inquiry , there was no exposure to any narcotics or muscle relaxants that might have contributed to her respiratory depression . she was in a supervised unit prior to the presentation , which deterred the possibility of any toxic exposure to the patient . two sets of blood cultures were negative while the wound culture revealed the same biotypes of mdr p. aeruginosa . neurological workup was unrevealing and ventilation / perfusion scan ( v / q scan ) for pulmonary embolism was low probability . colistin was stopped as it was thought to be the cause of renal failure and respiratory failure and the patient was started on imipenem . her respiratory and neurological status remained stable during hospitalization and tolerated imipenem and vancomycin for her osteomyelitis and chronic decubitus ulcer infection . our patient developed acute respiratory failure requiring intubation and mechanical ventilation 6 days after initiation of colistin . respiratory muscle paralysis , as depicted , is one of the manifestations of the neurotoxic effect of colistin . we applied the naranjo adverse drug reaction probability scale to our case , which indicated a probable relationship . the case is limited with the unavailability of arterial blood gas prior to the event as she was emergently intubated on presentation . also , since colistin is reserved for severely ill patients with multiple co - morbidities , it may be difficult to establish direct causal relationship for acute respiratory failure and colistin . colistin was most likely to be the culprit of her respiratory failure from the timing of apnea as well as rapid recovery after stopping colistin . acute renal failure leading to presumed high concentration of serum colistin may be another contributory factor for her respiratory failure . she had a chronic deep vein thrombus which is unlikely to cause acute respiratory failure since low probability for pulmonary embolism by v / q scan and she recovered within 24 hours without any intervention . reported cases of respiratory paralysis associated with polymyxin / colistin use since 1960 to present colistin is also called polymyxin e and is a part of polymyxin family which is a group of polypeptide antibiotics . it exerts its bactericidal effect by displacing the divalent cations magnesium and calcium , which stabilize anionic lipopolysaccharide molecules in the outer membrane of gram - negative bacteria . however , they were gradually abandoned in most parts of the world because of nephrotoxicity and neurotoxicity . the emergence of mdr gram negative bacteria led to the revival of polymyxins in more recent era as a valuable therapeutic option . in the last decade , intravenous polymyxins , particularly in the form of cms , have been used to treat serious p. aeruginosa and a. baumannii infections of various types , including pneumonia , bacteremia , and urinary tract infections . the major manifestations of colistin induced neurotoxicity include dizziness , weakness , facial and peripheral paresthesias , vertigo , visual disturbances , confusion , ataxia , and neuromuscular blockade . koch - weser et al . , reported the incidence of the neurotoxic manifestations to be about 7% , paresthesias being the most common . the proposed mechanism is a non - competitive myoneuronal presynaptic blockade of acetylcholine release , in contrast to the competitive blockade by most other antibiotics like neomycin , kanamycin , and streptomycin . a total of 32 cases of colistin / polymyxin - induced respiratory failure were found in the literature . recent studies have depicted the toxicities associated with colistin use to be less frequent than previously described . the most important risk factor known to be associated with respiratory failure with polymyxin use is the renal disease , which was present in 24 out of 29 patients reported . patients with preexisting renal insufficiency had a greater likelihood of developing nephrotoxicity during colistin therapy , compared to patients with normal baseline renal function . concomitant drug therapies also play a major role in potentiating the effect of neuromuscular blockade , which was noted in 11 out of 29 patients . this includes use of other neurotoxic drugs ( anesthetics , aminoglycosides , and paralytics ) , corticosteroids , narcotics and/or muscle relaxants . most of the cases had other neuromuscular symptoms like circumoral tingling , paresthesias , and restlessness prior to the development of respiratory failure . the duration of apnea lasted from few hours up to several days , maximum noted to be 11 days and 20 out of 29 patients recovered , indicating reversibility of the blockade . the number of doses prior to the respiratory failure seems to be variable and range from a single dose to 45 doses [ table 1 ] . the management of patient with respiratory failure induced by colistin involves discontinuation of the drug and respiratory support . intermittent monitoring of unassisted tidal volumes and forced vital capacity using a bedside spirometer will help determine recovery . monitoring arterial blood gas more importantly , one should be alert for the possibility of respiratory paralysis in any patients receiving this antibiotic and particularly in those patients with renal abnormality and with concomitant neurotoxic drug use . careful observation , regular neurological monitoring , and early appreciation of the symptoms of neuromuscular toxicity especially of dyspnea and restlessness may prevent respiratory arrest . neostigmine is not indicated in polymyxin or colistin - induced neurotoxicity and may be contraindicated as seen in earlier experiments that the blocking action of polymyxin b was shown to be neostigmine resistant . animal studies have noted the ability of polymyxin b to discharge histamine from tissue mast cells , which lead to deposition of chelated calcium along the nerve sheaths , leading to toxicity . higginbotham also reported use of heparin prevents death from respiratory arrest in mice receiving colistin . the use of antihistamines and heparin in polymyxin induced respiratory failure has not been studied in human subjects . as the use of colistin increased in recent years for treatment of life - threatening mdr infections , there are more reports of associated toxicities . clinicians should be aware of colistin - related adverse reactions , especially nephrotoxicity and neurotoxicity . since the formulation of the drug itself has changed , it has necessitated new data with the new formulation . regular monitoring of renal function as well as development of a tool to monitor neurological toxicity is essential to prevent and manage the toxicities associated with colistin use . variations in time , temperature , concentration , ph , and relative humidity may affect the sporicidal activity of various agents . accordingly , and especially for real - world situations , attention must be paid simultaneously to more than one controllable or uncontrollable factor . in tables 1 and 2 and in the discussion , we address some of the key ancillary factors . spores in liquid suspension exposed to flowing steam at 100c . rh , relative humidity ; conversions : 1 ppm = 1 mg / l ; mol / l = gram molecular weight / l ; 1 rad = 100 ergs / g ; and 1 watt = 10 ergs / s . boiling water for > 10 minutes , for example , can reduce b. anthracis spore counts by at least 10 ( table 1 ) . variations in time and temperature conditions required to reduce spore counts listed in table 1 can be attributed to differences in experimental conditions , strains of b. anthracis tested , or inoculum size . the u.s . environmental protection agency indicates that use of sodium hypochlorite as a sporicide is applicable under an emergency exemption ( section 18 : crisis exemption ; federal insecticide , fungicide , and rodenticide act ) ; as such , sodium hypochlorite may be used under the conditions specified ( 32 ) . given these conditions , the sporicidal effectiveness of hypochlorite solutions depends on the concentration of free available chlorine and ph . common household bleach ( sodium hypochlorite ) has a ph of 12 to prolong its shelf life . to achieve effective sporicidal activity , bleach must be diluted with water to increase the free available chlorine and acetic acid to change the ph of the solution to 7 ( 11 ) . ethylene oxide penetrates into porous material ( absorbed strongly by rubber and many plastics ) ; thus vapors are not readily eliminated by brief aeration . residual spores were not completely killed after a 30-minute exposure to chlorine dioxide at a relative humidity of 20% to 40% , whereas all spores were killed after a 15-minute exposure to chlorine dioxide with the addition of prehumidification at a relative humidity of 70% to 75% ( 21 ) . the amount of contamination , level of cleanliness of surfaces , and relative humidity will contribute to peracetic acid vapor s effectiveness as a sporicide ( 24 ) . the sporicidal property of ozone is affected by relative humidity : as relative humidity decreases , the time required for killing organisms increases ( 27 ) . decontamination of buildings from intentional release of b. anthracis is a new problem , and no accumulated scientific knowledge exists on the subject . two areas of prior scientific research may be relevant : food processing and laboratory decontamination . with modification based on further study , direct information on killing b. anthracis spores in foods by cooking is scarce , and the complexity of food matrices precludes easy extrapolation of the laboratory data into nonfood matrices . however , information on inactivating spores of bacterial species more resistant to environmental conditions than b. anthracis can provide guidance . the spores of clostridium botulinum are more resistant to heat inactivation than are b. anthracis spores ( 4 ) . the commercial retort process of canning achieves a 12-log reduction of c. botulinum spores , and by extension , should achieve a similar killing rate for b. anthracis spores . further research in this area is needed . historically , formaldehyde solution or gas has been used both as a disinfectant and chemical sterilant . formaldehyde was used to disinfect as early as the late 1880s and is still used to reprocess hemodialyzers for reuse on the same patient and to decontaminate biologic safety cabinets and laboratories ( 3537 ) . formaldehyde gas has been used for fumigation in the poultry industry and for disinfection of biologic safety cabinets and laboratories ( 38,39 ) . data from controlled experiments with b. globigii nctc 10073 spores have demonstrated the effect of humidity on formaldehyde concentration ( mg / m ) to obtain a > 8-log reduction in viable spores ( 15 ) . / l ) has also been used to treat a textile mill contaminated with b . gamma radiation was used in the 1960s and 1970s to disinfect b. anthracis contaminated imported bailed goat hair . a study by horne et al . suggested that a dose of 1.5 megarads from a 200,000-rad / hour cobalt source was sufficient to kill most resistant spores when mixed with goat hair ; after the intentional release of b. anthracis through the postal system in 2001 , pursuing a decontamination method for the undelivered mail was essential . gamma radiation was used to decontaminate all mail from contaminated facilities on the basis of these data . multiple technologies may be needed to decontaminate buildings and their contents . as in a laboratory , where some items are wiped , some items are autoclaved , and some spaces are treated with gas , more than one method may be required for decontamination . also , for certain decontamination tasks , e.g. , cleaning small heat - proof and water - proof objects , more than one option will be available . further , even within the context of one type of application ( e.g. , walls ; ducts for heating , ventilating , air conditioning , and refrigeration ; carpet ; and small objects ) , potentially conflicting priorities exist between bioefficacy , logistics , and safety . although transferring the methods used to decontaminate or sterilize laboratory or food industry settings to decontaminating buildings may be useful , this transfer of methods has not been scientifically tested . also , much of the data available is based on other bacillus species ; more testing with or correlation to b. anthracis contamination is suggested . second , choosing between technologies is a complex issue , and a formal decision process would be useful . various parties in the public and private sector have suggested numerous , sometimes disparate , methods for the inactivation of b. anthracis spores in contaminated environments . further research is needed regarding improved methods for remediation of environments contaminated with b. anthracis spores , and the literature summarized here provides a basis for that effort .

the emergence of multi - drug - resistant gram negative bacillary infections has regained popularity of ancient drugs such as polymyxins . we report a case of acute respiratory failure induced by use of intravenous colistimethate , which is one of the forms of polymyxin . the patient is a 31 year old female with paraplegia due to spina bifida who underwent excisional debridement of large lumbosacral decubitus ulcer with osteomyelitis infected with pan - resistant pseudomonas aeruginosa and mrsa . six days after initiation of intravenous colistimethate and vancomycin , she developed acute respiratory failure requiring mechanical ventilation . pan - culture was negative including a chest radiograph . v / q scan showed low probability for pulmonary embolism . echocardiogram showed normal right ventricle with no strain or pulmonary hypertension . colistimethate was discontinued . within 24 hours , she was extubated . in the early years after introduction of polymyxin , there were several reports of acute respiratory paralysis . the mechanism is thought to be noncompetitive myoneuronal presynaptic blockade of acetylcholine release . though a direct causal relationship for respiratory failure is often difficult to establish in current era with multiple co morbidities , the timeframe of apnea , acuity of onset as well as rapid recovery in our case clearly point out the causal relationship . in addition , our patient also developed acute renal failure , presumably due to colistimethate induced nephrotoxicity , a possible contributing factor for her acute respiratory failure . in summary , colistimethate can induce acute neurotoxicity including respiratory muscular weakness and acute respiratory failure . clinicians should consider its toxicity in the differential diagnosis of acute respiratory failure especially in critically ill patients . after the intentional release of bacillus anthracis through the u.s . postal service in the fall of 2001 , many environments were contaminated with b. anthracis spores , and frequent inquiries were made regarding the science of destroying these spores . we conducted a survey of the literature that had potential application to the inactivation of b. anthracis spores . this article provides a tabular summary of the results .

deep brain stimulators ( dbs ) are increasingly used to treat carefully selected cases of movement disorders , pain , and psychiatric disorders that respond poorly to medical therapy . anaesthesiologist may be involved in the implantation procedure or more frequently when these patients present for non - related medical or surgical illnesses . patients with long standing parkinson disease ( pd ) with dbs for surgery pose significant challenges to the anaesthesiologist in view of primary pathology , multisystem involvement , potential drug interactions , and risk of dbs malfunction by interference with monitoring and therapeutic electromagnetic devices . on reviewing the literature , we did not find any published report of shoulder arthroscopic surgery in a patient of pd with dbs and report the same . fifty - nine - year - old female presented to our hospital for arthroscopic repair of rotator cuff tear of right shoulder caused by a fall . she was a known case of pd since last 20 years with the history of slowing of movements , stiffness , speech disturbance , and tremors controlled only after the implantation of dbs 8 years back . she gave the past history of snoring , mild gastro - oesophageal reflux , depression , laparoscopic cholecystectomy , and three surgeries related to dbs . her medical therapy included tablet l - dopa plus carbidopa , bromocriptine , clonazepam , escitalopram , and trihexyphenidyl . patient weighed 80 kg , 155 cm tall ( body mass index 32 kg / m ) with a mouth opening of 4.5 cm , removable complete artificial denture , mallampati grade 3 , limited neck extension , and a thyromental distance of 6.2 cm . her pulse rate was 96/min , noninvasive blood pressure ( nibp ) 135/84 mmhg , breath holding time 20s , and chest auscultation was normal . examination of central nervous system revealed cog - wheel rigidity , short shuffling gait , and mild tremors . chest radiograph showed ipg with leads of dbs on the left side , which was otherwise normal [ figure 1 ] . she was assessed by a neurologist , dbs was interrogated , a procedure where battery life and device settings are evaluated and manipulated noninvasively . she was taken up for shoulder arthroscopy with informed high risk consent in view of long standing pd and dbs , kept nil per oral after midnight , and advised to continue all her medications till the morning of surgery along with tablet ranitidine 150 mg . operation room ( or ) and drugs were prepared ; difficult airway cart was kept ready . after placing standard monitors we gave iv midazolam 1 mg and oxygen by facemask . right - sided brachial plexus block by the interscalene approach using a peripheral nerve stimulator ( pns ) was given using 21 g , 50 mm insulated pns needle with 25 ml of 0.25% bupivacaine after eliciting contraction of muscles at and below shoulder at a current of 0.4 ma . after observing for 10 min , she was placed in ramp position ; general anaesthesia was induced with iv fentanyl 100 g , propofol 100 mg , vecuronium 8 mg and trachea was intubated . patient was placed in left lateral position taking care to pad all the pressure points including dbs skin site , eye , and breast care . anaesthesia was maintained with nitrous oxide ( 50% ) and isoflurane ( 0.6 - 0.8% ) in oxygen , iv fentanyl and vecuronium . her baseline heart rate was 80 beats per minute ( bpm ) and remained between 68 - 80 bpm throughout the procedure . baseline bp was 140/80 mm hg , sbp was maintained between 110 - 140 mm hg , dbp was maintained between 66 - 84 mm hg intraoperatively . thirty minutes after the onset of surgical procedure , patient 's bp rose to 190/90 mm hg which was controlled with nitroglycerine infusion ( 0.7 - 1 g / kg / min ) after ensuring adequate analgesia and muscle relaxation . postinduction iv dexamethasone 8 mg for prophylaxis of ponv and diclofenac sodium 75 mg was administered towards the end of surgery . surgeons used 26 1 ( litres ) of normal saline for irrigation of shoulder joint using arthroscopic infusion pump at a pressure of 70 mm hg . arthroscopic repair and subacromial decompression lasted 1.5 h. before closure of arthroscopic ports , fluid was vented out . after turning the patient supine , neck circumference was measured and was same as preoperatively . dbs was turned on by the trained personnel , reprogrammed to its original setting was functional . at return of spontaneous respiratory efforts , patient was conscious , haemodynamically stable and followed verbal commands with a visual analog score of 4 for pain . fifty - nine - year - old lady with pd with dbs in situ with complete rotator cuff tear was successfully managed using general anaesthesia with interscalene approach of brachial plexus block for arthroscopic repair . in contrast to the neurodestructive procedures practiced earlier like thalamotomy and palladotomy , the dbs offers the advantages of reversibility , adjustability , nondestructive nature , and long - term treatment . the high suppressive frequency ( 130 - 180 hz ) of dbs suppresses the overactivity in the pallidum , subthalamic nuclei or thalamus in pd patients . when these patients present for coincidental surgery , functionality of dbs should always be assessed in the preoperative period , precautions should be taken to prevent dbs malfunction intraoperatively and its function should be reassessed postoperatively . technical problems such as wire breakage , migration after insertion , twiddler syndrome ( malfunction of a device due to manipulation of either implantable generator or pacing leads ) should be ruled out . shoulder arthroscopy for the rotator cuff tear requires expansion of glenohumeral and subacromial area by irrigating large amounts of fluids for good visualization . as the subacromial area is uncapsulated , it communicates well via various anatomical planes to the soft tissues of the neck and chest with the risk of fluid extravasation into these areas . it can even cause external compression on the larynx and trachea with life - threatening airway obstruction . in our patient , dbs battery with its leads was located in close vicinity of the operative site . in the absence of the published literature , as an added precaution to prevent dbs malfunction we decided to switch off the dbs after inducing the patient to sleep as she had disabling tremor that was physically and psychologically distressing in the absence of dbs . , pns can be used safely for neuromuscular block assessment and for peripheral nerve blocks . an alternative is ultrasound - guided nerve block , but this modality was not available to us . interscalene block probably contributed to reduction of general anaesthetics as our patient required no vecurinium top - ups and minimal isoflurane . it also contributes to good surgical field with use of lesser amounts of irrigating fluids at a lower pressure , quick emergence from anaesthesia , and good postoperative analgesia . continuous delivery of antiparkinsonian medications during perioperative period is desirable to avoid off symptoms and increased peri- and postoperative complications . alternative routes of administration for continuous drug delivery such as nasogastric or intraduodenal levodopa , iv amantadine , subcutaneous apomorphine , transdermal rotigotine patch may be resorted to in situations where patients are not going to be started orally in the immediate postoperative period . drugs with extrapyramidal side effects such as metoclopramide , phenothiazines , and butyrophenones should be avoided . autonomic dysfunction in our patient with longstanding pd probably contributed to intraoperative rise of blood pressure in our patient . in the view of the presence of dbs , preoperative shoulder injury evaluation of our patient was done by ultrasound rather than mri that is the usual practice . powerful electromagnetic fields of mri can produce heating effects , movement of neurostimulator , induced stimulation , or dbs malfunction . ct scans , fluoroscopy , and plain x - rays can be performed as usual . defibrillator paddles and ground plate of unipolar diathermy should be placed as far from the neurostimulator as possible and perpendicular to it with use of lowest clinically appropriate energy output . after use of diathermy and external defibrillator , adequate function of neurostimulator should be confirmed . in our patient , radiofrequency ablation ( bipolar device ) was used for surgical haemostasis . as the number of patients with dbs are increasing , they can present even for emergency surgery when there is limited time and resources available for assessing the dbs . successful management of a patient of pd with dbs requires meticulous preoperative assessment , planning , continuation of antiparkinsonian drugs , avoidance of drugs precipitating parkinsonian symptoms , and adherence to precautions to avoid dbs malfunction . combination of general anaesthesia with interscalene approach brachial plexus block provides ideal conditions for these patients undergoing shoulder arthroscopy . the diagnoses of these disorders are primarily based on history , clinical features , and histopathology . the oral mucocutaneous lesions cause diagnostic problems , as these lesions resemble each other , and routine tissue biopsies may only offer a diagnosis of non - specific inflammation.1 for the past few years , immunofluorescence techniques emerged as an important tool in detecting the pathogenesis and diagnosis of oral mucocutaneous lesions ( and vesiculobullous disorders ) . two types of immunofluorescence techniques that commonly followed are direct immunofluorescence ( dif ) and indirect immunofluorescence ( iif ) . in some diseases , iif studies have revealed circulating autoantibodies2,3 whereas dif studies have revealed immunoglobulins , complement components and other protein substances within the affected tissue.4 these circulating and tissue - bound substances may have a role in the immunopathogenesis of these diseases . in addition detection of these immune reactive substances by dif aids in the diagnosis of many skin and mucosal diseases.1 the presence of characteristic fluorescent patterns in dif can definitely establish the diagnosis of pemphigus and pemphigoid and strongly indicate the likelihood of lichen planus ( lp ) and lupus erythematosus . the absence of these fluorescent patterns rules out these conditions , thereby strengthening the diagnosis of other oral mucosal diseases . the results of dif are sufficiently useful in the diagnosis of chronic ulcerative diseases of the oral mucosa.1 based on these facts , a prospective study of oral mucocutaneous lesions was designed to evaluate the consistency of the histopathology with dif . a retrospective analysis of the prevalence of oral mucocutaneous lesions namely oral lp ( olp ) , discoid lupus erythematosus ( dle ) , pemphigus vulgaris ( pv ) , and mucous membrane pemphigoid ( mmp ) were also studied with regards to demographic details and histopathological features ( hematoxylin and eosin stained sections ) that are characteristic of each disease were evaluated . twenty consecutive subjects in each of olp , dle , pv , and ten consecutive subject of mmp ( total 70 subjects ) were retrieved from the oral pathology files of tamil nadu govt . dental college and reviewed for salient features including age , gender , and intraoral site of the lesion . hematoxylin and eosin ( h and e ) stained sections of all the 70 subjects were reviewed , and the histopathological patterns were evaluated ( retrospective study ) . twelve subjects with chronic or recurrent ulcerative or erosive or vesiculobullous diseases of the oral mucosa were included for the prospective study . specimens were examined by routine histopathological methods ( h and e ) and dif . specimens for dif study were quickly washed in normal saline and stored immediately in michel s media and transported to the laboratory . the retrospective study comprised of 70 subjects - 20 subjects in each of olp , dle , and pv and 10 subjects of mmp . eleven males affected against 9 females ( 1:0:81 ) . fifth decade was predominantly involved - 30% ( 6 subjects ) . youngest involved was 11 years , and oldest was 72 years , mean age - 42.05 years . histopathologically , basal cell degeneration and subepithelial band of chronic inflammatory cells were seen in 100% of subjects . seven males affected against 13 females , sixth decade was predominantly involved - 45% ( 9 subjects ) . youngest individual was 33 years , and oldest was 78 years mean age - 52 years . histopathologically , epithelium ulcerated in 40% of subjects , intact basal cells attached to connective tissue seen in 100% of subjects , acantholytic ( tzanck ) cells seen in 85% of subjects and dense chronic inflammatory cells seen in the connective tissue in 55% subjects . seven males affected against 3 females ( 1:0.42 ) fifth and sixth decades were predominantly involved - 60% ( 6 subjects ) . youngest individual was 33 years , and oldest was 70 years , mean age - 48 years . histopathologically , subepithelial split was seen in 100% of the subjects , 40% showed inflammatory cells and red blood cells in the split , diffuse chronic inflammatory cells seen in the connective tissue in 90% of subjects and increased vascularity seen in 50% of subjects . five males affected against 15 females ( 1:3 ) , third and fourth decades were predominantly involved - 45% ( 9 subjects ) . youngest individual was 17 years , and oldest was 70 years mean age - 42 years . histopathologically , basal cell degeneration was seen in 80% and perivascular chronic inflammatory cells in 85% of subjects . histopathology showed 3 cases of lp , 4 cases of mmp and 5 cases were diagnosed as non - specific chronic inflammatory lesions . in dif study - 5 subjects were negative . 3 were reported as olp ( figure 1 ) , 2 were interpreted as pv ( figure 2 ) and 1 each of mmp ( figure 3 ) and lp pemphigoides ( figure 4 ) . histopathological diagnosis consistent with dif in 8 cases ( 66% ) ( 3 olp , 1 mmp and 4 non - specific inflammation ) . ( lichen planus ) , ( a ) hyperkeratosis , basal cell degeneration of epithelium along with subepithelial band of chronic inflammatory cells ( h and e , 10 ) , ( b ) direct immunofluorescence showing fibrin deposition along the basement membrane zone . ( pemphigus vulgaris ) ( a ) supra basal cleft with tzanck cells and intact basal layer ( h and e , 40 ) , ( b ) direct immunofluorescence showing intercellular space deposition of immunoglobulin g in the epithelium . ( mucous membrane pemphigoid ) ( a ) subepithelial cleft between epithelium and connective tissue ( h and e , 10 ) , ( b ) direct immunofluorescence showing smooth , linear , and continuous band of c3 deposit along the basement membrane zone . ( lichen planus pemphigoides ) direct immunofluorescence showing linear deposit of c3 along the basement membrane zone . the histopathological diagnosis and dif study were not consistent in four subjects in which one case diagnosed histopathologically as non - specific chronic inflammatory lesion showed positivity for lp pemphigoides in dif . among the other three cases , one diagnosed histopathologically as mmp was negative in dif and two cases which were diagnosed as mmp histopathologically were found to show positivity for pv in dif ( table 2 ) . the retrospective study comprised of 70 subjects - 20 subjects in each of olp , dle , and pv and 10 subjects of mmp . eleven males affected against 9 females ( 1:0:81 ) . fifth decade was predominantly involved - 30% ( 6 subjects ) . youngest involved was 11 years , and oldest was 72 years , mean age - 42.05 years . histopathologically , basal cell degeneration and subepithelial band of chronic inflammatory cells were seen in 100% of subjects . seven males affected against 13 females , sixth decade was predominantly involved - 45% ( 9 subjects ) . youngest individual was 33 years , and oldest was 78 years mean age - 52 years . histopathologically , epithelium ulcerated in 40% of subjects , intact basal cells attached to connective tissue seen in 100% of subjects , acantholytic ( tzanck ) cells seen in 85% of subjects and dense chronic inflammatory cells seen in the connective tissue in 55% subjects . seven males affected against 3 females ( 1:0.42 ) fifth and sixth decades were predominantly involved - 60% ( 6 subjects ) . youngest individual was 33 years , and oldest was 70 years , mean age - 48 years . histopathologically , subepithelial split was seen in 100% of the subjects , 40% showed inflammatory cells and red blood cells in the split , diffuse chronic inflammatory cells seen in the connective tissue in 90% of subjects and increased vascularity seen in 50% of subjects . five males affected against 15 females ( 1:3 ) , third and fourth decades were predominantly involved - 45% ( 9 subjects ) . youngest individual was 17 years , and oldest was 70 years mean age - 42 years . histopathologically , basal cell degeneration was seen in 80% and perivascular chronic inflammatory cells in 85% of subjects . histopathology showed 3 cases of lp , 4 cases of mmp and 5 cases were diagnosed as non - specific chronic inflammatory lesions . in dif study - 5 subjects were negative . 3 were reported as olp ( figure 1 ) , 2 were interpreted as pv ( figure 2 ) and 1 each of mmp ( figure 3 ) and lp pemphigoides ( figure 4 ) . histopathological diagnosis consistent with dif in 8 cases ( 66% ) ( 3 olp , 1 mmp and 4 non - specific inflammation ) . ( lichen planus ) , ( a ) hyperkeratosis , basal cell degeneration of epithelium along with subepithelial band of chronic inflammatory cells ( h and e , 10 ) , ( b ) direct immunofluorescence showing fibrin deposition along the basement membrane zone . ( pemphigus vulgaris ) ( a ) supra basal cleft with tzanck cells and intact basal layer ( h and e , 40 ) , ( b ) direct immunofluorescence showing intercellular space deposition of immunoglobulin g in the epithelium . ( mucous membrane pemphigoid ) ( a ) subepithelial cleft between epithelium and connective tissue ( h and e , 10 ) , ( b ) direct immunofluorescence showing smooth , linear , and continuous band of c3 deposit along the basement membrane zone . ( lichen planus pemphigoides ) direct immunofluorescence showing linear deposit of c3 along the basement membrane zone . the histopathological diagnosis and dif study were not consistent in four subjects in which one case diagnosed histopathologically as non - specific chronic inflammatory lesion showed positivity for lp pemphigoides in dif . among the other three cases , one diagnosed histopathologically as mmp was negative in dif and two cases which were diagnosed as mmp histopathologically were found to show positivity for pv in dif ( table 2 ) . mucocutaneous disorders ( lp , dle , pv , and mmp ) usually present themselves as chronic recurring ulcerations or vesiculobullous lesions , causing patients to seek diagnosis and treatment . though histopathology remains the gold standard for the diagnosis , in the past few year immunofluorescent techniques emerged as a valuable tool in the diagnosis of ulcerative and vesiculobullous mucocutaneous disorders.1 lp is predominantly a disease of the middle - aged and elderly with age ranging between 30 and 70 years.5 in the present study also , 65% of the patients were in the same age groups . lp affects both the sexes , although occasionally some survey have suggested a male predominance , majority suggested a female predilection.6,7 the slight male predilection in the present study ( m : f=1:0.81 ) may be due to the small sample size . similar to the most of the studies in the literature,5 the present study also showed buccal mucosal predominance . similar to most of the studies,6 histopathology of the present study also showed hyperorthokeratosis , basal all degeneration , and subepithelial band of chronic inflammatory cells in 60% , 100% , and 100% of the subjects , respectively . it is also considerably more common in women than men.6,8 the present study also showed most of the subjects in the third and fourth decades ( 45% ) with a male : female ratio of 1:3 . as with the other studies,8,9 lip is the most common site of involvement in the present study ( 85% ) . a survey of literature showed that dle lesions are characterized by hyperkeratosis , basal cell degeneration , perivasular lymphocytic infiltrate , and band like inflammatory cells in the lamina propria.10 the present study also showed basal cell degeneration , subepithelial band of chronic inflammatory cells , and perivascular chronic inflammation in 80% , 55% , and 85% , of the subjects , respectively . epithelial atrophy alternating with hyperplasia showed a high diagnostic value for dle against lp.10 this feature was seen in 35% subjects of the present study . pv predominantly seen between third and seventh decades and females were commonly involved . in the present study also 96% of the subjects were between third and seventh decades and female to male ratio was 1:0.53 . as with the majority of the studies6,8,11 in the present study also buccal mucosa was affected in 60% of the subjects . the formation of bulla is entirely intraepithelial just above the basal layer,8 tzanck cells and intact basal cells attached to connective tissue were seen in 80% , 85% , and 100% of the subjects , respectively . in contrast to other studies,8,11 present study showed dense chronic inflammatory cells infiltrate in 55% of subjects . mmp usually affects older persons ( fifth - eighth decade ) , females are commonly involved ( f : m : 2:1)6 in the present study also 70% of the patients were in the age group of fifth to eighth decades . more number of males ( 70% ) affected in the present study may be attributed to the small sample size . gingiva is the most common site of involvement followed by buccal mucosa.8,12 in the present study also gingiva was involved in 60% of subjects and the sub epithelial split and diffuse chronic inflammatory cell infiltrate were seen in 100% and 90% of the subjects , this is in accordance with the previous studies.8,12 in our prospective study of 12 subjects of oral mucosal lesions by dif , 5 were negative , 3 were lp , 2 were pv , and one each of mmp and lp pemphigoides . histopathological diagnosis was consistent with dif in 66% of subjects , and this was in accordance with the previous studies by daniels and carmen.1 histopathological and dif results were similar in 3 cases of olp , and one case of mmp and 4 subjects which were found to be non - specific inflammation were also found to be negative in dif study . negative results in dif will help us to rule out those diseases which produces specific fluorescent patterns and thereby strengthening the diagnosis of other oral mucosal lesions . though in some cases histopathology is not consistent with dif both were able to offer diagnosis in 2/3 of the subjects . early diagnosis of mmp is important since the prevalence of concurrent eye lesions is greater with oral lesions . dif is more specific and sensitive than histopathology and electron microscopy in diagnosing mmp.13 - 15 immunofluorescence analysis is also a helpful tool in monitoring therapeutic responses of these lesions by analyzing the lesion specific autoantibodies.16 although the histopathology remains the gold standard for the diagnosis of most of the diseases , it is inferred from this study that not all lesions are amenable to a definitive diagnosis . due to the accuracy of dlf study in diagnosing diseases , its report is taken as the final diagnosis , which ascertains the course and prognosis of the disease . however , the consistency of the dlf in this study can not be substantiated due to the limited sample size which is attributed to cost effectiveness of the dlf technique .

we describe the anaesthetic management of arthroscopic repair for complete rotator cuff tear of shoulder in a 59-year - old female with parkinson 's disease ( pd ) with deep brain stimulator ( dbs ) using a combination of general anaesthesia with interscalene approach to brachial plexus block . the dbs consists of implanted electrodes in the brain connected to the implantable pulse generator ( ipg ) normally placed in the anterior chest wall subcutaneously . it can be programmed externally from a hand - held device placed directly over the battery stimulator unit . in our patient , ipg with its leads was located in close vicinity of the operative site with potential for dbs malfunction . implications of dbs in a patient with pd for shoulder arthroscopy for anaesthesiologist are discussed along with a brief review of dbs . background : oral mucosa is often affected by many diseases including mucocutaneous disorders . the diagnoses of these disorders are primarily based on history , clinical features , and histopathology . for the past few years immunofluorescence techniques emerged as an important tool to study the pathogenesis and in the diagnosis of oral mucocutaneous and vesiculobullous disorders . the present study was designed to carry out retrospective and prospective analysis of oral mucocutaneous lesions to elucidate the clinicopathologic features and its immunofluorescence findings.materials and methods : a total of 70 subjects with oral mucocutaneous lesions were retrieved from the oral pathology files of tamil nadu govt . dental college and their clinical features were evaluated , and the histopathology was also evaluated with the help of hematoxylin and eosin stained sections . for the prospective study , biopsy from 12 subjects with oral mucocutaneous lesions was subjected to routine histopathological examination and dif to evaluate the consistency of the methods.results:in the retrospective analysis of 70 subjects with oral mucocutaneous lesions in relation to clinical features and histopathology , most of the findings were similar to the previous studies except for few criteria like male predilection in lichen planus and mucous membrane pemphigoid ( mmp ) and more prevalence of pemphigus vulgaris than mmp ( 2:1 ) . in the prospective analysis of 12 subjects , the histopathological diagnosis was consistent with dif study in 66% of cases.conclusion:the diagnostic efficiency of oral mucocutaneous lesions can be improved by modern tools like dif studies in addition to traditional methods like clinical and histopathology .

coevolutionary theories in the study of host parasite interactions indicate that antagonistic reciprocal selection pressures will lead to an arms race with a series of adaptations and counter - adaptations by the host and parasite ( thompson 1994 ) . such antagonistic interactions actually accelerate molecular evolution compared to selection pressures of environmental changes ( paterson et al . the evolutionary dynamics of a host parasite coevolution can lead to a relatively stable relationship , that is , fitness optimality for the host and/or the parasite , by means of a natural selection process ( schmid - hempel 2010 ) . the ectoparasitic mite , varroa destructor ( fig . 1 ) , causes relatively little harm to its original host the asian honey bee , apis cerana , as behavioral and physiological traits of the host limit the mite population growth ( reviewed in rath 1999 ) . varroa mites were introduced to the western honey bee , apis mellifera , over 30 years ago and has since become the largest threat to honey bees and apiculture around the world . the mite is in part responsible for the recent global honey bee colony losses that have caused ecological and economical pressures on plant biodiversity and crop production , respectively ( potts et al . 2010 ) . the coevolutionary process required for establishing a coexisting relationship between this parasite and its new host is lacking , both in time and in selective pressures because the selective disadvantage of being virulent is removed by apicultural practices aiming to control this damaging new mite pest . varroa destructor mite on the thorax of a male european honey bee ( apismellifera ) . the varroa mite is a highly specific parasite that relies completely on its host biology for its own survival and propagation . the mite feeds on bee hemolymph and reproduces in the brood cells of pupating bees . moreover , the mite is an important component in the virulence of certain honey bee viruses , additional microparasites in this complex system of host parasite interactions . in the absence of varroa mites , honey bee viruses can occur as symptomless covert infections within a colony but when varroa mites are present , a new transmission route is provided by this biological vector ( martin 2001 ; shen et al . 2005 ; gisder et al . according to theories in evolutionary epidemiology , vector - borne transmission often results in more virulent infections ( ewald 1994 ) . as the mite population grows within a colony , increasing opportunities for transmission will lead to the development of overt viral infections that ultimately result in colony mortality within one to three years if the mite population is not reduced by beekeepers ( martin 2001 ; boecking and genersch 2008 ) . therefore , the virulence of the mite is considered an indirect measure of its ability to vector these viruses . consequently , the viruses with a newly acquired vectorial transmission route will become more virulent , as their virulence is in general a measure of mite abundance . apis mellifera races in africa , a. m. scutellata , and in south and central america , of african origin ( africanized bees ) , are exceptions to the eventual mortality associated with mite infestation ( rosenkranz 1999 ; allsopp 2006 ) . wild and feral colonies under natural selective pressures in these populations have evolved resistance and are able to pass traits to managed colonies through natural mating events ( rosenkranz 1999 ) . wild and feral colonies are rare in europe and north america since the introduction of the mite ; however , a few subset populations of european honey bee races have been exposed to natural selective pressures of long - term mite infestation , as opposed to apicultural pressures or selective breeding programs . remarkably , these natural populations have been sustainably surviving mite infestation for extended periods , some over 10 years , without mite control treatments ( de jong and soares 1997 ; rinderer et al . 2001 ; kefuss et al . 2004 ; fries et al . 2006 ; le conte et al . 2007 ; seeley 2007 ) . parasite coevolution between european honey bees and varroa , though such studies have been lacking . recently , the long - term survival despite mite infestation of one such population on the island of gotland , sweden , has been confirmed to be a host adaptation rather than reduced virulence of the mite ( fries and bommarco 2007 ) and reduced mite reproductive success has been observed in this european honey bee populations have developed resistance to varroa mites and if so , has the same mechanisms of adaptive resistance occurred . the ability of a host to suppress the reproductive success and ultimately the population growth rate of a parasite has a strong adaptive value that would be an effective strategy toward establishing host resistance . successful mite reproduction requires the maturation of at least two eggs laid by a reproducing mother mite inside the brood cell : a male mite and a sister female mite , which must mate before bee eclosion ( rosenkranz et al . the male mite offspring will die when the bee hatches from the cell but any mated daughter mites will enter the colony 's mite population along with their mother to find a new brood cell for reproduction . this population has survived mite infestation without mite control treatment for an extended period of time ( i.e. , over 10 years ; le conte et al . varroa mite reproductive parameters are compared to findings of a previous study on the gotland population in sweden by locke and fries ( 2011 ) to determine whether adaptive strategies to resist varroa were similar or differed between geographically and genetically separate populations that are experiencing similar selective pressures . mite reproductive success is defined here as the ability of the mother mite to produce at least one viable mated female offspring before the developing bee pupa hatches as an adult . within a mite 's reproductive phase , a mother mite that lays no eggs , lays only one egg , produces no male offspring , or begins egg - laying too late in relation to larval development data for the avignon population that has survived since 1999 were collected in september 2009 and july 2010 in france . data from the mite - surviving population in gotland , sweden , used for comparison with the french population , were collected in july and august in both 2008 and 2009 ( locke and fries 2011 ) . both populations were compared first with mite - susceptible control colonies in the same location to determine any significant differences in the mite reproductive parameters . these control colonies were of different genetic background and receiving regular beekeeping management including varroa population control treatment during the autumn before experimentation . detailed descriptions of the methods for investigating mite reproductive success are found in locke and fries ( 2011 ) . in short , worker bee pupae older than approximately 190 h ( brown eyes and yellow body stage ) , but before pupal eclosion at approximately 280 h ( martin 1994 ) , were carefully removed in the laboratory . the developmental stage of each pupa was recorded based on the appearance description given by martin ( 1994 ) . complete mite families from cells infested with a single mother mite were removed using a fine brush , examined with a stereo - microscope and recorded . in france , 430 cells infested by a single mother mite were examined in 16 varroa - surviving colonies , and 211 such cells were examined in eight varroa - susceptible control colonies . in sweden , 614 cells were examined in 23 surviving colonies , and 592 cells were examined in 21 control colonies . observations were most often of 30 cells per colony but varied between 10 and 35 cells with lower numbers for those colonies where not enough mite - infested cells were found . within each pupal cell , the following parameters were recorded : the fertility ( whether the mother mite laid eggs ) ; the fecundity ( number of eggs laid ) ; the presence or absence of male offspring ; the number of dead offspring ; and the incidence of delayed egg - laying by mother mites ( identified by relating the developmental stage of mite offspring to the developmental appearance , and thus the determined age , of the infested pupa ) . the yellow thorax stage of the pupae , that is , the earliest stage of pupal exocuticle sclerotization , is the longest stage ranging from approximately 190230 h old . however , the male mite typically does not become an adult until the pupal age is around 210 h ( martin 1994 ) . therefore , to eliminate biased recordings , any yellow thorax stage infested pupa where no adult male mite was observed was recorded as uncertain since immature male mites are extremely difficult to distinguish from early stage immature female mite offspring . individual mite reproductive parameters recorded from france were compared to the results obtained in sweden ( locke and fries 2011 ) . a mixed - effects model ( sas proc mixed ) was used to test the effects that surviving colonies compared to control colonies had on the proportion of successfully reproducing mites and individual mite reproductive parameters ( = 0.05 ) . this model was also used to compare the mite reproductive parameters between the avignon and gotland populations . the random effects included in the model were date and location and a linear repeated - measures factor was used with the covariance structure selected according to aikaike 's information criteria ( littell et al . the assumption of normality and equal variance was verified by analysis of residuals ( littell et al . 1996 ) . the average proportion of successfully reproducing varroa mites was significantly lower in surviving colonies compared to control colonies in both avignon , france ( f1,21.9= 116.25 , p < 0.0001 ; fig . 2 ) , and in gotland , sweden ( f1,41.4= 75.78 , p < 0.0001 ; fig . 2 ) . interestingly , a similar between - colony difference in mite reproductive success was found in avignon ( surviving vs. control colonies ( ) 0.59 0.02 vs. 0.90 0.02 , respectively ) and in gotland ( 0.48 0.02 vs. 0.78 0.02 for surviving vs. control colonies , respectively ; fig . 2 ) . the random effects included in the statistical model , that is , date and location did not render significant differences in any of the comparisons . mean proportion of successfully reproducing mother mites in the varroa mite - surviving colonies and the mite - susceptible control colonies in avignon , france , compared to gotland , sweden , with standard error bars . investigations of the individual parameters involved in the mite 's overall reproductive success also revealed differences between surviving and control colonies at each location , as well as differences between the two mite - surviving populations ( table 1 ) . although all the parameters rendered statistically significant differences between groups in each location , a few are highly significant and biologically interesting . in avignon , the significantly high rates of infertility and secondly the high proportion of mites with delayed egg - laying seemed to be the most influential parameters in reducing the mite 's reproductive success ( table 1 ) . in gotland however , the proportion of mites with delayed egg - laying was the parameter most significantly different from control colonies with a high proportion of mite offspring mortality a secondary significant factor . fecundity was lower in the surviving colonies in both locations but this parameter does not necessarily contribute to the mite 's ability to reproduce successfully . instead , it represents only the number of eggs laid without accounting for the age of the offspring or the likelihood of them reaching maturity . therefore , fecundity may not be independent from the incidence of delayed egg - laying since any mother mite that begins laying eggs late may consequently lay fewer eggs . mean values and standard errors ( se ) of the different mite reproductive parameters investigated along with probability values of significant differences between the surviving colonies ( sc ) and control colonies ( cc ) within locations for each parameter investigated and probability values of significant differences between all surviving colonies and control colonies ( between locations ) . mite reproductive success was higher in the surviving population in avignon than in the surviving population in gotland ( f1,17.8= 17.57 , p= 0.0006 ; fig . however , comparing the two populations together shows significantly higher infertility rates and a lower mean fecundity in avignon than in gotland ( table 1 ) . the mite reproductive success was also higher in the control colonies in avignon than in the control colonies in gotland ( f1,26= 12.41 , p= 0.0016 ; fig . 2 ) and only delayed egg - laying was slightly significantly different in control colonies between locations . this result highlights that the surviving colonies in avignon and in gotland are unique from regular honey bee colonies and are distinctive from each other regarding the parameters involved in reduced mite reproductive success . in host parasite interactions , host tolerance is defined as the ability to reduce the effect of the parasite , while host resistance is the ability to reduce the fitness of the parasite ( schmid - hempel 2010 ) . until recently , the avignon and gotland populations have been considered tolerant to v. destructor since mites were still present but the damage of infestation was limited . in other words , colony mortality did not occur but the mechanisms behind the colonies survival were not understood . our study presents two honey bee populations that have in fact evolved resistant traits enabling them to reduce the mite 's fitness , measured as reproductive success . nevertheless , in most cases , resistance and tolerance are correlated ( lipstich et al . 1996 ; schmid - hempel 2010 ) . varroa mites are still present in these colonies at rates near the normal colony mortality thresholds and likely both tolerance and resistance may operate simultaneously to enable the long - term survival of these honey bee populations . in avignon , france and in gotland , sweden , varroa mite - resistant honey bee colonies reduce the average reproductive success of their infesting mites by about 30% compared to local control colonies . although these resistant populations are genetically unrelated and separated by over 2000 km , natural selection has in both cases resulted in the reduce reproductive success of this parasitic mite . the main cause of colony mortality related to high mite infestation is the virus infections vectored by the mite , particularly deformed wing virus for its close association with varroa ( martin 2001 ; boecking and genersch 2008 ) . the ability to suppress the mite 's reproductive success would delay or limit the mite 's population growth within the colony . this would consequently also limit the virus infections to less effective transmission pathways that rarely lead to colony mortality , such as vertical transmission through infected honey bee eggs and/or horizontal transmission to larvae by nurse bees through infected food ( chen et al . it is unknown what the observations from this study might mean for the evolution of the viruses . from an evolutionary perspective , the varroa mite 's strict dependence on its host 's biology causing a reduction in host fitness from parasitic infestation has imposed strong selective pressures leading to a coevolutionary arms race . in most cases of coevolution , parasites will have an evolutionary advantage above their host due to their faster evolution caused by a shorter generation time ( hafner et al . however , in this particular system , v. destructor is of clonal origin in europe with low genetic variation ( solignac et al . 2005 ) . in addition , the honey bee has 10 times higher genetic recombination levels than any higher order eu - karyote analyzed thus far ( beye et al . these aspects may have provided the honey bee with an evolutionary advantage in the arms race with v. destructor , an arms race that possibly is in the hosts favor , with mite adaptations limited . a counter - adaptation could be expected according to co - evolution theory ( thompson 1994 ; schmid - hempel 2010 ) but with the lack of genetic diversity among mites this may take a long time . on the other hand , the adapted resistance in these two honey bee populations has evolved incredible fast by natural selection . both the avignon and gotland populations have experienced similar selection pressures of natural mite infestation that is unique compared to most other european honey bee populations due to apicultural management and both have evolved a similar colony - level mite - resistant trait . however , these populations have different life - history traits and different environmental factors that would also be involved in their adaptive responses to the mite pressure . the evolved mechanisms behind the ability to suppress reproductive success of mites may differ between these two distinct populations . in general , one may expect different traits to be favored in different populations living in distinct environments even with similar natural selection pressures , especially in traits involved in coevolutionary relationships ( thompson 1999 ) . although the two populations have clearly both evolved the ability to reduce mite reproductive success , the between - population differences are less clear . therefore , more detailed investigations are necessary to identify and tease apart the possible mechanistic differences . a suggested mechanism involved in reducing the mite 's reproductive success could be for example , the adult bee behavior known as varroa - sensitive hygiene ( vsh ) , which involves the uncapping or removal of mite - infested brood ( harbo and harris 2005 ; ibrahim and spivak 2006 ) . it has been shown that bee colonies expressing this behavioral trait may selectively remove pupae with reproducing mites resulting in the remaining infested cells having a misrepresented higher proportion of infertile mites ( harbo and harris 2005 ; ibrahim and spivak 2006 ) . this could potentially be a mechanism of the avignon population , in light of the observed high mite infertility rates . since the gotland population does not demonstrate hygienic behavior ( locke and fries 2011 ) nor had significantly high proportions of infertile mites , there is no reason to suspect that they are expressing vsh . instead , the suppression of mite reproductive success in gotland may be due to another mechanism , such as pupal volatile compounds that can inhibit the initiation of egg - laying of mites ( garrido and rosenkranz 2003 ; milani et al . 2004 ) . besides suppressing mite reproduction , both varroa - resistant european honey bee populations in this study also share the fact that they have been unmanaged , enabling natural selection ( as opposed to artificial ) to shape the evolution of their mite resistance . this is an important consideration since it highlights the impact that apicultural practices otherwise have on these host parasite interactions ( fries and camazine 2001 ) , suggesting a human interference in co - evolution between species . this tri - layered complex host parasite system between honey bees ( a multilevel organism with high genetic recombination rates ) , the varroa mite ( with a fast generation time but low genetic variation ) , and the viruses ( vectored by varroa ) that infect both the bee and the mite ( de miranda and genersch 2010 ) , challenges basic coevolutionary theories and has not been fully exploited by evolutionary biologists as a model for host our hope is to stimulate interdisciplinary research between apicultural studies and evolutionary biology to provide new insight into parasitic interactions of this system . a deeper understanding of how honey bee colonies naturally coevolve with parasites , and understanding the mechanisms and traits behind such coevolution , is necessary for establishing new optimal and long - term sustainable honey bee health management strategies in apiculture . the research reports , accumulated since the fortieth of the last century , regarding the isotope effects in chemical exchange system proved that there are little differences in the chemical properties of the different isotopes of the same element . recently , researches in the isotopes separation field indicated that the isotopes of a given element may show some quantitative differences in chemical reaction equilibria and/or reaction rates ; the former is the equilibrium isotope effects and the later is the kinetic isotope effects . separation of isotopes by ion exchange chromatography is one of the most effective chemical exchange methods , which is based on the chemical equilibrium between isotopic species distributed between the stationary resin phase and the mobile solution phase . it has been applied successfully to the separation of isotopes of various elements in ligand exchange systems , in particular , those using hydroxycarboxylates as ligands , such as ce , gd , zn , eu , cu and nd [ 911 ] and in electron exchange systems such as eu and u . the first trial to explore the origin of the isotope effects in chemical exchange reactions was carried out by clewett and schaap , who suggested that the isotope effects in a chemical exchange reaction are due to a slight difference in the affinity of the isotopes for a given molecule or complex due to minor variances in the internal energies , mainly vibrational energy , of the molecule . based on the quantum molecular vibration energy , bigeleisen formulated the method to calculate the isotope exchange equilibrium constant from spectroscopic data . this method was used to calculate the equilibrium constant of the isotopic exchange of many elements ranging from hydrogen to uranium . unfortunately , this method could not explain the anomalous isotope effects of the odd isotopes u and u among the other uranium even isotopes . this anomaly was found to be similar to the odd even staggering of the isotope shift in the atomic spectra . according to the new theory derived by bigeleisen later on , similar odd even isotope effects were found in gd , zn [ 3,4,1921 ] , nd [ 911 ] and cd . lanthanides and actinides are known to have deformed nuclei , which cause the charge distribution effects in the isotope shifts of the atomic emission spectral lines . therefore , the field shift is expected to have a great effect on their isotope effects . in case of cd , the contribution of the nuclear field shift effect to the observed isotope enrichment factor was estimated to be 530% . another supporting proof for the importance of the field shift on isotope effects was given by the study of temperature effect on eu isotope effects . it was shown that the separation coefficient of eu isotopes increases with the increase in temperature , which could be explained by the field shift effects . kim et al . studied the isotope effects of uranyl complexes by means of ion exchange chromatography and reported that the malic acid eluent system had the largest separation coefficient among some selected uranyl carboxylate complexes . therefore , the purpose of the work is to study the isotope effects of neodymium in ligand exchange system using glycolic , malic and citric acids as mono , di and tri carboxylic acid to compare the effect of different ligands on the isotope effects of nd in nd - ligand exchange system . it is aimed to find the most suitable ligand that gives the highest separation coefficient and to get more information that may lead to more understanding of the theory of isotope effects . the cation exchange resin used in the ligand exchange system , lxs , was a macroporous strongly acidic cation exchange resin , ( sqs , 100200 mesh size ) obtained as a gift from asahi chemical co. japan , nd2o3 of purity 99.99% was supplied by alfa - aesar , usa , and converted to ndcl3 by dissolving in 2 m hcl solution followed by well gentle evaporation , drying the obtained solid salt , washing several times with distilled water followed by evaporation till neutrality , then used without further purification . neodymium isotope separation experiment based on the ligand complex formation was carried out with a cyclic displacement chromatography system which is composed of three glass columns , 0.8 cm i.d . 100 cm long , with water jacket , connected in series with teflon tubes , 1 mm inner diameter , so that they were repeatedly used in merry - go - round way for the desired migration length . the resin was pretreated with 2 m , mol / dm , hcl solution to remove impurities and to convert the resin into h form . this was followed by passing a solution of 0.1 m cucl2 to convert the resin into cu form . then a 0.05 m ndcl3 solution was fed into the first column at a constant flow rate by a peristaltic pump to form nd adsorption band . when the nd ion adsorption band had grown to an appropriate length the nd and cu adsorption bands were eluted by an eluent solution containing 0.2 m ammonium malate or 0.15 m ammonium citrate or 0.2 m ammonium glycolate + 0.1 m nh4no3 + 0.0002nan2 adjusted to ph 4.6 with nh4oh solution . the adsorption band of nd was visible , pink , in contrast with the preceding green cu band . when the nd adsorption band migration length reached to the desired length , it was eluted out from the last column . the effluent was collected in small fractions that were , thereafter , subjected to the concentration analysis and the isotopic analysis . the temperatures of the columns were kept constant at 25 0.2 c by circulating the thermostated water through the water jackets surrounding the columns . the uv visible spectra of lanthanides were scanned starting from a wavelength of 500 nm by means of uv visible spectrophotometer to check the interference with any possible other rare earth ions . the intense pink color solution of nd is the bases for the determination of nd concentration by photometry after dilution with 0.1 m hcl at wavelength 576 nm . the neodymium isotopic ratios of some selected samples were measured by using a joel high - resolution inductively coupled plasma mass spectrometer ( jms - plasma 2 ) . the samples were first burned completely to remove any residues for the carboxylic acids , then dissolved in nitric acid . the samples in the form of nd(no3)3 were supplied to the inlet system which consists of the peristatic sample inlet section of the icp - ms . the cation exchange resin used in the ligand exchange system , lxs , was a macroporous strongly acidic cation exchange resin , ( sqs , 100200 mesh size ) obtained as a gift from asahi chemical co. japan , nd2o3 of purity 99.99% was supplied by alfa - aesar , usa , and converted to ndcl3 by dissolving in 2 m hcl solution followed by well gentle evaporation , drying the obtained solid salt , washing several times with distilled water followed by evaporation till neutrality , then used without further purification . neodymium isotope separation experiment based on the ligand complex formation was carried out with a cyclic displacement chromatography system which is composed of three glass columns , 0.8 cm i.d . 100 cm long , with water jacket , connected in series with teflon tubes , 1 mm inner diameter , so that they were repeatedly used in merry - go - round way for the desired migration length . the resin was pretreated with 2 m , mol / dm , hcl solution to remove impurities and to convert the resin into h form . this was followed by passing a solution of 0.1 m cucl2 to convert the resin into cu form . then a 0.05 m ndcl3 solution was fed into the first column at a constant flow rate by a peristaltic pump to form nd adsorption band . when the nd ion adsorption band had grown to an appropriate length , the supply of the feed solution was stopped . the nd and cu adsorption bands were eluted by an eluent solution containing 0.2 m ammonium malate or 0.15 m ammonium citrate or 0.2 m ammonium glycolate + 0.1 m nh4no3 + 0.0002nan2 adjusted to ph 4.6 with nh4oh solution . the adsorption band of nd was visible , pink , in contrast with the preceding green cu band . when the nd adsorption band migration length reached to the desired length , it was eluted out from the last column . the effluent was collected in small fractions that were , thereafter , subjected to the concentration analysis and the isotopic analysis . the temperatures of the columns were kept constant at 25 0.2 c by circulating the thermostated water through the water jackets surrounding the columns . the concentration of neodymium was determined in each sample by using uv visible spectrophotometer . the uv visible spectra of lanthanides were scanned starting from a wavelength of 500 nm by means of uv visible spectrophotometer to check the interference with any possible other rare earth ions . the intense pink color solution of nd is the bases for the determination of nd concentration by photometry after dilution with 0.1 m hcl at wavelength 576 nm . the neodymium isotopic ratios of some selected samples were measured by using a joel high - resolution inductively coupled plasma mass spectrometer ( jms - plasma 2 ) . the samples were first burned completely to remove any residues for the carboxylic acids , then dissolved in nitric acid . the samples in the form of nd(no3)3 were supplied to the inlet system which consists of the peristatic sample inlet section of the icp - ms . the isotopes separation of certain element by ion exchange chromatography is best achieved by the band displacement technique . this operation is characterized by sandwiching a band of the ions of the element to be studied , nd , between two other chemical species bands , cu and nh4 + , maintaining self - sharpening band boundaries at both the migration band ends . during this operation , the band of the isotopic chemical species of the element to be separated is eluted through the column by a displacing eluent solution . the velocity of the band displacement is controlled by the eluent type and concentration in the solution phase , equilibrium between the solution phase and the resin phase as well as by the flow rate of the solution . the profiles of nd concentration in the effluent fractions , which correspond to the nd band profile in the column , after 11.58 m migration is shown in fig . 2 for glycolate system . the sharp boundaries of the band shown in this figure indicate that the chromatographic displacement was almost ideal at both boundaries . naturally occurring neodymium 3 shows the isotope distribution ratios for neodymium in nd- glycolate system at constant temperature of 25 c . it can be seen that the heavier isotopes nd , nd , nd , nd , nd and nd are enriched into the front part , or preferentially fractionated in the complex form in the solution phase . the degree of fractionation of neodymium isotopes takes the order ; nd 143nd144nd145nd146nd148nd. this tendency is the same as that observed in the chromatographic isotope separation of ce , gd , zn , cu and eu . since the heavier isotope is enriched in the complex species , the observed isotopic enrichment tendency accords with the theoretically expected direction of the isotopic effects in chemical exchange . the schematic diagram of the expected ion exchange mechanism under the above mentioned conditions , in the simplest form , is represented in fig . 4 . the chemical reactions involved in the present systems first takes place at the interface between nh4 + and nd adsorption bands . when ( nh4 ) n - ligand reached the rear boundary of nd adsorption band , the ligands are transferred to nd because of the large stability constant of the nd - ligand complex compared to that of ammonium ion - ligand complex . during the moving down of the solution phase , which contains nd - ligand complex species through the nd adsorption band in the column , the isotopic exchange reaction takes place between nd ions in the resin phase and nd - ligand complex species in the solution phase . after that the nd - ligand complex reaches the cu ion band , where ligand are transferred to cu ions and nd ions are adsorbed in the resin phase . the related chemical reactions for the three types of carboxylic acids , mono - basic ( glycolate ) , di - basic ( malate ) and tri - basic ( citrate ) ligands can be expressed , in the simplest form , as : for the mono - basic glycolate ligand(1)4nh4-lg+nd3++h+4nh4++nd-(lg)4-h(2)hnd3++lnd-(lg)4-hlnd3++hnd-(lg)4-h(3)nd-(lg)4-h+cu2++h+nd3++cu-(lg)4 - 2h for the mono - basic glycolate ligand for the di - basic malate ligand:(4)2(nh4)2-lm+nd3++h+4nh4++nd-(lm)2-h(5)hnd3++lnd-(lm)2-hlnd3++hnd-(lm)2-h(6)nd-(lm)2-h+cu2++h+nd3++cu-(lm)2 - 2h for the di - basic malate ligand : for the tri - basic citrate system:(7)(nh4)3-lc+nd3++h+3nh4++nd - lc - h(8)hnd3++lnd - lc - hlnd3++hnd - lc - h(9)nd - lc - h+cu2++h+nd3++cu - lc-2hwhere the underlines represent the species in the resin phase , l represents the ligand species ( where lg = glycolate , lm = malate and lc = citrate ) and nd and nd represent the heavy and the light neodymium isotopes , respectively . in fact , the chemistry of the system may be more complicated than that represented by the above equations . the exact complex structure and the different possibilities of nd and/or h2o hydrolysis are out of the scope of the present work . for the tri - basic citrate system : the single stage separation factor , = ( 1 + ) for each nd isotopes is defined here as:(10)=1+=142nd/hnd/142nd / hndwhere the underline represents the species in the resin phase and h can take the values 143 , 144 , 145 , 146 , 148 and 150 . the separation coefficients , s were calculated by using the isotopic enrichment curves of the front and rear boundaries according to the equation developed by spedding et al . and kakihana and kanzaki .(11)=qi|ri - ro|/{qro(1-ro)}where q is the amount of neodymium in the sample fraction , q is the total amount of sorbed neodymium in the column packed resin , ri is the isotopic ratio of nd / nd , and the subscripts i and o denoted the fraction number and the original feed , respectively . in general , the isotope exchange reaction effectively proceeds and reaches the equilibrium between two phases of the solution and the resin at lower flow rate condition . the mathematical averages of the two separation coefficient values obtained from the front and rear boundaries were taken to calculate the process separation coefficient ( ) . the average values of the separation coefficients of each isotope relative to nd for different ligands are given in table 3 with an estimated error factor of 5.0% . from the data shown in table 3 it can be easily noticed that the separation coefficient increases with the increase of the mass number . this trend agrees with the previous findings in case of u , zn [ 1921 ] , gd nd citrate system and nd malate system . the arrangement of the ligands takes the following direction with respect to the increasing capacity of each ligand to increase the separation coefficient of each isotope : malate ligand > citrate ligand > glycolate ligand the values of the separation coefficients were plotted as a function of the mass number at 25 c as shown in fig . a linear relationship was obtained between the mass number and the separation coefficient for the three ligands . the current discussion can not be extended to the odd even or mass anomaly phenomena due to the short migration of the bands that leads to high errors in the isotope ratio measurements carried out by icp mass . the odd even and mass anomaly phenomena were discussed for neodymium malate system elsewhere . 6 shows the structure of the three ligands compared in this study according to wikipedia site . the malate structure has up to 4 possible active sites for complexion with nd ions . this number is reduced to three only in case of citrate , as two sites were not available for complexion due to steric hindering . the number of possible complexion sits of the three ligands takes the order malate > citrate > glycolate , which agree with the order of isotope effects of the three systems as studied by the separation coefficients shown above . the plate height , hetp , is a very important factor in determining the performance of any chromatographic separation system . the smaller the value of hetp , the shorter the migration length needed for a specific separation task i.e. the higher the efficiency of the system . ( 12 ) and ( 13).(12)hetp=(/s)+(1/s2l)where l is the total migration length and s is the slope of the plots of ln ( ri ro ) vs. xi l , where ri is the neodymium isotopic ratio of nd / nd in the fraction , ro is the neodymium isotopic ratio of the feed solution , xi is the hypothetical distance of the sample fraction , calculated from the starting point at the time when the boundary is eluted from the column after migration distance of l. the hypothetical distance is calculated based on the effluent volume being proportional to the migration distance of the absorbed band:(13)xi=(vi / qt)*lwhere vi is the effluent volume of the sample fraction i , qt is the total effluent volume and l is the total migration length . xi l carried out at 25 c for nd at nd - malate system was shown in a previous article . the values of the hetp for each neodymium isotope for different ligands at constant temperature 25 c have been calculated using eq . it can be easily noticed from table 4 that the values of the ( hetp ) are small , which leads to a higher degree of separation and a better separation performance . the hetp values of neodymium isotope separation by ion exchange chromatography in ligand exchange system are of the same magnitude of hetp values of europium isotope separation , while it is 10 times larger than those of copper . this could be due to the larger size of the ions of the f electron element like eu and nd compared to cu ions . the isotopes separation of certain element by ion exchange chromatography is best achieved by the band displacement technique . this operation is characterized by sandwiching a band of the ions of the element to be studied , nd , between two other chemical species bands , cu and nh4 + , maintaining self - sharpening band boundaries at both the migration band ends . during this operation , the band of the isotopic chemical species of the element to be separated is eluted through the column by a displacing eluent solution . the velocity of the band displacement is controlled by the eluent type and concentration in the solution phase , equilibrium between the solution phase and the resin phase as well as by the flow rate of the solution . the profiles of nd concentration in the effluent fractions , which correspond to the nd band profile in the column , after 11.58 m migration is shown in fig . 2 for glycolate system . the sharp boundaries of the band shown in this figure indicate that the chromatographic displacement was almost ideal at both boundaries . naturally occurring neodymium 3 shows the isotope distribution ratios for neodymium in nd- glycolate system at constant temperature of 25 c . it can be seen that the heavier isotopes nd , nd , nd , nd , nd and nd are enriched into the front part , or preferentially fractionated in the complex form in the solution phase . the degree of fractionation of neodymium isotopes takes the order ; nd 143nd144nd145nd146nd148nd. this tendency is the same as that observed in the chromatographic isotope separation of ce , gd , zn , cu and eu . since the heavier isotope is enriched in the complex species , the observed isotopic enrichment tendency accords with the theoretically expected direction of the isotopic effects in chemical exchange . the schematic diagram of the expected ion exchange mechanism under the above mentioned conditions , in the simplest form , is represented in fig . 4 . the chemical reactions involved in the present systems first takes place at the interface between nh4 + and nd adsorption bands . when ( nh4 ) n - ligand reached the rear boundary of nd adsorption band , the ligands are transferred to nd because of the large stability constant of the nd - ligand complex compared to that of ammonium ion - ligand complex . during the moving down of the solution phase , which contains nd - ligand complex species through the nd adsorption band in the column , the isotopic exchange reaction takes place between nd ions in the resin phase and nd - ligand complex species in the solution phase . after that the nd - ligand complex reaches the cu ion band , where ligand are transferred to cu ions and nd ions are adsorbed in the resin phase . the related chemical reactions for the three types of carboxylic acids , mono - basic ( glycolate ) , di - basic ( malate ) and tri - basic ( citrate ) ligands can be expressed , in the simplest form , as : for the mono - basic glycolate ligand(1)4nh4-lg+nd3++h+4nh4++nd-(lg)4-h(2)hnd3++lnd-(lg)4-hlnd3++hnd-(lg)4-h(3)nd-(lg)4-h+cu2++h+nd3++cu-(lg)4 - 2h for the mono - basic glycolate ligand for the di - basic malate ligand:(4)2(nh4)2-lm+nd3++h+4nh4++nd-(lm)2-h(5)hnd3++lnd-(lm)2-hlnd3++hnd-(lm)2-h(6)nd-(lm)2-h+cu2++h+nd3++cu-(lm)2 - 2h for the di - basic malate ligand : for the tri - basic citrate system:(7)(nh4)3-lc+nd3++h+3nh4++nd - lc - h(8)hnd3++lnd - lc - hlnd3++hnd - lc - h(9)nd - lc - h+cu2++h+nd3++cu - lc-2hwhere the underlines represent the species in the resin phase , l represents the ligand species ( where lg = glycolate , lm = malate and lc = citrate ) and nd and nd represent the heavy and the light neodymium isotopes , respectively . in fact , the chemistry of the system may be more complicated than that represented by the above equations . the exact complex structure and the different possibilities of nd and/or h2o hydrolysis are out of the scope of the present work . for the tri - basic citrate system : the single stage separation factor , = ( 1 + ) for each nd isotopes is defined here as:(10)=1+=142nd/hnd/142nd / hndwhere the underline represents the species in the resin phase and h can take the values 143 , 144 , 145 , 146 , 148 and 150 . the separation coefficients , s were calculated by using the isotopic enrichment curves of the front and rear boundaries according to the equation developed by spedding et al . and kakihana and kanzaki .(11)=qi|ri - ro|/{qro(1-ro)}where q is the amount of neodymium in the sample fraction , q is the total amount of sorbed neodymium in the column packed resin , ri is the isotopic ratio of nd / nd , and the subscripts i and o denoted the fraction number and the original feed , respectively . in general , the isotope exchange reaction effectively proceeds and reaches the equilibrium between two phases of the solution and the resin at lower flow rate condition . the mathematical averages of the two separation coefficient values obtained from the front and rear boundaries were taken to calculate the process separation coefficient ( ) . the average values of the separation coefficients of each isotope relative to nd for different ligands are given in table 3 with an estimated error factor of 5.0% . from the data shown in table 3 it can be easily noticed that the separation coefficient increases with the increase of the mass number . this trend agrees with the previous findings in case of u , zn [ 1921 ] , gd nd citrate system and nd malate system . the arrangement of the ligands takes the following direction with respect to the increasing capacity of each ligand to increase the separation coefficient of each isotope : malate ligand > citrate ligand > glycolate ligand the values of the separation coefficients were plotted as a function of the mass number at 25 c as shown in fig . a linear relationship was obtained between the mass number and the separation coefficient for the three ligands . the current discussion can not be extended to the odd even or mass anomaly phenomena due to the short migration of the bands that leads to high errors in the isotope ratio measurements carried out by icp mass . the odd even and mass anomaly phenomena were discussed for neodymium malate system elsewhere . 6 shows the structure of the three ligands compared in this study according to wikipedia site . the malate structure has up to 4 possible active sites for complexion with nd ions . this number is reduced to three only in case of citrate , as two sites were not available for complexion due to steric hindering . the number of possible complexion sits of the three ligands takes the order malate > citrate > glycolate , which agree with the order of isotope effects of the three systems as studied by the separation coefficients shown above . the plate height , hetp , is a very important factor in determining the performance of any chromatographic separation system . the smaller the value of hetp , the shorter the migration length needed for a specific separation task i.e. the higher the efficiency of the system . ( 12 ) and ( 13).(12)hetp=(/s)+(1/s2l)where l is the total migration length and s is the slope of the plots of ln ( ri ro ) vs. xi l , where ri is the neodymium isotopic ratio of nd / nd in the fraction , ro is the neodymium isotopic ratio of the feed solution , xi is the hypothetical distance of the sample fraction , calculated from the starting point at the time when the boundary is eluted from the column after migration distance of l. the hypothetical distance is calculated based on the effluent volume being proportional to the migration distance of the absorbed band:(13)xi=(vi / qt)*lwhere vi is the effluent volume of the sample fraction i , qt is the total effluent volume and l is the total migration length . xi l carried out at 25 c for nd at nd - malate system was shown in a previous article . the values of the hetp for each neodymium isotope for different ligands at constant temperature 25 c have been calculated using eq . it can be easily noticed from table 4 that the values of the ( hetp ) are small , which leads to a higher degree of separation and a better separation performance . the hetp values of neodymium isotope separation by ion exchange chromatography in ligand exchange system are of the same magnitude of hetp values of europium isotope separation , while it is 10 times larger than those of copper . this could be due to the larger size of the ions of the f electron element like eu and nd compared to cu ions . the isotope effects of neodymium in nd - glycolate ligand exchange system were studied by using ion exchange chromatography . the heavier isotopes nd were clearly found to be enriched in the nd - glycolate species in the solution phase . the degree of fractionation takes the order , nd nd nd nd nd nd . the separation coefficients of neodymium isotopes , s , were calculated from the observed isotopic ratios at the front and rear boundaries of the neodymium adsorption band . the separation coefficients of neodymium isotopes , s , for the nd - glycolate ligand exchange system were compared with those of nd - malate and nd - citrate , which indicated that the isotope effects of neodymium as studied by the three ligands takes the following direction malate > citrate > glycolate . this order agrees with the number of available sites for complexation of each ligand . the plate height , hetp , values of nd in nd - ligand exchange systems were calculated and found to be of the same magnitude of eu , while it is 10 times larger than cu .

honey bee societies ( apis mellifera ) , the ectoparasitic mite varroa destructor , and honey bee viruses that are vectored by the mite , form a complex system of host parasite interactions . coevolution by natural selection in this system has been hindered for european honey bee hosts since apicultural practices remove the mite and consequently the selective pressures required for such a process . an increasing mite population means increasing transmission opportunities for viruses that can quickly develop into severe infections , killing a bee colony . remarkably , a few subpopulations in europe have survived mite infestation for extended periods of over 10 years without management by beekeepers and offer the possibility to study their natural host parasite coevolution . our study shows that two of these natural honey bee populations , in avignon , france and gotland , sweden , have in fact evolved resistant traits that reduce the fitness of the mite ( measured as the reproductive success ) , thereby reducing the parasitic load within the colony to evade the development of overt viral infections . mite reproductive success was reduced by about 30% in both populations . detailed examinations of mite reproductive parameters suggest these geographically and genetically distinct populations favor different mechanisms of resistance , even though they have experienced similar selection pressures of mite infestation . compared to unrelated control colonies in the same location , mites in the avignon population had high levels of infertility while in gotland there was a higher proportions of mites that delayed initiation of egg - laying . possible explanations for the observed rapid coevolution are discussed . the isotope effects of neodymium in nd - glycolate ligand exchange system were studied by using ion exchange chromatography . the separation coefficients of neodymium isotopes , s , were calculated from the observed isotopic ratios at the front and rear boundaries of the neodymium adsorption band . the values of separation coefficients of neodymium isotopes , s , for the nd - glycolate ligand exchange system were compared with those of nd - malate and nd - citrate , which indicated that the isotope effects of neodymium as studied by the three ligands takes the following direction malate > citrate > glycolate . this order agrees with the number of available sites for complexation of each ligand . the values of the plate height , hetp of nd in nd - ligand exchange systems were also calculated .

the world health organization has made prevention of visual impairment ( vi ) an international priority . in order to prevent vi , , priorities for prevention , treatment , and management can be identified , and strategies and resources allocated appropriately . for many years , diabetes has been a leading cause of vi in many countries , and still takes a major toll on vi . in england and wales , the uk national screening committee for diabetic retinopathy has set 18 service objectives and quality assurance standards for diabetic retinopathy screening services.1,2 the first service objective is to reduce new blindness due to diabetic retinopathy ( dr).2 it stipulates that local services will need to prospectively audit both certifications of visual impairment ( cvi ) and incidence of vi predominantly due to dr in order to establish a baseline . the standard acceptable annual registration rate for severe vi and vi due to dr is 9.5 and 9.3 per million per year for england and wales , respectively . these figures have been derived from national data in 19901991.3 the minimum standard achievable is a 10% reduction in severe vi and vi registration rate within 5 years of the start of the screening program . the desirable standard is 40% reduction . from 2006 and annually thereafter , dr screening programmes in england and wales will be required to submit an annual report to the national screening programme , containing general information about the service offered and information to support an assessment against the service objectives and quality assurance standards for the programme . all programmes in england and wales will be required to use a report template in order to facilitate the collation and comparison of data.4 locally derived data on new vi should be included in the annual report submitted to the national screening programme . prior to 2005 , screening for dr in cambridgeshire was largely hospital - based , with patients being referred by their general practitioners , diabetic physicians , or optometrists directly to ophthalmologists at either addenbrookes or hinchingbrooke hospital . optometrists have recently been trained to perform optometric based screening , and to refer patients in a timely manner with maculopathy ( m1 ) , preproliferative retinopathy ( r2 ) , and proliferative retinopathy ( r3 ) . in november 2003 , these apply to the function of the better eye ; people with good vision in one eye are not eligible for certification . this is the first study to obtain the severe vi and vi registration rates predominantly due to dr in south cambridgeshire , cambridge city , and huntingdonshire . it was performed to establish a reference point of vi registration due to dr at the beginning of the screening program . a retrospective review of all cvi for patients with cambridgeshire post codes over 24 months from january 1 , 2004 to december 31 , 2005 was conducted . the type of vi , cause of vi and patient s age were noted for each form . an attempt was made to validate the certificates of vi with the central list of all new registrations of vi held by cambridgeshire social services . population data was obtained from census 2001 and cambridgeshire primary care trusts to determine the total and diabetic populations of south cambridgeshire , cambridge city and huntingdon . we had to exclude east cambridgeshire and the fenlands as patients from these areas attend other local district general hospitals eg , queen elizabeth hospital king s lynn for vi assessment and registration . the annual registration rates of severe vi and vi due to dr were calculated per million population per year . in addition , the rate per million diabetic patients and prevalence of diabetes were calculated . from january 1 , 2004 to december 31 , 2005 , there were 367 cvi for patients from cambridgeshire , cambridge city , and huntingdonshire . each cause was noted which resulted in the number of causes ( 384 ) exceeding the number of registration forms ( 365 ) . the leading causes of vi registration were age - related macular degeneration in 224 or 61.4% of patients , glaucoma ( 43 or 11.8% ) , and optic neuropathy ( 27 or 7.4% ) , ( table 2 ) . according to census 2001 , the total population of south cambridgeshire the number of registrations predominantly due to dr was 18 ; 13 visually impaired and 5 with severe vi . the rates of severe vi and vi registration predominantly due to dr were 17.1 and 6.5 per million per year , respectively . the diabetic population of south cambridgeshire ( 2729 ) , cambridge city ( 3361 ) and huntingdonshire ( 4752 ) in june 2005 was 10,842 , and the prevalence of diabetes in this population of cambridgeshire is calculated to be 2.74% . the severe vi and vi registration rates in the diabetic population were 230 and 600 per million population with diagnosed diabetes per year , respectively . in our study , dr was the fifth leading cause of registered vi , superseded by age - related macular degeneration , glaucoma , optic neuropathy , and visual cortex disorder in that order . in persons of working age ( 1664 years ) , the leading cause of vi registration was optic neuropathy followed by retinitis pigmentosa ( table 2 ) . the specific causes of optic neuropathy were not stated in most instances on the cvi form . most optic neuropathies were congenital or secondary to demyelination and did not include diabetic or glaucomatous optic neuropathy . further studies are needed to clarify the types of optic neuropathy , by examining the patient records . a comparison of the demographic profile of our study population with that of england and wales reveals a younger age profile ( table 4 ) . there are 0.8% less persons aged 75 years and over , and 0.6% more persons in the 015 year age group of cambridge city , south cambridgeshire , and huntingdonshire . , dr does not appear to be the leading cause of cvi in the working age group in this population . this finding is contrary to that of bunce and colleagues5 who found that dr was the leading cause of vi in the working age group in england and wales in 19992000 . the severe vi registration rate of 6.5 per million per year meets the national standard of 9.5 per million per year . the vi registration rate was 17.1 per million per year , and almost doubles the current national standard of 9.3 per million per year . however , new data on national vi registration has emerged from a study carried out by bunce and colleagues5 in 19992000 . in this study the rate of certification of vi due to dr was 38.4 per million per year , compared to 19.9 per million per year in 19901991.3,6 these rates have doubled over the nine years , partly explained by increased vi registration rates during the time interval 19911999 , an aging population , and increasing prevalence of type 2 and type 1 diabetes.5 given these new figures , we recommend that the national standards of severe vi and vi registration rates be revised . a severe vi registration rate of 12 per million population per year has been reported for fife from 19901999,7 and a severe vi registration rate of 10 per million per year and a vi registration rate of 24 per million per year was reported for leeds for 2002.8 the prevalence of diabetes in south cambridgeshire , cambridge city , and huntingdonshire ( 2.74% ) is less than the national prevalence estimated as 4.26%.9 the prevalence in leeds is 2.9%.8 the region of cambridgeshire studied has about 5% more whites than the ethnic profile of england / wales , ( table 4 ) . likewise , our studied region has 2.8% less asians and 2.3% less blacks than england / wales . this difference might account for the slightly lower prevalence of diabetes than the national prevalence , as there is well documented evidence of an increased prevalence of diabetes in asians and blacks.10,11 the severe vi and vi registration rates per million diabetic patients per year were 230 and 600 respectively . these rates are lower than the corresponding rates in the diabetic population of leeds of 337 and 817 , respectively.8 the rates of registration in our study are also lower than the severe vi registration rate of 640 per million diabetics per year reported for fife between 19901999,7 and similar to the vi registration rate of 530 per million diabetics per year calculated for tayside in 1998.12 this interregional variation may be related to differing population profiles.13,14 addenbrookes hospital is a tertiary referral hospital in cambridge city and hinchingbrooke hospital is a secondary care centre which serves as a district general hospital for huntingdonshire and other surrounding subpopulations of cambridgeshire . we questioned whether our relatively low rates of registration of vi due to diabetes were due to the nature of these hospitals . geographically , addenbrookes hospital is the closest hospital to patients living in cambridge city and south cambridgeshire and being a tertiary referral centre , it would serve all patients in cambridge in addition to receiving referrals from outside cambridge . our results were cross - checked with the relevant primary care trusts and both sets of data correlated very well . the primary care trusts had no record of any patient living in the study population , who was registered visually impaired outside of addenbrookes or hinchingbrooke hospitals or from the private sector in the study period . the possibility of a large number patients being registered visually impaired in the private sector is unlikely . it is well known that there are limitations in using vi registration data to study rates of vi.15,16,17 vi registration data are hospital - based , not population - based.5 the patient must access the hospital eye service in order to be seen by a consultant ophthalmologist . between 43% and 58% of eligible outpatients are said to remain unregistered even after consultation with an ophthalmologist,15,16 and up to 40% of registered patients are inappropriately registered.15 patient attitudes to registration process ( patients are entitled to refuse the offer ) , and medical attitudes to registration ( there is no statutory requirement for it to be offered ) also affect registration rates.5,18 there is often a delay between onset of certifiable visual loss and offer of registration.5 studies have shown that certification of vi is biased towards severe visual loss , permanent , nontreatable causes , and those that affect central rather than peripheral vision.15,17 ethnic minorities are less likely to undergo registration.16,19 a prospective audit of incident vi would be more useful . despite these limitations , data on vi registration rates may be useful as estimates of incidence of vi and to establish a baseline for dr screening programmes . from our study it would appear that registration of severe vi secondary to dr is uncommon and does not appear to be the main cause of vi registration in the working age group in this population of cambridgeshire . these rates form a baseline for the region , to which future rates can be compared . we are unlikely to be able to achieve a lower rate of severe vi registration from dr . improvement in vi due to dr may depend on improving other aspects of diabetic care , such as control of blood glucose and blood pressure , particularly before retinopathy occurs , and timely referral of patients with sight - threatening retinopathy . genetic and epigenetic alterations that lead to the functional deregulations of several signaling and metabolic pathways are known to be the major driving forces behind carcinogenesis and cancer progression . those functional deregulations in cancer cells have been exploited for pathway - targeted anticancer therapy . small molecules and antibodies that directly inhibit critical nodes in oncogenic signaling networks , most notably kinases or enzymes , have been used to treat various cancers in humans , resulting in substantial improvement in clinical symptoms and outcomes in a subset of cancer patients . however , many critical nodes in oncogenic signaling networks may not be targeted directly by small molecules or antibodies . for example , functional losses in tumor suppressor genes caused by gene mutations or deletions may not be restored through small molecules . moreover , the functions of some intracellular oncogene products , such as ras and c - myc , have been found to be difficult to modulate directly through small molecules . nevertheless , functional alterations in nondruggable targets may lead to changes in signal transduction and metabolism that render the mutant cells more susceptible to functional changes in other genes or to pharmaceutical interventions aimed at other targets , providing an opportunity to selectively eliminate those mutant cells through synthetic lethality . synthetic lethality ( the creation of a lethal phenotype from the combined effects of mutations in two or more genes ) offers the potential to eliminate malignant cells by indirectly targeting cancer - driving molecules that are difficult to target directly with small molecules or antibodies . the concept of synthetic lethality is illustrated in figure 1a . the two genes a and b are synthetic lethal if the mutations in any one of them will not change the viability of a cell or an organism , but simultaneous mutations in both a and b genes will result in a lethal phenotype . this concept has has been used in genetic studies to determine functional interactions and compensation among genes for decades and has recently been exploited for the development of new genotype - selective anticancer agents , identification of novel therapeutic targets for cancer treatment , and characterization of genes associated with treatment response . for example , if gene a in figure 1b is mutated , small interfering rna ( sirna ) or small molecules targeting the genes x , y , or z would likely induce synthetic lethality in cells with an abberant a but not in the cells with a wild - type a. therefore , using paired isogenic cell lines with and without abberant a , one can screen for sirna or compounds that specifically kill the cells with an abberant a. concept and models of synthetic lethal interactions . ( a ) synthetic lethality between genes a and b. a and b represent wild types , while a and b represent mutants . synthetic lethality refers to a lethal phenotype observed only in the combination group of a and b. ( b ) an essential survival function is regulated by two pathways conducted by a , b , c and x , y , x , respectively . a functional change in either of these pathways is insufficient to induce viability changes . however , the simultaneous presence of mutations or dysfunctions in both pathways , such as a mutation in a and any mutation in x , y , or z , induces lethal phenotype . thus , a is synthetic lethal with x , y , and z , and vice versa . ( c ) an essential survival function is regulated by pathway a alone , in which a2 is a multiprotein complex composed of x , y , and z ; and a3 has homologues of , and . synthetic lethality may exist among x , y , and z and among a3 , , and . several models of interactions among genes and/or proteins have been proposed to account for synthetic lethality , including the components of parallel pathways that together regulate an essential biological function , the presence of homologous genes or protein isomers derived from the same ancestral gene ( paralogs ) , subunits of an essential multiprotein complex , and components of a single linear essential pathway ( figure 1b , c ) . studies in yeast revealed that synthetic lethal interactions occurred significantly more frequently among genes with the same mutant phenotype , among genes encoding proteins with the same subcellular localization , and among genes involved in similar biological processes , such as those in parallel or compensating pathways or bridging bioprocesses . for a particular tumor suppressor gene or oncogene , synthetic lethality can be identified by using isogenic cell lines to screen an sirna library for synthetic lethal genes or a chemical library for synthetic lethal compounds . this review discusses recent advances in the development of synthetic lethality based anticancer therapeutics . in particular , it emphasizes the development of anticancer agents that target dna damage response and oncogene ras pathways through a synthetic lethality approach . the tp53 gene , which encodes tumor suppressor protein p53 , a master transcriptional regulator of cellular response to dna damage , is commonly inactivated in about 50% of human cancers by either gene mutations or degradation through hdm2 . moreover , pathways involved in dna damage response are often constitutively activated in a majority of tumors , even in early stages of tumor development and in tumor specimens from untreated patients , presumably because of oncogene - mediated deregulation of dna replication . different mechanisms are used in cells in response to different types of dna damage . single - strand breaks ( ssbs ) activate poly adp - ribose polymerase ( parp ) and are repaired mainly by parp - mediated base - excision repair , while double - strand breaks ( dsbs ) are repaired by the mechanisms of homologous recombination ( hr ) and nonhomologous end joining ( nhej ) . parp can be activated by binding to ssbs , leading to ssb repair through base excision mechanisms ( figure 2 ) . however , if ssbs are not repaired , they will cause a blockage or collapse of dna replication forks during dna synthesis and the formation of dsbs . dsbs can also be incurred by endogenous and exogenous dna - damaging agents such as ionizing radiation . single - strand break ( ssb ) , double - strand break ( dsb ) , and single strand dna derived from dna damage or stalled replication fork are recognized by various sensor molecules ( marked yellow ) , leading to activation of signal transducers ( marked green ) , which in turn activate different dna repair pathways and checkpoint pathways , thereby preventing transmission of the genetic lesion to the daughter cells . those parallel pathways provide opportunities of eliminating some cancer cells with mutations in those pathways through synthetic lethality . the single - strand dna present at stalled replication forks or generated by processing of dsbs is recognized by replication protein a ( rpa ) . the assembly of those sensor molecules in the damaged dna sites leads to the recruitment and activation of signal transducers , including three phosphatidylinositol 3-kinase related kinases ( pikks ) ( ataxia telangiectasia mutated ( atm ) , atm- and rad3-related ( atr ) , and dna - dependent protein kinase ( dna - pk ) ) that in turn activate downstream effectors , resulting in the activation of checkpoint and dna repair pathways . the phosphorylation of h2ax by atm , atr , or dna - pk at s139 ( known as h2ax ) triggers the recruitment of dna repair proteins and leads to the assembly of dna repair complexes at the damaged dna sites . consequently , the cell cycle progression is arrested to permit the repair of the damaged dna , thereby preventing transmission of the genetic lesion to the daughter cells ( figure 2 ) . both brca1 and brca2 are required for the assembly of protein complexes for hr , including recruitment of recombinase rad51 to the dna dsbs . dsb repair through hr is mostly error free and can occur only in the late s and g2 phases of the cell cycle because it requires an intact sister chromatid as a template for repair . in contrast , nhej involves ku70/80 , dna - pk , xrcc4 , and dna ligase iv , often results in gene deletion or translocation , and can occur in all phases of the cell cycle . most nhej occurs independent of the mre11/rad50/nbs1 complex and atm activation because the ku70/ku80 heterodimer can directly bind to the ends of double - strand dna , recruit dna - pk to the site , and initiate an nhej process . in the absence of ku proteins , an alternative nhej initiates with the involvement of parp1 , xrcc1 , and ligase i / iii ( figure 2 ) . parp is a group of adp - ribose transferase enzymes that catalyze polyadp - ribosylation of proteins by transferring adp - ribose groups from the donor substrate nicotinamide adenine dinucleotide ( nad ) to glutamic acid , aspartic acid , and lysine residues in the acceptor proteins , thereby regulating the functions of those proteins . dna - dependent parp subfamily members parps 13 have been reported to regulate various dna damage response processes . the recognition and binding to damaged dna structures by parp1 and parp2 , either through their own dna binding domain or through interaction with damaged dna - binding protein 2 , lead to the activation of their enzymatic activity and the polymerization of adp - ribose units of a number of proteins , including parp1/2 , histones , topoisomerase , and dna - pk . the polyadp - ribose on those proteins provides a docking site for recruiting cell cycle checkpoint proteins and dna repair proteins ( e.g. , p53 , xrcc1 , dna - pk , ku70 , and atm ) to the sites of dna lesions , thereby regulating various processes of dna repair , including base - excision repair , hr , and nhej . parp1 regulates base - excision repair by interaction with xrcc1 , dna polymerase , and the base - excision repair enzymes apurinic / apyrimidinic endonuclease 1 and alc1 . parp1 also interacts with dna - pk and ku and is required for an alternative and parp - dependent nhej pathway . although parp1 embryonic stem cells and embryonic fibroblasts exhibit normal repair of dna dsbs and rad51 foci formation , parp1 mice have increased deletion mutations and insertions and/or rearrangements in vivo after treatment with the alkylating agent n - nitrosobis(2-hydroxypropyl)amine . interestingly , parp1 mice are viable and fertile and do not develop spontaneous tumors , possibly because of functional compensation from parp2 , as parp1 and parp2 double knockout is embryonically lethal . nevertheless , parp1 mice or cells exhibit defective dna ssb repair and increased hr , sister chromatid exchange , and chromosome instability . parp1 , but not parp2 , is required for the survival of cells with defects in the hr pathway because knockdown of both parp1 and brca2 significantly reduces the survival of human cells , whereas knockdown of both parp2 and brca2 has no effect on cell survival . the mechanisms underlying the synthetic lethality of parp1 and brca genes are still not clear , although evidence suggests that it might be caused by the deregulation of nhej , increased spontaneous dna breaks that need to be repaired by hr , or the suppression of brca and rad51 expression by e2f4/p130-mediated transcriptional repression caused by parp1 inhibition . the synthetic lethality of parp1 and brcr1/2 genes reported in 2005 by farmer et al . and bryant et al . sparked much interest in the concept of using parp inhibitors to selectively eliminate brca1 or brca2 mutant tumor cells . germ line mutations in brca genes predispose carriers to breast , ovarian , and other cancers in an autosomal dominant manner , with 5080% penetrance for breast cancer and 3050% for ovarian cancer . early studies revealed that parp1 activation facilitates dna repair and maintenance of genomic integrity and is required for recovery from dna damage in mice and in cells . however , excessive parp1 activation leads to cell death because of overconsumption and depletion of nad and atp in the cells , whereas genetic disruption or pharmaceutical inhibition of parp protects animals from ischemia - induced brain and heart damage . in addition , parp1 was found to regulate the transcriptional activity of nfb and other inflammation - related transcription factors , promoting the expression of inflammation mediators . thus , parp inhibitors have been investigated for the therapeutic benefits of protecting tissue from ischemia - induced injury , suppressing inflammation , and sensitizing cancer cells to dna damage - based anticancer therapy . briefly , parp inhibitors used in clinical investigation mostly compete with the adp - ribose donor substrate nad and inhibit both parp1 and parp2 . a binding assay with catalytic domains from 13 of the 17 human parp family members revealed that many of the best known parp inhibitors , including those used in clinical studies such as olaparib ( 1 ) , veliparib ( 2 ) , and rucaparib ( 3 ) ( figure 3 ) , bind to several parp family members , suggesting nonspecific activity of those inhibitors . structures of parp inhibitors . parp inhibitors are currently under intensive investigations as therapeutic agents for the treatment of cancers with deficiencies in brca or other dna repair proteins . an initial preclinical study by farmer et al . revealed that the brca1/2-defective cells were 57- to 133-fold more sensitive to parp inhibitors ku0058684 ( 4 ) and ku0058948 ( 5 ) than wild - type cells . at the same time , bryant et al . showed that hr - defective cells with a deficiency in xrcc2 , xrcc3 , or brca2 ( xrcc11 ) were killed by parp inhibitors such as nu1025 ( 6 ) and ag14361 ( 7 ) at concentrations that were nontoxic to normal cells . recent studies revealed that defects in other dna repair proteins , such as atm , mre11 , ercc1 , and p53 or pten , also induce synthetic lethality with the parp inhibitors . a high - throughput rna interference screen for 230 known and putative dna repair proteins revealed additional genes that have synthetic lethality with compound 5 , including atr , pcna , rad51 , and xrcc1 . a similar study with a novel potent parp1/2 inhibitor ( bmn 673 ) ( 8) and an sirna library targeting 960 genes , including kinases and kinase - related genes , tumor suppressors , and dna repair proteins , showed synthetic lethality of 8 with hr and dsb repair genes moreover , parp1 inhibitors induced synthetic lethality in cancer cells with positive e26 transformation - specific gene fusions and with egfr inhibitors . because of the critical roles of parp1 in the dna repair process , parp inhibitors have also been intensively tested for sensitizing radiotherapy and chemotherapy that induces dna damage . compounds 1 and 2 are the most extensively investigated inhibitors in clinical trials for the treatment of brca1/2-mutated cancers . other parp inhibitors under clinical trials are niraparib ( mk-4827 ) ( 9 ) , 3 , and 8 . a phase i study of the combination therapy of rucaparib with temzolomide in patients with advanced solid tumors showed that 3 at a dose of 12 mg / m inhibited 7497% of parp activity in peripheral blood mononuclear cells . a dose of 12 mg / m3 in combination with 200 mg / m temozolomide was tolerated , with a dose - limiting toxicity of myelosuppression . a phase ii study of intravenous administration of 12 mg / m3 with temozolomide in patients with metastatic melanoma showed that 150200 mg m day temozolomide can be safely given in the combination therapy , with a response rate of about 17% and an increase in progression - free survival over historical controls in metastatic melanoma patients . phase i clinical trials with compound 1 in recurrent / advanced cancer patients with or without brca1/2 mutations revealed that the maximum tolerated dose for 1 was 400 mg twice daily , with dose - limiting toxicities of mood alteration , fatigue , and thrombocytopenia . clinical benefit was observed in 3060% of brca1/2 mutant breast or ovarian cancer patients . the pharmacokinetics study revealed a maximum concentration of 2.64.8 g / ml at doses of 400 mg twice daily . the mean maximal parp inhibitions in peripheral blood mononuclear cells and tumor tissues were 50% and 70% , respectively . mean terminal half - life was about 711 h. subsequently , 1 has been evaluated in combination therapy with various conventional anticancer agents , including cisplatin and gemcitabine , topotecan , bevacizumab , dacarbazine , and the vegfr inhibitor cediranib . the overall response rates were variable , from 0% to 44% . a phase ii comparison study of 1 versus liposomal doxorubicin treatment for brca1/2 mutant ovarian cancer patients who had recurrent tumors after platinum therapy revealed overall response rates of 2531% , not significantly different from that of doxorubicin alone ( 18% ) . phase i trials of a combination of 2 ( half - life of about 5 h ) with cyclophosphamide or topotecan in refractory solid tumors and lymphomas showed promising activity in a subset of patients with brca mutations . the maximum tolerated dose was defined as 60 mg of 2 with 50 mg of cyclophosphamide once daily or 0.6 mg m day topotecan administered intravenously on days 15 and 10 mg of 2 twice daily on days 15 in 21-day cycles . parp activity was significantly inhibited in peripheral blood mononuclear cells ( by 50% ) and in tumor biopsies ( by 80% ) . a phase ii trial of the combination compared with single - agent cyclophosphamide is ongoing for cases of brca mutant ovarian cancer , triple - negative breast cancer , and low - grade lymphoma . a phase 1 dose - escalation trial of 9 in brca mutation carriers and patients with sporadic cancer revealed a maximum tolerated dose of 300 mg / day . the mean terminal elimination half - life was 36.4 h. pharmacodynamic analyses confirmed that parp inhibition exceeded 50% at doses greater than 80 mg / day , and antitumor activity was documented at doses beyond 60 mg / day . clinical benefit was observed in 4050% of brca1/2 mutation carriers with ovarian or breast cancer . antitumor activity was also reported in sporadic high - grade serous ovarian cancer , non - small - cell lung cancer , and prostate cancer . a phase i study of 8 in solid tumors showed that recist and/or ca-125 response occurred at doses of > 100 g / day in 11/17 brca carrier ovarian / peritoneal cancer patients . in summary , the results from these clinical studies revealed that myelosuppression is the major dose - limiting toxicity of various parp inhibitors and that clinical benefit is variable in the single - agent and combination therapies , with significant benefit observed in brca mutant cancers , consistent with the findings in the preclinical studies . the atm / checkpoint kinase 2 ( chk2 ) and atr / chk1 signaling pathways regulate many common downstream proteins , including p53 , cdc25 phosphatases , and wee1 kinase , thereby regulating g1 , s , and g2/m checkpoints ( figure 4 ) . because activation of oncogenes can cause replication stress and dna damage , inhibiting checkpoint pathways may trigger synthetic lethality in cancer cells by enhancing the dna damage - induced apoptosis or senescence , which may be modulated by p53 status . phosphorylation of p53 on s15 by atm / atr and/or on s20 by chk1/chk2 stabilizes p53 protein and up - regulates the expression of p21 ( also known as cip1/waf1 ) , an inhibitor of cyclin e / cyclin - dependent kinase 2 ( cdk2 ) that controls g1/s progression . in contrast , phosphorylation of cdc25 phosphatases by chk1/chk2 promotes degradation of cdc25 , which is required for dephosphorylation and activation of cyclin b / cdk1 kinases that control the transition of g2/m phases ( figure 4 ) . evidence has shown that atr / chk1 is critical in regulating the activity of cdc25 and s / g2 checkpoints . although p53 also regulates the g2 checkpoint , g2 arrest is normally induced in p53-deficient cancers , suggesting that some p53-independent mechanisms are sufficient for g2 checkpoint functioning . recent studies showed that p53-deficient cells rely on atm- and atr - mediated checkpoint signaling through the p38 mitogen - activated protein ( map ) kinase / map kinase - activated protein kinase 2 ( mk2 ) pathway for survival after dna damage . evidence has shown that p53-mediated apoptosis or cell cycle arrest is modulated by atm and chk2 activities . in cells and tumors that lacked a functional p53 pathway , inactivation of atm or its downstream molecule chk2 was sufficient to globally sensitize the cells to genotoxic chemotherapy with cisplatin or doxorubicin . in contrast , in p53 wild - type cells , the inhibition of atm or chk2 resulted in a substantial survival benefit , suggesting that a combination of cisplatin and doxorubicin with inhibitors of atm and chk2 could benefit patients with p53 mutant tumors . genome - wide small hairpin rna screening also revealed that inhibiting atm or met induced synthetic lethality with a p53 stabilizing / activating compound nutlin-3 ( 10 ) ( figure 5 ) and converted the cellular response from cell cycle arrest to apoptosis in various cancer cell types without affecting the expression of key p53 target genes . although atm inhibitors such as cp466722 ( 11 ) and ku59403 ( 12 ) do not have single - agent activity in cancer cell lines , they could sensitize cancer cells to ionizing radiation and/or genotoxic chemotherapeutics . dna damage caused by physical , chemical , and biological factors , such as oncogene activation , activates phosphatidylinositol 3-kinase related kinases atm and atr , leading to the activation of chk2 , chk1 , and p38/mk2 , which further regulate activity and stability of cell cycle regulators , including p53 and cdc25 . the loss of p53 function in cancer cells makes the cells addicting to the s and/or g2/m checkpoints for dna repaire and survival . inhibiting s and/or g2/m checkpoint regulators may induce synthetic lethality in p53 mutant cells when dna is damaged . arrow indicates activation , and the line terminating with a black circle indicates suppression . similar results have shown that inhibiting atr and chk1 exacerbates levels of oncogene - induced replicative stress , promoting the cell killing of oncogene - overexpressing cells and sensitizing tumor cells to dna - damaging therapy . inhibiting atr by a dominant negative construct selectively sensitizes g1 checkpoint - deficient cells to dna damage - induced lethal premature chromatin condensation . knockdown of atr expression selectively enhanced cisplatin sensitivity in human colorectal cancer cells with inactivated p53 , whereas the restoration of p53 in atr - deficient cells increased cell survival after cisplatin treatment . reduced atr expression prevented the development of myc - induced lymphomas or pancreatic tumors in mice by enhancing myc - induced replicative stress and apoptosis , which is more pronounced in p53-deficient cells . transgenic mice with diminished atr function in the skin have increased apoptosis after ultraviolet exposure and reduced ultraviolet - induced skin carcinogenesis , suggesting that the inhibition of the replication checkpoint function may have therapeutic and/or preventive benefits . inhibiting atr also induced synthetic lethality in xrcc1-deficient cancer cells and sensitized cancer cells to dna - damaging chemotherapeutic agents . synthetic lethality in xrcc1-deficient cancer cells is also elicited by the atm inhibitor ku55933 ( 13 ) and the dna - pk inhibitor nu7441 ( 14 ) . a cell - based screening of 623 pi3k inhibitors led researchers to identify an mtor- and atr - selective inhibitor , etp-46464 ( 15 ) , that induces replicative stress and synthetic lethality in p53-deficient or cyclin e - overexpressing cells . the ic50 values of 15 for mtor and atr are 0.6 and 14 nm , respectively , but > 36 nm for dnapk , pi3k , and atm . az20 ( 16 ) , an atr inhibitor derived from the mtor inhibitor sulfonylmorpholinopyrimidine , was reported to have single - agent activity when administered orally in mice bearing the xenograft established from colon cancer cell line lovo . through high - throughput screening and compound optimization , charrier et al . developed an atr - selective inhibitor , ve-821 ( 17 ) , with ic50 values for atr , atm , and dnapk of 12 nm , > 8 m , and > 8 m , respectively . in vitro study revealed that treatment with 17 alone induced selective killing of atm- or p53-deficient cancer cells but only reversibly limited cell cycle progression in normal cells . 17 also increased cisplatin potency 10-fold in hct116 cells , suggesting that inhibiting atr could potentiate the efficacies of radiotherapy and genotoxic drugs . ve-822 ( 18 ) , an analogue of 17 , was found to have in vivo activity in blocking atr and sensitizing pancreatic cancer cells to gemcitabine - based chemoradiation therapy . nevertheless , no clinical studies with atm or atr inhibitors have yet been reported . in comparison to the development of atm / atr inhibitors , the development of chk inhibitors , specifically chk1 inhibitors , is more advanced possibly because it is easier to perform enzymatic analysis on chk1/chk2 . chk1 and chk2 have a highly conserved kinase domain but have distinct overall protein structures . however , several small molecular inhibitors inhibit both chk1 and chk2 with a similar potency . in an e-myc - driving lymphoma model , inhibiting chk1 , chk2 , or both with small molecule inhibitors induced cell death , although the e-myc p53 null lymphoma cells were more sensitive to a dual chk1/chk2 inhibitor than to a chk1-specific inhibitor . azd7762 ( 19 ) ( figure 6 ) , a dual chk1/2 inhibitor , also enhances radiosensitivity- and chemotherapy - induced apoptosis in p53 mutant and/or p21-deficient tumor cells to a greater extent than in p53 wild - type tumor cells . the potentiation of dna damaging agent - induced apoptosis by chk inhibitors is possibly caused by abrogation of the g2 checkpoint and/or inhibition of hr dna repair . gene knockout studies revealed that knockout of chk1 or chk2 caused defects in the induction and/or maintenance of irradiation - induced g2 arrest . intriguingly , knockout of atm or chk2 is viable for cells and animals , although phenotypes of ataxia telangiectasia , chromosomal abnormality , and the development of thymic lymphomas are observed in atm knockout , whereas resistance to ionizing radiation - induced apoptosis is observed in chk2 knockout . in contrast , knockout of atr or chk1 is lethal for cells and embryos with defects in the g2/m dna damage checkpoint , indicating that the atr / chk1 pathway is essential for cell survival . therefore , inhibitors of atr and/or chk1 could be more potent and toxic than those of atm and/or chk2 . a synthetic lethality sirna screening of 572 kinases for improving gemcitabine or cisplatin response in pancreatic or ovarian cancer cells revealed the greatest potentiation by sirna targeting of atr and/or chk1 . similar results were obtained by inhibiting chk1 with small molecule inhibitors such as pd407824 ( a dual inhibitor of chk1 and wee1 ) ( 20 ) . a phase i study on 19 single - agent therapy and combination therapy with gemcitabine in patients with advanced solid tumors showed that the dose - limiting toxicities are cardiac and liver function abnormalities and myelosuppression . no objective responses were observed , although disease stabilization was observed in some patients . ucn-01 ( 7-hydroxystaurosporin ) ( 21 ) , a protein kinase c inhibitor that also inhibits many other kinases , including chk1 , and abrogates the g2 checkpoint has been reported to enhance the effectiveness of genotoxic agents in p53-deficient cells . a phase i study of 21 in combination with perifosine or irinotecan showed some partial response in p53-defective triple - negative breast cancer , whereas a phase ii trial in metastatic melanoma showed that 21 as a single agent is not active in refractory melanoma . both 19 and 21 have been found to be not favorable for further development because of pharmacokinetic and toxicity issues . mk-8776 ( sch900776 ) ( 22 ) and ly2603618 ( 23 ) are chk1 selective inhibitors ( 50- to 500-fold more active than on chk2 ) that are being used in clinical trials for the treatment of leukemias and solid tumors . cardiac toxicity and myelosuppression were observed in phase i trials of both 22 and 23 in combination therapy with cytarabine and pemetrexed , respectively.22 was used in combination with cytarabine to treat refractory acute leukemias . complete remissions occurred in 8 ( 33% ) of 24 patients , mostly at a dose of 40 mg / m or higher of 22 . the maximum tolerated dose for 22 was approximately 56 mg / m.23 in combination with pemetrexed ( 500 mg / m ) for the treatment of solid tumors revealed a maximum tolerated dose for 23 of 150 mg / m ; some partial response or disease stabilization was observed . some chk2-selective inhibitors have been identified , including pv1019 ( 24 ) and cct241533 ( 25 ) . both agents were reported to be highly selective against chk1 and have radioprotective effects in mouse thymocytes . as single agents , both 24 and 25 had mild antitumor activity but were found to potentiate the cytotoxicity of genotoxic agents and parp inhibitors , respectively . activating mutations in three oncogenic ras genes ( h- , n- , and k - ras ) is among the first and the most common genetic alterations identified in human cancers , occurring in approximately 30% of human tumors . the kras gene encodes two splicing isoforms , a major kras 4b and a minor kras 4a . therefore , mammals have four small ( 21 kda ) oncogenic ras proteins of about 190 amino acids in size , with the first 165 aa conserved in the n - terminal for all the ras proteins . the kras 4b , hras , and nras isoforms are ubiquitously expressed , whereas kras 4a is expressed mainly in kidney , liver , and gastrointestinal tissues . as a subfamily of small guanine nucleotide - binding proteins , ras proteins cycle between an active guanosine triphosphate ( gtp ) bound form and an inactive guanosine diphosphate ( gdp ) bound form . binding of ras with gtp is facilitated by guanine nucleotide exchange factors through catalyzing the release of gdp and is required for the interaction of ras with target proteins . the intrinsic gtpase activity that is enhanced by gtpase - activating proteins converts gtp to gdp , leading to a gdp - bound , inactive ras . ras mutations that diminish the gtpase activity or decrease the gdp binding capacity render ras in a constitutively active , gtp - bound status . in the absence of a ras mutation , increased ras activity is frequently detected in human cancer because of gene amplification , overexpression , an increase in upstream signals from tyrosine - kinase growth - factor receptors such as her2 and egfr , and altered expression of micro - rna such as let-7 . increased ras activity is associated with resistance to chemotherapy and radiotherapy , leading to a poor prognosis . as a key mediator in the signaling transduction for a variety of growth factors , cytokines , and hormones , ras proteins are transferred to the inner leaflet of the plasma membrane , where they interact with a diversity of membrane receptors and execute signal transduction in a variety of signaling pathways that govern cell growth , proliferation , differentiation , and death . several steps of posttranslational modifications are critical for trafficking ras proteins to the plasma membrane , including farnesylation at the cysteine residue of the carboxy - terminal caax motif and methylation of farnesyl cysteine at the c - terminal . the enzymes involved in these processes , such as farnesyltransferase , geranylgeranyltransferase , and isoprenylcysteine carboxyl methyltransferase , have been intensively investigated in preclinical and clinical trials for anti - ras therapy . small molecules binding irreversibly to the kras ( g12c ) mutant protein or interfering with ras effector protein interaction have recently been reported . targeting the ras downstream pathways , particularly the raf / mek / erk and pi3k / akt / mtor pathways , has also been investigated for inhibiting ras mutant cancer cells . a clinical trial with biomarker - integrated targeted therapy for lung cancer has revealed that sorafenib ( 26 ) , a pan - raf and vegfr inhibitor , has impressive benefits for kras mutant patients . however , selective inhibition of braf with a dominant negative construct in mice or with braf - selective inhibitors such as vemurafenib ( 27 ) in patients promoted the development and/or progression of ras mutant cancers possibly because of the activation of other raf isoforms , such as raf-1 . thus far , effective anti - ras therapeutics is not clinically available . indirect anti - ras therapy with the synthetic lethality - based approach has recently been investigated by several groups . it is noteworthy that expression of oncogenic ras in primary normal human or rodent cells often results in apoptosis or senescence , whereas expression of oncogenic ras in immortal cells or cells with inactivation of p53 , p16 , or the transcriptional activator interferon regulatory factor 1 leads to transformation and tumorigenesis , suggesting that ras transformed cells have additional signaling context for survival , which provides opportunity of synthetic lethality based anti - ras therapy . several studies on sirna library screens with isogenic cell lines harboring mutant and wild - type ras genes have revealed synthetic lethal interactions of oncogenic ras with some key nodes in cancer signaling network , including polo - like kinase 1 ( plk1 ) , a serine / threonine protein kinase that regulates cell mitosis ; the transcription factor wilms tumor 1 ( wt1 ) ; tank - binding kinase 1 ( tbk1 ) , a noncanonical ib kinase that regulates the stability of ib ; spleen tyrosine kinase ( syk ) ; and cdk4 ( figure 7 ) . some small - molecule inhibitors of plk1 , cdk4 , and syk are already being used in clinical trials for anticancer therapy . the synthetic lethality interactions of those targets with oncogenic ras suggest that ras gene mutation might be used as a marker to identify responders . receptor tyrosine kinases ( rtk ) activate ras and pi3k / akt / mtor pathways . ras in turn activates raf / mek / erk , pi3k / akt / mtor , and ralgef / ral pathways and crosstalks with rtk , wnt , c - jun n - terminal kinases ( jnk ) , reactive oxidative species ( ros ) , stat3 , and tumor necrosis factor ( tnf)/nfb pathways . several key nodes in those signaling networks , such as tbk1 , plk1 , cdk4 , syk , and wt1 ( colored tan ) , have been reported to have synthetic lethal interactions with oncogenic ras . arrow indicates activation , and the line terminating with a black circle indicates suppression . several innovative anticancer agents that selectively induce cytotoxic effects in cancer cells with ras mutations have been identified by screening chemical libraries , including triphenyltetrazolium ( 28 ) and a sulfinyl cytidine derivative ( 29 ) ( figure 8) that demonstrated approximately 6-fold selectivity for cell lines containing mutant kras ; erastin ( 30 ) , which exhibited lethal selectivity in human tumor cells harboring mutations in the hras , kras , or braf oncogenes by acting on mitochondrial voltage - dependent anion channels and inducing oxidative cell death ; lanperisone ( 31 ) ; and oncrasin-1 ( 32 ) . interestingly , 30,31 , and analogues of 32(161,162 ) all induced cell - killing effects in ras mutant tumor cells by triggering oxidative stress , although through different underlying mechanisms . while 30 may act on mitochondrial voltage - dependent anion channels and induce oxidative cell death,32 and its analogues act on rna polymerase ii , protein kinase c ( pkc ) , and stat3 . most of the molecules involved in oncrasin - induced antitumor activity are directly involved either in signal transductions in ras , myc , and stat3 pathways or in causing synthetic lethality in cells with elevated activity of ras or myc oncogenes . similar to the agents identified through other approaches , substantial efforts will be required for optimization of compounds identified through synthetic lethality screening . effort has been made in the developmental therapeutics program at the national cancer institute and in our own laboratory for compound optimization of oncrasin 1 . one of the most active analogue compounds , nsc-743380 ( 33 ) , was tested in vitro with the nci-60 cell panel and 50 non - small - cell lung carcinoma cell lines and was highly active ( ic50 between 10 nm and 1 m ) in 30 of 102 cancer cell lines tested . in 50 non - small - cell lung cancer cell lines tested , 16 ( 32% ) were highly responsive to this compound ( ic50 < 0.33 m ) , including many kras mutant cancer cell lines , suggesting that a large subset of cancer patients may potentially benefit from treatment with this agent . in vivo studies performed in our laboratory and by the national cancer institute s developmental therapeutics program have shown that intraperitoneal administration of 33 at doses of 67150 mg / kg caused complete tumor regression or significant growth suppression in some xenograft tumor models , suggesting that this compound has the potential to move to further development . nevertheless , most compounds developed by synthetic lethality screening for oncogenic ras are still at the preclinical evaluation stage . the enthusiasm for developing synthetic lethality based anticancer therapeutics has been increasing recently , with increasing numbers of publications on this topic . most potential therapeutic targets or therapeutic agents developed through the concept of synthetic lethality are identified by cell - based screening with pairs of isogenic cell lines . although this approach may facilitate development of genotype - specific anticancer therapeutics , several studies have demonstrated that synthetic lethality is likely cell context dependent . for example , kras mutant cancer cell lines have been characterized as either kras - dependent or kras - independent , on the basis of cell - killing effects induced by knockdown of the kras gene itself . when 45 genes that have synthetic lethality with egfr inhibitors in the cervical adenocarcinoma cell line a431 were tested for sensitization to erlotinib or cetuximab in seven other cell lines , none of the genes sensitized all tested cell lines , although several of the genes sensitized three to five of the cell lines . similarly , resistance to the synthetic lethality of parp inhibitors in brca1 and brca2 mutant cancers has been observed both in experimental tumor models and in clinical trials . the differences in genetic and/or epigenetic backgrounds in individual cells may explain the cell - context - dependent synthetic lethality observed in various studies . because a tumor may harbor an average of 3070 mutated genes , an individual primary tumor or established cancer cell line may carry multiple concomitantly activated oncogenes or inactivated tumor suppressor genes . the activation in other signaling pathways may provide redundant input that drives and maintains downstream survival signaling , resulting in resistance to therapeutic agents that target a particular genetic lesion either directly or indirectly through synthetic lethality . moreover , even though some passenger mutations in tumor cells may not contribute to tumorigenesis , these mutations may affect drug response because of altered drug metabolism or drug efflux . as a result , querying for the synthetic lethality partners of an oncogenic kras gene can identify different candidate genes or lead compounds in different cell lines . similarly , a synthetic lethality gene agent combination identified in one cell line may not necessarily be applicable to another cell line . mechanistic delineation of molecules or pathways that mediate responses to synthetic lethality anticancer therapeutics and the identification of predictive biomarkers for treatment responses will be critical for the successful development of synthetic lethality anticancer therapeutics . on the other hand , the concept of synthetic lethality has also been explored for characterizing mechanisms of resistance and for combination therapy or multimodality therapy to enhance the efficacy of anticancer drugs . simultaneous targeting the redundant survival pathways is expected to overcome resistance and/or enhance efficacy in anticancer therapy . our own experience in developing synthetic - lethality - based anticancer therapeutics suggests that mechanistic characterization and compound optimization could be challenging and time - consuming . the biological functions of compounds identified through cell - based synthetic lethality screening are largely unknown . technological developments in chemical biology , such as conjugating compounds to biotin or resin beads and performing affinity - based target precipitation and subsequent protein identification by mass spectrometry analysis , may facilitate target and mechanistic characterization . however , conjugating and immobilizing a small compound may change its biological function and protein - binding specificity . alternatively , target proteins may be enriched by protecting the targets from protease - mediated degradation through the binding of unmodified compounds to their targets . robust efforts on in vivo optimization of the lead compound are also essential in drug development , as compounds with similar chemical structures and in vitro activity may have dramatically different in vivo toxicity and efficacy profiles .

background / aims : the uk national screening committee ( nsc ) has set 18 standards for diabetic retinopathy ( dr ) screening services in england and wales , the first of which is to reduce new visual impairment ( vi ) due to dr by 10% within 5 years . this study examined the incidence of vi due to dr in cambridgeshire ( city , south , and huntingdonshire ) in order to establish a baseline rate of vi registration.methods:a retrospective review of all certificates of visual impairment ( cvi ) for 2004 and 2005 was conducted . hospital records of patients registered due to dr were reviewed to ascertain conformity to nsc standards . the incidence of vi registration due to dr was calculated.results:the number of registrations predominantly due to dr was 18 ; 13 visually impaired and 5 with severe vi . the rates of vi and severe vi predominantly due to dr were 17.1 and 6.5 per million per year , respectively . the vi and severe vi registration rates in the diabetic population were 600 and 230 per million per year , respectively.conclusion:the severe vi registration rate due to dr lies within the national standard . the vi registration rate exceeds 19901991 national standards but lies within 19992000 national figures . the concept of synthetic lethality ( the creation of a lethal phenotype from the combined effects of mutations in two or more genes ) has recently been exploited in various efforts to develop new genotype - selective anticancer therapeutics . these efforts include screening for novel anticancer agents , identifying novel therapeutic targets , characterizing mechanisms of resistance to targeted therapy , and improving efficacies through the rational design of combination therapy . this review discusses recent developments in synthetic lethality anticancer therapeutics , including poly adp - ribose polymerase inhibitors for brca1- and brca2-mutant cancers , checkpoint inhibitors for p53 mutant cancers , and small molecule agents targeting ras gene mutant cancers . because cancers are caused by mutations in multiple genes and abnormalities in multiple signaling pathways , synthetic lethality for a specific tumor suppressor gene or oncogene is likely cell context - dependent . delineation of the mechanisms underlying synthetic lethality and identification of treatment response biomarkers will be critical for the success of synthetic lethality anticancer therapy .

local anesthetics like lidocaine and bupivacaine , opioids like morphine , alpha2 adrenoceptor agonists like clonidine , dexmeditomidine , and magnesium sulphate have all been tried intra - articularly either as sole agents or in combination , to provide effective postoperative analgesia . studies have shown that dexamethasone increases the duration of regional blocks , when combined with local anesthetics . therefore , it is expected that such a beneficial effect of dexamethasone can be manifested when it is injected in combination with levobupivacaine to intra - articular spaces . this placebo - controlled , double - blind , prospective study is designed to assess the efficacy of intra - articular dexamethasone administered as adjuvant to local anesthetic levobupivacaine in patients undergoing arthroscopic knee surgery . the study protocol was approved by the institutional ethical committee and informed consent was obtained from every patient . fifty asa i - ii patients of either sex , aged 18 - 65 years , undergoing elective knee arthroscopy were randomly assigned to one of the two groups using computer - generated random numbers comprising of 25 patients each . before performing the study , we hypothesized that the beneficial effect of levobupivacaine alone ( in terms of prolonged analgesia ) will be evident in at least 25% of the patients , after reviewing related articles and our experience . considering an absolute improvement in the primary outcome by 40% ( by the addition of dexamethasone ) in the study group can be considered as clinically relevant and based on a type i error level of 0.05 , type ii error level of 0.2 , and a two - sided test , we needed 21 patients in each treatment groups . therefore , to account for probable drop outs , a number of 25 patients in each group was proposed . surgical procedures mainly consisted of meniscectomy and ligament repair . patients having history of cardiovascular , cerebrovascular , and respiratory diseases , pregnancy , receiving chronic pain treatment , diabetes , and acid peptic disease were excluded from the study . on preoperative rounds , patients were explained regarding the procedure and were also taught to interpret the visual analogue scale ( vas ) ( graded from 0 = no pain to 10 = maximum pain ) . ranitidine was repeated on the day of surgery 2 h before induction with sips of water . on the operation table , routine monitoring ( electrocardiography , pulse oximetry , noninvasive blood pressure ) was started and baseline vital parameters like heart rate ( hr ) , blood pressure ( systolic , diastolic , and mean ) , and arterial oxygen saturation ( spo2 ) were recorded . after preoxygenation for 3 min , induction of anesthesia was done by fentanyl 2 g / kg and propofol 2 mg / kg intravenously ( i.v . ) . trachea was intubated with appropriate size endotracheal tube after muscle relaxation with vecuronium bromide in a dose of 0.08 mg / kg i.v . anesthesia was maintained with 33% oxygen in nitrous oxide and isoflurane was adjusted to maintain a minimum alveolar concentration ( mac ) of 0.7 . hr and mean arterial pressure were maintained within 20% of baseline value by giving additional bolus doss of fentanyl 25 g and propofol 10 mg i.v . patients were randomly allocated using a computer - generated randomization list into two groups ( n = 25 ) . prefilled syringes were prepared with either levobupivacaine and saline or levobupivacaine and dexamethasone and kept in number coded sealed envelopes , made sterile by sterrad sterilization machine . the anesthesiologist and surgeon were unaware of the nature of the drug in each syringe . at the end of the surgical procedure , intraarticular solutions were injected into the knee joint through the cannular sheath after withdrawal of camera by the orthopedic surgeon before the arthroscope was removed . in group l , 18 ml of 0.25% levobupivacaine and 2ml isotonic saline [ total volume 20 ml ] was administered into the knee joint . similarly group d patients received 8 mg dexamethasone ( 2 ml ) added to 18 ml 0.25% levobupivacaine ( again making a volume of 20 ml ) . at the end of the operation , residual neuromuscular paralysis was reversed using intravenous glycopyrrolate and neostigmine and subsequently extubation was done . all patients were observed postoperatively by resident doctors who were unaware of the study group . patients were transferred to postanesthesia care unit and intensity of pain and vital parameters were assessed after 30 min and then an hourly interval for 24 h. diclofenac sodium ( 75 mg ) was administered i.v . as analgesic supplement if the recorded vas pain score was 4 or more and was repeated every 8 h , if required . was used as a rescue analgesic , if the patients continued to have pain after diclofenac administration . the time to the first analgesic requirement and the total diclofenac consumption during first 24 h after operation were also recorded . the primary outcome variable in the study was the duration of analgesia following the surgery and the secondary outcome variables were total diclofenac consumption and the requirement of rescue analgesic between the study groups . student 's t - test was employed to calculate the statistical differences in continuous variables between the groups , categorical variables were compared with chi - square test(or fisher 's exact test ; as applicable ) . a ssps ; version 16.0 ( spss , chicago , il , usa ) was used for analysis . the primary outcome variable in the study was the duration of analgesia following the surgery and the secondary outcome variables were total diclofenac consumption and the requirement of rescue analgesic between the study groups . student 's t - test was employed to calculate the statistical differences in continuous variables between the groups , categorical variables were compared with chi - square test(or fisher 's exact test ; as applicable ) . a ssps ; version 16.0 ( spss , chicago , il , usa ) was used for analysis . the two groups were comparable with regard to age , sex , body weight , and duration of surgery [ table 1 ] . the groups were also comparable regarding fentanyl and propofol consumption during intraoperative period ( p > 0.05 ) [ table 2 ] . intensity of pain was significantly less in group d compared with group l upto 9 h following surgery . however , from 10 h , intensity of pain was comparable in both groups [ table 3 , figure 1 ] . patientscharacteristics ( mean standard deviation ) fentanyl and propofol consumption in the intraoperative period ( mean standard deviation ) intensity of pain ( visual analogue scale ) in postoperative period intensity of pain in terms of visual analogue scale in postoperative period in patients belonging to group l and group d. * indicates p < 0.05 ; * * indicates p < 0.01 the mean duration of analgesia ( delay between the intra - articular injection and the first postoperative analgesic demand ) was longer in group d compared with group l ( 10.24 2.8 h vs. 5.48 1.6 h ; mean sd ; p < 0.01 ) [ table 4 ] . total diclofenac consumption in first 24 h was significantly less in group d compared with group l ( p < 0.01 ) [ table 4 ] . duration of analgesia and diclofenac consumption in the postoperative period ( mean standard deviation ) in an attempt to improve the recovery and early rehabilitation after arthroscopic knee surgery , research has been directed toward developing newer techniques for postoperative analgesia . dexamethasone a 9-derivative synthetic glucocorticoid was selected because of its highly potent anti - inflammatory property with minimal mineralocorticoid activity , thus found to be safer and devoid of potential side effects . local anesthetic agents can provide analgesia for limited period of time when used as single injection . as steroids block the transmission of impulse in nociceptive c fibers , we were interested in determining whether dexamethasone might prolong the duration of analgesia when administered intra - articularly along with local anesthetic agents . few preliminary studies reported that steroids significantly prolong the duration of analgesia in extremity nerve blocks . dexamethasone has been reported to result in significant prolongation of supraclavicular brachial plexus block when added as an adjuvant to local anesthetic bupivacaine . we assumed the similar probable mechanism regarding dexamethasone and levobupivacaine combination as bupivacaine and levobupivacaine were comparable regarding quality and duration of blockade when used in three - in - one blocks in a study by urbanek et al . the dense and prolonged block in the dexamethasone group is due to the synergistic action with local anesthetic levobupivacaine on blockade of nerve fibers . the block prolonging effect of dexamethasone is due to its local action , not a systemic one . it has been found that this effect of steroid is mediated via steroid receptors . when steroids alone were used in regional blocks , the blockade is not produced . steroids might bring about this effect by altering the function of potassium channels in excitable cells . the delay between intr - aarticular injection of levobupivacaine with dexamethasone and supplementary analgesic administration was 10.24 2.8 h in our study . we could not find studies using dexamethasone with levobupivaciaine intraarticularly for prolongation of postoperative analgesia after arthroscopic knee surgery . in a study performed by paul et al . , it was found that time period for first analgesic request for intra - articular bupivacaine and magnesium sulphate combination was 12.32 2.8 h , intra - articular bupivacaine and clonidine was 10.16 2.4 h. in another study by paul et al . , time for first analgesic request for intra - articular dexmedetomidine in combination with ropivacaine was 10.84 2.6 h. it seems that dexamethasone administered as adjuvant to levobupivacaine was able to provide analgesia which was comparable to other intra - articular agents used in previous similar studies . dexamethasone administered intra - articularly as an adjuvant to local anesthetic levobupivacaine improves the quality and duration of postoperative analgesia and reduces the consumption of diclofenac sodium in patients undergoing elective knee arthroscopy . disruptions in connectivity across distributed neural networks are common features of stress - related neuropsychiatric conditions , and understanding how they arise may yield new insights into mechanisms of resilience and vulnerability . stress has potent effects on apical dendrites and postsynaptic dendritic spines in multiple brain regions . in the hippocampus , which plays an important negative feedback role in hpa axis regulation , chronic stress causes atrophy of apical dendrites in ca1 and ca3 pyramidal cells and a decrease in the density of postsynaptic dendritic spines ( jacobson and sapolsky , 1991 , magarios and mcewen , 1995a , magarios and mcewen , 1995b , magarios et al . , 1996 , , 1997 , sousa et al . , 2000 , vyas et al . , other studies have identified associated behavioral deficits in spatial learning and memory tasks such as the radial arm and y mazes ( luine et al . , 1994 , conrad et al . , 1996 , in contrast , in the amygdala , which up - regulates hpa axis activity , chronic stress causes hypertrophy of dendritic arbors , accompanied by a facilitation of aversive learning and heightened fear and anxiety ( vyas et al . importantly , analogous effects have been observed in parallel rodent and human neuroimaging studies of the prefrontal cortex ( fig . 1 ) . many of these studies have focused on the dorsolateral prefrontal cortex in humans , and the medial prefrontal cortex in rodents , as these regions share important functional and neuroanatomical similarities ( ongur and price , 2000 , dalley et al . , 2004 ) , although it should be noted that rodents do have a dorsal prefrontal cortex , which may contribute to associated cognitive functions ( lai et al . , 2012 ) . in rats , pyramidal cells in layer ii / iii of the medial pfc show a pattern of structural changes similar to what has been observed in the hippocampus : retraction of apical dendritic branches and reduced spine density after repeated stress exposure ( cook and wellman , 2004 , radley et al . these changes were associated with specific deficits in an extradimensional attentional set shifting task that correlated with individual differences in the degree of dendritic atrophy ( liston et al . , 2006 ) . in another study , chronic stress caused deficits in spatial working memory that correlated with spine loss on the apical dendrites of prelimbic pyramidal cells ( hains et al . , 2009 ) . the apical dendrites of layer ii / iii pyramidal cells are important recipients of long - range corticocortical projections , so apical dendritic atrophy would be expected to impair functional connectivity across neuroanatomically distributed brain networks ( dehaene et al . , 1998 ) . this is exactly what was observed in a related functional neuroimaging study ( liston et al . , 2009 ) . here , chronically stressed but otherwise healthy human subjects were tested on an attention shifting task during fmri scanning . they exhibited deficits in fmri measures of functional connectivity between dorsolateral prefrontal cortex and a frontoparietal attention network that were correlated with stress levels and attention shifting impairments . similar effects were also observed in the medial prefrontal cortex in another human neuroimaging study , in which stressful life events were associated with decreased gray matter volume in the medial prefrontal , anterior cingulate , and subgenual cingulate cortex ( ansell et al . , 2012 ) . thus , chronic stress has been linked to deficits in structural and functional connectivity measures and associated attentional impairments in both rodent models and human neuroimaging studies . these studies also indicate that connectivity in cortical networks is highly plastic and is often capable of recovering after a change in stress exposure . in rats , four weeks after cessation of the stressor , spine densities fully recovered to unstressed levels ( radley et al . , 2005 ) . similarly , when the same human subjects were re - scanned after a month of rest and reduced stress , both functional connectivity deficits and attention shifting impairments normalized and were no different from unstressed control subjects ( liston et al . , the reversibility of these stress effects underscores the striking capacity for resilience that is evident in the healthy brain . while the healthy human brain demonstrates a remarkable capacity for adaptation and recovery from stressors in daily life , patients with neuropsychiatric disorders often do not . in a recent clinical neuroimaging study , we found that patients with depression exhibited a similar pattern of functional connectivity deficits between dorsolateral prefrontal cortex and a frontoparietal control network that may contribute to rumination , executive control deficits , and other cognitive symptoms ( liston et al . however , in contrast to the study of chronically stressed but otherwise healthy subjects , when depressed patients were re - scanned after five weeks of antidepressant treatment , frontoparietal connectivity deficits persisted ( liston et al . , these results are consistent with the hypothesis that stress - associated changes in connectivity in large - scale brain networks are an important feature of depression and other stress - related neuropsychiatric disorders , and that resilience and vulnerability may be determined in part by individual differences in the capacity for plasticity within these circuits . understanding the mechanisms by which stress alters connectivity in vulnerable circuits may reveal new avenues for treatment . undoubtedly , many factors are involved , and some of them have been reviewed elsewhere ( de kloet et al . , 2005b , arnsten , 2009 , jols and baram , 2009 , chen et al . , we focus on a factor that has received relatively little attention , namely , endogenous glucocorticoid oscillations and their role in regulating synaptic plasticity . glucocorticoids are hormones that are released from the adrenal gland in response to signals originating in the pituitary and hypothalamus , which receives projections from distinct circuits for detecting physiological and psychosocial stressors ( herman and cullinan , 1997 , ulrich - lai and herman , 2009 ) ( fig . , glucocorticoids serve to mobilize energy resources and facilitate sympathetic nervous system responses to maintain homeostasis and adapt to stress . in the long term , however , prolonged exposure to glucocorticoids in chronic stress states can have maladaptive effects , mediated in part by disruptions in negative feedback mechanisms ( mcewen , 1998 , mcewen , 2003 ) . glucocorticoid activity also oscillates with diurnal activity rhythms , independent of external stressors ( fig . 2b ) : glucocorticoid secretion tends to peak in the early morning in diurnal animals ( early evening in nocturnal animals ) , remains relatively elevated for most of the active period of the animal 's day , and becomes relatively suppressed for most of the night . in addition , recent reports ( stavreva et al . , 2009 , lightman and conway - campbell , 2010 ) have shown that an ultradian oscillation with a period of 12 h is superimposed on this circadian rhythm and has equally important consequences for glucocorticoid signaling ( reviewed below ) . in previous fixed tissue studies , stress and glucocorticoid effects on dendritic 2006 ) , which would imply that glucocorticoid oscillations occurring on a timescale of minutes to hours were unlikely to play a direct role in these changes . however , recent studies indicate that glucocorticoids and related signaling molecules can have much more rapid effects on dendritic spines than were previously suspected . for example , in hippocampal organotypic cultures , corticotropin - releasing hormone ( crh)a peptide hormone neurotransmitter and upstream regulator of glucocorticoid synthesis rapidly increased the retraction and elimination of apical dendritic spines after just 30 min of exposure ( chen et al . , 2008 ) , providing one potential mechanism for stress - induced deficits in memory recall ( chen et al . , 2010 ) . similarly , using transcranial two - photon microscopy to image the dynamic remodeling of postsynaptic dendritic spines in the living , developing cortex ( liston and gan , 2011 ) , we found that glucocorticoids have rapid effects on both spine formation and elimination within hours of exposure . surprisingly , low - dose dexamethasone ( 0.1 mg / kg ) , a synthetic glucocorticoid that inhibits endogenous corticosteroid synthesis without penetrating the blood / brain barrier ( karssen et al . , 2005 ) , effectively prevented developmental spine formation and pruning . it is important to note that studies in neuronal cultures and in the developing cortex are investigating spine remodeling under conditions of heightened plasticity , so additional work will be needed to understand how the results apply to the adult brain . however , these experiments indicate that glucocorticoids play an unexpected , necessary role in facilitating physiological spine maturation in the developing adolescent brain , acting on timescale of minutes to hours to facilitate spine remodeling . these unexpectedly rapid effects also suggest that circadian glucocorticoid oscillations may contribute to synaptic plasticity during learning and development . to test this hypothesis , we conducted a series of two - photon imaging studies in mice before and after training on a rotarod motor skill - learning paradigm , and found that circadian glucocorticoid peaks and troughs play critical , complementary roles in facilitating experience - dependent spine remodeling ( fig . 2c specifically , circadian glucocorticoid peaks enhanced spine formation rapidly in the hours after learning , acting through a glucocorticoid receptor - dependent , non - transcriptional mechanism . in accord with prior reports ( yang et al . , 2009 ) , training increased formation rates but only if it occurred during the circadian peak . in mice that were trained during the circadian trough , spine formation rates were equivalent to those of untrained mice , and memory retention was reduced one week later . furthermore , circadian troughs were necessary for stabilizing a subset of learning - related spines and pruning a corresponding set of pre - existing synapses . memory retention and the long - term survival of learning - related spines required intact circadian troughs in the days after learning , which enhanced learning - related spine pruning through a distinct , mineralocorticoid receptor - dependent , transcriptional mechanism . in this way , circadian glucocorticoid oscillations were critical for maintaining homeostasis in synaptic density , by balancing formation and pruning after learning to maintain relatively stable synaptic densities despite repeated bouts of learning - related remodeling . chronic stress states disrupt this balance . in the same rotarod motor skill - learning study ( liston et al . , 2013 ) , prolonged glucocorticoid exposure an important feature of chronic stress states disrupted circadian troughs , reducing the survival of newly formed spines while simultaneously increasing the elimination of pre - existing synapses . together , these two effects led to widespread spine loss and reduced spine densities , in striking contrast to the tight coupling between formation and pruning rates that was observed across all other experimental conditions in the study . related effects were observed on spine maturation across adolescence ( liston and gan , 2011 ) , and in a mouse model of chronic circadian rhythm disruption ( karatsoreos et al . notably , disrupted oscillations in chronic stress states have complex effects on synaptic remodeling that are modulated by the developmental trajectories of synapse formation ( fig . whereas transient glucocorticoid activity increases the pruning mostly of young , recently formed spines , prolonged glucocorticoid exposure disrupts circadian troughs , eliminating synapses that formed progressively earlier in development ( liston and gan , 2011 , liston et al . , 2013 ) . this finding may inform efforts to understand how stress effects interact with synaptic development across the lifespan of an organism . stress has varying effects on brain function , behavior , and psychiatric risk that depend on when during development the stressor occurs ( lupien et al . , this dependence may relate to the varying trajectories of synaptic development across different brain regions ( lupien et al . , 2009 ) . for example , during infancy and early childhood , the hippocampus is developing rapidly and may be particularly vulnerable to early - life stress , whereas protracted development in the prefrontal cortex during the transition from adolescence to early adulthood may increase its vulnerability during this period ( lupien et al . , 2009 ) . in accord with this hypothesis , a variety of early - life stressors can induce long - lasting changes in hippocampal corticosteroid receptor expression and hpa reactivity , heightened anxiety , and hippocampus - dependent memory deficits that persist into adulthood ( barbazanges et al . , 1996 , valle et al . , 1999 , lemaire et al . , 2000 , tsoory et al . , 2007 , eiland and romeo , 2012 , lui et al . , 2012 , importantly , glucocorticoid activity oscillates not only with the circadian cycle of day and night , but also on a much faster time scale with a period of 12 h ( stavreva et al . , 2009 , lightman and conway - campbell , 2010 ) . these ultradian oscillations , which are superimposed on the slower circadian cycle ( fig . recent studies have shown that ultradian glucocorticoid rhythms generate cycles of gr - mediated gene pulsing , which regulate gene expression on faster timescales than was previously suspected ( stavreva et al . , 2009 ) . facilitated by the rapid , chaperone - mediated recycling of nuclear grs , ultradian gene pulses trigger changes in gr - regulated promoter activity that are tightly coupled to physiological oscillations ( stavreva et al . , 2009 ) . ultradian glucocorticoid oscillations penetrate the blood / brain barrier and are preserved within stress - sensitive brain areas ( droste et al . , 2008 ) , where they probably play an important role in responding to stressors and other environmental stimuli in physiological circumstances . conversely , in chronic stress models , disruptions of the ultradian oscillation alter gene expression responses in these regions and cause correlated changes in locomotor activity and risk assessment behaviors ( sarabdjitsingh et al . , 2010a , sarabdjitsingh et al . , 2010b ) . whether and how these ultradian oscillations affect synaptic remodeling remains unclear , but they are likely to have important effects , acting potentially through both transcriptional and non - transcriptional mechanisms ( mcewen , 1991 , makara and haller , 2001 , lsel and wehling , 2003 , groeneweg et al . , 2011 ) . as mentioned above , glucocorticoids can increase spine formation in cortical pyramidal cells by ten - fold in just 20 min , acting through non - genomic signaling pathways ( liston et al . similarly , glucocorticoids can rapidly enhance the frequency of miniature excitatory postsynaptic potentials , increasing glutamate release probability by activating a non - genomic , mr - dependent signaling pathway ( karst et al . , 2005 ) . similarly rapid effects have been observed in other studies in the prefrontal cortex , hippocampus , amygdala , and hypothalamus ( di et al . , 2003 , , 2011 , popoli et al . , 2011 , tasker and herman , 2011 ) . the studies reviewed above indicate that stress and glucocorticoids have potent but complex effects on synaptic remodeling , and understanding the underlying molecular mechanisms is a rapidly emerging area of active investigation . these studies are challenging due in part to the fact that stress effects on dendritic remodeling , synaptic plasticity , and associated molecular signaling mechanisms vary with the region and developmental age under investigation ( lupien et al . , 2009 ) . however , one theme to emerge from this work is that glucocorticoids may engage distinct intracellular signaling mechanisms , depending on the timing of a stressor and the kinetics of the glucocorticoid response . for example , in response to an acute stressor , glucocorticoids promote memory consolidation and impair working memory ( mcgaugh and roozendaal , 2002 , barsegyan et al . , 2010 ) through a mechanism involving beta adrenergic- and camp - dependent activation of protein kinase a in the amygdala and prefrontal cortex ( roozendaal et al . , 2002 , barsegyan et al . , acutely , glucocorticoids also promote long - term memory retention by enhancing learning - dependent spine formation , acting through a glucocorticoid receptor - dependent non - genomic signaling mechanism that increases lim kinase and cofilin phosphorylation ( liston et al . , 2013 ) , which stabilizes actin polymers and promotes spine growth ( gu et al . , 2010 recent reviews underscore the point that acute glucocorticoid exposure modulates multiple additional molecular processes that are relevant in this context : acutely , glucocorticoids potentiate glutamate transmission by increasing presynaptic glutamate release and enhancing ampa and nmda receptor trafficking to postsynaptic membranes ; they activate mapk and camkii signaling pathways that have been linked to transcription - dependent mechanisms for memory consolidation ; and they enhance endocannabinoid signaling , which in turns modulates the release of glutamate and other neurotransmitters ( arnsten , 2009 , campolongo et al . , 2009 , in contrast , chronic glucocorticoid exposure engages a variety of molecular signaling mechanisms that are distinct from those engaged by an acute stressor . for example , chronic glucocorticoid exposure has effects on glutamate receptor expression that oppose those induced by an acute stressor , reducing the expression of the nmda receptor subunit nr2b and the ampa receptor subunits glur2/3 in the prefrontal cortex ( gourley et al . , 2009 ) . chronic stress effects on dendritic atrophy in the hippocampus and prefrontal cortex have also been linked to excessive protein kinase c signaling ( hains et al . , 2009 ) and reduced expression of neural cell adhesion molecules ( ncam-140 ) ( sandi , 2004 ) . and chronic glucocorticoid exposure suppresses bdnf transcription in the orbitofrontal cortex ( gourley et al . , 2009 ) and reduces trkb and erk1/2 signaling in the hippocampus ( gourley et al . , 2008 ) . although studies indicate that reduced activity - dependent bdnf secretion probably does not by itself cause spine loss or dendritic atrophy ( hill et al . , 2005 , magarinos et al . , 2011 ) , it is likely that altered bdnf signaling plays a role through interactions with other factors . stress especially chronic , uncontrollable stress is an important risk factor for depression , ptsd , and other anxiety disorders , and stress effects on glucocorticoid oscillations may contribute to this effect . stress has varying effects on hpa axis activity and glucocorticoid secretion that depend on the timing and nature of the stressor ; on the individual 's subjective perception of the situation ; and likely also on his genetic predisposition to developing stress - related psychiatric conditions ( miller et al . , 2007 ) . in a recent meta - analysis of 8521 subjects across 107 independent studies , the most consistent findings were that chronic stress increases the total daily output of cortisol ( the principal glucocorticoid in humans ) , flattens the diurnal rhythm , and reduces the amplitude of the circadian peak ( miller et al . , 2007 ) . . the disruption may be particularly significant during the circadian trough , as unopposed mr activity typically occurs only during the trough when glucocorticoid secretion is low ( reul and de kloet , 1985 , de kloet et al . , 1998b ) , and a relatively small increase in trough levels could have pronounced effects on glucocorticoid signaling . in conjunction with the studies cited above , these results suggest that chronic stress may predispose vulnerable individuals to a variety of neuropsychiatric disorders by disrupting the circadian oscillation and especially the circadian trough , reducing the survival of newly formed synapses , and destabilizing synapses formed early in development . converging evidence from both clinical studies and animal models lend support to this hypothesis . disrupted circadian glucocorticoid cycling is a relatively consistent feature in clinical studies of patients with depression or ptsd ( heim et al . , 2000 , holsboer , 2000 , yehuda , 2002 , miller et al . , 2007 ) . blunted circadian cortisol oscillations are a feature common to both ptsd and depression ( yehuda et al . . however , these two disorders appear to involve opposing changes in total cortisol secretion ( decreased in ptsd , variably increased in depression ) : in ptsd , blunted oscillations are driven primarily by reduced circadian peaks ( yehuda et al . , 1996 ) , while in depression , they are driven primarily by elevated cortisol secretion during the circadian trough ( yehuda et al . , 1996 ) , especially in psychotic depression ( sachar et al . , 1973 , keller et al . , 2006 ) . in both disorders , blunted corticol cycling is associated with hippocampal volume loss ( bremner et al . 1996 ) and partially overlapping alterations in functional connectivity ( davidson et al . , 2002 , , 2004 , greicius et al . , 2007 , sheline et al . , 2010 , yin et al . , 2011 , 2014 ) , which is consistent with results in animal models indicating that both peaks and troughs are necessary for balancing synaptic formation and pruning . multiple animal models of depression including chronic unpredictable stress , chronic social defeat stress , and early life stress recapitulate neuroendocrine abnormalities found in patients , including blunted glucocorticoid oscillations , elevated glucocorticoid activity , and disrupted circadian troughs ( willner , 1997 , meaney , 2001 , krishnan et al . , 2007 , nestler and hyman , 2010 ) . in at least one study , blunted circadian cycling was linked specifically to stress susceptibility : circadian rhythm amplitudes were blunted only in mice that exhibited a vulnerable behavioral phenotype in response to chronic social defeat stress , relative to resilient mice that did not develop depression - like symptoms ( krishnan et al . , 2007 ) . in other studies , circadian rhythm disturbances have been causally related to mood symptoms . in a genetic model , mice carrying a mutation in the clock gene showed a loss of circadian rhythms in locomotor activity and a host of mania - like behavioral symptoms ( roybal et al . , 2007 ) . and in an environmentally induced model of circadian rhythm disruption , mice that were housed on a shortened 20-h light dark cycle exhibited learning and structural connectivity deficits comparable to those seen in chronic stress states , including apical dendritic atrophy in mpfc pyramidal cells and pfc - dependent cognitive deficits ( karatsoreos et al . , dark cycle also developed metabolic problems , including obesity , increased leptin levels , and signs of insulin resistance . shift workers and frequent travelers who suffer from chronic jet lag may experience analogous cognitive and metabolic changes ( sack et al . , 2007 , lupien et al . , 2009 , mcewen , 2012 ) , and in susceptible individuals , travel across time zones may even trigger severe mood episodes requiring psychiatric hospitalization ( jauhar and weller , 1982 ) . an increasing awareness of the importance of circadian and ultradian glucocorticoid oscillations in learning - related synaptic remodeling may also have implications for efforts to optimize training regimens for promoting motor skill learning , which is known to vary with the time of day in both adolescents and adults ( atkinson and reilly , 1996 , miller et al . similarly , disruptions in circadian glucocorticoid oscillations may be an important factor to consider in patients undergoing treatment with corticosteroids , which are frequently used in the management of a variety of common autoimmune disorders . cognitive complaints and mood symptoms are extremely common but poorly understood side effects of treatment ( brown and suppes , 1998 , otte et al . , 2007 , cornelisse et al . , 2011 ) , which could potentially be mitigated by designing treatment regimens to preserve naturally occurring oscillations whenever possible . converging evidence from animal models and human neuroimaging studies indicates that stress - associated functional connectivity changes are a common feature of depression , ptsd , and other neuropsychiatric conditions and are associated with correlated structural changes in the prefrontal cortex , hippocampus , and other vulnerable brain regions . these , in turn , may be caused in part by circadian disturbances in glucocorticoid activity . circadian glucocorticoid peaks and troughs are critical for generating and stabilizing new synapses after learning and pruning a corresponding subset of pre - existing synapses . chronic stress disrupts this balance , interfering with glucocorticoid signaling during the circadian trough and leading to widespread synapse loss , dendritic remodeling , and behavioral consequences . these effects interact with synaptic development trajectories that vary across brain regions and may be particularly pronounced during the transition from adolescence to young adulthood , when major neuropsychiatric conditions most commonly emerge . together these findings provide a model for understanding how stress effects on circadian glucocorticoid oscillations may contribute to connectivity changes and ultimately to the pathophysiology of depression , ptsd , and other disorders . still , many questions remain , and we conclude by considering a few of them . perhaps most importantly , many of these links remain purely correlative , and it will be critical to test whether and how changes in synaptic remodeling directly affect the function of cortical microcircuits , the integration of information across neuroanatomically distributed networks , and the emergence of behavioral effects and psychiatric symptoms . to this end , the recent development of optogenetic tools for manipulating activity in specific neural circuits will be critical for establishing causal mechanisms ( yizhar et al . , 2011 , tye and deisseroth , 2012 , berndt et al . , 2014 ) . likewise , recently developed imaging modalities provide a means for testing how structural changes within a given microcircuit affect functional circuit dynamics another critical , unanswered question . these methods use genetically encoded calcium indicators ( tian et al . , 2009 ) to quantify neuronal activity with single - cell precision in the living organism . in combination with implantable optical devices ( flusberg et al . , 2008 , , 2009 , chia and levene , 2009 , andermann et al . , 2013 ) , these tools will extend the reach of conventional two - photon microscopy to enable in vivo imaging in the hippocampus , amygdala , medial prefrontal cortex , and other stress - sensitive limbic circuits , which tend to lie deep below the cortical surface . finally , in vivo imaging tools may also prove useful for investigating the role of ultradian oscillations , which are superimposed on the circadian glucocorticoid rhythm . what is clear is that these oscillations trigger pulses of gene expression every 12 h ( stavreva et al . , 2009 ) , and that glucocorticoids are capable of regulating synapse function and facilitating synapse formation on a comparably rapid timescale ( popoli et al . , 2011 , together , these emerging technologies will enable investigators to ask fundamentally new questions about the links between circadian rhythm disruptions , structural measures of synaptic remodeling , and their functional consequences .

background and aims : in an attempt to improve the recovery and early rehabilitation after arthroscopic knee surgery , various medications have been administered via intra - articular route to prolong the duration and improve the quality of postoperative analgesia . among the potentially effective substances , steroids like dexamethasone could be of particular interest.materials and methods : fifty patients undergoing elective knee arthroscopy were randomly assigned to one of the following groups containing 25 patients each . group d patients received 8 mg ( 2 ml ) of dexamethasone added to 18 ml of 0.25% levobupivacaine intra - articularly , ( total volume 20 ml ) . group l patients received 18 ml of 0.25% levobupivacaine and 2 ml of isotonic saline ( 20 ml in total ) intra - articularly . analgesic effect was evaluated by measuring pain intensity visual analogue scale score and duration of analgesia.results:a longer delay was observed between intra - articular injection of study medication and first requirement of supplementary analgesic in group d ( 10.24 2.8 hours ) compared with group l ( 5.48 1.6 h ) . total consumption of diclofenac sodium in first 24 h in postoperative period was significantly less in group d. no significant side effects were noted.conclusion:dexamethasone , used as adjunct to levobupivacaine in patients undergoing arthroscopic knee surgery , improves the quality and prolongs the duration of postoperative analgesia . stress especially chronic , uncontrollable stress is an important risk factor for many neuropsychiatric disorders . the underlying mechanisms are complex and multifactorial , but they involve correlated changes in structural and functional measures of neuronal connectivity within cortical microcircuits and across neuroanatomically distributed brain networks . here , we review evidence from animal models and human neuroimaging studies implicating stress - associated changes in functional connectivity in the pathogenesis of ptsd , depression , and other neuropsychiatric conditions . changes in fmri measures of corticocortical connectivity across distributed networks may be caused by specific structural alterations that have been observed in the prefrontal cortex , hippocampus , and other vulnerable brain regions . these effects are mediated in part by glucocorticoids , which are released from the adrenal gland in response to a stressor and also oscillate in synchrony with diurnal rhythms . recent work indicates that circadian glucocorticoid oscillations act to balance synapse formation and pruning after learning and during development , and chronic stress disrupts this balance . we conclude by considering how disrupted glucocorticoid oscillations may contribute to the pathophysiology of depression and ptsd in vulnerable individuals , and how circadian rhythm disturbances may affect non - psychiatric populations , including frequent travelers , shift workers , and patients undergoing treatment for autoimmune disorders .

the epidermal growth factor receptor ( egfr ) is over expressed in various solid malignancies including non small cell lung cancer ( nsclc ) . however , they are associated with a dermatologic side effects , which can occasionally be responsible for discontinuation of the egfr inhibitors . hence , we report a case of metastatic adenocarcinoma of lung who developed skin ulceration with gefitinib and responded to interruption of the drug and early intervention . the present case report is about a 50-year - old female patient who had been diagnosed as having lung adenocarcinoma with multiple bone metastases was initiated on gefitinib therapy at an oral dose of 250 mg / d . after 2 weeks of initiating therapy , the patient presented with ulcer over the palm [ figure 1 ] . the ulcers improved with stopping gefitinib for 2 weeks and also with the addition of topical steroids and antibiotics . non - small - cell lung cancer ( nsclc ) with sensitive mutations of the egfr is highly responsive to gefitinib . gefitinib is a small molecule tyrosine kinase inhibitor ( tki ) of egfr . since 2004 , it was clear that a substantial proportion of nsclc obtaining objective response when treated with gefitinib harboring activating mutations in the egfr gene . the occurrence of skin disorders ( dry skin and acneiform rash ) is explained by the fact that egfr is also expressed in the basal layer of the skin ; inhibition of the receptor will disturb normal biology and result in skin rash . skin rash is notorious as an adverse event of egfr - tki and is noted in up to two - thirds of patients receiving any of these agents although severe in only 5 - 10% who can develop pyogenic granuloma like lesions . very rarely the cutaneous inflammation is so pronounced that skin necrosis with black eschar formation and ulceration is seen . the cutaneous side - effects are treated with topical steroids and antibiotics with interruption of treatment for 2 - 4 weeks as in our case . to report a case of acute quinine poisoning , document acute and chronic macular changes with optical coherence tomography imaging and fluorescein angiography ( fa ) , and to review the literature on ocular toxicity of quinine . a 32-year - old white female presented to our emergency department after ingesting over 7.5 g of quinine . she underwent a complete ophthalmologic examination , fluorescein angiography , stratus time - domain optical coherence tomography ( oct ) , and electroretinography at 72 hours and 15 months postingestion . stratus time - domain and cirrus spectral - domain oct , fundus autofluorescence , and fa were obtained at 28 months postingestion . oct showed marked thickening of the inner retina bilaterally . at 15 and 28 months follow - up , fundus photography and fluorescein angiography demonstrated optic nerve pallor , severely attenuated retinal vessels while oct showed inner retinal atrophy . quinine toxicity as seen by oct reveals increased thickness with inner retinal hyperreflectivity acutely with development of significant retinal atrophy in the long - term . these findings suggest that quinine poisoning may produce a direct toxic effect on the inner retina in the acute phase resulting in long - term retinal atrophy . acute quinine poisoning is a devastating ocular event that can result in severe loss of vision.1 quinine is an antimalarial agent that is often used for the treatment of restless legs syndrome . the drug has a narrow therapeutic range and carries the risk of multiple side effects such as cinchonism , hypoglycemia , arrhythmias , hemolysis , gastrointestinal intolerance , fever , and nephritis.2 in early 2007 , the us food and drug administration restricted the use of quinine solely to the treatment of malaria . quinine has long been known to be toxic to the eye even at therapeutic levels , causing symptoms such as loss of vision , reduced color vision , and reduced visual fields.3,4 most visual symptoms occur after a quinine dose greater than 4 g and death can occur with doses exceeding 8 g.5 we describe a patient with profound visual loss after acute quinine poisoning . at 72 hours postingestion , erg , stratus time - domain optical coherence tomography ( oct ) , fundus photography , and fluorescein angiography ( fa ) were performed on the patient . at 15 months postingestion , time - domain oct and fa were again performed on our patient . at the 28 months postingestion visit , cirrus spectral - domain and stratus time - domain oct , fa , and fundus autofluorescence ( faf ) were performed . to the best of our knowledge , this is the first documentation in the literature of the retinal changes in acute quinine toxicity as seen by oct and faf . a 32-year - old white female presented to our emergency department five hours after attempting suicide by quinine overdose . the dose was estimated to be between 7.5 and 10 g. on initial examination the patient was complaining of tinnitus , nausea , vomiting , as well as total loss of vision . serum sodium and potassium were low at 133 meq / l and 3.6 meq / l , respectively , and the patient s ecg showed a prolonged qt interval . the patient was admitted to the medical intensive care unit of the ohio state university medical center for monitoring and support . the first ophthalmologic exam was performed at the bedside 23 hours postingestion and showed that the patient had no light perception in both eyes . fundoscopy revealed optic nerves with normal color and crisp margins , retinal vessels with normal course and caliber , and maculas with diffuse retinal thickening and bilateral cherry - red spots ( crs ) . the patient received hyperbaric oxygen therapy per the recommendation of the medical intensive care unit team . at 48 hours postingestion , examination revealed partial resolution of the crs with persistent retinal thickening . at 72 hours postingestion , fluorescein angiography showed normal filling times and no evidence of leakage or vascular staining ( figure 1c , d ) . stratus time - domain oct revealed inner retinal hyperreflectivity with thickening in both eyes ( figure 2a , b ) . central subfield thicknesses were 292 microns od and 293 os and macular volumes for a 6 mm diagonal were 8.43 mm od and 8.75 mm os . at this visit , bright flash erg yielded reduced a - waves and absent b - waves , consistent with previous reports on the electrophysiologic effects of quinine poisoning.6 the patient was subsequently lost to follow - up for several months , as she was repeatedly hospitalized in psychiatric institutions and underwent drug rehabilitation . her first ophthalmologic follow - up examination was performed 15 months postingestion . at this visit , the patient s uncorrected visual acuity ( va ) was 20/20 ou . although zeiss - humphrey 30 - 2 visual fields demonstrated normal foveal thresholds ou ( 33 db od , 35 db os ) , they were constricted to 15 degrees ou . dilated fundus examination revealed optic nerve pallor ou with retinal atrophy and severely attenuated retinal arterioles and venules ( figure 3a , b ) . however , normal arteriovenous transit times and arteriolar perfusion were demonstrated by fluorescein angiography ( figure 3c , d ) . stratus time - domain oct revealed severe atrophy of the neuroretina in both eyes with central subfield thickness being 199 microns od and 218 microns os . macular volumes for a 6 mm diagonal had decreased to 4.68 mm od and 5.06 mm os ( figure 4a , b ) . the patient was last seen at the ophthalmology clinic at 28 months postingestion . at this time , her uncorrected va was 20/20 od and 20/20 os . farnworth d-15 color vision testing demonstrated mild blue - yellow color deficiencies . there were no changes worth noting between the patient s humphrey visual fields or fa between this visit and her visit at 15 months postingestion . cirrus spectral - domain oct revealed central subfield thicknesses that were 283 microns od and 279 microns os and average retinal thicknesses that were 213 microns od and 211 microns os ( normal average retinal thickness is 264 21.9 microns).7 although the central retinal thicknesses on the cirrus oct were within the normal range , the average macular thicknesses were decreased , a finding consistent with retinal atrophy and to the peripheral constriction seen on the visual fields . faf photography ( trc-50dx ; topcon , oakland , nj ) revealed a generalized hyperfluorescent pattern without evidence of atrophic retinal pigment epithelial changes ( figures 5 , 6 ) . the presentation and ophthalmologic clinical findings of this patient appear typical of quinine - induced ocular damage . previous case reports have demonstrated that patients usually develop loss of vision between 10 and 24 hours after ingestion of toxic levels of quinine . most patients experience a return of central acuity with restricted visual fields over the next several weeks to months . vessel attenuation and disk pallor seen at our patient s 15- and 28-month examinations are consistent with the typical late fundus findings of quinine toxicity.5 despite the compound s well - established ocular toxicity , the mechanism behind the damage it mediates is not entirely known . towards this end , the acute oct and fa findings in our patient were enlightening . at 72 hours postingestion , this finding is consistent with oct performed on the ischemic retina during central retinal artery occlusion ( crao).8 however , in our patient the fa in the acute stage demonstrated no abnormalities in arteriovenous transit and retinal perfusion . cirrus spectral - domain oct demonstrates an intact external limiting membrane as well as inner and outer segment of the photoreceptor layer at 28 months . these findings suggest that quinine poisoning produces damage that is confined to the inner retinal layers . the presence of a crs during the acute phase of quinine poisoning in our patient is consistent with previous reports of quinine overdose . crs formation is seen with ischemia of the ganglion cell layer ( gcl ) in crao and in conditions where storage materials or metabolites accumulate in the gcl of the retina such as congenital gangliosidosis ( tay sachs disease and sandhoff s disease ) , sphingolipidosis ( niemann - pick s disease ) , glucocerebrosidosis ( gaucher s disease ) , and with patients on long - term parenteral nutrition.9,10 in addition to quinine , crs has also been observed with toxic interactions following intravitreal aminoglycoside injection , ingestion of dapsone , and ingestion of methanol.1113 in a histopathologic report on primates , conway et al reported that high doses of intravitreal gentamicin led to retinal toxicity with the formation of a crs . the retinal toxicity was primarily confined to the inner retina with relatively intact photoreceptor and retinal pigment epithelial layers . in the acute phase , there was nerve fiber and ganglion cell swelling with subsequent atrophy of these layers in the chronic phase . they postulated that retinal toxicity was primarily due to a direct neurotoxic effect on the inner retina.14 this mechanism appears to be most consistent with the clinical , oct , and faf findings in our patient . the optic nerves do not appear to be affected in the early stages of toxicity as their appearance was unremarkable and there was no papillary leakage or staining on fa at 72 hours . in the latter stages of follow - up the pattern of visual field loss was one of generalized constriction rather than a cecocentral scotoma , a pattern more consistent with diffuse retinal rather than optic nerve toxicity . quinine is a class ia anti - arrhythmic that blocks sodium and potassium channels in cardiac myocytes , causing a delay in the initiation of action potentials and increasing the action potential duration.15 various studies have shown quinine capable of blocking voltage - gated outward potassium channels , stimulating intracellular inositol-1,4,5-triphosphate production , and activating intracellular pathways via guanine nucleotide binding protein ( g - protein)-coupled receptors.1618 these pharmacologic actions of quinine could be responsible for the clinical observations of rapid onset bilateral vision loss and eventual neuronal damage . prolonged depolarization of the cell membrane can lead to depolarization of the mitochondrial membrane and stimulation of mitochondrial - mediated apoptosis.19,20 several proposed mechanisms of direct quinine - retina interaction exist in the literature . aloisi et al postulated that retina damage is a result of free radicals generated by the photoionization of quinine.21 it has long been suspected that quinine blocks acetylcholine transmission in the retina . recent evidence shows quinine to have an antagonistic effect on 910 nicotinic acetylcholine receptors elsewhere in the body.22 acetylcholine blockade can explain the immediate total blindness observed in quinine overdose , but does not explain how quinine leads to eventual cell death . perhaps the most promising research concerning the mechanism of quinine toxicity involves gap junctions and connexins . gap junctions are formed by structural proteins known as connexins and allow for intracellular electrical communication by linking the cytosol of two consecutive cells and are found in gastrointestinal smooth muscle , cardiac muscle , and retinal cells . studies in transfected mammalian cells have shown that quinine has the ability to act as a reversible , concentration - dependent gap junction channel blocker.23 furthermore , quinine has been shown to block specific connexins , including connexin 36 ( cx36 ) , which has been found in mouse and rat retinas.24,25 cx36 gap junctions are responsible for excitatory and inhibitory interneuron communications between retinal ganglion cells and amacrine cells.26 the interaction of quinine with these junctions may help explain the acute blindness seen in quinine toxicity and may also provide greater understanding of the mechanism of quinine s neurotoxic effect . our clinical experience and recent literature support the theory that quinine is directly neurotoxic to the inner retina . more research is needed to identify the exact mechanism by which quinine mediates direct neuronal cell death . interventions geared towards disrupting quinine s toxic neuronal activity would perhaps be able to prevent the devastating loss of vision associated with this agent .

we report a case of gefitinib - induced skin ulceration in a 50-year - old female with metastatic adenocarcinoma of lung who developed this adverse effect 2 weeks following initiation of gefitinib at a dose of 250 mg / day . the ulcer improved with stopping gefitinib for 2 weeks and also addition of topical steroids and antibiotics . we are reporting this case to create awareness among treating oncologists of this adverse effect and also prompt interruption of therapy and topical steroids / antibiotics is useful to treat this adverse event . purposeto report a case of acute quinine poisoning , document acute and chronic macular changes with optical coherence tomography imaging and fluorescein angiography ( fa ) , and to review the literature on ocular toxicity of quinine.methodsa 32-year - old white female presented to our emergency department after ingesting over 7.5 g of quinine . she underwent a complete ophthalmologic examination , fluorescein angiography , stratus time - domain optical coherence tomography ( oct ) , and electroretinography at 72 hours and 15 months postingestion . stratus time - domain and cirrus spectral - domain oct , fundus autofluorescence , and fa were obtained at 28 months postingestion.resultsfluorescein angiography at 72 hours postingestion revealed normal filling times and vasculature . oct showed marked thickening of the inner retina bilaterally . at 15 and 28 months follow - up , fundus photography and fluorescein angiography demonstrated optic nerve pallor , severely attenuated retinal vessels while oct showed inner retinal atrophy . fundus autofluorescence did not reveal any retinal pigmentary abnormalities.conclusionsquinine toxicity as seen by oct reveals increased thickness with inner retinal hyperreflectivity acutely with development of significant retinal atrophy in the long - term . fundus autofluorescence reveals an intact retinal pigment epithelial layer at 28 months . these findings suggest that quinine poisoning may produce a direct toxic effect on the inner retina in the acute phase resulting in long - term retinal atrophy .

developmental dyslexia is a neurobiological disorder characterized by a difficulty in reading acquisition despite adequate intelligence , conventional education , and motivation ( apa 1994 ) . the prevailing view supports the hypothesis that dyslexia results from a specific deficit of auditory - phonological perception , representation , and phonological memory ( see vellutino et al . children and adults with dyslexia show , indeed , deficits in the representation and manipulation of phonological information ( e.g. , poor speech - sound awareness , slow lexical retrieval and poor phonological short - term memory ; see ramus 2003 , for a review ) . these deficits could interfere with one of the most critical skills for successful reading acquisition , such as phonological decoding ( share 1995 ; ziegler and goswami 2005 ) . apart from their phonological difficulties , dyslexic subjects often suffer from a variety of subtle sensory and motor deficits . whether these deficits have any causal relation to the reading disorder , or are totally independent , is currently under debate . one hypothesis of a visual cause for dyslexia is that the reading disorder is caused by a deficiency in the magnocellular part ( also referred to as the transient system ) of the visual system ( stein and walsh 1997 ) . this hypothesis might seem controversial , since one would expect the parvo- rather than the magnocellular pathways to be largely involved with fine pattern vision and object discrimination that are essential for reading . however , studies have indicated a function for the magnocellular - dominated dorsal stream in selective attention ( motter 1993 ; vidyasagar 1998).vidyasagar and pammer ( 1999 ) suggested that if the magnocellular system ( m system ) is involved in gating all visual input going through the striate cortex , a deficit in this system would also affect the parvocellular system ( p system ) . this would become manifest in tasks in which there is intense competition for attentional resources and in which the supposed m - mediated attentional spotlight is essential for good performance . stein ( 2003 ) also suggested that a magnocellular deficiency may cause a type of visual attention deficit in dyslexia . this has made it important to assess visual attention in dyslexic readers in more detail . a recent and particularly interesting hypothesis that links the magnocellular deficit with reading problems is that dyslexics may have sluggish attentional shifting ( sas ) ( hari and renvall 2001 ) . the present study was motivated by the idea that sas could indeed provide a coherent framework to understand a variety of sensory problems that dyslexics encounter . the basic notion underlying sas is that sensory input is chunked and that attention of dyslexic subjects , once engaged on a chunk , can not be easily disengaged ( hari and renvall 2001 ) . this causes impairments in the shift or the focusing of attention ( hari and renvall 2001 ) , and it may result in a prolonged attentional dwell time and poor judgments of temporal order ( jskowski and rusiak 2008 ) . sas can affect fluent reading because the sensory input is prolonged , thereby degrading essential cortical representations ( for review , see hari and renvall 2001 ) . ( 2010 ) used an auditory and visual stream segregation task and reported that in order to process two successive stimuli separately , dyslexic participants with phonological impairments required a significantly longer inter - stimulus interval than controls regardless of sensory modality . another important prediction from sas is that dyslexics may profit from a transient sound because of a general alerting effect that improves the disengagement of attention . a number of methods have been used to measure visual attention in dyslexics , but so far , none has used sounds to improve search time . in the standard visual search task , participants search for a prespecified visual target among other distracters . previous reports are rather consistent with a visual attention deficit in dyslexics , reporting slower search times for dyslexic than normal readers ( williams et al . 1987 ; ruddock 1991 ; casco and prunetti 1996 ; vidyasagar and pammer 1999 ; sireteanu et al . pammer and vidyasagar ( 2005 ) and jones et al . ( 2008 ) argued that dyslexics may suffer from an impairment in the serial allocation of attention . according to sas , the slowness of dyslexics can be explained by an impairment in the disengagement of attention from an element in the search set . here we thought to add sounds to the visual search task , using the pip - and - pop these authors designed a visual search task in which a target ( a horizontal or vertical line ) was embedded in a cluttered display of distracters ( oblique lines ) . the targets and distracters changed , on randomly determined times color ( and , important from the perspective of the magnocellular system , also luminance ) from green - to - red or red - to - green . they found that a simple auditory pip could drastically decrease search times if the pip was synchronized with the color / luminance - change of the target : the pip then made the target pop - out . further studies have shown that a sound will only lead to benefits in visual search if the changes in the two signals are both synchronized and transient ( van der burg et al . if this condition is met , then the effect will also resist wide spatial misalignment ( fiebelkorn et al . , we used this paradigm to examine whether a sound would improve the visual search time of dyslexic readers more than it does in normal readers . if , as proposed in sas , dyslexic readers have problems with the disengagement of attention from the current fixation , one expects them in the tone - absent condition ( serial search ) to have longer search times than normal readers , and their slope of the search time per item should be steeper ( see also vidyasagar and pammer 1999 ; sireteanu et al . 2011 ) . in the sound - present condition , though , the pip can make the target pop - out ( parallel search ) , and search time may become independent of the set size of the distracters . this should be particularly helpful for dyslexic readers , as they may have a specific difficulty with serial , but not parallel , search ( sireteanu et al . ultimately , then , a single pip may compensate for the dyslexics visual attention deficit . fifteen young adults with developmental dyslexia ( five men and ten women ) and 15 age - matched controls without reading difficulties ( five men , ten women ) were tested . the dyslexic readers had been diagnosed with developmental dyslexia based on standard exclusion criteria ( apa 1994 ) . they were all formally assessed and diagnosed by clinical and educational psychologists . their reading achievements ( accuracy and/or speed ) were additionally assessed via standardized dutch reading tests for single word and nonword reading ( brus and voeten 1997 ; van den bos et al . dyslexic participants were selected on the basis of ( 1 ) normal or corrected - to - normal vision and hearing ; ( 2 ) absence of neurological and/or psychiatric disorders ; ( 3 ) absence of attention deficit disorder with hyperactivity ( because of the high comorbidity with dyslexia ) ; ( 4 ) absence of color blindness . the two groups were drawn from the same subject pool of university students , but were significantly different for both accuracy and speed of word and nonword reading ( see table 1 for details ) . participants were tested individually , were unaware of the purpose of the experiment , and received course credits or money for their participation . written consent was obtained from all participants according to the declaration of helsinki.table 1mean and standard deviation ( sd ) of age ( in years ) , word and nonword reading scores ( errors and speed in number of correctly read items ) in dyslexics ( n = 15 ) and age - matched normal reading controls ( n = 15)dyslexicscontrolscomparisonmeansdmeansdt(28)page21.52.220.71.71.10.28words reading errors1.31.10.30.63.090.004 speed77.810.393.516.43.160.004nonword reading errors8.93.02.32.96.73<0.001 speed69.916.298.514.05.17<0.001the bold values are with p < 0.05 mean and standard deviation ( sd ) of age ( in years ) , word and nonword reading scores ( errors and speed in number of correctly read items ) in dyslexics ( n = 15 ) and age - matched normal reading controls ( n = 15 ) the bold values are with p < 0.05 the stimuli were made as in van der burg et al . the auditory stimulus was a short white noise click of 68 ms presented at 74 db(a ) through the laptop speakers . the visual stimuli were presented on a 15-inch , 60-hz laptop monitor ( dell latitude e5500 ) , controlled by e - prime 1.2 ( psychology software tools , inc . ; http://www.pstnet.com/eprime ) . the visual search displays consisted of 24 or 48 red ( 20 cd m ) and green ( 11 cd m ) line segments ( length 0.88 visual angle ) against a dark background ( 0.05 cd m ) . the lines were randomly placed in an invisible 10 10 grid ( 10.5 6.5 ) centered on a white central fixation cross , with the constraint that the target was never presented at the four central positions , to avoid immediate detection . the target was a horizontal or vertical line , while for distracters line orientation deviated randomly by plus or minus 26.5 from horizontal or vertical . the distracters changed color ( from red - to - green or vice versa ) every 50 , 100 , or 150 ms . the number of distracters that changed simultaneously during a trial varied for the different set sizes ; in set size 24 , 1 , 2 or 4 distracters changed simultaneously , while in set size 48 , it was 1 , 4 or 7 distracters . the target changed color every 500 or 1,000 ms , and always changed alone . distracters did not change color from 150 ms before the target until 100 ms after the target had changed color . during the first 500 ms of a trial , the target also did not change color . a dynamic example of trials with and without sound can be found at http://www.psy.vu.nl / pippop/. participants were tested in a dimly lit and sound - proof cabin and were seated approximately 65 cm in front of the laptop screen . a white fixation cross was illuminated in the center of the screen at the beginning of each trial . after 150500 ms , the display with target and distracters appeared at the screen . in the sound - present condition , a change in the color of the target was always accompanied by a simultaneously presented sound . participants were instructed to search for the target and to press one of two buttons corresponding with the target orientation - or all participants were explicitly told that sounds , if present , were synced with a color change of the target , and that they thus could benefit from the sound because it signaled that the target had changed color . to encourage that participants reacted as fast and as accurately as possibly throughout the whole experiment , written feedback about accuracy and search time was given after each trial . a practice session preceded the experimental test that stopped until 10 consecutively correct answers were given . there were two within - subject factors : set size ( 24 or 48 ) and sound ( present or absent ) . the whole test consisted of 1 block of 80 experimental trials , in which each of the 4 unique conditions was presented 20 times . fifteen young adults with developmental dyslexia ( five men and ten women ) and 15 age - matched controls without reading difficulties ( five men , ten women ) were tested . the dyslexic readers had been diagnosed with developmental dyslexia based on standard exclusion criteria ( apa 1994 ) . they were all formally assessed and diagnosed by clinical and educational psychologists . their reading achievements ( accuracy and/or speed ) were additionally assessed via standardized dutch reading tests for single word and nonword reading ( brus and voeten 1997 ; van den bos et al . dyslexic participants were selected on the basis of ( 1 ) normal or corrected - to - normal vision and hearing ; ( 2 ) absence of neurological and/or psychiatric disorders ; ( 3 ) absence of attention deficit disorder with hyperactivity ( because of the high comorbidity with dyslexia ) ; ( 4 ) absence of color blindness . the two groups were drawn from the same subject pool of university students , but were significantly different for both accuracy and speed of word and nonword reading ( see table 1 for details ) . participants were tested individually , were unaware of the purpose of the experiment , and received course credits or money for their participation . written consent was obtained from all participants according to the declaration of helsinki.table 1mean and standard deviation ( sd ) of age ( in years ) , word and nonword reading scores ( errors and speed in number of correctly read items ) in dyslexics ( n = 15 ) and age - matched normal reading controls ( n = 15)dyslexicscontrolscomparisonmeansdmeansdt(28)page21.52.220.71.71.10.28words reading errors1.31.10.30.63.090.004 speed77.810.393.516.43.160.004nonword reading errors8.93.02.32.96.73<0.001 speed69.916.298.514.05.17<0.001the bold values are with p < 0.05 mean and standard deviation ( sd ) of age ( in years ) , word and nonword reading scores ( errors and speed in number of correctly read items ) in dyslexics ( n = 15 ) and age - matched normal reading controls ( n = 15 ) the bold values are with p < 0.05 the auditory stimulus was a short white noise click of 68 ms presented at 74 db(a ) through the laptop speakers . the visual stimuli were presented on a 15-inch , 60-hz laptop monitor ( dell latitude e5500 ) , controlled by e - prime 1.2 ( psychology software tools , inc . ; http://www.pstnet.com/eprime ) . the visual search displays consisted of 24 or 48 red ( 20 cd m ) and green ( 11 cd m ) line segments ( length 0.88 visual angle ) against a dark background ( 0.05 cd m ) . the initial color ( red or green ) was randomly determined for each item . the lines were randomly placed in an invisible 10 10 grid ( 10.5 6.5 ) centered on a white central fixation cross , with the constraint that the target was never presented at the four central positions , to avoid immediate detection . the target was a horizontal or vertical line , while for distracters line orientation deviated randomly by plus or minus 26.5 from horizontal or vertical . the distracters changed color ( from red - to - green or vice versa ) every 50 , 100 , or 150 ms . the number of distracters that changed simultaneously during a trial varied for the different set sizes ; in set size 24 , 1 , 2 or 4 distracters changed simultaneously , while in set size 48 , it was 1 , 4 or 7 distracters . the target changed color every 500 or 1,000 ms , and always changed alone . distracters did not change color from 150 ms before the target until 100 ms after the target had changed color . during the first 500 ms of a trial , the target also did not change color . a dynamic example of trials with and without sound can be found at http://www.psy.vu.nl / pippop/. participants were tested in a dimly lit and sound - proof cabin and were seated approximately 65 cm in front of the laptop screen . a white fixation cross was illuminated in the center of the screen at the beginning of each trial . after 150500 ms , the display with target and distracters appeared at the screen . in the sound - present condition , a change in the color of the target was always accompanied by a simultaneously presented sound . participants were instructed to search for the target and to press one of two buttons corresponding with the target orientation - or all participants were explicitly told that sounds , if present , were synced with a color change of the target , and that they thus could benefit from the sound because it signaled that the target had changed color . to encourage that participants reacted as fast and as accurately as possibly throughout the whole experiment , written feedback about accuracy and search time was given after each trial . a practice session preceded the experimental test that stopped until 10 consecutively correct answers were given . there were two within - subject factors : set size ( 24 or 48 ) and sound ( present or absent ) . the whole test consisted of 1 block of 80 experimental trials , in which each of the 4 unique conditions was presented 20 times . the overall mean error rate was low ( 5.8% for the dyslexic group and 4.4% for the control group ) and did not significantly differ between groups , t(28 ) = 1.32 , p > 0.05 . search time was measured from the onset of the search display until the response to the target . an overall repeated - measures anova was conducted with group ( dyslexics versus normal readers ) as between - subject factor , and set size ( 24 or 48 items ) and sound ( sound present or absent ) as within - subject factors . as expected , dyslexics had longer overall search times than normal readers , f(1,28 ) = 5.66 , p < 0.05 , p2 = 0.17 ( mean search time of 8,004 ms for dyslexics and 6,100 ms for normal readers ) . the search time was also faster for the small than large set size , f(1,28 ) = 144.94 , p < 0.001 , p2 = 0.84 ( mean search time of 4,759 ms for set size 24 and 9,351 ms for set size 48 ) , and search time of trials with sound was faster than without sound , f(1,28 ) = 13.58 , p < 0.01 , p2 = 0.17 ( mean search time of 7,929 ms for sound - absent conditions and 6,181 ms for sound - present conditions ) . there was an interaction between set size and group , f(1,28 ) = 7.15 , p < 0.05 , p2 = 0.20 , indicating that dyslexics had slower search times per item than normal readers ( dyslexics : 222 ms / item ; normal readers : 170 ms / item ) . most importantly , there was interaction between group , sound , and set size , f(1,28 ) = 5.275 , p < 0.05 , p2 = 0.16 . as is clearly visible in fig . 1 , and as predicted , both groups benefitted from sound , but the dyslexics profited more from sound than normal readers as their improvement in search times per set size was bigger ( an improvement of 1,000 ms for set size 24 , and 4,735 ms for set size 48 ) than that of the controls ( an improvement of 281 ms for set size 24 , and 976 ms for set size 48).fig . 1mean search time ( in seconds ) as a function of set size and presence of sound for the dyslexic ( left panel ) and normal reading ( right panel ) group . error bars represent 1 standard error of mean mean search time ( in seconds ) as a function of set size and presence of sound for the dyslexic ( left panel ) and normal reading ( right panel ) group . error bars represent 1 standard error of mean separate anovas on the sound - absent and sound - present conditions showed that in the sound - absent conditions , the dyslexics were significantly slower than the normal readers , f(1,28 ) = 9.15 , p < 0.01 , p2 = 0.25 , and this difference was bigger with the large set size ( set size group interaction effect , f = 9.52 , p < 0.01 , p2 = 0.25 ) . in stark contrast , in the sound - present condition , the search time of the dyslexic group was not different from the control group ( main effect of group , f < 1 , set size group , f < 1 ) . here we examined the effect of sound on visual search times of dyslexic versus normal readers , using the pip - and - pop task . our results confirmed , in accordance with predictions from sas , that ( 1 ) in the sound - absent condition , dyslexics search time was much longer than of normal readers , ( 2 ) in the sound - absent condition , dyslexics search time increased more strongly with an increase in set size than of normal readers , and ( 3 ) dyslexics search time improved drastically by the presence of a transient sound ; in fact their search time then became as good as normal readers . the general slowness of the dyslexic group is consistent with previous studies using visual search tasks ( e.g. , vidyasagar and pammer 1999 ; sireteanu et al . account of hari and renvall ( 2001 ) , according to which the dyslexics automatic attention system can not disengage fast enough from one item to move to the next . the improvement in performance by the presence of the sound leads to the question why a transient sound could speed up the orienting of attention of dyslexic readers as much as it does . first of all , it is important to note that the sound used in the experiment did not bias attention toward a specific direction or location . we used a static sound that was centrally presented , so the sound was not informative about the possible location of the target . the gain offered by the sound for dyslexic readers is in line with other findings , demonstrating that dyslexics have specific problems in serial , but not parallel , search . 2008 ) demonstrated that dyslexics , compared to normal readers , have difficulties in serial , but not parallel , search . 1987 ; ruddock 1991 ; casco and prunetti 1996 ; vidyasagar and pammer 1999 ) in that we found that in the sound - absent condition , the search time of dyslexic readers increased more with the number of distracters and was significantly slower than that of the normal readers . in the sound - present condition , it has been argued that the binding of synchronized auditory - visual signals occurs rapidly , automatically , and effortlessly , with the auditory signal attaching to the visual signal relatively early in the perceptual process . through this , a nonspatial auditory event ( pip ) can guide attention toward the location of a synchronized visual event that , without an auditory signal , is difficult ( van der burg et al . the pip then makes the target pop - out , changing a serial search into an automatic , parallel search . this then led to a decrease in dyslexics search times up to the level of normal readers . although it is clear that dyslexics were better in parallel search than serial search , one can still ask whether there were besides sas any other reasons why dyslexics benefited significantly more from sound than the normal readers . one clue for this may come from a visual search study by facoetti et al . these authors found that dyslexic children had a bigger pop - out effect , which is a characteristic of parallel processing . the authors argued that dyslexia is characterized by a difficulty to narrow the attentional focus , and dyslexics therefore tended to adopt a more distributed focus of attention . ( 2008 ) suggested that at least some distributed attention is necessary for observers to notice the synchronized event of the pip and pop . by combining these two notions , it may become understandable why dyslexics , presumably with a more distributed focus of attention , could profit more from sound than normal readers . plausible explanations for the substantial benefit of sound by dyslexics might also be derived from studies that examined the effect of sound on visual attention . robertson et al . ( 1998 ) studied patients with neglect and showed that the phasic alerting by a transient nonspatial sound can overcome their spatial deficits in visual awareness . this finding provides evidence that the phasic alerting alone can directly affect the speed of perceptual processing , rather than merely affecting motor readiness . ( 2008 , 2011 ) , however , found evidence for a very early multisensory interaction that ruled out that the pip - and - pop effect is due to increases of alertness , as the effect follows a time course that is quite different from alerting effects . doyle and snowden ( 2001 ) also examined the effects of simple auditory signals whose onset was synchronized with that of the visual target , but provided no information about the target location . their findings made them speculate that an auditory signal may promote attentional disengagement ( for similar reasoning , see keetels and vroomen ( 2011 ) ) . related to our study , the sound may thus have a facilitatory effect on the disengagement of attention of the dyslexic readers , making them process the visual target ( much ) sooner when accompanied by a sound relative to when visual information is presented alone . a final speculation about the reason for the bigger improvement by sound in the dyslexic group may be related to crossmodal temporal deficits that have been found in dyslexia . ( 2008 ) , in order to be effective , a sound needs to be presented in close temporal proximity to the visual target change . various authors , though , argued that dyslexics exhibit deficits in different sensory systems , which involve alterations in temporal information processing ( e.g. , tallal 1980 ; kinsbourne et al . the authors found support for the idea of altered crossmodal temporal processing in dyslexia , as they reported that dyslexic subjects showed an extended temporal window for binding visual and auditory cues . these findings could be potentially relevant for our results , because an enlarged temporal window may lead to a bigger pip - and - pop effect in the dyslexics , as they may profit over an extended period of time of the sound . it should be noted , though , that an enlarged temporal window of integration may also lead to more spurious binding between the sound and color changes of distracters . an enlarged temporal window would then interfere rather than being of help in visual search time . future studies that vary the soa between sound and target and the soa between sound and distractor change might give more detailed information on this and on the mechanisms of multisensory integration in dyslexic readers . to summarize , our results showed that dyslexic readers have problems with visual serial search . this is evidence for visual attentional abnormalities in dyslexia , more specifically in the disengagement and shifting of attention . it remains for future studies with other clinical populations who are thought to have difficulties with disengaging and shifting attention ( e.g. , high - functioning autistic individuals ) to further explore the effects of sound on visual attention . the diagnosed incidence of neuroendocrine tumors ( nets ) in the current decade has risen to 5/100,000 from about 1 to 2 cases per 100,000 , three decades ago . this can be attributed to advanced diagnostic radiological and pathological techniques available in medical science today . multi detector computed tomography ( ct ) techniques with dynamic scanning of the abdomen in the arterial phase to detect hypervascular lesions such as those found in net is routine protocol for patients who have a clinical suspicion of the tumor . similarly , functional imaging such as nuclear positron emission tomography ( pet ) scans with ga - dota - nai - octreotide ( ga - dotanoc ) has further narrowed the possibility of detecting these lesions . we present a perplexing case of a patient who presented with a solitary liver mass , mimicking hepatocellular carcinoma ( hcc ) , on imaging and cytopathology and was subsequently operated for the same . a fluorodeoxyglucose ( fdg ) pet scan done before the surgery did not detect the primary tumor . the patient was diagnosed to have a metastatic secondary in the liver from a primary net , only after histopathology and immunohistochemistry of the explanted liver was performed . further evaluation in the postoperative period with ga - dotanoc pet enabled the localization of the primary tumor in the midgut , which was then re - explored and excised with a follow - up surgery . this case highlights the importance of immunohistochemistry for suspected hypervascular mass lesions in the abdomen . in addition , it emphasizes the relevance of functional imaging with ga - dotanoc pet and pitfalls of fdg pet in the assessment of hypervascular tumors of the abdomen and gastrointestinal tract . the use of ga dotanoc pet in the first stage of evaluation of the liver mass , along with fdg pet could have pointed to the nature of the mass and thus saved the patient morbidity related to two consecutive major laparotomy procedures . on review of the literature , we came across only one case report , which showed a primary hcc metastasis to pancreas resembling a net , however no other such mimic cases have been reported . a 55-year - old male presented to our hospital with continuous vague pain in the upper abdomen for a month . no other positive history of chronic diseases , prodromal symptoms , altered bowel habits , weight loss or gastrointestinal bleed was elicited . ultrasound abdomen , performed at an outside hospital had revealed a space - occupying lesion in the liver for which he underwent a fine - needle aspiration and cytology ( fnac ) . the available report of the above ( done elsewhere ) showed hcc ; however no slides were made available for review . physical examination did not show any positive findings , and the patient underwent a contrast - enhanced computed tomography ( cect ) of the upper abdomen for further evaluation and staging of the mass lesion in the liver . the cect revealed underlying diffuse hepatomegaly with hepatosteatosis . an arterial phase enhancing , well defined lobulated , rounded , hypervascular lesion measuring approximately 10 cm in its largest diameter was present in the right lobe of the liver ( segments viii and v ) [ figure 1a d ] . a 55-year - old gentleman with solitary hypervascular mass in the right lobe of liver on dynamic computed tomography ( ct ) . ( a ) mildly hypodense mass ( * ) in the region of right lobe of liver on noncontrast ct scan . ( b ) the mass ( * ) shows arterial hypervascularity along its periphery ( bold arrow ) . ( c ) the lesion showed contrast washout on the portal venous phase and was seen to encase the anterior division of the right portal vein . ( d ) washout persistent in the equilibrium phase in the mass ( * ) in addition , a contrast - enhanced magnetic resonance imaging ( cemri ) with hepatocyte - specific contrast ( gadolinium bopta , bracco ) was performed for further characterization of the mass and to look for satellite lesions that may have been missed on ct . the cemri showed t1-weighted hypointense , t2-weighted hyperintense and diffusion restriction of the mass . with subsequent washout on portal venous and delayed hepatobiliary phase obtained at 90 min post injection [ figure 2a g ] . it showed hypervascularity of the lesion on arterial phase . a 55-year - old man with hypervascular hepatic neuroendocrine tumor secondary deposit . ( a ) the fat - suppressed t1-weighted fast spin - echo , magnetic resonance imaging ( mri ) image performed after the dynamic computed tomography ( ct ) shows a hypointense mass ( * ) in right lobe ( block arrow ) ( b ) the fat - suppressed t2-weighted fast spin - echo , mri image performed after the dynamic ct shows a hyperintense mass ( * ) in the right lobe of liver ( block arrow ) and in caudate lobe ( open white arrow ) . ( c ) the axial diffusion weighted image of b value 1000 shows restriction of the lesion ( * ) on t2-weighted ( block and open arrows ) . ( d ) the fat - suppressed t1-weighted fast spin - echo , dynamic mri in the arterial phase shows a hypervascular enhancing mass ( * ) in the right lobe of liver ( block arrow ) ( e ) fat - suppressed t1-weighted fast spin - echo , dynamic mri in the portal venous phase shows an encapsulated mass ( * ) with central washout in the right lobe of liver ( block arrow ) . ( f ) the fat - suppressed t1-weighted fast spin - echo , dynamic magnetic resonance imaging mri in the equilibrium phase shows an encapsulated mass ( * ) with better visualized central hypointensity in the right lobe of liver ( block arrow ) . ( g ) the fat - suppressed t1-weighted fast spin - echo , dynamic mri in the hepatocyte specific phase at 90 min shows an encapsulated mass ( * ) with central hypointensity in the right lobe of liver ( block arrow ) this suggested predominance of non hepatocyte cellular contents within the mass . differential diagnosis of primary hepatocellular cancer and solitary hypervascular metastasis from an occult primary was made . in view of the above findings and previously conducted fnac , the diagnosis of hcc was primarily considered , and an fdg pet scan was conducted to rule out occult primary or distant secondaries before subjecting the patient to hepatectomy . no obvious evidence of fdg pet avid lesion or extrahepatic disease was found on the pet scan . with a provisional diagnosis of primary hcc without extrahepatic spread , right hepatectomy specimen was sent for histopathological analysis [ figure 3a and b ] on gross pathology examination ; transection margin was found to be 1 cm away from the tumor mass and was free from the tumor [ figure 3c ] . explanted right hepatectomy specimen with tumor . ( c ) gross pathology specimen with tumor ( * ) the uninvolved liver parenchyma was non cirrhotic . on microscopic examination of the sections from the tumor , lobular architecture comprising of thickened trabeculae nests , insular pattern and acini separated by fine endothelial network with a mild amount of interspersed fibrous stroma was found . on immune histochemical analysis , tumor cells showed strong immunostaining with synaptophysin and chromogranin - a with ki-67 immunolabelling index of < 2% . a diagnosis of metastatic neuroendocrine carcinoma , with small and large vessels invasion , was made . in view of the metastatic neuroendocrine nature of the liver lesion , a dotanoc pet scan was done to look for the primary , as well as any other occult secondary deposits in the abdomen . the scan was suggestive of nodular activity in the distal small bowel mesentery , in the right lower abdomen with an associated reactive focus in the ileum [ figure 4a c ] . ( a ) axial section of dota - nai - octreotide ( dotanoc ) positron emission tomography / computed tomography ( pet / ct ) showing reactive focus in the small bowel mesentery ( * ) . ( b , c ) coronal section of dotanoc pet / ct ( bold arrow ) and isotope scan ( outlined arrow ) showing reactive focus in the small bowel mesentery subsequently the patient was posted for a follow - up surgery at 4 weeks from the previous hepatectomy . the repeat laparotomy revealed a stricture in the long segment of the distal ileum with multiple small nodules and ulceroproliferative lesions in the mucosa for a span of approximately 30 cm . an additional small mass lesion in the terminal ileum with mesenteric invasion and desmoplastic reaction was localized which was subjected to en block resection with end to end anastomosis of the ileum . on immunohistochemistry , the tumor was synaptophysin , chromogranin - a positive with ki-67 index < 2% with the final diagnosis of multicentric neuroendocrine carcinoma ( pathological staging of iv ) . the patient had an uneventful postoperative period and was free at a year 's follow - up . the term neuroendocrine in nets derives from precursor cells of these mass lesions , which are essentially endocrine cells with nerve cell antigens . these cells normally evolve into endocrine glands which are an essential element of the neuroendocrine system and are classically found in the small bowel , where they are involved in gut motility as well as secretion of metabolically active substances such as serotonin , chromogranin , kinins , prostaglandins and various other growth factors . nets are most commonly seen in the midgut ( ileum and jejunum ) , pancreas as well as large bowel . many of these tumors are incidentally detected during the course of investigations or at the time of autopsy . as the name suggests , nets are comprised of endocrine cells that may or may not secrete hormones and hence are classified as functional or nonfunctional tumors . the nonfunctioning nets are usually asymptomatic and difficult to diagnose based on the clinical history . for example , in the case discussed above , the patient did not report any endocrine or hormonal imbalances , hence the primary was not suspected . functional tumors manifest with clinical symptoms pertinent to the hormone secreted by the tumor for example serotonin , insulin , glucagon , etc . , which are likely to cause endocrinological imbalance . the world health organization ( who ) recently introduced a novel classification system for nets on the basis of tumor grading in 2010 . this system utilizes histopathological markers of ki-67 index , mitotic count , and tumor necrosis and is now being used as standard criteria for histopathological diagnosis and staging of these tumors . nets are associated with syndromes like von - hippel - lindau , multiple endocrine neoplasia type 1 and neurofibromatosis - type 1 . midgut nets are second most common malignancy after adenocarcinoma 's in the small bowel and are notorious for being asymptomatic or presenting as nonspecific symptoms . they have a relatively good prognosis and a 5 year survival rate of > 50% even with synchronous metastatic disease . the hepatic involvement by nets is seen as : metastatic spread ( in 2590% ) , mixed hcc / adeno - neuroendocrine carcinoma and the primary net of liver ( < 0.5% of all net 's ) in the same order . there is an increase by 5 times in the detection rate of net 's in the last two decades , partly due to improved radiological techniques . the latest epidemiological survey from a south asian country like japan shows that almost 20% of patients have distant spread of the disease at presentation . carcinoid syndrome which is characterized by flushing , diarrhea , and abdominal pain is observed in approximately 3% of the gut tumors . abdominal ct with contrast is the most readily available and simplest method of the preliminary investigation of suspected midgut net 's or tumors . ct enterography using positive or neutral oral contrast with adequate small bowel distension and multi - planar reformatting post acquisition facilitates viewing of the small bowel loops . the sensitivity of ct and mr enteroclysis to detect small bowel net 's is 85% and 86% respectively and appears limited , however distant spread and extra - tumoral involvement is well - demonstrated by the above . at our center , we use the ct enterography technique where oral neutral contrast along with methylcellulose is given to distend the small bowel without any discomfort of inserting the enteroclysis tube . in cases where the primary site of the tumor is unidentifiable , such as seen in our patient , whole - body scintigraphy using somatostatin receptor scintigraphy ( ssrs ) for pet / ct ( using gallium - labeled somatostatin analogs ) or it has been established that receptor subtypes , sstrs 2 and 5 are useful for diagnostic workup of primary net 's . dotanoc binds to sstr2 , sstr3 , and sstr5 receptors whereas dota octreotide ( dotatoc ) and dota octreotate bind to sstr2 and sstr5 . these analogues accumulate in the tumor nodules within 100120 min and can be rapidly imaged . the demonstration of somatostatin receptor status by in - octreotide or ga - labelled peptide pet / ct imaging not only serves as a diagnostic tool but also helps in predicting the response to somatostatin analogue therapy . ga - dotatoc has higher sensitivity ( 96% ) and specificity ( 92% ) to ct or ssrs for occult primary work up , staging and follow - up of the disease process . dotatoc is superior in diagnosis of systemic and bony spread however has been found inadequate for detection of liver and lung secondaries where ct is recommended . the unavailability of nuclear scanning on a daily widespread basis and its practical cost implications are the major disadvantage of this technique . it also has its limitations in detection of low receptor density and small ( < 1 cm ) lesions . recent advances in use of newer agents like 18f - dopa for pet / ct has shown even higher sensitivity in metastatic nets ( almost 100% ) . premedication with carbidopa for reduction of artifacts due to normal physiological activity in the peripancreatic tissues has been recommended . 18f - fdg pet / ct is mostly useful for diagnosis and staging of poorly differentiated net 's which essentially are who grade 3 and 4 . poorly differentiated midgut lesions with reduced hormone production , and a high cellular proliferative activity have a higher propensity to take up 18f - fdg as do most other this may be the reason that the tumor in distal small bowel ( which was not poorly differentiated ) in the case described above , could not be detected on fdg pet on the initial screening . this fact suggests that all tumors that have not been histologically defined should undergo both fdg and a dotanoc pet so as not to miss occult and well differentiated net 's . also in cases where the clinical suspicion of an net is high and fdg pet is unable to diagnose the primary , dotanoc pet or alternate scintigraphy scans may be considered so as not to miss the primary lesion . other modalities such as meta - iodobenzyl guanidine scans can be used to investigate patients with metastatic disease who may be potential candidates for treatment with radionuclide therapy . debulking of the small bowel primary tumor and subsequent surgical anastomosis is generally recommended in case of midgut net 's because of future beneficial effect in decreasing small bowel and vascular complications . a multivariate analysis of the study population in the united kingdom by niederle mb et al . showed that age at diagnosis , ki-67 level and surgical removal of the primary tumor were standalone predictors of survival in patients with midgut net and associated hepatic secondaries . this is especially true for the nonfunctioning midgut tumors where somoatostatin analogs and systemic chemotherapy has limited or essentially no role to play . the chances of lymph nodal spread of the disease is seen in almost 60% of small bowel primary net 's . associated probability of hepatic secondaries at the time of diagnosis is about 30% . as discussed in the guidelines of management of midgut net 's by cheung et al . resection of the primary tumor and associated mesenteric lymph nodes is mandatory to treat small bowel net 's , irrespective of the presence of hepatic secondaries or not . in addition , this group recommended , that patients who are found to have small bowel nets , after laparotomy and histopathology , such as in our patient 's case , are candidates for further surgery which is beneficial for future prognosis . the modality for follow - up imaging should ideally be the one which was able to detect and diagnose the lesion pretreatment . ssrs imaging should be performed for sstr avid tumors with ct and mri wherever indicated . ct and mri are the only means of follow - up when tumor is sstr negative . frequency of assessment depends on tumor growth rate , and mri may be used to prevent excessive radiation exposure . the term neuroendocrine in nets derives from precursor cells of these mass lesions , which are essentially endocrine cells with nerve cell antigens . these cells normally evolve into endocrine glands which are an essential element of the neuroendocrine system and are classically found in the small bowel , where they are involved in gut motility as well as secretion of metabolically active substances such as serotonin , chromogranin , kinins , prostaglandins and various other growth factors . nets are most commonly seen in the midgut ( ileum and jejunum ) , pancreas as well as large bowel . many of these tumors are incidentally detected during the course of investigations or at the time of autopsy . as the name suggests , nets are comprised of endocrine cells that may or may not secrete hormones and hence are classified as functional or nonfunctional tumors . the nonfunctioning nets are usually asymptomatic and difficult to diagnose based on the clinical history . for example , in the case discussed above , the patient did not report any endocrine or hormonal imbalances , hence the primary was not suspected . functional tumors manifest with clinical symptoms pertinent to the hormone secreted by the tumor for example serotonin , insulin , glucagon , etc . , which are likely to cause endocrinological imbalance . the world health organization ( who ) recently introduced a novel classification system for nets on the basis of tumor grading in 2010 . this system utilizes histopathological markers of ki-67 index , mitotic count , and tumor necrosis and is now being used as standard criteria for histopathological diagnosis and staging of these tumors . nets are associated with syndromes like von - hippel - lindau , multiple endocrine neoplasia type 1 and neurofibromatosis - type 1 . midgut nets are second most common malignancy after adenocarcinoma 's in the small bowel and are notorious for being asymptomatic or presenting as nonspecific symptoms . they have a relatively good prognosis and a 5 year survival rate of > 50% even with synchronous metastatic disease . the hepatic involvement by nets is seen as : metastatic spread ( in 2590% ) , mixed hcc / adeno - neuroendocrine carcinoma and the primary net of liver ( < 0.5% of all net 's ) in the same order . there is an increase by 5 times in the detection rate of net 's in the last two decades , partly due to improved radiological techniques . the latest epidemiological survey from a south asian country like japan shows that almost 20% of patients have distant spread of the disease at presentation . carcinoid syndrome which is characterized by flushing , diarrhea , and abdominal pain is observed in approximately 3% of the gut tumors . abdominal ct with contrast is the most readily available and simplest method of the preliminary investigation of suspected midgut net 's or tumors . ct enterography using positive or neutral oral contrast with adequate small bowel distension and multi - planar reformatting post acquisition facilitates viewing of the small bowel loops . the sensitivity of ct and mr enteroclysis to detect small bowel net 's is 85% and 86% respectively and appears limited , however distant spread and extra - tumoral involvement is well - demonstrated by the above . at our center , we use the ct enterography technique where oral neutral contrast along with methylcellulose is given to distend the small bowel without any discomfort of inserting the enteroclysis tube . in cases where the primary site of the tumor is unidentifiable , such as seen in our patient , whole - body scintigraphy using somatostatin receptor scintigraphy ( ssrs ) for pet / ct ( using gallium - labeled somatostatin analogs ) or it has been established that receptor subtypes , sstrs 2 and 5 are useful for diagnostic workup of primary net 's . dotanoc binds to sstr2 , sstr3 , and sstr5 receptors whereas dota octreotide ( dotatoc ) and dota octreotate bind to sstr2 and sstr5 . these analogues accumulate in the tumor nodules within 100120 min and can be rapidly imaged . the demonstration of somatostatin receptor status by in - octreotide or ga - labelled peptide pet / ct imaging not only serves as a diagnostic tool but also helps in predicting the response to somatostatin analogue therapy . ga - dotatoc has higher sensitivity ( 96% ) and specificity ( 92% ) to ct or ssrs for occult primary work up , staging and follow - up of the disease process . dotatoc is superior in diagnosis of systemic and bony spread however has been found inadequate for detection of liver and lung secondaries where ct is recommended . the unavailability of nuclear scanning on a daily widespread basis and its practical cost implications are the major disadvantage of this technique . it also has its limitations in detection of low receptor density and small ( < 1 cm ) lesions . recent advances in use of newer agents like 18f - dopa for pet / ct has shown even higher sensitivity in metastatic nets ( almost 100% ) . premedication with carbidopa for reduction of artifacts due to normal physiological activity in the peripancreatic tissues has been recommended . 18f - fdg pet / ct is mostly useful for diagnosis and staging of poorly differentiated net 's which essentially are who grade 3 and 4 . poorly differentiated midgut lesions with reduced hormone production , and a high cellular proliferative activity have a higher propensity to take up 18f - fdg as do most other this may be the reason that the tumor in distal small bowel ( which was not poorly differentiated ) in the case described above , could not be detected on fdg pet on the initial screening . this fact suggests that all tumors that have not been histologically defined should undergo both fdg and a dotanoc pet so as not to miss occult and well differentiated net 's . also in cases where the clinical suspicion of an net is high and fdg pet is unable to diagnose the primary , dotanoc pet or alternate scintigraphy scans may be considered so as not to miss the primary lesion . other modalities such as meta - iodobenzyl guanidine scans can be used to investigate patients with metastatic disease who may be potential candidates for treatment with radionuclide therapy . debulking of the small bowel primary tumor and subsequent surgical anastomosis is generally recommended in case of midgut net 's because of future beneficial effect in decreasing small bowel and vascular complications . a multivariate analysis of the study population in the united kingdom by niederle mb et al . showed that age at diagnosis , ki-67 level and surgical removal of the primary tumor were standalone predictors of survival in patients with midgut net and associated hepatic secondaries . this is especially true for the nonfunctioning midgut tumors where somoatostatin analogs and systemic chemotherapy has limited or essentially no role to play . the chances of lymph nodal spread of the disease is seen in almost 60% of small bowel primary net 's . associated probability of hepatic secondaries at the time of diagnosis is about 30% . as discussed in the guidelines of management of midgut net 's by cheung et al . resection of the primary tumor and associated mesenteric lymph nodes is mandatory to treat small bowel net 's , irrespective of the presence of hepatic secondaries or not . in addition , this group recommended , that patients who are found to have small bowel nets , after laparotomy and histopathology , such as in our patient 's case , are candidates for further surgery which is beneficial for future prognosis . the modality for follow - up imaging should ideally be the one which was able to detect and diagnose the lesion pretreatment . ssrs imaging should be performed for sstr avid tumors with ct and mri wherever indicated . ct and mri are the only means of follow - up when tumor is sstr negative . frequency of assessment depends on tumor growth rate , and mri may be used to prevent excessive radiation exposure . neuroendocrine tumors are a unique group of tumors pertaining to their etiology , origin and function . they may or may not be easily detected on routine ct and mri investigations especially if they are nonfunctional . distant spread to the liver may sometimes be the only manifestation of the tumor and despite the frequency of this presentation ; it may be a clinically diagnostic challenge . multidisciplinary approach , in which nuclear medicine techniques play an important role in the detection , staging , follow - up and response of these tumors , is essential . net metastases to the liver can mimic primary hcc on imaging , fnac as well as core biopsy . it is crucial to be aware of this pitfall while evaluating solitary hypervascular liver lesions and to develop a robust strategy with judicious use of ga - dotanoc and 18f - fdg pet / ct to reach the correct diagnosis as well as staging which has a major role in treatment management and patient prognosis .

we examined whether developmental dyslexic adults suffer from sluggish attentional shifting ( sas ; hari and renvall in trends cogn sci 5:525532 , 2001 ) by measuring their shifting of attention in a visual search task with dynamic cluttered displays ( van der burg et al . in j exp psychol human 34:10531065 , 2008 ) . dyslexics were generally slower than normal readers in searching a horizontal or vertical target among oblique distracters . however , the addition of a click sound presented in synchrony with a color change of the target drastically improved their performance up to the level of the normal readers . these results are in line with the idea that developmental dyslexics have specific problems in disengaging attention from the current fixation , and that the phasic alerting by a sound can compensate for this deficit . neuroendocrine tumor metastases to the liver can mimic primary hepatocellular carcinoma ( hcc ) on imaging , cytology , and core biopsy . we present a case study along with the literature review of a patient who presented as a solitary liver mass mimicking hcc and subsequently underwent a partial hepatectomy . the histopathology and immunohistochemisrty of the resected specimen revealed metastatic neuroendocrine carcinoma . positron emission tomography ( pet ) scan with 68ga - dota - nai - octreotide ( 68ga - dotanoc ) localized the primary tumor in the ileum . a curative follow - up surgery for resection of the small bowel containing the primary tumor was carried out . this case illustrates the shortcomings of routine imaging methods , utility of immunocytochemistry and the importance of 68ga - dotanoc pet in determining the metastatic spread as well as the origin of neuroendocrine tumors ( nets ) . this case report attempts to highlight the current imaging paradigms and management strategy of midgut and other net 's at the point of detection , staging and follow - up .

exposure to moderate to heavy amounts of alcohol during pregnancy remains a leading preventable cause of intellectual disabilities in north america ( abel and sokol , 1986 ; may and gossage , 2001 ; chudley et al . , 2005 ; may et al . , 2009 ) . children with fetal alcohol spectrum disorder ( fasd ) face a broad spectrum of cognitive and behavioral challenges , including deficits in sensory processing , attention , working memory and executive function ( mattson et al . , 1998 ; , 2010 ; mattson et al . , 2011 ; riley et al . , 2011 ) . although facial dysmorphia is characteristic of fetal alcohol syndrome ( fas ) , the severe end of the fasd spectrum , many children with fasd lack physical abnormalities and hence are often not diagnosed , even though sharing many of the same cognitive and behavioral issues ( mattson et al . , 1998 ; kodituwakku , 2009 ; this latter group of children , labeled as showing alcohol related neurodevelopmental disorder ( arnd ) , is known to display aberrant neural functions and subtle neuroanatomical differences in neuroimaging studies . delayed and/or abnormal brain development may contribute to deficits in cognitive function and increased behavioral issues in adolescents with fasd ( lebel et al . , 2011 ; triet et al . , 2013 ) . brain regions particularly vulnerable to alcohol 's teratogenicity include the frontal and parietal cortex , posterior sensory cortex , caudate , hippocampus , and cerebellum ( for a review , see roussotte et al . , 2010 ) . although reduction in total brain volume is a common feature in fasd ( astley et al . , 2009 ; lebel et al . , 2011 ) , increased local cortical thickness has been reported in the parietal and frontal cortices ( sowell et al . , 2002 ; sowell et al . , 2008 ) and in the inferior frontal , superior temporal , and middle temporal cortex ( yang et al . , 2012 ) . , 2012 found that even children with arnd showed volume reductions in multiple brain regions , including the frontal , parietal , and temporal lobes , although these volume reductions were due to reduced cortical surface area rather than thickness . abnormal and delayed development of white matter tracts have also been seen in fasd in the posterior corpus callosum , anterior posterior fiber bundles , superior longitudinal fasciculus and superior and inferior fronto - occipital fasciculus , and cerebellar peduncles ( cf . 2011 ; triet et al . , 2013 ; green et al . , 2013 ) . functional neuroimaging studies reveal that typically developing adolescents exhibit weaker modulatory control from frontal areas compared to adults ( hwang et al . , 2010 ) . in a recent fmri study of children from 714 years of age , fasd showed reduced activation bilaterally in the frontal , parietal and temporal cortex compared to controls , suggesting an impact of alcohol exposure in utero on maturation ( gautam et al . , 2014 ) . increased frontal and parietal activation has been reported during the performance of spatial working memory task in arnd and go / no - go tasks in fasd ( malisza et al . , 2012 ; norman et al . , 2013 ; although differences observed in microvascular networks in a mouse model of fas / partialfas suggest some caution in the interpretation of these fmri data ( jgou et al . , 2012 ) , electroencephalographic ( eeg ) and magnetoencephalographic ( meg ) studies also support differences in measures of brain function in fasd . deficits in auditory stimulus classification and inhibition have been reported in adolescents with fas / partialfas ( steinmann et al . , 2011 ) . latency and amplitude differences of the p300 auditory event - related potential ( erp ) in an oddball task discriminated between children with down syndrome , fasd , and typically developing controls ( kaneko et al . auditory processing delays have also been observed in a meg study of preschool children with fasd ( stephen et al . , 2012 ) . the present study utilized meg to investigate brain dynamics of adolescents with fasd during the performance of an auditory oddball task . perceptual expectation for a set of repeated ( standard ) tones , detection of target tones that elicit behavioral responses and processing of novel digital sounds in cortico - hippocampal circuits ( halgren et al . , 1998 ) . sex - related differences in brain structure are known to emerge in typically developing adolescents , with increased volume of the amygdala in males and of the hippocampus in females ( for a review , see blakemore , 2012 ) . since prenatal ethanol exposure is known to produce sex - specific deficits in hippocampus in rodent models ( coleman et al . , 2012 ; see also helfer et al . 2014 ) , we hypothesized that meg measures of brain activation in the oddball data may reveal sex - specific differences for adolescents with fasd . consideration of potential sexual dimorphism in neuroimaging and neurophysiological studies in adolescents with fasd is rare ( although see kaneko et al . , 1996a ) . the present effort will contribute novel information on sex - specific effects on brain function and motivate further attention to potential sexual dimorphism in studies of adolescents with fasd . forty eight adolescents and young adults in the age range 1222 years were recruited for this study . for the participants utilized in the meg analysis , twenty two of the participants ( 9 male , age 15.0 yrs , sd = 3.6 yrs ; 13 female , age 15.5 yrs , sd = 2.8 yrs ) were identified as fasd according to the modified institute of medicine criteria ( stratton et al . , , 9 were diagnosed with fas ( 6 male and 3 female ) and 13 with alcohol - related neurodevelopmental disorder ( arnd ) ( 3 male and 10 female ) . twenty - two age- and sex - matched healthy individuals ( hc ; 9 male , m age 13.5 yrs , sd = 4.7 yrs ; 13 female , m age 16.8 yrs , sd = 3.3 yrs ) with no history of prenatal alcohol exposure , developmental delays , significant psychiatric or neurological problems served as controls . all of the participants were right handed ( edinburgh handedness inventory : oldfield , 1971 ) and none of the participants had significant sensory problems ( e.g. poor vision or hearing ) or difficulty understanding the task demands . participants with fasd were recruited at the university of new mexico fetal alcohol diagnostic and evaluation clinic and healthy controls ( hc ) through flyers and word of mouth . this study was approved by the university of new mexico health sciences center institutional review board and was in full compliance with the code of ethics of the world medical association ( declaration of helsinki ) . informed consent was obtained from the parents / legal guardians and/or participants dependent on the age and assent from minors in accord with the institutional review board guideline of the university of new mexico . cognitive assessment was performed at the mind research network in conjunction with the meg / mri scans . two subtests from the wechsler abbreviated scale of intelligence ( wasi , vocabulary and matrix reasoning ) were utilized to compute full scale iq . tone bursts ( 80% ) , 1.5 khz target tone bursts ( 10% ) and novel digital sounds ( 10% ) presented binaurally through plastic tubes placed into the ear canals . the duration of all stimuli was 200 ms with a pseudorandom stimulus onset asynchrony between the stimuli of 13 s ( mean 1.5 s ) and intensity of 85 db . the stimuli were grouped into four equal - duration blocks , with brief rest intervals ( 15 min ) between blocks . the total number of stimuli in all four blocks was 784 ( standard ) , 98 ( target ) and 98 ( novel ) . participants were asked to press a response button with the right index finger upon perception of the 1.5 khz target tones . presentation software ( http://www.neurobs.com ) was used to deliver stimuli and record motor responses to target stimuli . magnetoencephalographic ( meg ) data were acquired at the mind research network ( mrn , albuquerque , nm ) . participants were seated inside a double - layer magnetically shielded room ( vacuumschmelze ) under a 306-channel meg array ( elekta neuromag ) and monitored throughout the scan . prior to the scan , a polhemus 3d position tracker was used to record the location of four head positioning ( hpi ) coils on the left and right mastoid and bilaterally on the forehead and the left and right preauricular and nasion fiducial points . the meg data were sampled at 1200 hz and band - pass filtered online at 0.1330 hz . the location of the participant 's head with respect to the meg array was recorded throughout the scan through activation of the hpi coils . bipolar horizontal and vertical electro - oculograms and a bipolar electrocardiogram were recorded throughout the scan . high - resolution t1-weighted anatomic images were acquired using a 3 t siemens trio tim with a multi - echo magnetization prepared rapid gradient echo ( mprage ) sequence ( echo time [ te ] = 1.64 , 3.5 , 5.36 , 7.22 , and 9.08 ms ; repetition time [ tr ] = 2530 ms ; inversion time [ ti ] = 1200 ms ; flip angle = 7 ; number of excitations = 1 ; slice thickness = 1 mm ; field of view [ fov ] = 256 mm ; resolution = 256 256 mm ) . the scan session lasted approximately 30 min and also included functional mri ( fmri ) and diffusion tensor imaging ( dti ) acquisition , which are not included in this manuscript . the average image ( root - mean - square ) across the five echoes of the mprage was used for co - registration with meg data and for construction of a boundary element model ( bem ) for each participant . elekta neuromag maxfilter software was used to compensate for head movement under the scanner and to remove artifacts originating outside the cranial volume ( taulu and simola , 2006 ) . all data for each subject were co - located to a common subject - specific head position . signal - space projectors were created from these data and used to further suppress blink and cardiac signals on an individual subject basis ( tesche et al . , 1995 ; tesche et al . , the data were down - sampled at 600 hz and averaged time - locked to stimulus presentation for each stimulus type ( standard , target , novel ) for epochs of 500 ms before to 1000 ms after auditory stimulus presentations . the evoked - response averages were baseline corrected from 500 to 0 ms before stimulus presentation . since activity at 1000 ms after stimulation occurred immediately before the earliest possible presentation of a subsequent stimulus ( the minimum isi between all stimuli was 1000 ms ) , the evoked - response waveforms were terminated at 1000 ms to avoid contamination with the processing induced by subsequent stimuli . surface tessellation of the cerebral and of the cerebellar cortex was extracted from each participant 's mr images using brainsuite ( http://brainsuite.org ) . the cerebral and cerebellar cortical tessellations were merged to form a brain - based source space for meg data inversion . a similar cortico - cerebellar tessellation was constructed from mr images for the collin 27 adult brain ( collins , 1998 ) which was used as a common source space for all between - subject analyses . brainstorm ( tadel et al . , 2011 ) was used to compute average sensor waveforms and global field power plots from the meg sensor data and to extract brain - based waveforms for specific brain regions . brainstorm is a documented and freely available package downloaded online under a gnu general public license ( http://neuroimage.usc.edu/brainstorm/ ) . the meg data were co - localized with the structural mri data on an individual participant basis . source - space waveforms were then computed for each participant from all sensor evoked - response data ( utilizing both gradiometer and magnetometer sensor data ) and the individual brain surface tessellation using a weighted minimum norm estimate ( wmne ) . the p50 m and n100 m responses were determined for each participant from their individual meg / mri data . an initial region of interest was selected in and near the posterior ramus of the lateral sulcus . a waveform of activation for this roi was then extracted from the participant 's evoked - response data and the latency of the peak response noted . the initial roi was adjusted on an iterative basis to encompass the region of activation at the peak latency . this procedure was followed for activation between 40 and 70 ms for the p50 m response and independently at 80120 ms for the n100 m response . when these cortical areas corresponding to the p50 m and n100 m responses were projected onto the colin 27 brain , variable blurring and shifting of the rois across participants were observed . more representative individual latency values used in the present study were determined using each participant 's mri before the transformation of the source data into the colin 27 brain . group - averaged brain activation patterns were determined by projection of each participant 's source - space data onto a common surface derived from the collin 27 brain . averages of the absolute value of individual activation patterns within this source space were determined for the standard , target and novel trials separately for male and female fasd and the sex- and age - matched controls . meg data for this subject were projected directly onto the collin 27 brain using a polhemus digitization of the participant 's face and scalp . brain activation patterns were determined for all participants at 100 , 250 and 350 ms after stimulus presentation . waveforms of brain activity were computed within brainstorm from group averages of the absolute value of source activation data . the following regions were selected as salient for stimulus processing and novelty detection : the posterior ramus of the lateral sulcus and the inferior frontal sulcus as described in the destrieux cortical atlas ( destrieux et al . , 2010 ) and the hippocampus as determined from the collin 27 images within brainstorm . comparisons of fasd and hc were calculated for neuropsychological measures , response times to target stimuli and age ( table 1 ) . all statistics were calculated two - tailed and the critical value for rejecting the null hypothesis was p 0.05 . to assess performance on the oddball task , response times ( rts ) and accuracy to the target stimuli the raw scores for the vocabulary and matrix reasoning subtests from the wasi were t scored and used to determine the participant 's iq score . fasd and control group performance on the oddball task , and wasi performance was further explored by comparing sex differences by group and then within group . to test for a difference in age an independent samples t - test was performed . to test for difference in sex , a chi - squared test was performed . independent samples t - tests were performed to evaluate the differences between fasd and hc groups for latency measures of the auditory evoked - response to standard stimuli ( p50 m , n100 m , and n100 m onsets ) ( table 2 ) . differences in latencies were further explored by comparing sex differences by group and then within group . a 2 3 repeated measures anova was performed to compare the left hemisphere p50 m , n100 m , and n100 m latencies by group . a separate 2 3 repeated measures anova was performed to compare the right hemisphere p50 m , n100 m , and n100 m latencies by group . within subject lateralization differences were tested using paired - sample t - tests . pearson 's correlations were executed to examine relationships between the auditory evoked - response latencies and the behavioral , neuropsychological and age data . these relationships were further explored by comparing sex differences both between groups and within group . brainstorm ( tadel et al . , 2011 ) was used to compute brain activation patterns for fasdm , fasdf , hcm and hcf ( figs . the data were averaged for standard , target and novel trials at 0 , 100 , 250 and 350 ms following stimulus presentation . in brainstorm , the visualization parameter amplitude was set to 50% and the minimum size was set to 1 ( all features of every size are displayed ) . comparison of activation plots for fasdm vs. hcm and for fasdf vs. hcf was also computed in brainstorm ( figs . the activation plots for each participant were normalized to baseline values from 500 to 0 ms prior to computation of group - averaged data . the comparisons between groups of evoked - response waveforms were performed using a matched - pair ( sex and age ) t - test on each sampled instant ( figs . 9 , 11 , 13 ) . epochs of significant differences ( p < 0.01 ) were computed from the data and are indicated as black bars on the horizontal axes of the figures . the time frequency plots ( figs . 10 , 12 , 14 ) were computed without normalization or thresholding of the results . a matched - pair ( sex and age ) t - test was used for comparison between groups with p - value threshold of 0.05 . five participants did not complete the cognitive assessment . the remaining 39 participants ( 46.15% male ) were assessed for a relationship between response time to target auditory stimuli and demographic ( age , sex ) and cognitive measures ( iq , wasi vocabulary , wasi matrix reasoning ) . no significant between - group differences were detected for age ( t(44 ) = 0.919 , p = .36 ) or sex ( (1 , n = 39 ) = 0.022 , p = .882 ) . large and significant differences were detected for iq mainly due to a substantial difference in vocabulary ( see table 2 ) . in addition , a significant association was detected between wasi vocabulary and response time ( r = .350 , p = .039 ) . there was no significant effect of sex on response time ( rt ) , the number of false positives , the ratio of incorrect / correct , on rt weighted by hits or on the change of rt or false positives across the 4 blocks . significant differences in iq and vocabulary scores were also observed in male and female group comparisons . in the male comparisons by group , iq scores were significantly different [ t(15 ) = 4.42 , d = 2.28 , p = 0.001 ] , with the control males having higher iq scores . vocabulary scores were also significantly different [ t(15 ) = 5.97 , d = 3.08 , p = 0.00003 ] , with the control males having higher vocabulary scores . the female comparisons followed the same trend with iq scores being significantly different [ t(22 ) = 4.27 , d = 1.82 , p = 0.0003 ] , with the control females having higher iq scores . vocabulary scores were also significantly different [ t(21 ) = 4.53 , d = 1.98 , p = 0.0002 ] , with the control females having higher vocabulary scores . again within group comparisons did not reveal any significant differences between sexes for any of the measures . the p50 m and n100 m peak latencies and the onset of the n100 m for the standard stimuli were determined for each participant from waveforms for activity in primary / secondary auditory cortex ( fig . an independent samples t - test for p50 m and n100 m latencies and for the n100 m onset in the left and in the right hemisphere revealed significant differences between fasd and hc in the left n100 m onset [ t(36 ) = 2.267 , p = 0.029 , d = 0.735 ] and left n100 m latency [ t(39 ) = 2.368 , p = 0.023 , d = 0.739 ] . a repeated measures anova was performed to compare the latencies of the p50 m , n100 m , and n100 m evoked responses in the left and right hemisphere . this yielded a significant difference between hemispheres [ f(1,34 ) = 5.382 , p = 0.026 , partial = 0.195 ] . both fasd and hc showed differences in latency that depended on sex ( fig . an independent samples t - test revealed that in hc , males and females were significantly different for the left hemisphere n100 m onset [ t(17 ) = 4.137 p = 0.001 , d = 1.922 ] , left hemisphere n100 m latency { t(18 ) = 3.536 p = 0.002 , d = 1.613 } , right hemisphere p50 m latency [ t(17 ) = 2.448 , p = 0.026 , d = 1.137 ] , and right hemisphere n100 m latency [ t(19 ) = 2.834 , p = 0.011 , d = 1.273 ] . in fasd , males and females were significantly different for the left hemisphere n100 m onset [ t(17 ) = 2.275 , p = 0.036 , d = 1.082 ] , right hemisphere p50 m latency [ t(16.151 ) = 5.905 , p = 0.000021 , d = 2.743 ] , and right hemisphere n100 m onset [ t(9 ) = 2.790 , p = 0.021 , d = 1.69 ] . however , an independent t - test comparison of female fasd and female hc revealed no significant differences . an independent t - test comparison of male fasd and male hc revealed significant differences in the left hemisphere n100 m onset [ t(13 ) = 2.537 , p = 0.025 , d = 1.313 ] , and left hemisphere n100 m latency [ t(14 ) = 2.229 , p = 0.043 , d = 1.115 ] . lateralization differences were observed in hc and tested using paired - sample t - tests . onset of n100 m in the left hemisphere at 72 ms lagged onset in the right at 66 ms [ t(15 ) = 2.199 , p = 0.044 , d = 0.55 ] . n100 m latency on the left at 100 ms lagged the right at 91 ms [ t(19 ) = 3.158 , p = 0.005 , d = 0.706 ] . the difference between the left p50 m at 52 ms was almost significant compared to the right p50 m at 47 ms [ t(16 ) = 1.95 , p = 0.069 , d = 0.472 ] . in contrast no significant differences were observed between the hemispheres in p50m / n100 m latency and n100 m onset in fasd . correlations between the p50 m , n100 m onset and n100 m component latency and neuropsychological tests ( wasi : iq score , vocabulary , and matrix reasoning ) were also investigated . no significant correlations were found with the control males , fasd males , nor with all male scores grouped together . with all female scores combined , there was a correlation between the left n100 m latency and iq [ r(21 ) = 0.427 , p = 0.042 , r = 0.18 ] and with the left n100 m latency and matrix reasoning [ r(20 ) = 0.459 , p = 0.032 , r = 0.21 ] . there was also a correlation between the right n100 m latency and vocabulary [ r(13 ) = 0.522 , p = 0.046 , r = 0.27 ] , but not with the left n100 m latency . there were also no correlations found in the combined control group . in the fasd group , there were no correlations with the left and right p50 m and n100 m onset . there was a correlation between the right n100 m and vocabulary [ r(14 ) = 0.512 , p = 0.043 , r = 0.26 ] , but not in the left n100 m . 2 shows group - averaged meg sensor waveforms and global field power ( gfp ) for standard , target and novel trials . there are also a series of peaks from 250 to 450 ms for standard , target and novel trials , and also sustained activity to 800 and 1000 ms for target and novel trials , respectively . figs . 35 show group - averaged patterns of brain activation represented on the cortical surface as the absolute value of current flow at 0 , 100 , 250 and 350 ms following presentation of the standard , target and novel stimuli . responses for standard stimuli were strongly right lateralized for both fasdm and hcm but not for fasdf and hcf at 100 , 250 and 350 ms . increased frontal and parietal activation responses following target stimuli showed a similar pattern of strongly right - lateralized activation for fasdm and hcm , but not for fasdf and hcf . increased frontal and parietal activation for fasdm and fasdf responses following novel stimuli showed right - lateralized activation for all groups , with frontal activation at 100 ms and temporal activation at 350 ms for fasdm . 68 show group - averaged patterns of brain activation for fasdm vs. hcm and fasdf vs. hcf at 0 , 100 , 250 and 350 ms following presentation of the standard , target and novel stimuli . the data for each of the participants were normalized to baseline values ( from 500 to 0 ms before stimulus presentation ) prior to computation of the mean activation patterns for each group . 68 reflect both activation at the indicated time epochs and also baseline activation levels . in fig . 6 , the t - test plots at 100 ms reveal scattered regions of significant differences between fasdm and hcm , including increased activation of the right parietal cortex for standard stimuli and of the right cerebellum for target stimuli in fasdm . novel stimuli elicited significant increases in bilateral frontal , parietal and cerebellar cortex for fasdm compared to hcm . there was also increased activation of bilateral frontal cortex in fasdf compared to hcf for all three stimulus types . in fig . 7 the t - test plots at 250 ms reveal significant increases for fasdm in left temporal and left inferior frontal activation and decreases in bilateral medial frontal and right frontal activation compared to hcm for standard stimuli . responses for target stimuli also showed significant increases in left temporal activation and decreases in bilateral medial frontal and right frontal activation . there was also significant increases for fasdf in bilateral frontal activation to standard stimuli , right frontal activation to target stimuli and bilateral activation to novel stimuli compared to hcf . novel stimuli also elicited increases in medial frontal and decreases in parietal activation in fasdf compared to hc . both target and novel stimuli elicited significant increases in the cerebellum for fasdm compared to hcm , but not for fasdf compared to hcf . in fig . 8 the t - test plots at 350 ms for standard stimuli show significant decreases in bilateral frontal and parietal cortex and increases in bilateral temporal cortex for fasdm compared to hcm . target and novel stimuli elicited significant increases in bilateral frontal and temporal cortex for fasdm . there were also significant increases in right frontal activation to standard stimuli and increases in bilateral frontal activation to target and novel stimuli for fasdf compared to hcf . decreased activation of bilateral parietal cortex 914 show group - averaged waveforms and power time frequency ( tf ) plots for current flow within and near the left and right primary auditory cortex ( posterior ramus of the lateral sulcus ) , the inferior frontal sulcus and the hippocampus for the standard , target and novel conditions . although there are intermittent epochs of significant differences between fasd and hc both before and after stimulus presentation , differences in the amplitude of the n100 m response to standard and target stimuli are not significant . interestingly , the evoked - response waveforms following standard and target stimuli reveal sustained activity from 0.5 to 1 s for both fasd and hc . the activity at 1 s occurs just prior to the earliest possible presentation of a subsequent stimulus . in contrast , hcm , hcf and fasdf waveforms for novel trials return to baseline in the left and right hemispheres during the same epoch . only waveforms for fasdm show a tendency for sustained activity in the right hemisphere . 10 for current flow in the auditory cortex reveal stimulus - locked alpha - band ( 813 hz ) activity for both fasd and hc . right - lateralization of responses is seen for hcm for standard stimuli and for fasdm for all stimulus types . right - lateralization of responses for standard stimuli is seen also for hcf , and for target stimuli for fasdf . the t - test plots for the right auditory cortex show significant increases in alpha - band activity for fasdm compared to hcm both prior to and after stimulus presentation for target stimuli . in contrast , the t - test plots for fasdf vs. hcf show significant decreases in alpha - band activity for fasdf both prior to and after stimulus presentation in the left and right auditory cortex for all stimulus types . the t - test plots for standard trials in the left but not the right auditory cortex also show significant increased alpha , beta ( 1335 hz ) and gamma ( 3560 hz ) activities for fasdm compared to fasdf . increased gamma - band activity in the left auditory cortex is not seen in hcm vs. hcf . the t - test plots for the right auditory cortex show significant increased alpha - band activity for fasdf compared to fasdm for target stimuli . fig . in contrast to the waveforms for activity within the left and right auditory cortex , fig . 11 reveals multiple epochs of significant differences between responses for fasd compared to hc , with amplitudes for fasd exceeding that for hc for all stimulus categories , particularly in the right hemisphere . there is also sustained activity from 0.5 to 1 s following standard and target stimuli for fasd and hc , and sustained activity from 0.5 to 1 s in the right inferior frontal sulcus following novel stimuli for fasd . significant differences in baseline activity are also seen in the data for both male and female fasd compared to controls . 12 for current flow in the inferior frontal sulcus are similar to those for the auditory cortex , in that stimulus - locked alpha - band activity is more right - lateralized for both fasd and hc following standard and target stimuli . in contrast to the results for the auditory cortex , alpha - band activity for novel stimuli is more right - lateralized for fasdm and more left lateralized for fasdf . the t - test plots show significantly increased alpha - band activity for fasdm compared to hcm both prior to and after stimulus presentation in the right inferior frontal sulcus for target and novel stimuli . in contrast to the results for the auditory cortex , the t - test plots for fasdf vs. hcf for standard stimuli show significant increases in alpha- , beta- and gamma - band activities for fasdf after stimulus presentation in the left hemisphere and both prior to and after stimulus presentation in the right hemisphere . gamma - band increases for fasdf compared to hcf can also be seen for target and novel trials . the t - test plots for fasdm vs. fasdf for standard and target trials show decreased alpha- , beta- and gamma - band activities in the left hemisphere for male compared to female fasd . the t - test plots for hcm vs. hcf for standard and target trials also show decreased alpha- and beta - band activities in the left hemisphere for males compared to female hc . in contrast to responses in the auditory cortex , there are few significant differences for either fasdm vs. fasdf or for hcm vs. hcf in the right hemisphere for standard and target stimuli , or in either hemisphere for novel stimuli . fig . there are multiple epochs of significant differences between responses for fasd compared to hc , with waveform amplitudes for fasd exceeding that for hc for all stimulus categories in both the left and right hemispheres . fasdm waveforms show a pronounced , bilateral and prolonged p300-type response to novel stimuli with a sustained response to ~800 ms . there are also significant differences between fasd and hc in the sustained activity from 0.5 to 1 s following standard and target stimuli . differences in baseline activity are also seen in the data for all conditions with the exception of the left hemisphere responses in the hippocampus for fasdf compared to hcf . the tf plots in fig . 14 for current flow in and near the hippocampus show increased bilateral alpha - band activity for fasdm compared to hcm following standard and target stimuli , and increased right - hemisphere alpha - band activity following novel stimuli . differences in the tf plots for fasdf and for hcf are less pronounced , although interesting stimulus - locked beta- and gamma - band activities can be seen for both fasdf and hcf . the t - test plots for fasdm vs. hcm for standard and target stimuli reveal significant increases in alpha- , beta- and gamma - band activities for fasdm . alpha - band activity is also significantly increased for fasdf compared to hcf , although the differences are not as pronounced . the t - test plots for standard stimuli also reveal significantly less alpha- , beta- and gamma - band activation for fasdm compared to fasdf in the left and right hippocampus , but only minor differences for hcm compared to hcf . the present study revealed differences in brain activation and dynamics in adolescents with fasd compared to typically - developing controls during the performance of an auditory oddball task . this task probes development of top - down perceptual expectation for repeated ( standard ) tones , detection of target tones that elicit a behavioral response and processing of novel digital sounds in cortico - hippocampal circuits ( halgren et al . , 1998 ) . the main finding was widespread sex - specific differential activation of the frontal and medial temporal cortex in adolescents with fasd compared to typically developing controls . significant differences in evoked - response and time frequency measures of brain dynamics for fasd were observed for all stimulus types in the auditory cortex , inferior frontal sulcus and hippocampus . these results underscore the importance of considering the influence of sex when analyzing neurophysiological data in children with fasd . although fasd responded more slowly to target tones compared to hc in the oddball task , there was no effect of sex on the response times to target stimuli . in contrast , significant differences were observed in the latencies of the auditory n100 m response to standard stimuli between male and female participants with fasd . in a previous meg study of 10 preschool children with fasd and 15 healthy controls , stephen and colleagues ( 2012 ) found significant delays in fasd in the n100 m and n200 m evoked - response peaks following presentation of a train of 1000 hz tone bursts . this interesting study suggested that auditory evoked - response latency differences may evidence alcohol - induced brain damage in this age group . in the present study on 22 adolescents with fasd and 22 typically developing controls , latency differences between participants with fasd and healthy controls were observed in both the p50 m and n100 m auditory evoked responses . in contrast to the stephen study , latencies of the p50 m and n100 m were shorter for adolescent fasd compared to healthy controls . ( 1996a ) reported no significant n100 latency differences for standard stimuli between children from 4 to 15 years with fas and healthy controls . suggested that the discrepancy between their results and that of kaneko et al . may be due in part to maturational differences in auditory processing between younger and older children . maturation of the cortical auditory evoked response in typically developing children is known to shorten the latency of n1 and p2 in eeg scalp recordings ( wunderlich et al . , 2006 ) . in the context of fasd , studies performed in alcohol - exposed rats reveal that abnormalities in auditory brainstem responses observed in the newborn ( church , 1987 ) tend to dissipate in adulthood ( church et al . , 2012 ; leigland et al . , these data suggest that , although neurophysiological measures of auditory processing delays may have potential as markers for compromised brain function in very young children with fasd , these measures may not be useful for older children . another possible contributor to the observed differences between the young children in prior work ( kaneko et al . , 1996a ; stephen et al . , 2012 ) and the present study could be in the nature of the auditory stimuli presented to each group . the young children in the stephen study were passively exposed to trains of 1000 hz tones . children in the kaneko study listened to standard , rare and noise - burst stimuli but did not make a behavioral response , in contrast , adolescents in the present oddball study were requested to make a motor response to the ( rare ) target stimuli . perceptual expectation for the repeated standard tones and processing of novel digital sounds in cortico - hippocampal circuits but also generation of behavioral responses to target tones and inhibition of behavioral responses to both standard and novel stimuli . thus participants needed to attend to all of the oddball stimuli in order to successfully perform the requested motor response . stimulus - driven attention is known to increase the speed of auditory processing ( folyi et al . , 2012 ) . hence the latency differences observed for the standard stimuli between fasd and hc in the present oddball study may reflect not just maturation but also a contribution from differences in top down attentional and inhibitory functions between the two groups . in support of this notion , we observed widespread activation , not only of auditory , but also of frontal cortex as early as 100 ms after stimulus presentation . activation of the frontal cortex continued until immediately prior to presentation of the subsequent stimuli with significant differences in brain activation between male and female fasd and sex - matched hc . the shorter p50 m and n100 m auditory evoked - response latencies in the auditory cortex in fasd did not translate into reduced response times to the target stimuli . this result supports the notion that , although differences in early processing in the auditory cortex may evidence the effects of alcohol exposure in fasd , processing in other cortical and/or subcortical regions must also have been impacted by alcohol exposure . prenatal ethanol exposure is known to affect both the frontal cortex and hippocampus ( for a review , see roussotte et al . , 2010 ) , and also to produce sex - specific deficits in the hippocampus in rodent models ( coleman et al . , 2012 ; sickmann et al . , 2014 ) . our results indicate that the application of high - temporal - resolution neuroimaging techniques such as meg with the potential to characterize brain dynamics in frontal - hippocampal circuitry can play a valuable role in characterizing neurophysiological deficits seen in fasd . in the present study , significant differences were observed between fasd and hc and between males and females in the evoked - response waveforms and in the time frequency ( tf ) plots of stimulus - locked oscillatory activity for regions of interest centered in the primary / secondary auditory cortex , inferior frontal sulcus and hippocampus . although the relationship between evoked - response waveforms , ongoing ( endogenous ) alpha , induced ( stimulus - locked ) alpha and stimulus - evoked alpha in meg data are complex and controversial ( for a review , see mazaheri and jensen , 2010 ) , oscillatory activity is believed to play a critical role in the sculpting and coordination of brain function . alpha - band ( 813 hz ) activity has been implicated in timing and attention ( for a review see klimesch , 2012 ) and in the inhibition of neuronal populations irrelevant to task performance ( jensen and mazaheri , 2010 ) . although alpha oscillations can be found throughout the frontal and posterior cortex , the most prominent alpha - band activity ( eyes - closed alpha ) occurs near the parieto - occipital sulcus in the absence of visual stimuli . reduction of left - hemisphere eyes - closed alpha has been reported in a previous eeg study in fas ( kaneko et al . meg detection of stimulus - induced alpha in the auditory cortex ( tau ) has been reported by several groups ( tiihonen et al . , 1991 ; in the present study , differences between fasd and hc and between males and females were more apparent in the stimulus - locked alpha - band activity than in the evoked - response waveforms . the significant increases in the alpha - band activity seen in the left hemisphere for hcf compared to fasdf , for fasdf compared to fasdm and for hcf compared to hcm suggest that measures of induced alpha may be salient markers for abnormalities in inhibitory processes in the auditory cortex in fasd . the motivation for selecting the inferior frontal sulcus as a region of interest in the present study is the known role of the ventrolateral prefrontal cortex ( vlpfc ) in the processing of acoustic features of complex sounds ( for a review , see plakke and romanski , 2014 ) . auditory association areas , including the auditory belt and superior temporal gyrus project to vlpfc areas 12/47 , 45 , and 12 orbital . a more general role of the right inferior frontal cortex in auditory processing is somewhat more controversial ( cf . swick and chatham , 2014 ; aron et al . , 2014 ) , although recent fmri data suggest that inferior frontal cortical regions participate to spatially distributed networks in the processing of infrequent stimuli ( erika - florence et al . , 2014 ) . in the present study , in contrast to responses seen in the auditory cortex , there was a consistent increase in evoked - response and alpha - band activity in the inferior frontal sulcus for standard and target stimuli in fasd compared to hc . in male fasd , increases were seen primarily in the right hemisphere . in female fasd , increased activity was more bilateral and accompanied by prominent right - hemisphere gamma oscillations . a deficit in inhibitory interneuron development , hypothesized to be a long - term consequence of developmental ethanol exposure ( sadrian et al . , 2013 ) , may contribute to this increased evoked - response , alpha- and gamma - band activities seen in fasd . in pioneering passive oddball eeg study of children with fas , kaneko , riley and collaborators found latencies of the p300 evoked response over the parietal cortex to novel stimuli to be increased in fas compared to controls ( kaneko et al . , 1996a ) . the oddball paradigm in the eeg literature is utilized to characterize attention and novelty detection , with the p3a component of the p300 associated with involuntary orientation of attention to novel stimuli in fronto - hippocampal circuits , and the p3b component associated with voluntary detection of task - salient target stimuli ( for a review see soltani and knight , 2000 ; polich , 2007 ) . in the present meg study , significant differences emerged between fasd and hc in and near the hippocampus . standard , target and novel stimuli all elicited significant increases in evoked - response amplitudes in fasd compared to hc at 300 ms , with sustained activation to 1000 ms for both standard and target , but not novel , stimuli . tf plots revealed increased alpha , beta and gamma - band activities for fasd vs. hc and for fasdm vs. fasdf for standard and target stimuli , and for fasdf vs. hcf for novel stimuli . the elevated activation to all stimulus types was also seen in the right inferior frontal sulcus in fasd . hyperactivity has been reported in olfacto - hippocampal circuits in the alcohol - exposed rodent during both the spontaneous ( resting ) state and in sensory - evoked responses ( wilson et al . , 2011 ) . although a detailed analysis of cortico - hippocampal network dynamics is not reported here , these results suggest a common auditory - evoked and alpha - band hyperactivity in both the inferior frontal sulcus and hippocampus in adolescents with fasd . observation of both evoked - response and alpha - band differences in fasd enlarges the potential set of neurophysiological measures for the impact of alcohol exposure on brain function . the main finding of this meg study was widespread sex - specific differential activation of the auditory , frontal and mesial temporal cortex in adolescents with fasd compared to typically developing controls during the performance of an auditory oddball task . this task probed development of top - down perceptual expectation for repeated ( standard ) tones , detection of ( rare ) target tones that elicit a behavioral response and processing of novel digital sounds in cortico - hippocampal circuits . auditory n100 m evoked - response latencies for fasd compared to healthy controls were opposite to those reported previously for very young children . significant sex - related differences in evoked - response and oscillatory time frequency measures of brain dynamics were observed for all stimulus types . these results underscore the importance of considering the potential effect of sex when analyzing neurophysiological data in adolescents with fasd and expand the potential set of neurophysiological markers for this disorder . nowadays color doppler study of corpus cavernosum ( cc ) with intracavernosal injection ( ici ) of vasoactive agents is the first step to investigate etiologic causes of male erectile dysfunction ( ed ) ( 1 , 2 ) . diagnostic criteria are based mostly on the peak systolic velocity ( psv ) and end diastolic velocity ( edv ) values of the cavernosal artery spectral waveform that were first measured 5 minutes after injection and then repeated every 5 minutes afterward up to 30 minutes ( 1 - 4 ) . however , erection is a multi - stage neurovascular phenomenon that needs the fine balance of contractile properties of trabecular smooth muscles in the cc . it consists of these phases : filling , congestion , rigidity and full erection ( 1 ) . noteworthy to know is that in most of the patients , maximum psv happens in the first 5 minutes ; moreover , in a minority of patients all the process of changes happen in the first 5 minutes ( 5 ) . for better description of these dynamic changes , we got an idea from the study of fitzgerald al . ( 5 ) and tried to designate dynamic patterns for spectral waveform changes of cavernosal arteries in each patient . in this study , we tried to classify our patients based upon their patterns of changes in a dynamic study and to subsequently better describe their underlying problem . this case series study was conducted in hasheminejad urology center , iran university of medical sciences ( iums ) , from april 2011 till april 2012 . we evaluated 59 men who were referred from a urology clinic for post - ici color doppler investigation of suspected ed . the stages of the test and possible role of the study in their therapeutic plan were described comprehensively for each patient and then a written consent was obtained . the demographic data and medical history including duration of ed , history of diabetes , hypertension , cigarette smoking , drug history and previous interventions for vascular diseases were recorded . one of this study investigators ( gharib , mh . ) performed all examinations with a linear ( 5 - 10 mhz ) probe of esaote technos device ( italy , 2002 ) . the probe was placed over the ventral aspect of the penis and scans were performed in longitudinal and transverse planes before injection of the vasoactive agent . we used papaverine hydrochloride because of the availability and acceptable price . for post - ici assessment , first a maximum dose of 40 mg papaverine was injected into the right corpus covernosum , at the junction between the middle and distal third of the penile dorsal aspect . if maximum psv did not reach 30 cm / s in 5 minutes , further injections of 20 mg doses of papaverine were also performed , up to a maximum total dose of 80 mg . scanning started about one minute after the injection and each of the cavernosal arteries was evaluated continuously thereafter until either erection happened or psv started to decline . parameters such as psv , edv , minimum of resistive index ( ri ) and time to reach maximum psv were recorded . if full erection happened , the patient was under observation and if erection persisted for more 3 hours , the patient was sent to the urologic emergency room for appropriate interventions . based on temporal changes in the waveform of cavernosal arteries , new patterns were defined . patients were classified based on previously known etiologic categories ( i.e. arterial insufficiency , venous leak , mixed type , and normal response ) and also categorized into these new patterns . fifty - nine patients with the mean age of 45.6 13.1 ( 24 to 74 ) years were enrolled into this study . twenty - two cases revealed normal response ( non - organic causes as 37.3% ) , 27 were classified as venous leakage , eight with arterial insufficiency and two were considered as mixed type . the mean time to reach maximum psv was 3.4 0.8 minutes ( median 3 , range 2 - 5 minutes ) in 51 patients without arterial insufficiency . we defined 8 patterns for the temporal changes in cavernosal arterial waveform in our patients ( table 1 ) . for classification , we considered four different aspects in our study : sufficiency of cavernosal arteries [ for judgement of this sufficiency , we considered points such as maximum psv during the study ( cut point of 30 cm / s ) , amount of difference of psv between the two sides ( below or above 10 cm / s ) , acceleration time of each waveform ( cut point of 80 ms ) and pattern of blood filling in the arteries ] competent filling of sinusoidal blood pools [ including qualitative assessment of edv ( considered as satisfactory or not ) and duration of edv rise ( at least one minute ) ] veno - occlusive capability of corpus cavernosum [ even though a clinically assessed full erection is the best determinant . in doppler study , long time persistence of vigorous diastolic flow ( edv more than 5 cm / s ) would be the indicator ] . finally , how long did it take the study to be finished ( below or above 5 minutes ) . we defined a psv level more than 40 cm / sec as a requisite to consider the patient as having venous leak with no arterial component based on benson et al . those patients with a psv between 30 and 40 who did not elicit erection were placed in category 2 under the label of mixed type . patterns 3 , 4 and 5 denote to previously known venous leak group and patterns 6 , 7 and 8 are patients with normal response . table 2 shows the patient classification based on various patterns of arterial spectral waveform changes . among all , 8 patients completed all phases of erection in the first 5 minutes ( one with pattern 3 and 7 with pattern 6 ) . pattern 5 was considered as the common pattern of venous leak ; while patterns 3 and 4 were considered as the uncommon group . in our study , six patients demonstrated the uncommon patterns of venous leak ( 22.2% of all patients with venous leak ) . the mean of psv was not different between these two groups ( 57.7 vs. 52.5 cm / s , p = 0.38 ) . mean age and minimum recorded ri were higher in the uncommon group , although they did not show statistical significance ( p = 0.14 and 0.22 , respectively ) . hypertension was more prevalent in the uncommon pattern of venous leak ( p = 0.01 ) . smoking was more prevalent in patients with uncommon patterns ; however , the difference was not statistically significant ( p = 0.29 ) . we detected one patient with micropenis who demonstrated normal pattern of changes in response to papaverine even though with a maximum psv under 30 cm / s . htn , hypertension ; psv , peak systolic velocity ; ri , resistive index . in this study , we investigated continuous assessment of cavernosal arterial waveforms of patients with ed in comparison with its routine doppler evaluation . the conventional method of post ici of papaverine color doppler study includes recording psv and edv values in the cavernosal artery spectral waveform 5 minutes after injection which is repeated every 5 minutes afterward up to 30 minutes ( 1 , 4 ) . we found that in most patients ( 92.2% ) , maximum psv happened before the fifth minute . fitzgerald et al . also showed this finding in a considerable percent of their patients ( 76% ) ( 5 ) . this emphasizes the need for early initial assessment of flow in cavernosal artery after the injection of papaverine . they are filling , tumescence , rigidity and full erection ( 1 , 3 , 4 , 8) . it is obvious that these events will result in a sequence of changes in the waveform of cavernosal artery . a process that can be chased meticulously in a continuous color doppler protocol ( figure 1 ) . conventionally four groups of responses have been defined : arterial insufficiency , venous leak , normal response and a mixed pattern ( 9 ) . for better delineation of dynamic changes during ultrasonography , we got the idea of categorizing waveform pattern changes during continuous assessment from the study conducted by fitzgerald et al . ( 5 ) and defined 8 patterns of changes for arterial spectral waveform . in our classification , we considered subgroups for patients with the uncommon pattern of venous leak and those with normal response based on the response within the first 5 minutes after injection or thereafter ( patterns 3 and 4 ; and patterns 6 and 7 respectively ) . the left column of the image shows tumescent phase in this patient 2 minutes after injection . in the middle column , finally , the right column depicts waveforms in the rigidity phase in the 6th minute . we noticed that all phases of the response to papaverine occurred within the first 5 minutes in 13.8% of our patients ( 1 with pattern 3 and 7 with pattern 6 ) . in conventional practice , for patients with pattern 3 , we might need to repeat the injection or just judge based on the clinical fact that erection has not happened . systolic velocity starts to decline and the arterial system , especially helicine arterioles become progressively less visible ( 4 ) . so here , we were not able to designate a normal response based on the findings of the study and just the occurrence of full erection would be the clue for our normal reports . these findings again clarify the importance of careful observation of changes both continuously and from the early beginning . we used to designate 4 groups to our patients based upon findings in the conventional study . in fact in the conventional study , the sufficiency of the arterial system and veno - occlusive mechanisms of corpus cavernosum are the mainstay of classification ; however , we think that one more aspect should be delicately followed in the process of erection , and that is the appropriate reaction of sinusoidal blood pools in each cc . an important outcome of this dynamic study is distinction between patients formerly designated as having venous leak . normally , a marked increase in psv should result in concomitant significant rise in the diastolic blood flow . we believe that patterns 3 and 4 are related to a more decreased amount of trabecular smooth muscles and/or a change in the fibroelastic composition of cc , what we considered as the competency of filling of sinusoidal blood pools or in other words , effective congestion . consequently , in color doppler study , the diastolic flow will not rise to a favorable amount for an acceptable duration ( figure 2 ) . these patients , although few ( 6 patients in this study ) , seem to have more aggravated problems and would not have a satisfactory response to common medications used for venous leak and should be directed for prosthesis placement . the meanage and the lowest recorded ri are higher in this group of venous leak , which we labeled arbitrarily as the uncommon group , compared to the common group ( 54.5 vs. 46.3 and 77.7 vs. 74.3 , respectively ) . although possibly due to the small number of patients , they did not show statistical significance ( p value 0.14 and 0.22 , respectively ) . the probable extensive changes in tissue texture of patients with uncommon venous leak would result in a lesser degree of diastolic flow compared to the common pattern . as the patient grows older , the ratio of collagen type 3 ( the elastic one ) counts down and collagen type 1 gradually replaces it . moreover , the content of trabecular smooth muscle also decreases up to 35% ( 1 ) . hypertension and smoking have been introduced as other important factors involved in the extent of changes in the tissue texture of cc ( 1 ) . however , in our study the prevalence of smoking was still not statistically significant in the uncommon venous leak group ( p = 0.29 ) . we think that the amount and duration of smoking ( quantified in pack - year ) may alter the pattern of response in patients with venous leak . as the pack - year of smoking inflates , we had one patient that showed erection with psv values below 30 cm / sec . the amount of blood flow directed to cc depends on the volume of recipient sinusoidal blood pools . it is obvious that in a patient with micropenis , a lesser amount of flow is needed to result in erection and the maximum psv ( one important determinant of flow ) would be lower compared to other normal responders with a normal - sized penis , even below the required post injection psv of more than 30 cm / s . while , in a continuous study , we can follow the normal sequence of changes in cc and easily denote that the patient has no vascular problem . in conclusion , we highlight a considerable role of early and continuous evaluation started one minute after ici for better description of underlying pathologies of ed especially in patients with venous leak etiology . this study was just a case series to obtain a general concept about the benefits of a dynamic study . the patterns described here are just those seen in these 59 cases . in further studies with a larger sample size , it may be possible to declare new patterns or more sharply elucidate the characteristics of the aforementioned groups . the two left columns are waveforms of a patient with the common pattern of venous leak ( pattern 5 ) in the 3rd and 8th minutes , respectively . the two right columns are for a patient with an uncommon pattern of venous leak ( pattern 4 ) in approximately similar times . note the prominent diastolic flow ( edv = 19cm / s ) in the left side patient , while the diastolic flow in the right side patient is not as much vigorous ( edv = 8 cm / s ) throughout the dynamic study .

children exposed to substantial amounts of alcohol in utero display a broad range of morphological and behavioral outcomes , which are collectively referred to as fetal alcohol spectrum disorders ( fasds ) . common to all children on the spectrum are cognitive and behavioral problems that reflect central nervous system dysfunction . little is known , however , about the potential effects of variables such as sex on alcohol - induced brain damage . the goal of the current research was to utilize magnetoencephalography ( meg ) to examine the effect of sex on brain dynamics in adolescents and young adults with fasd during the performance of an auditory oddball task . the stimuli were short trains of 1 khz standard tone bursts ( 80% ) randomly interleaved with 1.5 khz target tone bursts ( 10% ) and novel digital sounds ( 10% ) . participants made motor responses to the target tones . results are reported for 44 individuals ( 18 males and 26 females ) ages 12 through 22 years . nine males and 13 females had a diagnosis of fasd and the remainder were typically - developing age- and sex - matched controls . the main finding was widespread sex - specific differential activation of the frontal , medial and temporal cortex in adolescents with fasd compared to typically developing controls . significant differences in evoked - response and time frequency measures of brain dynamics were observed for all stimulus types in the auditory cortex , inferior frontal sulcus and hippocampus . these results underscore the importance of considering the influence of sex when analyzing neurophysiological data in children with fasd . background : erection is a dynamic multi - stage neurovascular phenomenon consisting of 4 phases . conventional protocol of color doppler study can easily overlook these ongoing dynamic events.objectives:here , we tried to designate patterns for these dynamic spectral waveform changes of cavernosal arteries in patients with erectile dysfunction and subsequently better describe the extent of their underlying problem.patients and methods : we evaluated 59 men who were referred for post - intracavernosal injection ( ici ) color doppler investigation of suspected erectile dysfunction ( ed ) . the demographic data and medical history were recorded . afterwards , first scan injection was done . then scanning of cavernosal arteries was started about one minute after the injection and was continued thereafter . for better description of temporal changes in the waveform of cavernosal arteries , new patterns were defined and used . patients were also classified based on previously known etiologic categories ( i.e. arterial insufficiency , venous leak , mixed type , and normal response).results : the mean age was 45.6 13.1 ( 24 to 74 ) years . twenty - two were normal responders [ considered as non - organic causes ( 37.3% of all patients ) ] , 27 were classified as venous leakage , eight had arterial insufficiency and two were mixed type . maximum psv occurred before the fifth minute in 47 patients ( 92.2% ) . eight patients completed all phases of erection in the first 5 minutes . we defined 8 patterns for the temporal changes in cavernosal arterial waveform . pattern 5 was the most common pattern of venous leak ; while , patterns 3 and 4 were considered as the uncommon group . six patients demonstrated the uncommon patterns of venous leak ( 22.2% ) . hypertension was more prevalent in the uncommon pattern of venous leak.conclusions:we highlight the considerable role of continuous evaluation starting one minute after intra cavernosal with ici injection of the vasoactive agent for better description of the underlying pathologies of ed especially in patients with venous leak etiology .

crohn 's disease ( cd ) is an autoimmune inflammatory disorder that may involve different sites along the gastrointestinal tract . the disease tends to be more severe and widespread in children and young adults [ 1 , 2 ] . during the last 2 decades , the incidence of cd in childhood has considerably increased [ 2 , 3 ] . pulmonary involvement in cd is well described , and though it is rarely reported in pediatric patients , it may be more common than previously reported . pulmonary cd can be latent or symptomatic , with symptoms manifesting before , during or after the diagnosis of cd . down syndrome ( ds ) has been associated with increased frequency of malignancies , autoimmune diseases and infections . it has been hypothesized that this is due to abnormal precocious aging . however , recent evidence suggests that it is more likely that the immune system in ds is deficient from the very beginning , with immune dysregulation causing a tendency towards autoimmune diseases . in addition to other congenital defects , individuals with ds are also more likely to have structural and functional disorders of the gastrointestinal tract . associations between cd and ds have been reported , though very rarely [ 9 , 10 , 11 , 12 , 13 ] . in our search of the literature , we did not find any reported case of ds with pulmonary cd to date . a 5-year - old girl with known ds and a history of chronic intermittent abdominal pain presented to the emergency department with worsening symptoms . her abdominal pain has previously been attributed to underlying chronic constipation and possible irritable bowel syndrome . workup for her abdominal pain included serologic testing for celiac disease , which showed increased anti - gliadin iga and igg but normal tissue transglutaminase iga . on presentation to the emergency department , an abdominal x - ray was done due to suspicion of constipation , and a chest x - ray confirmed left lower lobe pneumonia . she then returned to the hospital due to persistent fevers and abdominal pain and was admitted for 5 days for intravenous antibiotics . on serial chest x - rays during the hospitalization and after discharge , she seemed to have persistent left - sided and perhilar infiltrates that remained essentially unchanged in appearance . a pediatric pulmonologist was consulted , who felt that she had no symptoms suggestive of asthma , aspirated foreign body , cystic fibrosis , primary ciliary dyskinesia or immunodeficiency . a chest computed tomography ( ct ) scan was done that showed multiple noncalcified pulmonary nodules bilaterally , mainly peripheral in location . the largest nodule measured 8.0 mm and was in the posterior medial aspect of the superior segment of the left lower lobe adjacent to the pleura . the second largest was 7.0 mm in the right pulmonary apex , and another 2 nodules measuring 4.0 mm were seen in the right upper lobe ( fig . 1 ) . two additional 4.0-mm nodular densities were seen in the posterior periphery of the superior segment of the left lower lobe just caudal to the dominant lesion . there were localized patchy reticular nodular infiltrates in an extreme superior aspect of the superior segment of the left lower lobe and similar extensive infiltrates extending from the suprahilar region on the right side into the right pulmonary apex . lymphocyte subsets , immunoglobulin levels and vaccine responses to tetanus and pneumococcus were done , as well as a neutrophil oxidative burst assay for chronic granulomatous disease . neutrophil function showed 74% activity at 48 h , which was considered a normal response and not compatible with a diagnosis of chronic granulomatous disease . her immunoglobulin assay showed an igg level of 1,130 mg / dl ( range 2261,870 ) , an iga level of 218 mg / dl ( range 3155 ) and an igm level of 45 mg / dl ( range 12219 ) . her tetanus antibody level was 0.97 iu / ml , which was considered a normal immunization response . results showed a cd19 b - cell count of 127 cells/l , a cd3 t - cell count of 1,033 cells/l , a cd4 cell count of 605 cells/l , a cd8 cell count of 395 cells/l , and a cd56/16 natural killer cell count of 99 cells/l ; the cd4/cd8 ratio was normal at 1.5 . after a relatively extensive infectious workup , she underwent a bronchoscopy and ct - guided needle biopsy of the lung nodules . the branching pattern also was normal ; however , the right upper lobe bronchus gave only 2 branches compared with the usual 3 branches . broncheoalveolar lavage ( bal ) analysis was performed , and specimens were sent for bacterial , fungal and mycobacterial culture analysis as well as for cytology . no acid - fast bacilli were seen on fluorochrome stain , and culture results showed no organisms even after 56 days of incubation . ct - guided core lung biopsies showed extensive necrosis with surrounding palisading histiocytes , fibrosis and chronic inflammation ( fig . 2 ) . acid - fast bacillus and grocott 's methenamine silver stains were negative for any organisms . these findings , along with the history of chronic abdominal pain , raised concern for primary cd with secondary involvement of the lungs . the esophagogastroduodenoscopy showed localized mild mucosal abnormality characterized by congestion , erosion and inflammation in the 3rd part of the duodenum . a benign - appearing diffuse , severe mucosal abnormality characterized by congestion , discoloration , erythema , erosion , friability , granularity , inflammation and nodularity was found in the entire examined stomach . on colonoscopy , patchy , mild inflammation characterized by congestion ( edema ) , erythema and aphthous ulceration was found in the cecum , ascending colon , transverse colon and descending colon . normal mucosa was seen in the rectum . the gastric biopsy exhibited moderate to focally marked expansion of the lamina propria by a mixed leukocytic population . also present were scattered areas of neutrophils , some of which were present in the surface epithelium . a giemsa stain was performed and demonstrated the presence of bacteria ( these bacteria , however , did not have the usual morphological features of helicobacter ) . both the cecum and ascending colon biopsies exhibited cryptitis as well as eosinophilia of the lamina propria . also present were focal aggregates of histiocytes ( although well - formed epithelioid granulomas were not observed ) . in contrast , the transverse colon and descending colon biopsies showed similar cryptitis and eosinophilia and , in addition , there were scattered epithelioid granulomas . acid - fast bacillus and grocott 's methenamine silver stains were performed on these specimens , and no organisms were seen . she was then started on mesalamine and later on infliximab and methotrexate . with the start of therapy , to our knowledge , this is the first reported case of pulmonary cd in a child with ds . the natural history of pulmonary cd in the pediatric population is not very well studied . furthermore , since ds has been well known to be associated with increased frequency of malignancies and autoimmune conditions due to immune dysregulation , it is difficult to predict the severity and possible complications in this patient . a 28-year - old man who was known to have fhi in the right eye was referred for secondary iol implantation . six years before referral , his right eye had undergone cataract surgery which was complicated by the capsular rupture and vitreous prolapse , for which the patient received complete anterior vitrectomy with removal of all capsular remnants . the patient was left aphakic and was prescribed with aphakic contact lens ; however , he developed contact lens intolerance over time . on presentation , his uncorrected visual acuity was 20/20 in the left eye and counting finger in the right eye which could be corrected to 20/20 with aphakic correction . slit - lamp examination of the right eye revealed diffuse fine keratic precipitates over the entire corneal endothelium and mild iris stromal atrophy with notable heterochromia . advantages and unknown risks of the surgery were thoroughly explained for the patient and he consented to have secondary iol implantation . under general anesthesia , an iris - claw iol ( artisan , ophtec , groningen , the netherlands ) was implanted in right eye through a limbal incision followed by a superior peripheral iridectomy . enclavation of the iol haptics was easily performed ; no intraoperative complication including hyphema was noted . the latter was prescribed as 0.1% betamethasone every 2 hours while awake for 1 week and then four times a day which was tapered within 6 weeks . postoperative follow - up examinations were performed at 1 , 2 , 3 , 5 , and 7 days , then weekly for 1 month , monthly for 3 months , and every 23 months thereafter until 1 year . postoperative course was uneventful with no significant anterior chamber inflammation ( more than 1 + cellular reaction ) or fibrin formation . on the first postoperative day , the examination showed 1 + cellular reaction and pigments in the anterior chamber which disappeared within 2 weeks . no subsequent exacerbation of the intraocular inflammation was observed during 12 months of postoperative follow - up ; therefore , no additional course of steroid was required . occasional cells in the anterior chamber were seen at some visits which were left untreated . one month after surgery , the patient achieved a best - corrected visual acuity of 20/20 in the right eye which was maintained for 12 months of follow - up . the iol remained stable with no subsequent iris atrophy at the enclavation sites , subluxation , or pupil ovalization . furthermore , the patient did not develop any anterior or posterior segment complication including glaucoma , vitreous inflammation , or clinical cystoid macular edema . secondary implantation of iris - claw artisan intraocular lens ( iol ) in an eye with fuchs heterochromic iridocyclitis . during 12 months of postoperative follow - up , no remarkable anterior chamber inflammation was observed in the right eye ( a ) and there were only few deposits on the iol surface ( b ) . the heterochromia in the involved eye is most obvious compared with the normal left eye ( c ) although secondary iol implantation in the ciliary sulcus has been reported to be safe in fhi , angle- and iris - supported iols have been feared because of the possible risk of postoperative uveitis , glaucoma , and hyphema . to the best of our knowledge , there has been no previous report of implantation of iris - claw artisan iols in eyes with fhi . even though our patient only received topical steroids , he did not show any significant postoperative inflammation or fibrinous reaction neither at the early postoperative period nor during 12 months of follow - up . therefore , it may suggest that in eyes with fhi the uveal irritation by iris - claw artisan iols is less than expected and the iol is more tolerable , even though recurrent or chronic anterior chamber inflammations has previously been reported in some eyes with these iols without preexisting uveitis . however , this lack of exacerbated postoperative inflammation in fhi may not be extrapolated to eyes with other more severe forms of uveitis . on the other hand , although fhi - associated iris atrophy in severe cases may theoretically make enclavation more difficult or compromise the long - term stability of an iris - claw iol , neither did develop in our case . therefore , it seems iris - claw iols , which have been shown to be safe in aphakic eyes without uveitis , may be an option in aphakic patients with fhi who do not have capsular support . however , studies on large number of patients with long - term follow up are required to determine the safety of these iols in eyes with uveitis including fhi .

crohn 's disease ( cd ) is an autoimmune inflammatory disorder that primarily affects the gastrointestinal tract . it may have pulmonary involvement , which has been rarely reported in pediatric patients . down syndrome ( ds ) has been associated with increased frequency of autoimmune diseases . however , associations between cd and ds have been rarely reported . we present the case of a 5-year - old girl with known ds and a history of chronic intermittent abdominal pain who presented with persistent pneumonia . her workup included a chest computed tomography ( ct ) scan that showed multiple noncalcified pulmonary nodules . an extensive infectious workup was done that was negative . ct - guided needle biopsy of the lung nodules showed necrotizing granulomas . this raised concern for primary cd with extraintestinal pulmonary manifestation . an esophagogastroduodenoscopy and colonoscopy were performed , and colon biopsies showed scattered epithelioid granulomas . based on this information , there was consensus that her lung nodules were secondary to cd . she was started on standard therapy for cd , and her abdominal and respiratory symptoms gradually improved . however , she continues to have mild residual lung calcification and fibrosis . to our knowledge , this is the first reported case of pulmonary cd in a child with ds . the natural history of pulmonary cd in the pediatric population is not very well studied . furthermore , since ds has been well known to be associated with increased frequency of malignancies and autoimmune conditions due to immune dysregulation , it is difficult to predict the severity and possible complications in this patient . implantation of iris - claw artisan intraocular lens ( iol ) is a surgical option for correction of aphakia ; however , these iols have not been used in eyes with uveitis including fuchs heterochromic iridocyclitis ( fhi ) due to possible risk of severe postoperative intraocular inflammation . in the case reported here , we secondarily implanted an artisan iol in a 28-year - old man with fhi who had aphakia with no capsular support due to a previous complicated cataract surgery . enclavation was easily performed and no intraoperative complication was noted . postoperative course was uneventful with no significant anterior chamber inflammation during 12 months of follow - up . although there were few deposits on the iol surface , the patient achieved a best - corrected visual acuity of 20/20 without developing glaucoma or other complications . therefore , artisan iol may be considered for correction of aphakia in patients with fhi . however , studies on large number of patients are required to evaluate safety of the procedure .

the world health organization ( who ) , in 2012 , indicated there were an estimated 8.6 million new cases of active tb and 1.3 million tb deaths . the world health organization reported saudi arabia as having a moderate tb incidence rate , with 15 for every 100,000 . tb remains an important public health problem in saudi arabia , affecting all age groups and regions , and is associated with higher mortalities among saudis . since pulmonary tb this has become even more important with the development of drug - resistant tb , making effective treatment even more difficult . tuberculosis was accepted as a diagnosis if the sputum culture was positive for mycobacterium tuberculosis ( mtb ) . the interferon alpha release assay ( qft - g ) is a new diagnostic test for latent tuberculosis infection ( ltbi ) . qft - g is similar to the tuberculin skin test ( tst ) , but can not differentiate between ltbi and active tb . however , despite the limitation of qft - g in the diagnosis of active disease , it has been recommended by some investigators and it has been used in the diagnosis of active tuberculosis in the private sector . , using qft - g , was performed for the diagnosis of active tuberculosis in 13 studies , and there were 13 studies on cases with known active tuberculosis . the overall sensitivity of qft - g in the diagnosis of active tuberculosis was 6983% . another meta - analysis by dai y et al . revealed the overall sensitivity and specificity of qft - g in the diagnosis of active tuberculosis to be 85 and 84% . few other studies recommended the use of qft - g for ruling out active tuberculosis , especially in high - income countries where the prevalence of tuberculosis was low . legesse m et al . documented in their study that the sensitivity and specificity of qft - g , using the manufacturer 's cut - off value , was very low in the diagnosis of active tuberculosis in tuberculosis - endemic regions . the qft - g - tb gold kit ( cellestis limited , melbourne , australia ) was approved in 2009 for use in king abdulaziz medical city - riyadh ( kamc - r ) for the diagnosis of latent tb . local saudi guidelines on the management of latent tuberculosis did not recommend using qft - g for the diagnosis of active tuberculosis . the clinical use of qft - g has expanded dramatically in the diagnosis of tuberculosis disease . this expanded use was not based on local studies , which confirmed or refuted such practice in saudi arabia . it was clear that the use of qft - g in the diagnosis of active tuberculosis should be based on the local prevalence of the disease , local data , and many other logistic and resource considerations . the aim of this study was to assess the sensitivity and specificity of the qft - g test in the diagnosis of tuberculosis in patients admitted with pneumonia , when it was ordered to rule out tuberculosis . a retrospective study was conducted at kamc - r saudi arabia from january 2009 to december 2013 . the study included all patients admitted with a diagnosis of pneumonia , and qft - g was ordered to rule out active tuberculosis . all cases with pneumonia or a final diagnosis of tuberculosis , but with no qft - g results available , only patients aged 18 years or older with available qft - g tests were included in the analysis . the qft - g results were divided into three categories : positive , indeterminate , and negative . the procedures for the qft - g test in our hospitals have been explained in detail in our previous published study . the following data were extracted from the hospital records - demographic and clinical characteristics and qft - g results . descriptive statistics such as means and standard deviation , mean sd , were used to describe the age of the patients . frequencies and percentages n(% ) were used to describe the gender and the qft - g test results . the diagnostic performance of the qft - g test was assessed using sensitivity , specificity , and positive / negative predictive values . we used independent sample t - tests to compare the patients ages across the tuberculosis and pneumonia cases . the chi - square tests were used to assess the associations between the demographic characteristics and groups ( tuberculosis and pneumonia cases ) . the percentage of positive qft - g results in each disease group and gender group was assessed by the chi - square tests . we examined whether the age was significantly different between the positive and negative qft - g results of the two groups . statistical analyses were performed using sas 9.2 ( sas institute , cary , nc , usa ) . over the study period , 6907 patients were admitted with diagnoses of pneumonia , and 639 ( 9% ) of them were diagnosed with tuberculosis . the results of qft - g test were available in 368 patients : 142 ( 38.6% ) patients with active tb and 226 ( 61.4% ) patients with pneumonia . table 1 demonstrates the patients demographic and clinical characteristics in relation to the disease status . out of 368 patients , 188 ( 51.1% ) were female and 180 ( 48.9% ) were male . most of the 368 patients were saudis : 363 ( 98.6% ) . in both groups ( tuberculosis and pneumonia ) , the results of the qft - g tests were observed to be negative in 183 cases ( 49.7% ) , positive in 143 cases ( 38.9% ) , and indeterminate in 42 cases ( 11.4% ) . when the 368 patients were classified by the disease status , the qft - g test appeared to have a higher positivity rate among the tuberculosis cases ( 68.3% ) than the pneumonia cases ( 20.4% ) , p - value = 0.001 . the result of the qft - g test was observed to be negative in 33 cases ( 23.2% ) and indeterminate in 12 cases ( 8.5% ) in the tuberculosis group , whereas , the qft - g test was observed to be negative in 150 cases ( 66.4% ) and indeterminate in 30 cases ( 13.3% ) in the pneumonia group [ table 1 and figure 1 ] . the patients ages were significantly higher among the pneumonia group ( 64.4 17.4 , range 19107 years ) than the tuberculosis group ( 54.9 19.4 , range 1892 ) , p - value = 0.001 . there were no differences between males and females across the two groups ( p - value = 0.755 ) . demographic characteristics and quantiferon results in relation to disease status ( n = 368 ) diagnostic accuracy of the quantiferon test among cases with pneumonia or tuberculosis table 2 shows the patients demographic and disease status in relation to qft - g results , excluding 42 patients with indeterminate results . the 42 patients who had indeterminate results were excluded because no follow - up qft - g test was performed on these patients . the mean age for the negative qft - g ( n = 183 ) results was shown to be 62.50 18.0 years , which was significantly higher than that for the positive qft - g ( n = 143 ) results ( 57.7 19.7 years ) , p - value = 0.021 . there was no significant difference between the positive qft - g results comparing males ( 42.9% ) and females ( 44.7% ) , p - value = 0.749 . the positive results of qft - g were higher in patients with tuberculosis disease than in patients with pneumonia disease ( 74.6 vs. 23.5% , p - value = 0.001 ) . table 3 shows the sensitivity , specificity , and positive / negative predictive values of the qft - g test . the sensitivity of the qft - g test was 74.6% [ 95% ci : 66.09 to 81.65% ] , while its specificity was 76.53% [ 95% ci : 69.85 to 82.15% ] . the positive predictive value of the qft - g test was 67.83% [ 95% ci : 59.43% , 75.25% ] and the negative predictive value was 81.97% [ 95% ci : 75.46% , 87.10% ] . among 130 tuberculosis cases , the false negative rate was 25.4% ( 33/130 tuberculosis cases ) . among 196 pneumonia cases , the false positive of the qft - g test was 23.5% ( 46/196 pneumonia cases ) . furthermore , figure 2 shows how well the qft - g test separates the two groups , tuberculosis and pneumonia , with an area under the curve of 75.60% [ 95% ci : 70.1081.10% ] . demographic characteristics in relation to quantiferon results , excluding 42 patients with indeterminate results ( n = 326 ) diagnostic accuracy of the qft - g test , excluding 42 indeterminate results ( n = 326 ) receiver operating characteristic ( roc ) curve of qft - g test early diagnosis of active tuberculosis is essential to ensure early treatment and prevention of infection . sputum culture with microbiological confirmation remains the gold standard for the clinical diagnosis of active tb , but requires a long follow - up . qft - g held a promise to provide a rapid and reliable test to diagnose active tb . in this study , the sensitivity of qft - g for the diagnosis of tuberculosis was 74.6% and specificity was 76.53% . however , several studies subsequently showed only a modest sensitivity of the qft - g test to detect tb , although it performed better than tuberculin skin test ( tst ) . lai cc et al . reported 66% sensitivity and specificity ( 76% ) , which was comparable to our findings . in their meta - analysis of 26,680 participants , rangaka et al . concluded that qft - g did not have high accuracy for the prediction of active tuberculosis , although the use of qft - g in high - risk populations might reduce the number of people considered for preventive treatment . equally , the qft - g has suboptimal accuracy for confirming or ruling active tuberculosis disease . in a meta - analysis comparing commercial interferon - gamma release assays for detecting active tb , the authors found that commercial qft - gs were superior to tsts for detecting confirmed active tb disease , especially when performed in developed countries . the overall pooled sensitivity and specificity of quantiferon tb gold was 6985% and 5184% , which was similar to our study . a study from china reported a higher overall sensitivity of 81.4% and up to 86.6% in smear - positive cases , while having a specificity of 97% , which was comparable to our findings . another study from indonesia revealed the sensitivity of qft - g to be 88.7% in active tb cases , which was higher than our study . our study had three advantages : first it was from an intermediate tb - prevalent region and included large numbers of culture - positive , confirmed , tb cases , therefore , it was probably a better representation of the region , with an intermediate prevalence of tb compared to other studies . pre - test probability is a very important factor for a better interpretation of a positive qft - g test in cases of active tb suspicion . interferon- release assays can be used more reliably in excluding tb , particularly extrapulmonary , in patients originating from areas of low tuberculosis incidence . other factors influencing discordant results of the qft - g test in patients with active tb include older age , lower lymphocyte count , total protein and albumin levels , and high crp levels . in both groups , the elderly patients were more likely to have negative results of qft - g ( 62.50 18.0 years ) than positive results of qft - g ( 57.7 19.7 years ) . nevertheless , no gender differences between pneumonia and tuberculosis groups were found in our study . there was some evidence that the qft - g response could be diminished in cases with advanced tb . the advantage of our study was having a large number of patients with a confirmed culture positive tuberculosis the major limitations included the fact that it was a retrospective study with known limitations . we did not include tst as an additional test to the qft - g and we did not include pre - test probability of active tuberculosis when ordering the qft - g test . the indeterminate results of the qft - g tests were not common in tuberculosis ( 8.5% ) compared to pneumonia ( 13.2% ) , which was similar to other studies . it is imperative to indicate that there are two commercial qft - g tests ( tb gold and tb spot ) . they differ in their positive and negative predictive values , and the concordance of the two tests is modest in both immunocompetent and immunocompromised patients , for both latent and active tb . although our findings that quantiferon tb gold can be used as an adjunct tool in the diagnosis of active tb , it certainly can not be used solely and indiscriminately , separate from other clinical epidemiological and radiological factors , and the pre - test probability remains very important for the interpretation of these tests . there is considerable interest in understanding the role of vitamin d in cancer in general and in ovarian cancer in particular . experimental studies have shown that vitamin d administration reduces proliferation and promotes apoptosis in ovarian cancer cell lines and animal models [ 16 ] . ovarian cancer incidence and mortality rates are higher in northern latitudes , where sun exposure , which is required for the initiation of vitamin d synthesis in the skin , is lower [ 710 ] . an inverse association between dietary vitamin d intake and risk of ovarian cancer has been reported in one epidemiologic study , although others have not supported this finding [ 1216 ] . studies of the effect of dietary vitamin d intake are limited because estimation of dietary vitamin d intake does not capture cutaneous production of vitamin d. thus , circulating vitamin d , which reflects both cutaneous production and dietary / supplement intake [ 1720 ] , is considered the best indicator of overall vitamin d status . however , its concentration is about 1000 times lower than that of 25(oh)d in circulation , due to its shorter half - life and local production in target tissues , such as the ovaries [ 21 , 22 ] . thus , 25(oh)d is thought to better reflect overall vitamin d status than 1,25(oh)2 d [ 23 , 24 ] . to date , only one epidemiologic study has examined the relationship between prediagnostic levels of vitamin d and risk of ovarian cancer . did not find an association of ovarian cancer risk with the 25(oh)d or 1,25-dihydroxyvitamin d ( 1,25(oh)2d ) plasma levels . polymorphisms in the vdr gene may influence the ability of 1,25(oh)2d to activate vitamin d target genes , including those involved in growth regulation and apoptosis . in a previous manuscript , we reported an overall null association between four common polymorphisms in the vdr gene ( bsm1 , apa1 , taq1 , and fok1 ) and risk of ovarian cancer , although a retrospective multiethnic study of 313 cases and 574 controls and a large pooled analysis including 1473 cases and 2006 controls from four studies ( one retrospective case - control study and three case - control studies nested in prospective cohorts ) found an association with the fok1 snp among caucasian women . mixed results for this and other vdr snps may be due to chance findings or limited power to detect associations . it is also possible that genetic variants influence ovarian cancer risk differently , depending on an individual 's vitamin d status . the objective of the present study was to examine the relationship between circulating levels of 25(oh)d and risk of invasive epithelial ovarian cancer and to assess the combined effect of circulating 25(oh)d and vdr polymorphisms on ovarian cancer risk . descriptions of the university of ume northern sweden health and disease study ( nshds ) and the new york university women 's health study ( nyuwhs ) have been provided previously [ 2931 ] . briefly , since 1985 the nshds has enrolled approximately 53 756 women aged 3065 through local health promotion intervention programs in northern sweden . the nyuwhs enrolled 14 274 healthy women aged 3465 years at a breast cancer screening center in new york city between 1985 and 1991 . each study cohort collected information about medical history , reproductive history , family history of cancer , medications , smoking history , and diet during enrollment and/or followup . blood collected at enrollment and any subsequent visits was processed according to standardized procedures within each cohort . in the nshds cohort , blood was collected , centrifuged , and plasma aliquots were frozen at 80c and transferred within 1 week to a 80c central storage facility ( the northern sweden medical research biobank ) . in the nyuwhs cohort , blood was drawn , collection tubes were kept covered at room temperature ( 2125c ) for 15 minutes , then at 4c for 60 minutes to allow clot retraction , and then centrifuged for 25 minutes . after centrifugation , serum samples were divided into aliquots and immediately stored at 80c at the local site . participants who reported being pregnant or using exogenous hormones within 6 months of enrollment were not eligible for the nyuwhs cohort or for case - control selection from nshds . in the nshds , cohort linkages to regional and national cancer registries and to all - cause mortality registries were used to capture cases of incident invasive epithelial ovarian cancer . in the nyuwhs cohort , case ascertainment was achieved through self report on followup questionnaires and through linkages with state tumor registries in new york , new jersey , and florida . medical records were obtained to verify reported events . as of november 1 , 2005 , a total of 192 invasive ovarian cancer cases ( 107 from the nshds and 85 from the nyuwhs ) had been identified . twenty - two nonepithelial ovarian cancer cases ( 8 from the nshds and 14 from the nyuwhs ) were excluded from this study because they did not meet the criteria of being invasive epithelial ovarian cancer . for the current study , there were a total of 170 invasive eoc cases ( 71 from nyuwhs and 99 from nshds ) . however , two cases from the nshds cohort had only dna available for analysis , and were only included in the main vdr genotyping analysis . for each case , two controls were randomly selected from cohort members who were alive and free of cancer at the time of diagnosis of the case . controls were also matched to the case on cohort ( nyuwhs or nshds ) , age at entry ( 6 months ) and date of blood donation ( 15 days ) . seventeen cases from the nyuwhs cohort had only one eligible matched control because 10 controls had a complete bilateral oophorectomy before the diagnosis date of the case and seven cases could not be sufficiently closely matched to a second control on date of blood donation ( strict matching is required to control for seasonal effects of vitamin d ) . of the potential 323 eligible controls in total ( 198 from the nshds , 125 from the nyuwhs ) , seven were excluded from the nsdhs because either the control or matching case did not have plasma available . the institutional review boards of new york university school of medicine and the regional ethical committee of the university of ume , sweden , and the swedish data inspection board reviewed and approved this study . serum 25(oh)d was measured at the university of ume using a gamma - b 25-hydroxy vitamin d radioimmunoassay ( immunodiagnostic systems ( ids ) , inc . ) . acetonitrile extraction of 25(oh)d was followed by incubation with radioionidated tracers and sheep antibodies to 25(oh)d . samples from each cohort and case - control set were assayed together in the same laboratory batch . quality controls and study samples were distributed randomly throughout the batches and the laboratory personnel were blinded to the samples ' case - control status . the intra- and interbatch coefficients of variation were 17.7% and 24.6% for nshds , respectively . for nyuwhs , the intra- and interbatch coefficients of variation were 16.6% and 16.2% , respectively . 25(oh)d values were log 2 transformed to reduce departures from the normal distribution ( the log 2 transformation is useful for estimating odds ratios associated with a doubling in vitamin d levels ) . linear regression and anova were used to test whether 25(oh)d levels differed according to baseline characteristics . conditional logistic regression , which allows for the matched design , was used to evaluate the association between serum concentration of 25(oh)d and risk of eoc . because cases were individually matched to controls for laboratory batch and date of sampling ( i.e. , season ) , these variables were controlled for by design when 25(oh)d was modeled as a continuous variable . to create meaningful tertiles of 25(oh)d for categorical analysis , however , the 25(oh)d values needed to be laboratory batch and season , to prevent individuals from being arbitrarily included in a tertile simply on the basis of their season of blood draw or inclusion in a particular batch . the adjustment for laboratory batch was done by first regressing the log - transformed 25(oh)d values on season , age , and bmi ( the three variables that were associated with 25(oh)d ) and computing the residuals within each cohort . we then computed the mean residual for each laboratory batch and subtracted the appropriate batch mean from each individual 's log - transformed vitamin d value to center each batch at the grand mean for each cohort . we then adjusted for seasonal effects by performing a nonparametric local regression ( proc loess , sas ) of 25(oh)d values on day of the year of blood donation and used the residuals to create cohort - specific tertiles . potential confounders , specifically reproductive history ( parity , number of full - term pregnancies , and age at first full - term pregnancy ) , age at menarche , menopausal status at enrollment , history of oral contraceptive use at baseline , body mass index at enrollment , and smoking status at baseline ( never , current , former ) were considered in the logistic regression models . however , the only variables significantly different between cases and controls were parity and oral contraceptive use ( both categorized as ever / never ) . therefore , multivariate models included only these two variables ( age and date of blood donation which were controlled for by the matched design and use of the conditional logistic regression model ) . an indicator variable for sample type ( serum versus plasma ) also was examined as a potential interaction term . we also conducted analyses excluding individuals diagnosed within five years of blood donation , and evaluated the vitamin d - ovarian cancer relationship within histological subtypes , and by stage ( i - ii versus iii - iv ) , grade , bmi ( dichotomous variable < 25 versus 25 ) , oral contraceptive use ( ever versus never ) , and vdr snp genotype ( assuming a codominant model ) . haplotypes were estimated from genotype data using phase version 2.1.1 ( http://www.stat.washington.edu/stephens/phase.html ) and odds ratios for the interaction between having zero , one , or two copies of the haplotype and 25(oh)d levels were determined using conditional logistic regression . we used sas software ( version 9.1 , sas institute , cary , nc ) for all statistical analyses . the characteristics of the study participants from each cohort at baseline have been described previously . the median age at enrollment was 52 and 55 years for nshds and nyuwhs participants , respectively . nshds cases had a median of 4.6 years between blood donation and diagnosis and nyuwhs cases were diagnosed an average of 7.0 years after blood donation . nshds cases were less likely than nshds controls to have ever been pregnant ( 78% versus 89% , p = .01 ) and to have ever taken oral contraceptives ( 33% versus 45% , p = .03 ) . nyuwhs cases and controls were not significantly different with regard to their baseline characteristics . regarding differences between cohorts , nyuwhs controls were more likely than nshds controls to be nulliparous ( 31% versus 11% , p < .0001 ) , more likely to be premenopausal at baseline ( 42% versus 28% , p = .01 ) , less likely to have used oral contraceptives ( 32% versus 45% , p = .03 ) , more likely to have smoked ( 61% versus 43% , p = .01 ) , and had higher 25(oh)d levels ( 45.8 versus 39.4 nmol / l , p = .04 ) . cases and controls did not differ with regard to genotype frequency at any of the four vdr snp sites for either of the cohorts . table 2 shows 25(oh)d by characteristics of controls for the nshds and nyuwhs cohorts . in both cohorts , median 25(oh)d levels were higher in women who were older at enrollment and in women who had lower bmi , and were somewhat higher in women who were ever parous and had used oral contraceptives . however , the two cohorts differed with regard to smoking status : in the nyuwhs , current smokers had higher median 25(oh)d levels ( 50 nmol / l in current versus ~45 nmol / l in former and never smokers ) , while smoking in the nshds showed a somewhat opposite relationship with 25(oh)d ( ~38 nmol / l in current and former versus 40 nmol / l in never smokers ) . 25(oh)d was only modestly related to vdr genotype in the nshds cohort at the bsm1 ( p = .16 ) and taq1 ( p = .19 ) loci ( table 3 ) . in both cohorts , women with two copies of the bat haplotype ( associated with the bsm1 g , apa1 g , and taq1 t allele combination ) had higher levels of circulating 25(oh)d , although the tests for trend were not statistically significant ( table 3 ) . we did not observe an overall association between vitamin d and ovarian cancer when 25(oh)d was modeled on the continuous ( multivariate adjusted or for a doubling in vitamin d levels = 1.1 , 95% ci : 0.71.7 ) or categorical scale ( table 4 ) . there was some evidence of interaction between 25(oh)d and cohort ( p = .07 ) , though cohort - specific odds ratios do not support an association with risk in either cohort ( table 4 ) . the lack of association between vitamin d and ovarian cancer remained after controlling for parity and oral contraceptive use . we did not observe any significant differences in the relationship between 25(oh)d and ovarian cancer by season of blood collection , bmi ( < 25 versus 25 kg / m ) , menopausal status at enrollment , vdr snp genotype or haplotype , tumor stage ( i - ii versus iii - iv ) , grade , or histological subtype ( serous versus mucinous , clear cell , and endometriod ) ( data not shown ) . odds ratios for 25(oh)d and risk of ovarian cancer did not change appreciably in analyses restricted to 74 matched sets in which cases were diagnosed five or more years after blood donation ( or for the highest versus lowest tertile = 0.9 , 95% ci : 0.461.72 ) . in the current study , we did not observe an association between circulating levels of 25(oh)d and risk of epithelial ovarian cancer . the lack of an association remained after controlling for potential confounders , excluding cases diagnosed within 5 years of blood donation , and considering subgroups defined by genetic variation in the vdr , tumor characteristics ( i.e. , stage , grade , and histological subtype ) , menopausal status , and bmi . initial support for a protective role for vitamin d was provided by ecological studies showing an inverse association between uv - b exposure and ovarian cancer incidence or mortality rates [ 710 ] . however , research conducted with this type of study design can not elucidate the temporal association between exposure and disease . case - control and cohort studies of dietary vitamin d intake and risk of ovarian cancer have been inconsistent [ 1116 ] . the results of our study are in agreement with the overall results of tworoger et al . , the only other prospective study of ovarian cancer to report on circulating 25(oh)d to date . our study similarly found that the lack of association between serum 25(oh)d and risk of ovarian cancer did not differ by vdr genotype . however , tworoger et al . found an inverse association for 25(oh)d among women with bmi 25 kg / m , which was not observed in the current study . consistent with previous studies [ 3337 ] , higher bmi was associated with lower circulating 25(oh)d in both cohorts . vitamin d is fat - soluble and studies in obese persons and mice have shown increased storage and decreased bioavailability of vitamin d in fat cells [ 38 , 39 ] . we also observed that parity was associated with higher 25(oh)d ; which is in agreement with a similar observation in the plco cancer screening trial . contrary to some previous reports [ 37 , 40 , 41 ] , we observed a positive relationship between age and 25(oh)d , which showed increased levels of vitamin d in the 5060 year age group as compared with younger women . this may be due to an increased intake of calcium and vitamin d - rich foods and supplements around the time of menopause in an effort to prevent or treat osteoporosis . oral contraceptive use was associated with higher median 25(oh)d levels in our study ; previous studies have found a similar relationship in current oc users [ 17 , 42 , 43 ] . we also found that the bsm1 g , apa1 g , and taq1 t haplotype , in the vdr gene was modestly associated with higher 25(oh)d . publications on the relationship between vdr genotype and levels of 25(oh)d have been fairly inconsistent . in the physicians health study cohort , the bsm1 genotype was not related to total 25(oh)d levels , although the bb genotype was associated with higher index of free 25(oh)d and with higher 1,25(oh)2d levels in older men . the bb genotype was also associated with higher 25(oh)d in a small finnish study ( n = 93 ) that collected all blood samples in the winter when skin synthesis of vitamin d is very low . however , a large study of 2845 women found no association between any vdr snps genotype and 25(oh)d . individuals with two copies of the bat haplotype ( bsm1 g , apa1 t , and taq1 t ) had lower levels of 25(oh)d than individuals with 0 or 1 copy ( p - trend = .09 ) . individuals without any copies of bat ( bsm1 g , apa1 g , and taq1 t ) in the current study ( which would include all individuals with two copies of the bat haplotype ) had lower 25(oh)d levels , thus providing indirect support for the previous finding . this study limitations include the use of only one sample to determine each participant 's 25(oh)d status . however , in a preliminary reproducibility study of 25(oh)d among 16 nyuwhs participants with at least 3 annual visits , the intraclass correlation coefficient was quite high ( 0.71 ) , suggesting stability of this metabolite from visit to visit , which has been observed by others [ 47 , 48 ] . the relatively high coefficient of variation for the 25(oh)d assay adds random error to the measurements and may have attenuated the association . although we had sufficient sample size to detect small differences between cases and controls ( a priori , we had 80% power to detect a mean difference of 6 nmol / l between cases and controls ) , we had limited ability to explore interactions . previous studies have demonstrated that vitamin d deficiency is more common in europe than the united states , and that in the united states , the latitude of residence is inversely associated with vitamin d ( reviewed in ) . we anticipated that the ranges of 25(oh)d values in the new york and northern sweden populations would be lower than those reported from lower latitudes [ 50 , 51 ] . in the highest tertile , nmol / l for nyuwhs and 51.6 nmol / l for nshds ; somewhat lower than the 75 nmol / l , that is , considered to be optimal for multiple health outcomes . nmol / l , thus we can not rule out the possibility that women with very high 25(oh)d levels may have a reduced risk of ovarian cancer . strengths of the study include the use of samples collected before diagnosis , limiting the potential for existing disease to influence vitamin d levels , the use of circulating 25(oh)d as a composite measure of vitamin d overall status , careful consideration of seasonal effects , and the use of a nested case - control design , which helps ensure that the controls are comparable to the cases . in conclusion , our findings do not provide support for the hypothesis that circulating vitamin d levels are associated with risk of invasive epithelial ovarian cancer later in life . larger studies are needed to evaluate gene - environment interactions and potential subgroups which may benefit from vitamin d chemoprevention .

background : the utility of quantiferon - tb gold in - tube ( qft - g ) test in the diagnosis of tuberculosis disease has been validated in high and low tuberculosis - prevalent ( tb ) countries . aim : the aim of this study is to assess the performance of the qft - g test in the diagnosis of tuberculosis disease among tuberculosis patients in an intermediate prevalent country.setting and design : a retrospective study at the king abdulaziz medical city - riyadh ( kamc - r)materials and methods : we retrospectively reviewed all the patients with a diagnosis of pneumonia , including tuberculosis , admitted to kamc - r between 1 january 2009 and 31 december 2013 . we included only patients with an available result of the qft - g test . a total of 142 tuberculosis cases and 226 pneumonia cases were studied , to assess the utility of the qft - g test in diagnosing tuberculosis cases.results:among the tuberculosis ( n = 142 ) cases , the qft - g tested positive in 68.3% , negative in 23.2% , and indeterminate in 12 cases ( 8.5% ) . of the 226 pneumonia cases , the qft - g tested positive in only 20.4% , while a majority of 66.4% tested negative , with 30 cases ( 13.3% ) being indeterminate . when we excluded 42 patients with indeterminate results , the qft - g test achieved a sensitivity of 74.6% [ 95% ci : 66.09 to 81.65% ] and specificity of 76.53 % [ 95% ci : 69.85 to 82.15% ] in the diagnosis of tuberculosis cases.conclusions:this study concludes that the qft - g test is a useful tool for detecting tuberculosis disease when used as an adjunct tool for the diagnosis of active tb cases . it certainly can not be used solely and indiscriminately , separate from other clinical and radiological information , in the diagnosis of active tuberculosis cases . we conducted a nested case - control study within two prospective cohorts , the new york university women 's health study and the northern sweden health and disease study , to examine the association between prediagnostic circulating levels of 25-hydroxy vitamin d ( 25(oh)d ) and the risk of subsequent invasive epithelial ovarian cancer ( eoc ) . the 25(oh)d levels were measured in serum or plasma from 170 incident cases of eoc and 373 matched controls . overall , circulating 25(oh)d levels were not associated with the risk of eoc in combined cohort analysis : adjusted or for the top tertile versus the reference tertile , 1.09 ( 95% ci , 0.592.01 ) . in addition , there was no evidence of an interaction effect between vdr snp genotype or haplotype and circulating 25(oh)d levels in relation to ovarian cancer risk , although more complex gene - environment interactions may exist .

for a century , the cardinal features of alzheimer 's disease ( ad ) , amyloid plaques and neurofibrillary tangles , were thought to underlie this chronic neurological disorder . however , based on the evidence accumulated over the past ten to fifteen years , the toxicity of these lesions has been questioned . instead , the emerging soluble aggregation - intermediate forms of amyloid - beta ( a ) and tau proteins , which compose plaques and tangles , are now believed to underlie the synaptic and neuronal losses observed in ad . studies focusing on oligomeric a assemblies [ 14 ] have paved the way for other amyloid proteins including tau , alpha - synuclein [ 68 ] , and the prion protein prp in the field of neurodegenerative disorders . this principle simply revolutionized our understanding of ad , parkinson 's disease , frontotemporal dementias , and prion diseases , opening new avenues for therapeutic strategies . in what might seem like an all rosy affair , this paradigm shift also contributed to complicating even more the putative sequence of biological events responsible for these diseases . in ad , the classical view of the amyloid hypothesis postulated that amyloid plaques are altering the physiological function of neurons , which in turn disrupts tau biology leading to the demise of the cell . the modern view of the amyloid hypothesis suggests the involvement of a multitude of endogenous bioactive a molecules that include a dimers , trimers , a*56 , annular protofibrils , and amyloid plaques , as opposed to a single culprit ( i.e. , plaques ) . this notion appears to be consistent with the myriad cell surface receptors and signaling pathways that have been described as specifically activated by putative endogenous soluble a oligomers . if this scenario was not entangled enough , numerous studies aiming at elucidating the function of oligomeric a ( oa ) use oligomeric preparations of synthetic a peptides whose folding conformation and posttranslational modifications might not accurately reflect to these found in biologically relevant systems ( i.e. , brain , cerebrospinal fluid , blood , and primary neurons ) . in the end , this increased complexity of the problem coupled with a lack of adequate experimental descriptions of the oa used and specific detection tools ( e.g. , antibodies specific to a single a assembly ) renders interpretation and comparison of the observed phenomena between different research groups arduous and impedes on our progress to better understand the role of a oligomers in ad . a clear example of this issue plaguing our field is illustrated by the controversial debate surrounding the role of the cellular form of the prion protein ( prp ) in mediating the deleterious effects of oligomeric a. in 2009 , lauren and colleagues reported that prp was acting as a receptor for synthetic a oligomers also called a-derived diffusible ligands ( addls ) [ 13 , 14 ] . addls have been characterized by denaturing electrophoresis ( sds - page ) , transmission electron microscopy ( tem ) and size - exclusion chromatography ( sec ) coupled with static light scattering ( sls ) , but each technique generated inconsistent and contradicting results . first , addls ran as an undefined smear ranging from ~25 to 200 kda using sds - page followed by western blotting with the sole 6e10 antibody detecting a116 . additional bands were detected as putative monomers , trimmers , and tetramers in the addl preparation but since these same immunoreactive bands were also detected in freshly resuspended synthetic a peptides , they are likely a result of the presence of sds in the experimental conditions . tem revealed that addls contained spheroidal structures of various sizes ; the most abundant form appeared to correspond to 5 - 6 nm spheroids . it is important to note that short filamentous structures were also clearly visible possibly corresponding to protofibrils . finally , liquid phase chromatography coupled with sls revealed the presence of only two elution peaks under nondenaturing conditions , a broad trailing peak detected shortly after the void volume containing a molecules of ~500 kda mass and a well - defined sharp peak corresponding to monomeric a peptides . the authors concluded that the preparation of addls used was approximately made of an assembly composed of 50 to 100 a monomers . based on the aforementioned data , it seems reasonable to conclude that these addls are not stable under denaturing conditions as previously reported and that the exact composition of the synthetic a oligomers used remains inconclusive . despite the apparent inconsistency of the observations characterizing the oa used in this study , prp appeared to be necessary to mediate the inhibition of long - term potentiation ( ltp ) induced by oa . as expected , this study stimulated several independent groups to reproduce these findings using various sources and preparations of a [ 1720 ] . a team led by gianluigi forloni first reported that prp was not required to mediate the cognitive impairments induced by synthetic a oligomers . synthetic a peptides were prepared to generate addls following the same groundwork established by william klein and his colleagues at northwestern university [ 13 , 21 ] . analyses using atomic force microscopy ( afm ) and sec defined the addls and obtained and confirmed the presence of mixed structural species ( i.e. , spherical assemblies and protofibrils ) by afm and the presence of two elution peaks following sec ( a sharp peak close to or within the void volume and a smaller peak containing putative a monomers ) . while these elements could suggest at first glance that the addls generated at yale and at the mario negri institute are similar , it bears to mention here that the columns used in both studies greatly differed ( a sequential connection of superdex 200 , superdex 75 , and superdex peptide , 10/30 , hr sec columns for the yale group and a single superdex 75 column for the italian group ) raising the possibility that in fact both addl preparations were different . to further demonstrate the involvement of prp in a-induced deficits , the role of prp was examined in middle - aged appps1e9 transgenic mice used to model alzheimer 's disease expressing or deficient for the prnp gene . gene deletion of prnp had no apparent effect on soluble and insoluble monomeric a levels as measured by western blot analyses using 6e10 despite a ~20% reduction in amyloid burden , indicating potential discrepancies in a measurements and quantification . behaviorally , ablation of prnp resulted in rescuing synaptic loss , app - induced premature mortality , and spatial learning and memory compared to appps1 mice . puzzlingly , ca1 ltp was not altered in appps1e9 hippocampal slices , possibly suggesting that the endogenous a species responsible for ltp inhibition are not present or that these mice might develop homeostatic compensations in response to synaptic injury induced by a. in addition to the apparent inconsistency in the a levels , the nature and characterization of the a molecules in 12-month - old appps1 and appps1xprnp were not mentioned , begging the question as to whether the same a species initially found to interact with prp are the same as the hypothesized a oligomers present in vivo . a few months later , two independent studies published at the same time challenged the conclusions that prp is a mediator of a toxicity [ 18 , 19 ] . prp was not found to be required for a-induced synaptic deficits in hippocampal slices transfected with a carboxyl terminal domain of the amyloid precursor protein appct100 and for addl - induced ltp inhibition . in the former paradigm , it is unknown whether oligomeric a species are present in appct100-expressing slices [ 19 , 24 ] , and if they were , the information pertaining to their characterization was not discussed . in the second experimental condition although the method used to generate addls was identical to the one used by lauren and coworkers , gene deletion of the prnp gene failed to rescue the ltp inhibition induced by addls . it is important to note that the characterization of the a oligomers formed only included one western blot analysis with an unspecified antibody following sds - page and revealed the presence of a poorly resolved smear ranging from ~35 to ~180 kda and monomers . in addition , the concentration at which the mixtures were used ( 1 m ) was greater than those used by the original study ( 20200 nm ) , possibly adding an additional confounding factor when comparing the experimental designs . due to the absence of data describing the aggregation state of the a used in these paradigms , it is difficult to conclude that the results presented invalidate the findings of the initial study by laurn et al . . the role of prp in mediating a-induced ltp deficits was investigated in hippocampal slices of 2 to 4-month - old appps1l166p mice that were genetically manipulated to express 2 , 1 , or 0 copies of the prnp gene . contrary to earlier findings , ltp was impaired in an age - dependent fashion in appps1l166p slices , but prnp copy numbers did not influence the observed ltp deficits . neither full - length app and carboxyl - terminal fragments of app ctf and ctf nor soluble a42 levels were altered by prnp genotypic differences indicating that prp does not alter app / a metabolism in this mouse model . despite these rigorous analyses of app derivatives , the exact nature and relative abundance of soluble a assemblies present in 4-month - old appps1l166p mice were not addressed . in light of these disparate observations , nature neuroscience published an editorial in april 2011 entitled state of aggregation which reiterated the critical need to clearly describe the initial state of the protein , its source , and its stoichiometry in order to maximize the success of independent groups that want to reproduce observed phenomena . shortly thereafter , freir and colleagues confirmed that prp is required for ltp inhibition induced by addls and by protein lysates of ad brain tissue containing a . a major reason as to why this study stood out relies on the fact that synthetic oa preparations were carefully characterized by sec , analytical ultracentrifugation , electron microscopy , and by sds - page and that all techniques produced results that were intrinsically consistent . sec and auc analyses of addls and biotinylated addls ( baddls ) confirmed the presence of 2 peaks reminiscent of these described by laurn et al . however , leading and trailing shoulders in the sec elution peaks were observed suggesting the presence of species ranging from 90 to 400 kda in the mixture , which was confirmed by auc . astutely , the authors also noticed that the addition of a biotin residue to a artificially enriched the abundance of high - molecular weight species compared to unbiotinylated addls . using em , both spherical and short filamentous structures were observed consistent with the profile obtained in the original study . finally , sds - page followed by 6e10 immunoblotting analyses confirmed that addls are not sds resistant and predominantly migrate as experimental artifacts as a monomers , dimers , trimers , and tetramers following denaturation . when this mixture was applied to hippocampal slices , altogether , based on these biophysical observations , prp appears to be mediating the inhibition of ltp induced by one or several unidentified synthetic a oligomers . more importantly , a similar rescue of ltp inhibition was observed in prnp slices when tris - buffered saline ( tbs ) soluble protein extracts from an ad brain were applied . biochemical analysis of tbs fractions from ad and control brains by immunoprecipitation / western blotting revealed the presence of putative sds - stable a dimers ( ~7 kda ) and monomeric a in ad tbs extracts , while no a species were detected in control tbs lysates . it is difficult to determine whether other a assemblies were present as there was substantive nonspecific background in the no protein condition ranging from 18 to 80 kda and because only one antibody was used to detect a ( presumably 6e10 ) . integrating the observations from the studies mentioned above , it seemed reasonable at the time to conclude that prp is required for the inhibition of ltp induced by a mixture of soluble brain - derived a species . after two years of intense investigation , we still did not know the answers to the most crucial questions related to oa if one aims to use this knowledge to develop diagnostic and therapeutic tools : ( 1 ) which endogenous a assembly is binding to prp ? ( 2 ) where is this interaction occurring ? ( 3 ) when do endogenous oa engage prp ? ( 4 ) how does prp mediate the deleterious effect(s ) of oa ? we sought to answer these questions combining in vivo experiments using human , transgenic mouse brain tissues and in vitro paradigms using primary neurons derived from various mouse lines . to ascertain the relevance of the study , all soluble a species were purified from human ad brain tissue or conditioned media of transgenic cortical neurons in liquid phase experiments ( i.e. , immunoaffinity capture in suspension followed by sec ) and characterized by immunoprecipitation / western blot using a panel of 4 antibodies detecting the n - terminal region ( 6e10 ) , the central domain ( 4g8 ) , or the c - termini of a ( 40- and 42-end specific antibodies mab2.1.3 and mab13.1.1 , kind gifts from pritam das , mayo jacksonville ) . in a reproducible fashion , we isolated endogenous a monomers , dimers , trimers , a*56 , and protofibrillar species migrating at ~175180 kda in absence of any additional detectable a species using our panel of a antibodies . of note , we also used the oligomer - specific antibody a11 to further confirm the nature of human a*56 ( data not shown ) . moreover , none of the purified soluble a species displayed aberrant migration profiles induced by sds - page analysis ( i.e. , apparent monomers , dimers , trimers , and tetramers comigrating in the same lane ) , and all soluble a captured were eluted at the predicted molecular weight during sec , arguing against the possibility that the assemblies detected are gel artifacts . finally , putative a dimers and trimers could be found in the conditioned medium of primary mouse cortical neurons expressing the swedish mutant form of human app disproving that these apparent a oligomers are induced by lysis or the presence of detergents . because we thoroughly characterized and documented the initial or current state of the endogenous oa present in our biological specimens , we believed we could address the who / where / when / how . briefly , we identified that prp formed a complex with fyn / caveolin-1 in ad brain tissues and that a dimers were the only low - molecular oligomer that coimmunoprecipitated with this complex . using 84 human brain specimens from the religious orders study ( ros ) , we also demonstrated that both prp and active fyn ( phosphorylated at y416 , pfyn ) proteins were abnormally elevated in ad compared to age - matched controls and that fyn activation was correlated to prp expression levels . we next applied a mixture of oa purified from ad brain tissue containing a monomers , dimers , trimers , a*56 , and protofibrils onto protein extracts enriched in membrane proteins derived from control subjects with no detectable a species . upon prp pulldown , only a dimers were visibly captured further validating the coimmunoprecipitation findings previously obtained using ad brain . to determine where oa could interact with prp , we performed triple - labeling immunofluorescence colocalization experiments using sections from ad and control brain and confocal imaging and image reconstruction . soluble a was identified as punctae along the neuronal processes , colocalized with prp at dendritic spines in ad but not control brain tissue , which accounted for ~22% of oa present at dendritic spines labeled with fyn . although the data were slightly higher ( ~36% ) , analyses performed on tg2576 primary cortical neurons expressing a monomers , dimers , and trimers generated similar results . importantly , pfyn was also observed to colocalize with a and prp most notably at synaptic varicosities traditionally considered to reflect alterations in microtubule organization . since tau is a microtubule - associated protein and believed to mediate a-induced deficits , we analyzed tau phosphorylation status and cellular localization when prp / fyn / oa were engaged into forming an active complex . consistent with the synaptic varicosities , tau was hyperphosphorylated at y18 , a well - known target phosphorylation site for fyn , and abnormally accumulated at postsynaptic sites reminiscent of phenomena associated with synaptic dysfunction [ 30 , 31 ] . it then appeared that a dimers could bind to prp engaging the activation of fyn at dendritic spines , but knowing when this pathological event took place remained unknown . to address this question , we examined the role of aging on oa in appps1l166p mice . in 2-month - old appps1l166p , in contrast , very abundant a monomers and putative a dimers and trimers were observed at 14 months of age . these results were consistent with earlier reports considering that a dimers are associated with plaques [ 4 , 32 ] and that amyloid deposition occupies ~10% of the neocortical areas at 8 months in appps1l166p mice . further supporting the hypothesis that a dimers activate the prp / fyn complex , fyn activation was remarkably elevated in aged appps1l166p mice while undetectable in young animals . in addition , the electrophoretic migration pattern for oa did not appear to differ substantially between appps1l166p mice expressing prp and appps1l166p mice deficient for prnp ( appps1l166pxprnp ) . as predicted by our hypothesis , fyn phosphorylation was reduced by ~50% at 14 months of age in appps1l166pxprnp mice suggesting that oa , and presumably a dimers induced the activation or prp / fyn in aged appps1l166p mice when amyloid burden is well established . finally , we sought to establish how prp mediated the effects of oa. to this end , we applied isolated a monomers , dimers , trimers , a*56 , and protofibrils at equimolar concentrations ( 5 nm ) onto primary cortical neurons . since a trimers did not appear to interact with prp based on our coimmunoprecipitations , our results pointed to a dimers as the major soluble endogenous a ligand for prp in vitro . these findings were also in agreement with our in vivo data showing that prnp gene deletion partly abolished fyn activation in aged appps1l166p mice . tau , known to mediate a-induced deficits , was hyperphosphorylated at y18 in neurons treated with a dimers and trimers . in aged appps1l166p mice , removing both copies of prnp diminished tau hyperphosphorylation by ~40% and missorting by ~65% compared to appps1l166pxprnp mice . in contrast , overexpressing prp in appps1l166p mice ( appps1l166pxtga20 ) led to an ~60% increase in tau phosphorylation at y18 and 80% in tau missorting to the postsynaptic density . accompanying this apparent potentiation of the prp / fyn pathway activation in old appps1l166pxtga20 mice , the expression of postsynaptic but not presynaptic proteins including the postsynaptic scaffold protein pds-95 was reduced by ~35% adding weight to the suggestion that increasing prp expression was potentiating a dimer - induced toxicity in vivo . the publication of our study was preceded by a few months by a study from the strittmatter group whom reported that oa binds to postsynaptic prp to activate fyn and impair neuronal function . here , synthetic oa were used as previously described as well as tbs - soluble extracts from individuals diagnosed with ad . despite using 4 antibodies to identify prp - oa complexes ( namely , 2454 , 82e1 , nu-4 , and ab5306 ) on immobilized prp molecules , the characterization of the species detected with these antibodies in both preparations was not documented thereby hampering our ability to put clothes on the emperor to borrow the expression employed by benilova et al . . instead , i am convinced that we , as a field , need to dedicate more efforts into better defining what oligomeric amyloid species are employed if we want to leapfrog towards a more comprehensive knowledge of the disease . i think we can do better than describing a subset of peptide with deleterious actions on neurons and synapses . a recent study from the ashe and lesn groups provides support to the need of distinguishing oligomeric forms of a from each other as opposed to considering them as a pool of molecules triggering the same biological effect . if correct , the findings suggest that the mixture of soluble a species present in the continuum of aging ad is evolving contrasting with the determined mixture of synthetic oa preparations . specifically , a*56 was most prominent in preclinical phases of ad , a trimers were elevated in early symptomatic phases ( i.e. , mild - cognitive impairment ) , and a dimers dominated in late symptomatic phases of ad . if longitudinal studies can confirm these changes , knowing the pathophysiological function of each a oligomer in the brain could be crucial in designing therapeutic interventions . such vision could be envisioned particularly at a time when personal medicine is emerging and when our population is aging very quickly . in addition , another important advance in our knowledge of ad will be to decipher where each oligomeric a assembly is coming from , that is , intracellularly or extracellularly . for these reasons , i believe we should encourage better characterization of the soluble forms of a we use experimentally and pursue initiatives to develop new reagents specific to each oligomeric a assembly ( which might also allow us to identify the formation and location of a oligomers in situ ) in the hope that together we can soon break the code of the a oligomer enigma . physicians have long recognized that certain patients develop postoperative cognitive dysfunction ( pocd ) , especially after cardiac surgery . recently , as investigators have begun to examine the incidence of pocd after major non - cardiac , non - carotid , non - neurosurgical procedures , certain risk factors have been identified . monk and colleagues studied 1064 patients aged 18 years and older before and after non - cardiac surgery and found that pocd was present at hospital discharge in 117 ( 36.6% ) young ( 18 to 39 years ) , 112 ( 30.4% ) middle - aged ( 40 to 59 years ) , and 138 ( 41.4% ) elderly ( 60 years and older ) patients . the difference in incidence was significant between the age groups themselves and between each age group and 210 age - matched ( but not disease - matched ) controls . at 3 months after surgery , the presence of pocd , while similar between young and middle - aged patients and age - matched controls , was significantly higher in older patients as compared with older controls ( p 0.001 ) . the independent risk factors for pocd at 3 months identified by monk and colleagues included older age ( perhaps owing to a diminution in cognitive reserve ) , lower level of education , history of previous cerebrovascular accident without residual impairment , and presence of pocd at hospital discharge . postoperative delirium and increased use of opioid analgesics correlated with pocd at hospital discharge but not at 3 months . patients who had pocd were also at increased risk of death in the first year after surgery . other risk factors for the development of pocd include major but not minor surgery , a history of alcohol abuse in older patients , and postoperative changes in thyroid hormones as demonstrated by the occurrence of the euthyroid sick syndrome - reduced serum t3 and t4 concentrations without an increase in thyroid - stimulating hormone secretion - within hours after major surgery . modifications in thyroid hormone functioning may take place as a result of the psychophysical stress caused by surgery and the reduced conversion of t4 into t3 by a liver engaged in the metabolism of anesthetic drugs . as there is a possible association between thyroid abnormalities and alzheimer disease because of the interrelationship between thyroid hormones and the cholinergic system , which is selectively affected in alzheimer disease , postoperative changes in thyroid hormones may play a role in the pathogenesis of pocd . bickel and colleagues , in their study of 200 patients , 60 years and older , undergoing hip surgery , reported that postoperative delirium was a strong independent predictor of the development of subsequent cognitive impairment , subjective memory decline , and the need for long - term care . a similar correlation was noted by rudolph and colleagues between delirium and early ( 7 days ) but not late ( 3 months ) pocd in 1,218 patients , 60 years and older , undergoing elective non - cardiac surgery . kat and colleagues also reported that the risk of dementia or mild cognitive impairment in 112 hip surgery patients , 70 years and older , at 30 months after discharge was almost doubled in patients who had postoperative delirium ( 77.8% ) as compared with at - risk patients who had not developed delirium ( 40.9% ) . cerebral trauma ( even in the absence of symptoms ) and preoperative cognitive decline have also been reported to be risk factors for the development of postoperative delirium and pocd in the elderly . tan and colleagues investigated 103 patients who had pocd and 103 cognitively normal controls and reported that pocd was induced by the use of patient self - controlled intravenous analgesia ( pcia ) . a history of cerebral trauma was an independent risk factor for pocd induced by the use of pcia , but a high education level seemed to be protective . ggenur and colleagues , in their study of the cognitive function in 36 patients before and 4 days after major abdominal surgery , reported that pocd was associated with inferior sleep quality and more awakenings , but not with postoperative disturbances in circadian rhythm . interestingly , what has not been shown to correlate with the development of pocd is whether the patient had regional or general anesthesia [ 17 - 19 ] . deeper levels of general anesthesia , as assessed by the use of the bispectral index ( median of 50.7 versus 38.9 ) , however , were associated with better cognitive function , especially the ability to process information , 4 to 6 weeks after surgery . most of the studies of pocd , however , have methodological difficulties , as pointed out by newman and colleagues in their review of more than 40 studies . the definition used to classify individuals as having pocd was not standardized among investigators , nor was there uniformity in the number , range , and diversity of neuropsychological tests , their sensitivity to change and learning , and the intervals between testing . there was also no standardization in experimental design with variations in the type of surgery and anesthesia , presence or absence of controls , type of controls , and statistical methods used to analyze the data , all of which make comparisons among studies very difficult . a significant limitation of the studies of pocd is that they do not include a standardized preoperative neurological examination in addition to the neuropsychological testing . this makes it almost impossible to separate out the relationship between surgery and anesthesia and subsequent cognitive decline and death from the cognitive decline and death that occur among older adults without surgery . the relationship between aging and cognitive decline and mortality rate in the absence of surgery was demonstrated almost a decade ago . more recently , inzitari and colleagues developed a 15-minute neurological examination to elicit subtle but clinically detectable neurological abnormalities ( snas ) , defined as abnormalities demonstrated in the absence of patient complaints and not associated with any neurological disease . follow - up examination at 4 and 8 years revealed that patients who had three or more snas at initial evaluation had an increased risk of cognitive decline , cerebrovascular events , and death , possibly because the snas signaled a concomitant decline in cognitive reserve . the authors did not track which patients had surgery and anesthesia during the follow - up period . in light of the demonstration by inzitari and colleagues of the progression of neurological deterioration , it is essential that future studies of pocd include a uniform neurological examination of all patients and control subjects . the neurological examination must have good inter - rater agreement and reproducibility . without prior knowledge of snas , it is impossible to assign causation to the cognitive dysfunction that may occur after surgery and anesthesia . is it the normal progression of a condition , however subtle , that is present preoperatively ; is it the effect of surgery and anesthesia alone ; or have surgery and anesthesia worked in some way to hasten the progression of the preoperatively present condition ? clearly , whatever else the experimental protocols include to achieve uniformity and facilitate interstudy comparison ( age- and disease - matched controls in every study and uniformity of operation , anesthesia , neuropsychological tests , testing intervals , statistical analysis , and control subjects ) , neurological examination for both gross and subtle neurological abnormalities must be included too . even in the absence of pocd study protocols , preoperative information about snas would facilitate the discussion of informed consent when older adults are contemplating surgical procedures as neurological abnormalities , even in the absence of overt symptoms , have been correlated with pocd . thus it may be advisable for an sna score as developed by inzitari and colleagues with their brief neurological examination , to be included with the other vital signs measured in older adults before surgery . it is important to note that the studies of pocd are , as yet , in their infancy . until research protocols achieve sufficient power and uniformity , it will be difficult to draw conclusions applicable to patient care . as suggested by newman and colleagues , the very term pocd as a binary definition of what may be , in reality , a continuous process may require modification to reflect the necessity of examining cognitive change as a continuum that marches through discrete events such as surgery and anesthesia . this will necessarily influence the methodology by which changes in test performance are analyzed . while central nervous system dysfunction after surgery and anesthesia has been demonstrated to occur , especially in older adults , it is important to bear in mind that , as silverstein and colleagues point out , any illness requiring hospitalization may be associated with cognitive decline . this introduces the possibility that cognitive decline occurs as a concomitant of generalized illness rather than being causally related to surgery and anesthesia and speaks to the need for not only age - matched controls but also disease - matched and hospitalization - matched clinical cohorts as well . in addition , the observation made by a number of investigators that cognitive decline itself increases the risk of mortality in older adults further confounds interpretation of the correlation between pocd and mortality reported by monk and colleagues . newman and colleagues also direct our attention to the problem inherent in conducting adequately - powered studies in this era of scarce health care resources . they suggest that investigators develop a consensus regarding the experimental protocols to enable them to pool data across studies to achieve power in secondary analyses . even in the absence of uniform , adequately - powered studies of pocd for example , postoperative delirium , a correlate of pocd , is associated with increased intraoperative blood loss and postoperative transfusions , a postoperative hematocrit of less than 30% , and severe postoperative pain . the hospital elder life program has developed interventions to decrease the incidence and severity of delirium . these include the frequent presentation of orienting information , physical activity , cognitive stimulation activities , use of visual aids and auditory amplifying devices , sleep inducement through nonpharmacological methods , assistance with alimentation , geriatric - psychiatric consultations , and patient and family education .

departing from the original postulates that defined various neurodegenerative disorders , accumulating evidence supports a major role for soluble forms of amyloid proteins as initiator toxins in alzheimer 's disease , parkinson 's disease , frontotemporal dementias , and prion diseases . soluble multimeric assemblies of amyloid- , tau , -synuclein , and the prion protein are generally englobed under the term oligomers . due to their biophysical properties , soluble amyloid oligomers can adopt multiple conformations and sizes that potentially confer differential biological activities . therein lies the problem : with sporadic knowledge and limited tools to identify , characterize , and study amyloid oligomers , how can we solve the enigma of their respective role(s ) in the pathogenesis of neurodegenerative disorders ? to further our understanding of these devastating diseases , the code of the amyloid oligomers must be broken . postoperative cognitive dysfunction ( pocd ) refers to a deterioration in cognition noted to occur after surgery and anesthesia . recent studies have demonstrated a number of correlates and risk factors for this condition , although much remains to be elucidated in terms of the true incidence , etiology , prevention , and treatment .

although perioperative mortality from tonsillectomy and submucosal uvulopalatopharyngoplasty ( smuppp ) with tonsillectomy has decreased significantly , postoperative bleeding is still an important complication . the incidence of bleeding after tonsillectomy is approximately 0.5%-10% , with death occurring in 1 in 20,000 patients . primary bleeding refers to that occurring within the first 24 h postoperatively , and bleeding occurring 5 - 10 days after surgery has been classified as secondary or delayed bleeding . factors such as age , gender , and surgical technique have been evaluated as risk factors for posttonsillectomy bleeding . to date , no study has specifically compared the rate of bleeding after tonsillectomy and smuppp . we reviewed the medical records of all patients at our institution who underwent unipolar cautery tonsillectomy alone or in conjunction with smuppp during the last 10 years to compare the incidence of secondary , delayed postoperative hemorrhage between the two procedures . in this retrospective study , following approval from the institutional review board , we reviewed the medical records of 404 patients who underwent tonsillectomy and smuppp procedures at our institution between january 2001 and december 2010 . the patients were divided into two groups according to the surgical intervention they had gone through . group 1 , who underwent tonsillectomy , consisted of 198 patients ; group 2 , who underwent smuppp , consisted of 206 patients . patients younger than 18 years of age or those who underwent a tonsillectomy as part of the treatment of carcinoma of the head or neck were excluded from the study . all postoperative secondary , delayed ( 5 - 10 days after surgery ) hemorrhage which required intervention , such as electrocautery under local anesthesia or a second surgery under general anesthesia for both groups was recorded . it is based on standard surgical principles of epithelial preservation and tension - free closure of the epithelium . most of the mucosa of the soft palate and the anterior and posterior pillars are preserved . elimination of redundant palatal and pharyngeal tissue is accomplished by subepithelial and muscular stretching and closure . the smuppp technique comprises two steps : step 1 , tonsillectomy and excision of the soft palate and redundant tissue ; and step 2 , reconstruction . a detailed description of the technique is as follows . the patient is orally intubated , and the head is hyperextended with a crowe - davis tonsillectomy mouth gag in place . this helps to assess the lateral extent of the tonsil and the proper plane of dissection . the needle tip is then used in 35 w cutting mode to expose the tonsil capsule via a curvilinear incision . to further identify the capsule , yankauer suction the dissection proceeds using a bovie in 35 w coagulation mode to separate the tonsil capsule from the superior constrictor muscle and to cauterize the blood vessels entering the tonsil . constant traction of the tonsil is maintained , allowing the tissue to be separated as it is cauterized by the bovie . the removal of the tonsil proceeds within its anatomical boundaries , with the musculature of the tonsil fossa left intact . dissection proceeds inferiorly toward the base of the tongue until the tonsil is free of all attachments . at the end of the tonsillectomy , after the removal of both tonsils , the palate is addressed . the uvula is grasped with allis forceps and retracted anteriorly for an optimal approach to the posterior surface of the soft palate . a curvilinear horizontal incision is made on the mucosa at the base of the uvula posteriorly , preserving almost the entire posterior soft palate mucosa . using the needle tip in cautery coagulation mode , the mucosa is separated from the muscle , releasing the posterior soft palate mucosa . using the needle tip in cutting mode , a trapezoid incision is outlined at the anterior mucosa of the soft palate at a level previously identified below the dimpled area , as visualized preoperatively on the awake patient . the incision is extended bilaterally and horizontally across the soft palate to the downward slope of the anterior pillar . the uvula and the submucosal tissue of the lower edge of the soft palate are excised . the redundant pharyngeal folds are eliminated by approximation of the muscular tissue of the tonsillar fossa and the soft palate using interrupted 3 - 0 vicryl sutures through these muscles . the mucosal flap edges are loosely approximated , without undermining , using 3 - 0 vicryl sutures . particular attention is paid to the inferior parts of the tonsillar fossa , where the majority of secondary bleeding occurs . bipolar cautery is used to cauterize these parts , after which the inferior parts of the tonsillar fossas are sutured using inferolateral pharyngoplasty . inferolateral pharyngoplasty involves at least four interrupted sutures with 3 - 0 vicryl to tack the inferior pharyngeal redundancy laterally . this is accomplished by suturing the muscle layer of the inferior tonsillar fossa laterally to the base of the tongue as the first layer . the second layer of mucosal epithelial interrupted sutures then overlies this layer . during soft palate excision , particular attention is paid to the base of the uvula on both sides where the uvular artery enters the soft palate . bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have decreased the incidence of postoperative bleeding compared wth routine tonsillectomy . the tonsillectomy technique used in group 1 patients is also the same electrocautery technique and instrumentation described by drezner , and the tonsillar fossae are left intact for secondary healing [ videos 1 - 2 ] . the test was used for statistical analysis . except in cases with expected sample sizes of fewer than five , postoperative secondary bleeding from the tonsillectomy regions were recorded and compared . bleeding from the palate was excluded . the smuppp technique used in this study it is based on standard surgical principles of epithelial preservation and tension - free closure of the epithelium . most of the mucosa of the soft palate and the anterior and posterior pillars are preserved . elimination of redundant palatal and pharyngeal tissue is accomplished by subepithelial and muscular stretching and closure . the smuppp technique comprises two steps : step 1 , tonsillectomy and excision of the soft palate and redundant tissue ; and step 2 , reconstruction . a detailed description of the technique is as follows . the patient is orally intubated , and the head is hyperextended with a crowe - davis tonsillectomy mouth gag in place . this helps to assess the lateral extent of the tonsil and the proper plane of dissection . the needle tip is then used in 35 w cutting mode to expose the tonsil capsule via a curvilinear incision . to further identify the capsule , yankauer suction the dissection proceeds using a bovie in 35 w coagulation mode to separate the tonsil capsule from the superior constrictor muscle and to cauterize the blood vessels entering the tonsil . constant traction of the tonsil is maintained , allowing the tissue to be separated as it is cauterized by the bovie . the removal of the tonsil proceeds within its anatomical boundaries , with the musculature of the tonsil fossa left intact . dissection proceeds inferiorly toward the base of the tongue until the tonsil is free of all attachments . at the end of the tonsillectomy , and the muscle fibers are still covered by fascia . after the removal of both tonsils , the uvula is grasped with allis forceps and retracted anteriorly for an optimal approach to the posterior surface of the soft palate . a curvilinear horizontal incision is made on the mucosa at the base of the uvula posteriorly , preserving almost the entire posterior soft palate mucosa . using the needle tip in cautery coagulation mode , the mucosa is separated from the muscle , releasing the posterior soft palate mucosa . using the needle tip in cutting mode , a trapezoid incision is outlined at the anterior mucosa of the soft palate at a level previously identified below the dimpled area , as visualized preoperatively on the awake patient . the incision is extended bilaterally and horizontally across the soft palate to the downward slope of the anterior pillar . the uvula and the submucosal tissue of the lower edge of the soft palate are excised . the redundant pharyngeal folds are eliminated by approximation of the muscular tissue of the tonsillar fossa and the soft palate using interrupted 3 - 0 vicryl sutures through these muscles . the mucosal flap edges are loosely approximated , without undermining , using 3 - 0 vicryl sutures . particular attention is paid to the inferior parts of the tonsillar fossa , where the majority of secondary bleeding occurs . bipolar cautery is used to cauterize these parts , after which the inferior parts of the tonsillar fossas are sutured using inferolateral pharyngoplasty . inferolateral pharyngoplasty involves at least four interrupted sutures with 3 - 0 vicryl to tack the inferior pharyngeal redundancy laterally . this is accomplished by suturing the muscle layer of the inferior tonsillar fossa laterally to the base of the tongue as the first layer . the second layer of mucosal epithelial interrupted sutures then overlies this layer . during soft palate excision , particular attention is paid to the base of the uvula on both sides where the uvular artery enters the soft palate . bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have decreased the incidence of postoperative bleeding compared wth routine tonsillectomy . the tonsillectomy technique used in group 1 patients is also the same electrocautery technique and instrumentation described by drezner , and the tonsillar fossae are left intact for secondary healing [ videos 1 - 2 ] . the test was used for statistical analysis . except in cases with expected sample sizes of fewer than five , the mean age of group 1 patients was 38.1 ( 2.58 ) years , and that of group 2 was 37.7 ( 2.2 ) years . no statistically significant difference in age or gender distribution was found between groups 1 and 2 ( p > 0.05 ) . there were 198 patients in the tonsillectomy with unipolar electrocautery group ( group 1 ) and 206 in the smuppp group ( group 2 ) . the incidence of secondary , delayed hemorrhage was 5.05% ( 10 patients ) in group 1 and 1.45% ( 3 patients ) in group 2 ( p = 0.05 ) showing statistically significant difference . the incidence of delayed hemorrhage requiring surgical treatment was 4.54% ( nine patients ) in group 1 and 0.97% ( two patients ) in group 2 [ graph 1 ] . the rate of post - tonsillectomy hemorrhage reported in the literature is variable due to the lack of consensus on what is considered significant postoperative hemorrhage . in many studies , only if surgical intervention under general anesthesia is required to control the bleeding will a case be considered as having significant hemorrhage , and the studies that take this view find lower hemorrhage rates . but in our study , all patients who required medical attention for control of bleeding were considered as having postoperative hemorrhage , regardless of whether they required hemorrhage control under local anesthesia or full surgery under general anesthesia . posttonsillectomy hemorrhage incidence in adults is reported in the literature to be 1.5%-18.0% , but the more commonly given range is between 3.0% and 5.0% . in our study , the postoperative bleeding rate of the tonsillectomy group was similar to the rates mentioned in the literature . rates of hemorrhage after smuppp have not been studied as extensively as those after tonsillectomy . the range encountered in the literature varies from 0.6% to 14.0% , and in our study , the bleeding rate after smuppp was 1.45% which falls within that range . in the present study , when electrocautery tonsillectomy and smuppp groups were compared , a significant difference in the rate of hemorrhage was apparent . although the tonsillectomy portion of the procedures was performed in an identical manner for both of our study groups , the postoperative bleeding rates differed . some studies have found an increased rate of posttonsillectomy hemorrhage in males compared with females , but the gender distribution did not differ between study groups , which agrees with several other studies . the increased rate of hemorrhage in the tonsillectomy group could not be attributed to any factor other than the operative techniques used . however , the bleeding rates for tonsillectomy and smuppp patients remained significantly higher among tonsillectomy patients whose muscular tissue of the tonsillar fossa was not approximated using interrupted 3 - 0 vicryl sutures through the tissue . suturing both the muscular and mucosal layers of the tonsillar fossa in smuppp seemed to decrease the incidence of postoperative bleeding . furthermore , bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have . decreased the incidence of postoperative bleeding . suturing of the tonsillar pillars is the only influencing factor detected that may have lowered the bleeding incidence . double - layer closure and approximating muscular and mucosal layers after tonsillectomy might help postoperative oropharyngeal healing , resulting in decreased bleeding incidence . medical attention for control of bleeding were considered as having postoperative hemorrhage , regardless of whether they required hemorrhage control under local anesthesia or full surgery under general anesthesia . posttonsillectomy hemorrhage incidence in adults is reported in the literature to be 1.5%-18.0% , but the more commonly given range is between 3.0% and 5.0% . in our study , the postoperative bleeding rate of the tonsillectomy group was similar to the rates mentioned in the literature . rates of hemorrhage after smuppp have not been studied as extensively as those after tonsillectomy . the range encountered in the literature varies from 0.6% to 14.0% , and in our study , the bleeding rate after smuppp was 1.45% which falls within that range . in the present study , when electrocautery tonsillectomy and smuppp groups were compared , a significant difference in the rate of hemorrhage was apparent . although the tonsillectomy portion of the procedures was performed in an identical manner for both of our study groups , the postoperative bleeding rates differed . some studies have found an increased rate of posttonsillectomy hemorrhage in males compared with females , but the gender distribution did not differ between study groups , which agrees with several other studies . the increased rate of hemorrhage in the tonsillectomy group could not be attributed to any factor other than the operative techniques used . however , the bleeding rates for tonsillectomy and smuppp patients remained significantly higher among tonsillectomy patients whose muscular tissue of the tonsillar fossa was not approximated using interrupted 3 - 0 vicryl sutures through the tissue . suturing both the muscular and mucosal layers of the tonsillar fossa in smuppp seemed to decrease the incidence of postoperative bleeding . furthermore , bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have . suturing of the tonsillar pillars is the only influencing factor detected that may have lowered the bleeding incidence . double - layer closure and approximating muscular and mucosal layers after tonsillectomy might help postoperative oropharyngeal healing , resulting in decreased bleeding incidence . submucosal tonsillectomy , which is a part of smuppp , has a lower incidence of postoperative delayed hemorrhage compared with tonsillectomy using unipolar cautery in adult patients . we suggest routine use of the suturing technique for smuppp in all tonsillectomy patients to reduce the rate of postoperative hemorrhage . acute pancreatitis ( ap ) is an acute inflammatory process of the pancreas with variable severity . it can be severe and associated with systemic or local complications , especially those with infected pancreatic or peripancreatic necrosis ( ipn ) . gastrointestinal ( gi ) fistula is one of the well - recognized complications secondary to severe ap . depending on the sites of fistula , it may involve the stomach , duodenum , jejunum , ileum , and colon , either in combination or separately . it may result from direct erosion from digestive enzymes released by the inflamed pancreas on adjacent gi tract , or it could occur as a consequence of the intestinal necrosis due to vascular thrombosis in an area of inflammation and infection . although previous studies suggest the fistula of gi tract occurs infrequently in ap patients , ipn and the operative managements for ipn would increase the frequency . it has been reported gi fistulas may cause serious clinical consequences such as hemorrhage and exacerbation of infection with fatal outcome . in this retrospective study we aimed to evaluate the incidence and clinical outcome of gi fistulas in the setting of ap with ipn clinical data from patients with gi fistula secondary to ap between january 1 , 2010 and december 31 , 2013 in our center were retrospectively reviewed . ap was diagnosed according to clinical presentation ( typically abdominal pain ) , laboratory parameters ( serum amylase or lipase levels exceeding 3 times the upper limit of normal ) and abdominal imaging by contrast - enhanced computed tomography ( ct ) . contrast - enhanced ct was performed for all patients within 72 hours since admission . ipn was diagnosed according to the positive gram stain and culture results of pancreatic or peripancreatitic necrotic tissue obtained by means of ct guided fine needle aspiration , or from the first percutaneous drainage or operation . the severity was defined according to the determinant - based classification of acute pancreatitis severity . fistula was defined as pathological communications that connect any portion of gi tract with the necrotic cavity , the peritoneal space , the retroperitoneal areas , or another internal organ . diagnosis of gi fistula was based on fistulography , digestive endoscopy , or operative findings . data pertaining to the severity of ap , the severity score , ct severity score , interval between onset of ap and admission to our center , interval between onset of ap and detection of gi fistula , location of gi fistula , intervention for gi fistula and ipn , and outcomes were recorded . continuous data were compared using a student t test , and categorical data were analyzed using a chi - square test . a p - value of < 0.05 was regarded as statistically significant . the data analyses were performed using spss 20.0 ( ibm spss statistics ; ibm corporation ; armonk , ny ) . a total of 928 patients with ap admitted between january 1 , 2010 and december 31 , 2013 were screened for potential inclusion and 311 of them developed ipn in the disease course . of the 311 patients , 209 ( 67.2% ) had been diagnosed with ipn before admission , of which 87 ( 28.0% ) patients had percutaneous drainage , 39 ( 12.5% ) patients had open necrosectomy . only 83 ( 8.9% ) of all ap patients were admitted in our center within 24 hours since onset of symptoms , and 7 of these 83 patients developed ipn . one hundred nineteen of all ap patients were diagnosed with gi fistula and all of them were ipn patients . all these patients were classified as critical or severe ap and most of them developed a critical course ( 98 , 82.4% ) . their mean ( sd ) age was 45.1 ( 13.7 ) , and 79 ( 66.4% ) of them were male . the median interval between onset of ap and admission to our center was 38 days . table 1 shows the comparison of baseline characteristics between the patients with gi fistula and those without gi fistula . moreover , they likely had higher ct severity score with a certain trend toward significance ( p = 0.08 ) . comparison of baseline characteristics of ap patients with ipn in total , 119 patients developed 160 gi fistulas . sixty - one ( 45.9% ) patients of 133 ipn patients who had drainage and operative before admission in our center were found with fistulas . of the other 178 patients who did not have drainage and operative in other centers , 58 ( 15.7% ) patients developed gi fistulas . the colonic fistula was the most common form which was found in 72 patients , followed by duodenal fistula which could be seen in 53 patients . fistula occurred in duodenum in isolation in 28 patients and in colon alone in 42 patients . isolated gastric and jejunal fistula were observed in only 8 and 5 patients , respectively . the remaining patients demonstrated a combination of fistulas and duodenal combined with colonic fistula was the dominant form unsurprisingly ( table 2 ) . the median ( quartile ) interval between onset of ap and detection of gi fistula was 54 ( 3778 ) days . one hundred one ( 84.9% ) patients were detected with gi fistula beyond 1 month . fifty - four ( 45.4% ) patients were detected with gi fistula after performing first open necrosectomy . distribution of gastrointestinal fistulas fistulas were managed by a similar step - up approach which included percutaneous drainage , cnpi , and open necrosectomy . all fistulas of upper digestive tract , jejunum , and ileum were managed by nonsurgical procedure . seventy - four ( 84.1% ) of these 88 fistulas closed spontaneously over time after the source of infection was controlled . twenty - one of the other 25 colonic fistulas managed by nonsurgical procedure closed spontaneously . parenteral nutrition was used for a transition period and after the sinus tract 's formation enteral nutrition was performed continuously ( table 3 ) . nineteen of these died of multiorgan failure , and the other 15 of septic shock . table 4 shows the comparison of outcomes between the ipn patients with and without gi fistula . although patients with gi fistula stayed longer in intensive care unit ( icu ) and hospital , the mortality between the 2 groups did not differ significantly however , the death rate in patients with colonic fistula was 25 ( 34.7% ) out of 72 , which was significantly higher than that in patients without gi fistula ( p = 0.039 ) . treatment and outcomes of gastrointestinal fistulas in acute pancreatitis comparison of outcomes of ap patients with ipn sixty - one ( 45.9% ) patients of 133 ipn patients who had drainage and operative before admission in our center were found with fistulas . of the other 178 patients who did not have drainage and operative in other centers , 58 ( 15.7% ) patients developed gi fistulas . the colonic fistula was the most common form which was found in 72 patients , followed by duodenal fistula which could be seen in 53 patients . fistula occurred in duodenum in isolation in 28 patients and in colon alone in 42 patients . isolated gastric and jejunal fistula were observed in only 8 and 5 patients , respectively . the remaining patients demonstrated a combination of fistulas and duodenal combined with colonic fistula was the dominant form unsurprisingly ( table 2 ) . the median ( quartile ) interval between onset of ap and detection of gi fistula was 54 ( 3778 ) days . one hundred one ( 84.9% ) patients were detected with gi fistula beyond 1 month . fifty - four ( 45.4% ) patients were detected with gi fistula after performing first open necrosectomy . fistulas were managed by a similar step - up approach which included percutaneous drainage , cnpi , and open necrosectomy . all fistulas of upper digestive tract , jejunum , and ileum were managed by nonsurgical procedure . seventy - four ( 84.1% ) of these 88 fistulas closed spontaneously over time after the source of infection was controlled . twenty - one of the other 25 colonic fistulas managed by nonsurgical procedure closed spontaneously . parenteral nutrition was used for a transition period and after the sinus tract 's formation enteral nutrition was performed continuously ( table 3 ) . nineteen of these died of multiorgan failure , and the other 15 of septic shock . table 4 shows the comparison of outcomes between the ipn patients with and without gi fistula . although patients with gi fistula stayed longer in intensive care unit ( icu ) and hospital , the mortality between the 2 groups did not differ significantly . however , the death rate in patients with colonic fistula was 25 ( 34.7% ) out of 72 , which was significantly higher than that in patients without gi fistula ( p = 0.039 ) . treatment and outcomes of gastrointestinal fistulas in acute pancreatitis comparison of outcomes of ap patients with ipn although gi fistula is a well - recognized complication of ap , it has been reported in limited literature recently . in this study , we reported our experience with gi fistula in ap with ipn in our center . the incidence of gi fistula varied from 3% to 47% in different studies depending on different study population , but all these studies included small number of cases . in the present retrospective study , we found 119 ( 38.3% ) patients with gi fistula among 311 ap patients with ipn , and the overall incidence in all ap patients in our center was 12.8% . furthermore , enzyme - rich fluid and necrosis can lead to vascular thrombosis , which compromise the blood supply of the segmental gi tract , leading to bowel necrosis eventually . in our study , all the patients with gi fistula had necrotizing pancreatitis with ipn . with regard to the time of occurrence of gi fistula during the course of ap , 84.9% patients had gi fistula beyond 1 month , which suggests that development of gi fistulas is associated with the long - term effects of the pancreatic or peripancreatic inflammation and infection . it was reported that frequent debridement and controlled open drainage may increase the incidence of intestinal fistulas in 1989 . and a study published in 1995 from mayo clinic showed that 19 out of 61 ( 31.2% ) patients developed gi fistulas after surgical management while only 4 ( 6.6% ) were found at the time of initial operation . during recent years , the mainstream management of ap with ipn has changed from open necrosectomy to minimally invasive techniques based step - up approach . our patients were treated by step - up approach but with a similar incidence of gi fistula and 54.6% patients were detected with gi fistula before performing first open necrosectomy . it was also observed previously that the approach for ipn management did not affect incidence of gi fistula . it suggests that at present time step - up approach might not reduce the risk of iatrogenic gi injury . in addition , it had been recommended that severe ap should be treated in multidisciplinary specialist units , which included icu specialists , interventional endoscopists , diagnostic and interventional radiologists , and surgeons . our results showed that 133 of ipn patients had operative procedures before transfer and 45.9% developed fistula as opposed to 15.7% of those transferred in who did not have operation . gi fistula could involve the stomach , duodenum , jejunum , ileum , and colon , either in combination or separately . same to other studies , colonic fistula was the commonest form of gi fistulas in our study , which was found in 72 patients and duodenal fistula the second ( 53 patients ) . the underlying mechanisms of this phenomenon may include : first , the anatomy characteristics of peritoneal reflection results in colon and duodenal more frequently affected directly . second , gut ischemia , as a result of low - flow state or hemodynamic response to sepsis , may be more commonly associated with colon . furthermore , the stomach and small intestine have a much better blood supply , which could provide a protective mechanism for preventing the formation of fistula . it has been reported that most upper - gut fistulas could close with time and colonic fistulas may require active intervention . in our center , fistulas of upper gi tract , jejunum , and ileum usually closed spontaneously with time if infected source could be well controlled while approximately 7 out of 10 colonic fistulas needed to be managed by enterostomy . however , a study which reviewed 43 reports suggested that colonic fistulas which became apparent after percutaneous pancreatic drainage might close spontaneously . moreover , several reports showed colonic fistula could close with the aid of nonsurgical techniques such as percutaneous drainage and endoscopic therapy . in our study , 21 colonic fistula closed spontaneously with conservative management . our experience is that those excrement can be completely drained by nonsurgical techniques would close spontaneously . gi fistula may cause clinical consequences such as gi hemorrhage and sepsis which might lead to death eventually . in our study , patients with gi fistula were associated with more severe disease course and suffered more organs failure . on the other hand , gi fistula could potentially benefit the patient by draining ipn into gi tract . in persistence to previous studies that suggested gi fistula did not increase mortality , the death rate of patients with gi fistula in our study did not differ significantly from those without gi fistula . however , subgroup analyses showed mortality of patients with colonic fistula was significantly higher than that in patients without gi fistula , namely , colonic fistula was related with higher mortality . the actual incidence of gi fistula could be lower than ours because the patients admitted to our center were so complicated that most of them had been treated in other facilities for a long time , even with percutaneous drainage or open necrosectomy , and some patients had been diagnosed with gi fistula before transferred . the heterogeneity of our patients and variable lengths of time to referral to our center would impact the reliability of conclusions regarding identification of factors associated with fistula formation and predictors of death . and some data in previous facilities were incomplete . additionally , it was a single - center retrospective study . in conclusion , colonic and duodenal fistulas are the two commonest forms of gi fistula in ipn patients , and most of them could close spontaneously with proper nonsurgical management except approximately 70% of colonic fistulas needing surgical intervention . for the clinical outcome , the occurrence of colonic fistula is associated with worse outcome .

background : the aim of this study was to compare the incidence of postoperative secondary hemorrhage for tonsillectomy and submucosal uvulopalatopharyngoplasty ( smuppp).materials and methods : in this retrospective case series , the medical records of 404 patients who underwent tonsillectomy with unipolar electrocautery and smuppp at our institution between january 2001 and december 2010 were reviewed . the patients were divided into two groups : group 1 ( 198 patients ) underwent tonsillectomy ; group 2 ( 206 patients ) underwent smuppp . main outcome measures were incidence of bleeding or complications after tonsillectomy and smuppp and the need for revision surgery.results:the mean age of group 1 patients was 38.1 ( 2.58 ) years and that of group 2 was 37.7 ( 2.25 ) years . males were 51.3% of group 1 and 46.7% of group 2 . no statistically significant difference in age or gender distribution was found between groups 1 and 2 . the incidence of secondary , delayed hemorrhage was 5.05% ( 10 patients ) in group 1 and 1.45% ( three patients ) in group 2 ( p = 0.05 ) . the incidence of delayed hemorrhage requiring surgical treatment was 4.54% ( nine patients ) in group 1 and 0.97% ( two patients ) in group 2.conclusion:in adults , smuppp , which includes tonsillectomy , has a lower incidence of postoperative delayed hemorrhage than does tonsillectomy with unipolar cautery . abstractgastrointestinal ( gi ) fistula is a well - recognized complication of acute pancreatitis ( ap ) . however , it has been reported in limited literature . this study aimed to evaluate the incidence and outcome of gi fistulas in ap patients complicated with infected pancreatic or peripancreatic necrosis ( ipn).between 2010 and 2013 ap patients with ipn who diagnosed with gi fistula in our center were analyzed in this retrospective study . and we also conducted a comparison between patients with and without gi fistula regarding the baseline characteristics and outcomes.over 4 years , a total of 928 ap patients were admitted into our center , of whom 119 patients with ipn were diagnosed with gi fistula and they developed 160 gi fistulas in total . colonic fistula found in 72 patients was the most common form of gi fistula followed with duodenal fistula . all duodenal fistulas were managed by nonsurgical management . ileostomy or colostomy was performed for 44 ( 61.1% ) of 72 colonic fistulas . twenty - one ( 29.2% ) colonic fistulas were successfully treated by percutaneous drainage or continuous negative pressure irrigation . mortality of patients with gi fistula did not differ significantly from those without gi fistula ( 28.6% vs 21.9% , p = 0.22 ) . however , a significantly higher mortality ( 34.7% ) was observed in those with colonic fistula.gi fistula is a common finding in patients of ap with ipn . most of these fistulas can be successfully managed with different procedures depending on their sites of origin . colonic fistula is related with higher mortality than those without gi fistula .

all forms of mercury viz . , organic , elemental , and mercury salts are toxic and manifestations depend on nature , intensity and the chemical form of mercury . most human exposure results from fish consumption ( organic mercury ) or dental amalgam ( metallic mercury ) . kidneys are the prime target of mercury toxicity as it is primarily excreted through them . here , we describe a patient who consumed mercuric chloride with suicidal intent , and presented with typical manifestation such as acute kidney injury ( aki ) and gastrointestinal erosion . in addition , he had disseminated intravascular coagulation ( dic ) , a rare complication of mercury poisoning . a 36-year - old male was admitted with 2 days history of oliguria progressing to anuria , facial puffiness , edema legs , bleeding gums , hematochezia , and fever . he gave a history of consumption of unknown substance ( around 500 mg ) a week back used for folk remedies and rituals . his heart rate was 92/min , respiratory rate was 15/min , and blood pressure was 130/90 mm of hg . laboratory investigations showed hemoglobin 11.2 g / dl ; total count 6000/mm ; platelet count 100,000/mm ; blood urea 124 mg / dl ; serum creatinine 6.7 mg / dl ; sodium 135 meq / l ; and potassium 6.1 meq / l ; arterial blood gas analysis showed high anion gap metabolic acidosis . his coagulation profile showed prothrombin time 18 s , inr 1.6 ; activated partial thromboplastin time 60 s ; fibrin degradation products 10 mg / ml ; d - dimer levels 1 mg / ml ; and serum fibrinogen 250 mg / dl . diagnosis of overt dic was made as per criteria proposed by the international society of thrombosis and hemostasis with total score of five at admission . he received eight sessions of hemodialysis over 2 weeks after which his urine output started improving . renal biopsy done 2 weeks after admission showed markedly dilated tubules with sloughed off epithelium and cell debris within lumen [ figure 1 ] , interstitial edema , and mild inflammatory infiltrate in the interstitium consistent with acute tubular necrosis . consumed substance brought by the patient 3 weeks later was white colored powder , called . toxicological analysis of the compound revealed it to be mercuric chloride . at the end of 2 months , there are three classes of mercury : metallic elemental mercury , inorganic mercurial salts ( mercurous and mercuric salts ) , and organic mercurials . acute poisoning of this leads to corrosive bronchitis , pulmonary edema / fibrosis , diarrhea , renal dysfunction , visual and neuropsychiatric disturbances , and in severe cases , death due to respiratory failure . organic mercury compounds are absorbed completely from the intestine , converted to inorganic forms , and possess similar toxic properties . mercuric chloride is still used as wood preservative , photographic intensifier , disinfectants and also in indigenous drug formulation , and folk remedies in asian countries . once ingested , mechanism of mercury toxicity include ( a ) mercuric ions precipitate proteins that cause direct necrosis of tissues . about 8590% of mercury in the body accumulate in the kidneys causing acute renal failure due to necrosis of the proximal tubular epithelium . ( b ) inorganic mercury complexes sulfhydryl groups and causes metabolic acidosis , vasodilatation , and shock . though acute tubular necrosis is the most common lesion , tubulointerstitial nephritis and immune - mediated glomerular damage can also occur . rarely , it can present as hypertensive encephalopathy especially in children , nephrotic syndrome , chronic tubulointerstitial nephritis , or with isolated tubular dysfunction . our patient consumed mercuric chloride with suicidal intention and developed gastrointestinal erosion , anuric renal failure , and dic . franco et al . reported one patient with mercuric chloride poisoning who developed two consecutive episodes of acute renal failure by two different mechanisms , one toxic and the other immunological . renal biopsy done in that patient , showed acute tubular necrosis initially and granulomatous interstitial nephritis in the second biopsy . the international society of thrombosis and hemostasis criteria , which has 91% sensitivity and 97% specificity , was used for the diagnosis of overt dic . a score of five or higher is compatible with dic while a score below five is suggestive of dic . the possible explanation for dic was the lowered fibrinolytic activity due to inhibition of plasma plasminogen activator or the inhibition of plasminogen activation reaction catalyzed by this enzyme as demonstrated in experimental rat models . measurement of mercury levels in blood ( > 3.6 mg / dl ) and urine ( > 15 mg / dl ) may be helpful in diagnosis . chelation therapy should be considered for any symptomatic patient with a history of acute elemental mercury exposure . chelating agents include dimercaprol ( bal ) , 2,3-dimercaptopropane-1-sulfonate ( dmps ) , dimercaptosuccinic acid , and penicillamine . hemodialysis is not effective in removing mercury , but can enhance the removal of the dimercaprol - mercury complexes . the outcome depends on the form of the mercury compound and severity of the exposure . we did not do urine mercury levels , and the patient had not received any chelating agents as toxicological analysis was done much later . we conclude that mercury poisoning should be considered in case of aki and dic though it is a rare complication . prompt treatment with chelating agents guided by measurement of mercury levels will have an impact on the favorable clinical outcome . hair dyes are perhaps among the most commonly used cosmetics by elderly and the young alike - for concealing gray hair by the former or just for a fashion statement by the latter . the popularity of hair colouring can be gauged from the fact that the median age for debut was 16 years and nearly 75% of women and 18% of men had dyed their hair at some point in their lives according to a danish population - based study . this rising trend of hair colouring has resulted in an increased prevalence of hair dye - associated adverse effects accounting for 5.3% of danish persons who dyed their hair . similarly , 42% of the respondents reported adverse reactions from use of hair dyes and the mean age of hair dye debut was 27 years in an indian study . hair dyes include a variety of coloring agents such as 2,7-naphthalenediol , 2-aminomethyl - p - aminophenol hydrochloride , 2-chloro - p - phenylenediamine , n - phenyl - p - phenylenediamine , and o - aminophenol , p - aminophenol . although all these agents can cause allergic contact reactions , p - phenylenediamine ( ppd ) remains the most commonly implicated agent for the contact sensitivity . it is an effective compound for hair dyes due to its low molecular weight , high protein- binding capacity , and ability to penetrate the hair shaft . apart from being a common constituent in varying concentrations of nearly 70% of the hair colorants available worldwide , it is also present in dyes for henna tattoos , textiles , leather and fur , and black rubbers . being a member of 1,4-substituted benzenes , it cross - reacts with para - amino benzoic acid ( paba ) , sulphonamides , para - amino salicylic acid , ester anesthetics , thiazides , sesquiterpene - lactone mix , and azo dyes . it is a potent contact sensitizer even in low concentration and considered a best indicator and useful patch test screening allergen for hair dye dermatitis . the reported prevalence of positive patch test reactions to ppd among dermatitis patients is 4.4% in asia , 4.1% in europe , 6.0% in north america , and 11.5% in india . the clinical features of hair dye dermatitis vary from mild contact dermatitis localized to one body site ( hand dermatitis ) or disseminated generalized dermatitis to severe life - threatening complications such as contact urticaria / angioedema , rhinitis / bronchospasm / asthma , and renal toxicity . the most common clinical presentations include contact dermatitis localized to sites of contact or photoexposed skin , periorbital dermatitis , airborne contact dermatitis , hand dermatitis , and rarely erythema multiforme - like lesions . in this pilot study , we present our clinico - epidemiological observations and patch test results of ppd contact sensitivity in patients suspected to have hair dye dermatitis . eighty ( m : f 47:33 ) consecutive patients suspected to have hair dye allergy were enrolled for the study after obtaining informed consent . patients having acute dermatitis were patch tested after control of their dermatitis , when they were off systemic corticosteroids or the dose of prednisolone was < 20 mg / day . details about age , gender , occupation , personal or family history of atopy ( nasobronchial allergy , asthma , childhood eczema ) , use of hair dye and its duration , onset , duration , and distribution of dermatitis were noted . the enrolled patients were patch tested by finn chambers method with indian standard patch test series including ppd ( 1.0% pet ) recommended by contact dermatitis and occupational dermatoses forum of india . patches were applied on the upper back and the patients were asked to return for results after 48 hrs ( d2 ) and 72 hrs ( d3 ) . the results were graded according to the international contact dermatitis research group criteria . only reactions persisting on d3 were considered positive for final analysis . side effects such as adhesive tape reaction , discomfort and itching , flare of dermatitis , angry back phenomenon , active sensitization , and pigment alteration at test site , when they occurred , were recorded . the study included 47 ( 58.8% ) males aged between 19 and 74 years and 33 ( 41.2% ) females aged between 18 to 74 years . the majority , that is , 57 ( 71% ) patients were older than 40 years , while whereas 23 ( 29% ) were younger than 40 years , the youngest being a 19-year - old male . forty six ( 57.5% ) patients were from an urban background and 34 ( 42.5% ) patients belonged to a rural background . among the males , 21 ( 44.7% ) were office goers , 9 ( 19.1% ) shopkeepers , and 6 ( 12.7% ) defence personnel . among females 30 ( 90.9% ) were housewives and 3 ( 9.09% ) were students . the duration of hair dye usage varied from less than one month in 11 ( 13.8% ) to more than three years in 17 ( 21.2% ) patients . other 52 ( 65% ) patients were using hair dyes for variable period of 1 month to 3 years . the duration of dermatitis varied from < 1 month in 11 ( 13.8% ) patients and > 1 year in 27 ( 33.7% ) patients ; forty - two ( 52.5% ) patients had dermatitis between one month and one year . clinically , characteristic acute ( erythematous , edematous , oozy , crusted eczematous plaques ) , subacute and chronic dermatitis ( hyperpigmented , lichenified eczematous lesions ) involving multiple sites such as scalp , scalp margins , forehead , eyelids , beard , neck , upper back , and/or hands was observed in all cases . additionally , the most common patterns of hair dye dermatitis observed were contact dermatitis localized to scalp and/or scalp margins in 49 , followed by dermatitis of face ( including beard and periorbital skin ) and neck in 23 and hands and/or feet in 12 patients , respectively . other patterns observed were airborne contact dermatitis in 9 , hand dermatitis in 5 , eyelid dermatitis in 4 , and discoid dermatitis and disseminated dermatitis in 1 ( 1.85% ) patient each . one patient had generalized diffuse dermatitis and another patient showed erythema multiforme - like lesions . none of the patients had followed the manufacturer 's instructions to perform sensitivity testing prior to applying the hair dye . baseline characteristics of the patients studied only 54 ( 67.5% ) patients comprising 33 ( 61% ) males and 21 ( 39% ) females aged between of 19 and 74 years showed positive results to patch testing with ppd . eight of these patients also showed positive patch test reaction to other allergens ; fragrance mix in 5 , thiuram mix in 3 , and paraben mix and colophony in 1 patient each . thirty ( 55.5% ) patients belonged to urban populations and 24 ( 44.5% ) patients were from rural background . the common clinical patterns noted were : ( a ) dermatitis localized to the scalp , face , neck or hands , ( b ) airborne dermatitis , ( c ) chronic actinic dermatitis ( actinic reticuloid ) , ( d ) generalized diffuse dermatitis , and ( e ) erythema multiforme - like dermatitis [ figures 1 to 5 ] . ppd contact dermatitis localized to face , cheeks , nose , neck , and dorsal hands in photodistributed pattern ppd contact dermatitis localized to eyelids and periorbital skin ppd contact dermatitis localized over neck . she also had dermatitis involving the scalp skin ( a ) chronic actinic dermatitis ( actinic reticuloid ) due to ppd . ( b ) patch test shows 3 + reaction to ppd ppd induced erythema multiforme - like lesions over ( a ) upper back and ( b ) palms . the use of hair colorants has increased exponentially over the last few decades due to increased societal pressure and changing fashion trends . over the years , the colouring agents have diversified in nature with the arrival of oxidative hair dyes that include many synthetic and natural agents . the more commonly used hair dye agents include resorcinol , henna , hydroquinone , lead acetate , m - aminophenol , n - phenyl - p - phenylenediamine , o - aminophenol , p - aminophenol , and ppd or related para - amino compounds such as toluene-2,5-diamine sulfate . nearly 5% of 21,515 individuals were allergic to ppd in a multicentric european study . similarly , ppd was the commonest sensitizer among cosmetics in two separate indian studies . hairdressers and textile dye workers are particularly at risk and have high prevalence of ppd contact dermatitis varying between of 15% and 45% or as high as 58% because of high occupational exposure . apart from hair colorants , sensitization may also occur from ppd in textile or fur dyes , black rubber , temporary tattoos , and photocopying and printing inks . similarly , hair dye dermatitis is observed among urban dwellers more often than in their rural counterparts ; a prevalence of 84% versus 16% among urban versus rural population has been observed in a previous study . this trend may be because of easy availability of hair dyes , increased awareness , and peer pressure to look younger among the urban population . sensitization from ppd can occur at any age including among children as it depends mainly upon exposure to the allergen . the weighted average of ppd sensitization in the general population is estimated to be 1.0% . however , the prevalence of ppd sensitivity varies according to cultural and occupational practices and a higher prevalence is seen in asia ( 2%12% ) , particularly among asian males . most cases occur in middle - aged individuals between 30 and 50 years of age and males are affected two times more than the females . this increased prevalence in middle - aged people has been attributed to the frequent use of hair dye in order to camouflage their greying hair . higher sensitization to hair dye among males is probably due to the more frequent applications that are required for facial ( beard ) hair as compared to scalp hair . clinically , hair dye dermatitis can present as both irritant dermatitis and allergic contact dermatitis . it may also cause severe clinical contact reactions ranging from anaphylaxis to oedema of the scalp , face , and eyelids . scalp and scalp margins , and beard skin are the reported sites that are commonly affected due to hair dye usage , while hands and other body sites may be affected among persons not using gloves while dying hair . mild to moderate contact reactions such as erythema , oozing , and ulceration typically at the scalp margin , and on the ears and neck are common as a result of direct contact . ho et al . observed that hair dye dermatitis primarily involved the face and neck ( 28% ) , and hands ( 25% ) , whereas widespread dermatitis occurred in 17% of patients . airborne contact dermatitis , irritant contact dermatitis , photocontact dermatitis , periorbital eczema , hand eczema , lichenoid lesions , and lichen planus pigmentosus - like pigmentary changes were the commonly observed clinical patterns of hair dye dermatitis , whereas contact leukcoderma , contact urticaria , lymphomatoid papulosis , erythema multiforme - like or prurigo nodularis - like lesions , and anaphylaxis have occurred atypically with ppd . localized contact dermatitis was observed in nearly all our patients , particularly involving the scalp and/or scalp margin in 49 patients , face , beard , and periorbital skin in 23 , and hands and feet in 12 patients . uncommon patterns such as airborne contact dermatitis ( 9 patients ) , hand dermatitis ( 5 patients ) , eyelid dermatitis ( 4 patients ) , disseminated dermatitis and erythema multiforme - like lesions ( 1 patient each ) were also observed in this series . although ppd is not considered photosensitizing , dermatitis localized to photoexposed skin and chronic actinic dermatitis ( actinic reticuloid ) in a few patients suggests its possible photosensitizing potential . it is also possible that persistence of allergen and photoaggravation of dermatitis is responsible for photolocalization of ppd induced dermatitis in some individuals . contact sensitivity to fragrance mix , colophony , paraben mix , and thiuram mix , observed in eight patients was perhaps due to prior sensitisation as they are present in most topical medications , cosmetics , perfumes , and pharmaceuticals . prohaptens and their metabolites in the skin , particularly the p - benzoquinone , are considered responsible for its allergenicity and cross - reactivity . another postulation is that bandrowski 's base , a trimer of ppd formed on exposure , is perhaps responsible for its allergenicity . however , patch testing with these agents individually is negative in a sizeable population of patients who had tested positive to for ppd . a genetic polymorphism in the cutaneous metabolism of ppd too has been suggested to explain why only a few persons using hair dyes develop contact sensitivity or variable allergic responses from to it . reported a personal history of atopy in 24% of their patients with cosmetic contact sensitivity . atopic individuals are perhaps at an enhanced risk of for cosmetic contact sensitivity because of increased allergen penetration through impaired skin barrier . a personal history of atopy was observed in 18 ( 33% ) patients in our study . the european union cosmetic directive allows a maximum concentration of 6% ppd in hair dyes , while the bureau of indian standards has set a maximum permissible limit of 30% and not less than 3% ppd after dilution of the powder form and a maximum of 4% and not less than 1.2% after dilution of the oxidation liquid type . however , many manufacturers fail to comply with these standards , and a strict legislation is perhaps needed for adherence to safe limits of ppd in hair dyes . it is also desirable that manufacturers print directions in bold text over the product package regarding safe use of the hair dye . there is also an urgent need to increase awareness among consumers regarding the adverse effects of hair dyes , the available safer alternatives , and the significance of performing sensitivity testing prior to actual use , in adherence of usage instructions . the hair dyes usually have two separate components , the colorant and the developer that are mixed before application . we did not patch test these agents in our study and that might have been responsible for negative patch test results in 26 of our patients . unavailability of hair dye allergens other than ppd , the small number of patients , and not testing with patient 's own hair colorant or other cosmetics may have resulted in our missing some cases of compound allergy . the hair dyes usually have two separate components , the colorant and the developer that are mixed before application . we did not patch test these agents in our study and that might have been responsible for negative patch test results in 26 of our patients . unavailability of hair dye allergens other than ppd , the small number of patients , and not testing with patient 's own hair colorant or other cosmetics may have resulted in our missing some cases of compound allergy .

mercury is a toxic heavy metal and occurs in organic and inorganic forms . inorganic mercury includes elemental mercury and mercury salts . mercury salts are usually white powder or crystals , and widely used in indigenous medicines and folk remedies in asia . inorganic mercury poisoning causes acute kidney injury ( aki ) and gastrointestinal manifestations and can be life - threatening . we describe a case with unknown substance poisoning who developed aki and disseminated intravascular coagulation ( dic ) . renal biopsy showed acute tubular necrosis . later , the consumed substance was proven to be mercuric chloride . his renal failure improved over time , and his creatinine normalized after 2 months . background : the contact allergic reactions from p - phenylenediamine ( ppd ) in hair dyes vary from mild contact dermatitis to severe life- threatening events ( angioedema , bronchospasm , asthma , renal impairment).objectives : to study the clinical patterns and ppd contact sensitivity in patients with hair - dye dermatitis.materials and methods : eighty ( m : f 47:33 ) consecutive patients aged between 18 and 74 years suspected to have contact allergy from hair dye were studied by patch testing with indian standard series including p - phenylenediamine ( ppd , 1.0% pet).results:54 fifty - four ( m : f 21:33 ) patients showed positive patch tests from ppd . eight of these patients also showed positive patch test reaction from fragrance mix , thiuram mix , paraben mix , or colophony . fifty - seven ( 71% ) patients affected were aged older than 40 years . the duration of dermatitis varied from < 1 month to > 1 year with exacerbation following hair coloring . forty - nine patients had dermatitis of scalp and/or scalp margins and 23 patients had face and neck dermatitis . periorbital dermatitis , chronic actinic dermatitis , and erythema multiforme - like lesions were seen in 4 , 2 , and 1 patients , respectively.conclusions:hair dyes and ppd constitute a significant cause of contact dermatitis . there is an urgent need for creating consumer awareness regarding hair - dyes contact sensitivity and the significance of performing sensitivity testing prior to actual use .

eosinophilic granuloma ( eg ) , a benign local bone disease , is classified as the mildest form of langerhans ' cell histiocytosis ( lch ) . eg predominantly affects children , adolescents , and young adults , and the skull is the most common site of involvement.5 ) the pathologic diagnosis of eg can be confirmed by positive stains of cd1a antigen and protein s-100 or when intracytoplasmic organelles ( birbeck granules ) are detected using electron microscopy.11 ) the most common symptom of eg is a tender , growing scalp mass , but it can be asymptomatic and incidentally found on skull x - rays . the clinical course is benign with a tendency to spontaneous regress ; however , eg may also recur . it is common to prove the antecedent of a mild head trauma preceding clinical symptoms . a-6-year - old boy was referred to our hospital with a soft mass on the midline of his forehead . he had undergone surgery for eg in the vertex 19 months ago ( figure 1 ) . one month prior to minor head trauma , a follow - up computed tomography ( ct ) ( figure 2 ) was performed to check for recurrence , and none was found . one week before his arrival at the hospital , he had hit his head , and the resulting mass grew rapidly after the trauma . the findings of the neurological examination were unremarkable , except for a non - tender , soft , and immobile mass . the three - dimensional ( 3d ) ct showed a lytic bony defect on the frontal bone without hemorrhage ( figure 3a and b ) and the enhanced magnetic resonance imaging ( mri ) revealed a 1.4 cm - sized growing mass on the midline of the forehead ( figure 3c and d ) . the level of erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) was within normal limits . the protruding mass extended from the scalp through the defect in the frontal bone , and was visualized when the scalp flap was reflected ( figure 4a ) . after removal of the bone flap and tumor , it was noted that there was no hematoma and the dura mater was intact without invasion ( figure 4b ) . the tumor was removed completely and the bony defect was replaced with bone cement ( figure 4c ) . the histopathological examination revealed numerous oval langerhans ' cells and eosinophils ( figure 5a and b ) . the langerhans ' cells were stained for cd1a and s-100 ( figure 5c and d ) . the patient was followed - up six months after the operation and had a normal neurologic examination at that point . lch is a unifocal or multifocal disorder of the bone or soft tissue , most often appearing as a lytic lesion of the skull . eg is the mildest form of lch.5 ) the exact incidence of eg is not well known but vandenberg and coley12 ) found that it accounted for only 2.4% of benign skull tumors . in the series of cases studied by jelsma and ross,4 ) the most common symptom of eg is a tender , enlarged scalp mass on the parietal and frontal bones . eg may be asymptomatic and only discovered on a skull x - ray by chance . the x - ray radiologic findings consist of a punched - out , skull lesion with sharply defined margins . as the clinical and radiologic findings are not specific enough to determine the diagnosis , cytology is very helpful in diagnosing eg . the key feature is the identification of characteristic langerhans cells in a background of eosinophils and red blood cells.5 ) there is no definite disease etiology for eg . however , autoimmune diseases , inflammatory processes , and uncontrolled langerhans cell replication can be considered to be etiological factors.3 ) also , minor trauma is considered an etiologic factors . there have been six reported cases of eg associated with epidural hematoma ( table 1).1678910 ) the possible mechanisms of epidural hematomas in the six cases consisted of a rupture of the tumor cyst , communicating with the epidural vein and the sinus pericranii , or a head trauma - related intratumoral hemorrhage , which subsequently penetrated into the epidural space . all six cases were associated with epidural hemorrhages , while our case was not . in our case , no hematoma or hemorrhage was found in the ct / mri image , or in intraoperative and pathologic findings . the patient in our case regularly visited the clinic for follow - up appointments due to his history of eg . the ct scan taken one month prior to the trauma showed no definite recurrence ; however , a newly developed eg lesion was found one week after the trauma . this indicates that the development of eg may be associated with trauma . in this case , it is thought that eg developed through a local inflammatory response due to a minor trauma . trauma may be a trigger factor of the inflammatory response instead of the etiology of the disease . the elevated levels of esr and crp are found in some , but not all , cases of eg . this is because eg lesions are limited to local level and the time between diagnosis and trauma varies . in the present case , the author finds the period between the trauma and diagnosis to be relatively long . factors related to hematoma formation may include the trauma 's intensity and the time interval after the trauma . for example , in one case , a patient with an epidural hematoma had a two - week time interval , with a fluid level confirmed four weeks later . in the management of solitary eg , surgical excision is recommended and radiotherapy can be considered as an adjuvant therapy if necessary . some authors advocate surgical excision followed by postoperative treatment that includes low - dose radiotherapy , intralesional application of corticosteroids , or a combination of chemotherapy and radiotherapy.11 ) other studies found that solitary calvarial eg in skeletally immature patients 14 years of age or younger resolved fully without any recurrence.2 ) eg is responsive to steroid treatment and this indicates that the etiology of eg is associated with local inflammatory processes . to avoid surgical procedures , the prognosis depends on the age of the patient at the time of diagnosis and the number of lesions . the local recurrence rate is 6% , and new lesions appear in about 22% of patients , therefore , long - term follow - up is recommended.311 ) our case suggests that minor trauma may be an aggravating factor for the progression of eg , based on the radiological findings before and after the trauma . thus , careful observation with regular follow - up is needed for patients with eg after trauma . situs inversus totalis , a congenital anomaly present in approximately 0.01% of the population , results in complete mirror - image reversal of all the thoracic and abdominal organs . the laparoscopic literature has several reports of successful bariatric procedures in patients with situs inversus despite the extremely low prevalence of this condition.[13 ] while several equivalent alternatives are available in the bariatric algorithm , more recently the laparoscopic sleeve gastrectomy ( sg ) has been gaining traction as an effective means of weight loss in patients with morbid obesity.[47 ] a 39-year - old woman who had previously undergone an open gastric banding procedure 20 months earlier for morbid obesity presented with increased weight gain . her weight at presentation was 106 kg ( body mass index 42 kg / m ) , a percentage excess weight loss ( ewl ) of only 1.9% . her medical history was significant for hypertension , noninsulin - dependent diabetes mellitus , hypothyroidism , obstructive sleep apnea , and situs inversus totalis . the presence of situs inversus was initially discovered during her first weight loss operation and confirmed by computed tomography of the abdomen [ figure 1 ] . the operating surgeon began the case laparoscopically , but upon recognizing the altered anatomy made the decision to convert to an open gastric band placement . given the failure of this first procedure to produce meaningful weight loss , the patient was scheduled for laparoscopic removal of the gastric band with concurrent sg . coronal computed tomographic scan of the abdomen and pelvis with oral and intravenous contrast demonstrating the mirror - image anatomy . panel a displays a cross - section of the band in its position on the right side of the abdomen . panel b further demonstrates the altered anatomy with left - sided liver and right - sided stomach the patient was positioned supine on the operating room table . secondary to the presence of situs inversus , the veress needle was placed in the right upper quadrant to avoid the liver . a 12-mm trocar was placed five fingerbreadths above the umbilicus , just to the right of midline . next , a 5-mm trocar was placed in the left lower quadrant approximately two fingerbreadths below the umbilicus in the midclavicular line . a 12-mm port was then placed under direct vision in the left midclavicular line approximately five fingerbreadths above the umbilicus . a 12-mm port was placed in the right upper quadrant in the midclavicular line , and a 5-mm port was placed in the right upper quadrant one fingerbreadth below the costal margin in the midaxillary line [ figure 2 ] . exploration revealed extensive adhesions as a result of her previous open procedures . at this point , the stomach was identified and noted to be severely adhered to the liver , which was , in turn , adherent to the abdominal wall . the stomach was mobilized , the band was identified , and the band buckle was cut and removed . the previous fundoplication was taken down using a 3.5-mm depth 45-mm length stapler , and the greater curvature and short gastric vessels were divided using a harmonic scalpel . after performing the dissection all the way to the angle of his and exposing the left crux of the diaphragm , a 34-french bougie was placed into the stomach with the tip of the bougie sitting in the proximal duodenum . the formation of the gastric sleeve with a 4.8-mm depth 45-mm length stapler was started from a point 5 cm proximal to the pylorus muscle and directed toward the angle of his [ figure 3 ] . eight total firings were used , thus dividing the stomach completely and forming a gastric tube over the 34-french bougie . at the most proximal aspect of the stomach , several dense adhesions to the diaphragm were encountered and eventually taken down . following the formation of the gastric tube , the staple line was oversewn with 2 - 0 absorbable suture using an endo stitch ( covidien inc . , after submerging the gastric tube underwater , air was pumped into the scope , and subsequently no leak was appreciated . before removal of the last trocar , the detached stomach was placed into a specimen retrieval bag and removed from the peritoneal cavity via the 12-mm port in the right upper quadrant . the same trocar site was slightly extended to allow for removal of the port and band . finally , a jackson pratt drain was placed by the sleeve for surveillance purposes . novel port placement ( copyright sages 2010 , soper nj , swanstrom ll , eubanks ws , eds . mastery of endoscopic and laparoscopic surgery ) intraoperative image ( white arrow indicates the angle of his ; blue arrows indicate several gastrohepatic adhesions , and black arrows indicate the greater curvature of the stomach ) the postoperative course was complicated by a leak resulting in a 3.9 2.8 cm collection in the gastrosplenic ligament adjacent to the surgical suture line [ figure 4 ] . a jackson pratt drain was left alongside the sg at the time of surgery as this is the surgeons protocol for revisional surgery . the patient underwent an upper endoscopy and placement of a 12-cm removable polyflex stent ( boston scientific inc . , natick , ma ) for management of the leak [ figure 5 ] . over the next few weeks further imaging demonstrated interval migration of the stent into the stomach with no change in the size or location of the perigastric collection [ figure 6 ] . the patient was managed conservatively despite the stent movement and had eventual resolution of her symptoms . the stent was subsequently removed without any complications . currently , the patient is 2 years out from the laparoscopic sg . postoperative upper gi series demonstrating extravasation of contrast at the most proximal aspect of the staple line completion radiograph following endoscopic placement of a removable stent across the anastamotic leak site coronal computed tomographic scan of the abdomen and pelvis with oral and intravenous contrast demonstrating complete migration of the stent into the body of the stomach secondary to the presence of situs inversus , the veress needle was placed in the right upper quadrant to avoid the liver . a 12-mm trocar was placed five fingerbreadths above the umbilicus , just to the right of midline . next , a 5-mm trocar was placed in the left lower quadrant approximately two fingerbreadths below the umbilicus in the midclavicular line . a 12-mm port was then placed under direct vision in the left midclavicular line approximately five fingerbreadths above the umbilicus . a 12-mm port was placed in the right upper quadrant in the midclavicular line , and a 5-mm port was placed in the right upper quadrant one fingerbreadth below the costal margin in the midaxillary line [ figure 2 ] . exploration revealed extensive adhesions as a result of her previous open procedures . at this point , the stomach was identified and noted to be severely adhered to the liver , which was , in turn , adherent to the abdominal wall . the stomach was mobilized , the band was identified , and the band buckle was cut and removed . the previous fundoplication was taken down using a 3.5-mm depth 45-mm length stapler , and the greater curvature and short gastric vessels were divided using a harmonic scalpel . after performing the dissection all the way to the angle of his and exposing the left crux of the diaphragm , a 34-french bougie was placed into the stomach with the tip of the bougie sitting in the proximal duodenum . the formation of the gastric sleeve with a 4.8-mm depth 45-mm length stapler was started from a point 5 cm proximal to the pylorus muscle and directed toward the angle of his [ figure 3 ] . eight total firings were used , thus dividing the stomach completely and forming a gastric tube over the 34-french bougie . at the most proximal aspect of the stomach , several dense adhesions to the diaphragm were encountered and eventually taken down . following the formation of the gastric tube , the staple line was oversewn with 2 - 0 absorbable suture using an endo stitch ( covidien inc . , after submerging the gastric tube underwater , air was pumped into the scope , and subsequently no leak was appreciated . before removal of the last trocar , the detached stomach was placed into a specimen retrieval bag and removed from the peritoneal cavity via the 12-mm port in the right upper quadrant . the same trocar site was slightly extended to allow for removal of the port and band . finally , a jackson pratt drain was placed by the sleeve for surveillance purposes . novel port placement ( copyright sages 2010 , soper nj , swanstrom ll , eubanks ws , eds . mastery of endoscopic and laparoscopic surgery ) intraoperative image ( white arrow indicates the angle of his ; blue arrows indicate several gastrohepatic adhesions , and black arrows indicate the greater curvature of the stomach ) the postoperative course was complicated by a leak resulting in a 3.9 2.8 cm collection in the gastrosplenic ligament adjacent to the surgical suture line [ figure 4 ] . a jackson pratt drain was left alongside the sg at the time of surgery as this is the surgeons protocol for revisional surgery . the patient underwent an upper endoscopy and placement of a 12-cm removable polyflex stent ( boston scientific inc . , natick , ma ) for management of the leak [ figure 5 ] . over the next few weeks further imaging demonstrated interval migration of the stent into the stomach with no change in the size or location of the perigastric collection [ figure 6 ] . the patient was managed conservatively despite the stent movement and had eventual resolution of her symptoms . postoperative upper gi series demonstrating extravasation of contrast at the most proximal aspect of the staple line completion radiograph following endoscopic placement of a removable stent across the anastamotic leak site coronal computed tomographic scan of the abdomen and pelvis with oral and intravenous contrast demonstrating complete migration of the stent into the body of the stomach to our knowledge , this is the first published report of a laparoscopic sg after open gastric banding in a patient with situs inversus totalis . however , modifications in the standard trocar placement can help make the procedure more technically feasible . awareness of the inherited condition before undertaking the operation allows for advanced planning and preparation . in this case , the history of prior open gastric banding made the operation especially tricky , primarily due to the numerous adhesions encountered . revisional surgery is always difficult , but when performed in a patient with situs inversus may be even more challenging . after encountering the initial disorientation , experienced we believe the postoperative leak occurred secondary to the difficult dissection around the gastric fundus , independent of the altered anatomy . these anastamotic leaks can be classified as early or late , depending on how soon postoperatively they occur . in the early period ( <3 days ) , many advocate primary repair . washout and drainage is also an excellent alternative in patients with severe inflammation , signs of sepsis , or hemodynamic instability . the endoscopic placement of polyester or nitinol covered stents is a fairly recent innovation , allowing patients to be maintained on oral enteral nutrition . there have been several reports in the literature supporting their use in both early and late leaks.[911 ] the accepted leak rate for sg is 1.6% at the esophagogastric junction and 0.8% at lower points along the staple line . laparoscopic vertical sg after open gastric banding in a patient with situs inversus can be performed safely by experienced laparoscopic surgeons . while our case yielded a good result , other similar cases are needed to establish the reproducibility and long - term efficacy of the procedure .

the authors present a case of rapidly progressing eosinophilic granuloma ( eg ) of the skull without hemorrhage after minor trauma . a 6-year - old boy presented with a soft mass on the midline of his forehead . he had a surgery for eg 19 months ago . one month earlier , computed tomography ( ct ) and bone scans were performed to evaluate the possible recurrence of eg , and there was no evidence of recurrence in ct . however , a slightly increased uptake in the bone scan was noted on the midline of the forehead . a rapid growing mass developed in a new spot after a minor trauma 7 days before the patient arrived at the clinic . his physical examination was unremarkable , except for a non - tender , soft , and immobile mass . a plain skull x - ray and ct showed a lytic bony defect on the midline of the frontal bone . magnetic resonance imaging showed a 1.4 cm sized enhancing mass . surgical resection and cranioplasty were done . the role of trauma in the development of eg is unclear . however , our case suggests that minor trauma is an aggravating factor for eg formation . careful observation with regular follow - up is necessary in patients with eg after minor trauma . while several equivalent alternatives are available in the bariatric algorithm , more recently the laparoscopic sleeve gastrectomy ( sg ) has been gaining traction as an effective means of weight loss in patients with morbid obesity . we present the case of a 39-year - old woman with situs inversus totalis , who was taken to the operating room for laparoscopic sg . the patient had previously undergone a failed open gastric banding procedure 20 months earlier . awareness of the inherited condition before performing the operation allows for advanced planning and preparation . subsequent modifications to the standard trocar placement help make the procedure more technically feasible . to our knowledge , this is the first published report of a laparoscopic sg after open gastric banding in a patient with situs inversus totalis . after encountering the initial disorientation , we believe experienced laparoscopic surgeons can perform this procedure successfully and safely .

all 53 scz families were recruited from the afrikaner population in south africa and heritage was established by surname and by having 4 afrikaans - speaking grandparents . family history was obtained from the proband , each participating parent , and additional relatives as needed , by two independent raters , a nursing sister , who recorded pedigree information , and by the clinical interviewer , who inquired in detail about family history during the clinical interview . for additional cohort characteristics , the control cohort consisted of 22 families ( triads ) with established afrikaner heritage recruited from the afrikaner community . paternity and maternity were confirmed prior to sequencing for all case and control families via the affymetrix genome - wide human snp array 5.0 as well as via a panel of microsatellite markers . exome enrichment was conducted using the sureselect human all exon target enrichment system ( agilent technologies ) based on the methodology described in . briefly , 3 ug of genomic dna was fragmented by sonication using the covaris s2 to achieve a uniform distribution of fragments with a mean size of 300 bp . the sonicated dna was purified using agencourt s ampure xp solid phase reversible immobilization paramagnetic bead ( spri ) followed by polishing of the dna ends by removing the 3 overhangs and filling in the 5 overhangs resulting from sonication using t4 dna polymerase and klenow fragment ( new england biolabs ) . a-base was added to the 3 end of the dna fragments using klenow fragment ( 3 to 5 exo minus ) . this prepares the dna fragments for ligation to specialized adaptors that have a t-base overhang at their 3ends . the end - repaired dna with a single a-base overhang is ligated to the illumina paired - end adaptors in a standard ligation reaction using t4 dna ligase and 2 um 4 um final adaptor concentration , depending on the dna yield following purification after the addition of the a-base . following ligation , the samples were purified using spri beads , quality controlled by assessment on the agilent bioanalyzer and then amplified by 6 cycles of pcr to maintain complexity and avoid bias due to amplification . 500 ng of amplified , purified dna ( dna library ) was prepared for hybridization by adding the dna library to agilent blocking reagents , denaturing at 95 c and incubating at 65 c . hybridization buffer was added to the prepared library and the entire mix was then added to an aliquot of the agilent sureselect capture library and mixed . the dna library and biotin - labeled capture library were hybridized by incubation at 65 c for 24 hours . following hybridization , streptavidin coated magnetic beads were used to purify the rna : dna hybrids formed during hybridization . the rna capture material was digested via acid hydrolysis following elution from the purification beads . the neutralized captured dna was purified , desalted and amplified by 12 cycles of pcr using herculase ii fusion dna polymerase . the libraries were purified following amplification and the library was assessed using the agilent bioanalyzer . a single peak between 300 400 bp indicates a properly constructed and amplified library ready for sequencing . final quantitation of the library was performed using the kapa biosciences real - time pcr assay and appropriate amounts of the library were loaded onto the illumina flowcell for sequencing by paired - end 50 nt sequencing on the illumina hiseq2000 instrument . following dilution to 10 nm final concentration based on the real - time pcr and bioanalyzer results , the final library stock was then used in paired - end cluster generation at a final concentration of 6 8 pm to achieve a cluster density of 600,000/mm ( on the illumina hiseq2000 instrument ) . following cluster generation , raw sequencing data for each individual were mapped to the human reference genome ( build hg19 ) using the burrows - wheeler aligner ( bwa v0.5.81536 ) . the bwa aligned sequencing reads were processed by picard ( http://picard.sourceforge.net/ ) to label the pcr duplicates . the genome analysis toolkit ( gatk , version 5091 ) was then used to remove duplicates , perform local realignment and map quality score recalibration to produce a cleaned bam file for each individual . snp calls were made by the unified genotyper module in gatk using the cleaned bam files in batch fashion ( 90 samples per batch ) . the resulting variant call format ( vcf , version 4.0 ) files were annotated using the genomicannotator module in gatk to identify and label the called variants that are within the targeted coding regions and overlap with known and likely benign snps reported in dbsnp v132 ( ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606/vcf/v4.0/00-all.vcf.gz ) . the annotated vcf files were then filtered using the gatk variant filter module with a hard filter setting and a custom script for initial filtering . variant calls that failed to pass the following filters were eliminated from the call set : i ) mq0 > = 4 & & ( ( mq0/(1.0 * dp ) ) > 0.1 ) ; ii ) qual < 30.0 || qd < 5.0 || hrun > 5 || sb > 0.00 ; iii ) cluster size 10 ; iv ) contain dbsnp i d ; v ) outside the targeted regions . combined vcf files were then split into individual files and variants in each offspring were compared to variants present in parents using a custom script pipeline in order to determine the inheritance pattern and annotate de novo mutations . because the gatk unified genotyper is set to maximize the sensitivity of variant calls , it allows for a significant portion of false positives among candidate variants even following the initial filtering process . to address this issue and eliminate potential false positive calls in the offspring and false negative calls in the parents , we took advantage of the inheritance information provided by our family design and revalidated all variants identified using the mpileup module in the sam tools ( http://samtools.sourceforge.net/ ) according to the following rules : i ) the forward reference ( fr ) count ( i.e. the number of forward reads that match the reference base at this locus ) , the reverse reference ( rr ) count ( i.e. the number of reverse reads that match the reference base at this locus ) , the forward non reference ( fnr ) count ( i.e. the number of forward reads that do not match the reference base at this locus ) and the reverse non reference ( rnf ) count ( i.e the number of reverse reads that do not match the reference base at this locus ) in the offspring must be 2 or greater ; ii ) total read depth in both parents must be 10 or greater ; iii ) both fr and rr count in both parents must be 2 or greater ; iv ) either fnr or rnr count in both parents must be 0 ; v ) the fnr and rnr count to total count ratio in the parental population ( defined as all 150 parental samples sequenced ) must be less than 1/2n , where n is the population size ; vi ) if any of rules 15 was violated , the sequence information was considered insufficient to make a de novo call at this locus . in / del calls were made by the dindel software using one cleaned bam file per run . the resulting vcf files were used to determine inheritance patterns using the same procedure described above for point mutations . to determine potential mutations at splice - donor or acceptor sites , gatk variant calls were made in a batch fashion ( with 90 samples per batch ) that covered each target coding region and 50 bp flanking segments in each direction . the polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) online server was used to determine the non - synonymous and synonymous nature of the mutations and predict their functional impact by further classifying them as non - tolerated ( damaging ) or benign at a given site . the grantham score for each coding variant the phylop score for each coding variant was extracted from the phylop46wayall table in the ucsc table browser ( http://genome.ucsc.edu/cgi-bin/hgtables ) . the human splicing finder ( hsf , version 2.4.1 ) software ( http://www.umd.be/hsf/ ) was used to predict potential functional impact of the mutations at splice sites . the kolmogorov - smirnov test ( ks - test ) was used to compare the distribution of phylop and grantham scores among de novo or private inherited mutations in cases . fisher s exact test or chi - square test with yates correction was used for the analysis of contingency tables depending on the sample sizes . candidate de novo variants were tested using standard sanger sequencing on an abi 3730xl dna analyzer to validate presence of each mutation in the subjects and absence in the parental genomes , by designing custom primers ( sigma ) based on ~500 bp of genomic sequence flanking each variant . de novo occurrence of mutations was not confirmed in 6 out of 46 and 2 out of 9 candidate alterations in cases and controls , respectively . all 53 scz families were recruited from the afrikaner population in south africa and heritage was established by surname and by having 4 afrikaans - speaking grandparents . family history was obtained from the proband , each participating parent , and additional relatives as needed , by two independent raters , a nursing sister , who recorded pedigree information , and by the clinical interviewer , who inquired in detail about family history during the clinical interview . for additional cohort characteristics , the control cohort consisted of 22 families ( triads ) with established afrikaner heritage recruited from the afrikaner community . paternity and maternity were confirmed prior to sequencing for all case and control families via the affymetrix genome - wide human snp array 5.0 as well as via a panel of microsatellite markers . exome enrichment was conducted using the sureselect human all exon target enrichment system ( agilent technologies ) based on the methodology described in . briefly , 3 ug of genomic dna was fragmented by sonication using the covaris s2 to achieve a uniform distribution of fragments with a mean size of 300 bp . the sonicated dna was purified using agencourt s ampure xp solid phase reversible immobilization paramagnetic bead ( spri ) followed by polishing of the dna ends by removing the 3 overhangs and filling in the 5 overhangs resulting from sonication using t4 dna polymerase and klenow fragment ( new england biolabs ) . a-base was added to the 3 end of the dna fragments using klenow fragment ( 3 to 5 exo minus ) . the end - repaired dna with a single a-base overhang is ligated to the illumina paired - end adaptors in a standard ligation reaction using t4 dna ligase and 2 um 4 um final adaptor concentration , depending on the dna yield following purification after the addition of the a-base . following ligation , the samples were purified using spri beads , quality controlled by assessment on the agilent bioanalyzer and then amplified by 6 cycles of pcr to maintain complexity and avoid bias due to amplification . 500 ng of amplified , purified dna ( dna library ) was prepared for hybridization by adding the dna library to agilent blocking reagents , denaturing at 95 c and incubating at 65 c . hybridization buffer was added to the prepared library and the entire mix was then added to an aliquot of the agilent sureselect capture library and mixed . the dna library and biotin - labeled capture library were hybridized by incubation at 65 c for 24 hours . following hybridization , streptavidin coated magnetic beads were used to purify the rna : dna hybrids formed during hybridization . the rna capture material was digested via acid hydrolysis following elution from the purification beads . the neutralized captured dna was purified , desalted and amplified by 12 cycles of pcr using herculase ii fusion dna polymerase . the libraries were purified following amplification and the library was assessed using the agilent bioanalyzer . a single peak between 300 400 bp indicates a properly constructed and amplified library ready for sequencing . final quantitation of the library was performed using the kapa biosciences real - time pcr assay and appropriate amounts of the library were loaded onto the illumina flowcell for sequencing by paired - end 50 nt sequencing on the illumina hiseq2000 instrument . following dilution to 10 nm final concentration based on the real - time pcr and bioanalyzer results , the final library stock was then used in paired - end cluster generation at a final concentration of 6 8 pm to achieve a cluster density of 600,000/mm ( on the illumina hiseq2000 instrument ) . following cluster generation , raw sequencing data for each individual were mapped to the human reference genome ( build hg19 ) using the burrows - wheeler aligner ( bwa v0.5.81536 ) . the bwa aligned sequencing reads were processed by picard ( http://picard.sourceforge.net/ ) to label the pcr duplicates . the genome analysis toolkit ( gatk , version 5091 ) was then used to remove duplicates , perform local realignment and map quality score recalibration to produce a cleaned bam file for each individual . snp calls were made by the unified genotyper module in gatk using the cleaned bam files in batch fashion ( 90 samples per batch ) . the resulting variant call format ( vcf , version 4.0 ) files were annotated using the genomicannotator module in gatk to identify and label the called variants that are within the targeted coding regions and overlap with known and likely benign snps reported in dbsnp v132 ( ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606/vcf/v4.0/00-all.vcf.gz ) . the annotated vcf files were then filtered using the gatk variant filter module with a hard filter setting and a custom script for initial filtering . variant calls that failed to pass the following filters were eliminated from the call set : i ) mq0 > = 4 & & ( ( mq0/(1.0 * dp ) ) > 0.1 ) ; ii ) qual < 30.0 || qd < 5.0 || hrun > 5 || sb > 0.00 ; iii ) cluster size 10 ; iv ) contain dbsnp i d ; v ) outside the targeted regions . combined vcf files were then split into individual files and variants in each offspring were compared to variants present in parents using a custom script pipeline in order to determine the inheritance pattern and annotate de novo mutations . because the gatk unified genotyper is set to maximize the sensitivity of variant calls , it allows for a significant portion of false positives among candidate variants even following the initial filtering process . to address this issue and eliminate potential false positive calls in the offspring and false negative calls in the parents , we took advantage of the inheritance information provided by our family design and revalidated all variants identified using the mpileup module in the sam tools ( http://samtools.sourceforge.net/ ) according to the following rules : i ) the forward reference ( fr ) count ( i.e. the number of forward reads that match the reference base at this locus ) , the reverse reference ( rr ) count ( i.e. the number of reverse reads that match the reference base at this locus ) , the forward non reference ( fnr ) count ( i.e. the number of forward reads that do not match the reference base at this locus ) and the reverse non reference ( rnf ) count ( i.e the number of reverse reads that do not match the reference base at this locus ) in the offspring must be 2 or greater ; ii ) total read depth in both parents must be 10 or greater ; iii ) both fr and rr count in both parents must be 2 or greater ; iv ) either fnr or rnr count in both parents must be 0 ; v ) the fnr and rnr count to total count ratio in the parental population ( defined as all 150 parental samples sequenced ) must be less than 1/2n , where n is the population size ; vi ) if any of rules 15 was violated , the sequence information was considered insufficient to make a de novo call at this locus . in / del calls were made by the dindel software using one cleaned bam file per run . the resulting vcf files were used to determine inheritance patterns using the same procedure described above for point mutations . to determine potential mutations at splice - donor or acceptor sites , gatk variant calls were made in a batch fashion ( with 90 samples per batch ) that covered each target coding region and 50 bp flanking segments in each direction . the polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) online server was used to determine the non - synonymous and synonymous nature of the mutations and predict their functional impact by further classifying them as non - tolerated ( damaging ) or benign at a given site . the grantham score for each coding variant the phylop score for each coding variant was extracted from the phylop46wayall table in the ucsc table browser ( http://genome.ucsc.edu/cgi-bin/hgtables ) . the human splicing finder ( hsf , version 2.4.1 ) software ( http://www.umd.be/hsf/ ) was used to predict potential functional impact of the mutations at splice sites . the kolmogorov - smirnov test ( ks - test ) was used to compare the distribution of phylop and grantham scores among de novo or private inherited mutations in cases . fisher s exact test or chi - square test with yates correction was used for the analysis of contingency tables depending on the sample sizes . candidate de novo variants were tested using standard sanger sequencing on an abi 3730xl dna analyzer to validate presence of each mutation in the subjects and absence in the parental genomes , by designing custom primers ( sigma ) based on ~500 bp of genomic sequence flanking each variant . de novo occurrence of mutations was not confirmed in 6 out of 46 and 2 out of 9 candidate alterations in cases and controls , respectively . animal models with a human hematopoietic and immune system are now important tools for studying the human immune system as well as human - specific infections , such as hiv-1 [ 1 , 2 ] . recent approaches have involved the use of genetically immunodeficient mice that have been reconstituted using human - derived hematopoietic stem cells ( hscs ) ( known as humanized mice ) . several immunodeficient mice have been developed and nod / scid mice with nk defective genetically modified mice are mainly used for this approach [ 35 ] , because they exhibit deficiencies in macrophage and dendritic cell function , and complement as well as t and b lymphocyte deficiencies . rag-2c mice are also commonly used for establishing humanized mice [ 7 , 8 ] , and it is known that the genetic background of rag-2c mice is influenced by the efficiency of human cell engraftment . jak3 is a non - receptor - type tyrosine kinase crucial for mediating signaling from the common -chain of cytokine receptors , and jak3-deficient mice and common -chain - deficient mice show the same phenotype . based on these findings , we generated rag-2jak3 mice with c57/bl6 and balb / c genetic backgrounds . balb / c rag-2jak3 mice showed high efficiency of both human cd34 hematopoietic stem cell ( hsc ) and peripheral blood mononuclear cell ( pbmc ) transplantation compared with c57/bl6 rag-2jak3 mice . our data show that the genetic background of the mice influences human cell engraftment and balb / c rag-2jak3 mice are becoming an alternative source of humanized mice . c57/bl6 rag-2/jak3 double - deficient mice were established by crossing rag-2-deficient mice ( obtained from the center for animal resources and development , kumamoto university , japan ) and jak3-deficient mice ( kindly provided by dr . balb / c rag-2-deficient ( rag-2 ) mice and balb / c jak3-deficient ( jak3 ) mice were established by crossing rag-2 mice or jak3 mice with the balb / c strain for 10 generations , respectively . balb / c rag-2/jak3 double - deficient ( rag-2jak3 ) mice were established by crossing balb / c rag-2 mice and balb / c jak3 mice , and were housed and monitored in our animal research facility according to institutional guidelines . all experimental procedures and protocols were approved by the institutional animal care and use committee at kumamoto university . peripheral blood and umbilical cord blood samples were collected after obtaining written informed consent and approval from the ethics committee of kumamoto university faculty of medical and pharmaceutical sciences . cd34 cells were isolated using the cd34 progenitor cell isolation kit ( miltenyi biotec , sunnyvale , ca ) and transplanted into the liver of irradiated ( 4.0 gy ) newborn mice . at 12 weeks after transplantation , peripheral blood was obtained and analyzed by flow cytometry . peripheral blood mononuclear cells ( 2 10 cells ) were transplanted into 5.0 gy irradiated adult mice ( 810 weeks old ) as described . at 2 weeks after transplantation , mouse spleen cells were stained with dx5-fitc ( pan nk marker ) , mcd122 ( il-2r)-pe , mcd19-apc , and mcd3-pe / cy7 ( ebiosciences , san diego , ca ) to detect murine lymphocytes . peripheral blood samples were obtained from the retro - orbital sinus of transplanted mice , and red blood cells were lysed with an ammonium chloride - potassium buffer and stained with antimouse cd45-apc / cy7 and antihuman cd45-pb ( biolegends , san diego , ca ) . cells were washed and resuspended in pbs supplemented with 3% fcs , 0.02% nan3 , and 1 g / ml propidium iodide and were analyzed using lsr ii ( bd biosciences , san jose , ca ) . k562 cells ( obtained from riken cell bank , tsukuba , japan ) were cultured in rpmi1640 medium ( sigma ) supplemented with 10% fbs , and were transplanted subcutaneously into the right flank of c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice . the generated c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice survived and bred well under the specific pathogen - free conditions . to confirm the predicted immunophenotype of c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice , single - cell suspensions from spleen cells were labeled with fluorescent antibodies against mouse dx-5 ( pan nk marker ) , cd122 ( il-2r ) , cd3 ( t - cell marker ) , and cd19 ( b - cell marker ) . in contrast to wild - type mice , no b ( cd20 positive ) and t ( cd3 positive ) lymphocytes or nk cells ( dx-5 and cd122 double - positive cells ) were detected in either c57/bl6 or balb / c rag-2jak3 mice ( figure 1 ) . to determine the engraftment efficiency of human hematopoietic stem cells , 2 10 cord blood - derived cd34 cells from normal volunteers were injected intrahepatically into c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice as described [ 5 , 7 ] . levels of human cell engraftment were determined 12 weeks later ( figure 2(a ) ) . of the engrafted c57/bl6 rag-2jak3 mice , 18 of 28 mice had less than 0.1% of human cd45 cells , and only 2 had > 10% human cd45 cells in the peripheral blood ( 1.7 4.5% , n = 28 ) . in contrast , balb / c rag-2jak3 mice were highly engrafted and reconstituted with human cells ( 37.8 21.5% , n = 24 ) ( figure 2(b ) ) . to determine the engraftment efficiency of human primary lymphocyte populations , 2 10 pbmcs from normal volunteers were injected intraperitoneally into c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice . although human cells ( human cd45 cells ) were detected in c57/bl6 rag-2jak3 mice , the degree of engraftment was very low ( 1.7 4.5% , n = 13 ) . in contrast , balb / c rag-2jak3 mice were highly engrafted with human cells ( 37.8 21.5% , n = 12 ) ( figure 3 ) . subcutaneous administration of k562 cells resulted in solid tumor formation in both strains of rag-2jak3 mice . the weight of tumors in both strains showed no significant difference ( c57/bl6 : 0.59 0.26 g , n = 7 , balb / c 0.63 0.33 g , n = 7 , p > in this study , we established c57/bl6 and balb / c strains of rag-2jak3 mice , and showed that balb / c rag-2jak3 mice supported a high level of human lymphohematopoietic cell engraftment , whereas c57/bl6 rag-2jak3 mice achieved little engraftment . interestingly , both strains supported the engraftment of human hematopoietic malignancy . humanized mice with reconstituted human hematopoietic and immune cells are becoming a powerful tool for the investigation of human biological systems and for translational research . nod / scid mice have been used for these purposes ; however , human hematopoietic stem cell engraftment is not sufficient and functionally mature lymphocytes are not developed in these mice , mostly due to residual nk activity in this mouse . to overcome these limitations , nod / scid mice were intercrossed with nk defective genetically modified mice , and several strains of mice suitable for humanized mice were established [ 35 ] . among them , nod / scid/c ( nog and nsg ) mice are becoming the gold standard for generating humanized mice . recently , spira gene polymorphism has been identified as a determinant influencing the efficacy of human hematopoietic stem cell engraftment into the nod / scid strain . meanwhile , stroma cells of the balb / c strain have been shown to support human hematopoiesis to a lesser extent but not other strains ( c57/bl6 , c3h , icr ) , indicating that other mechanisms support human cell engraftment in immunodeficient mice than spira gene polymorphism . in fact , manz and colleagues described the reconstitution of human acquired and innate immunity in balb / c rag-2c mice . although no study has compared reconstitution efficiency directly between nod / scid/c mice and balb / c rag-2c mice , the efficiency of human hematopoietic stem cell engraftment seems to be higher in nod background from the previous studies [ 1 , 2 ] . however , the efficiency of reconstitution in balb / c rag-2c mice is high enough for human immune and hematopoietic cell research ( figures 2 and 3 ) and balb / c background has several advantages compared with nod / scid background . mice with the scid mutation lack functional t and b lymphocytes resulting from the absence of gene recombination events in t - cell receptors ( tcrs ) and immunoglobulins ; however , they are known to show a leaky phenomenon in which functional t and b lymphocytes are produced with aging or ionized irradiation [ 13 , 14 ] . in addition , the prkdc gene disrupted by the scid mutation is expressed broadly and is involved in dna repair , while expressions of rag-1 and rag-2 are limited to hematopoietic cells and are involved only in dna recombination of t- and b - cell receptor genes , indicating that scid mice are more sensitive to radiation - induced dna damage than their rag-1 or rag-2 counterparts . established radioresistant nod rag-1c mice and showed that they support a similar level of human lymphohematopoietic cell engraftment , which could overcome the disadvantages of the scid mutation . the nod / scid strain has a predisposition to thymic lymphoma due to an endogenous ectopic provirus ( emv-30 ) . nod / scid and nod / scid/2 m mice have a short mean life span for this reason [ 6 , 17 ] , while nod / scid/c ( nog and nsg ) mice do not develop thymic lymphoma for an unknown reason . balb / c strain could overcome these disadvantages of nod / scid strain , although efficiency of human hematopoietic stem cell engraftment is not better than nod / scid strain . as the strain background is known to affect human - cell engraftment and to function markedly in scid mice [ 1 , 2 , 18 ] , we established and directly compared c57/bl6 and balb / c strains of rag-2jak3 mice . rag-2jak3 mice showed the same phenotype as rag-2c mice lacking t , b , and nk cells ( figure 1 ) . the efficiency of human hematopoietic stem cell and pbmc engraftment in c57/bl6 rag-2jak3 mice was extremely poor , although balb / c mice and rag-2jak3 mice appeared to have similar levels as balb / c rag-2c mice ( figures 2 and 3 ) . the breeding performance of balb / c rag-2c mice was the same as balb / c mice in our study . in addition , balb / c rag-2c mice were relatively radioresistant compared with nod / scid strains . thus , balb / c rag-2jak3 mice are a very useful strain of immunodeficient mice derived from animals with defined molecular lesions and have distinct advantages over nod / scid - based immunodeficient mice . in conclusion , we established c57/bl6 and balb / c strains of rag-2jak3 mice and showed that balb / c rag-2jak3 mice enabled high human hematopoietic cell engraftment .

despite high heritability , a large fraction of cases with schizophrenia do not have a family history of the disease ( sporadic cases ) . here , we examine the possibility that rare de novo protein - altering mutations contribute to the genetic component of schizophrenia by sequencing the exome of 53 sporadic cases , 22 unaffected controls and their parents . we identified 40 de novo mutations in 27 patients affecting 40 genes including a potentially disruptive mutation in dgcr2 , a gene removed by the recurrent schizophrenia - predisposing 22q11.2 microdeletion . comparison to rare inherited variants revealed that the identified de novo mutations show a large excess of nonsynonymous changes in cases , as well as a greater potential to affect protein structure and function . our analysis reveals a major role of de novo mutations in schizophrenia and also a large mutational target , which together provide a plausible explanation for the high global incidence and persistence of the disease . immunodeficient mice are becoming invaluable tools in human stem cell and tumor research . in this study , we generated rag-2/jak3 double - deficient ( rag-2/jak3/ ) mice with a c57/bl6 and balb / c genetic background and compared the human lymphohematopoietic cell engraftment rate . human cord blood - derived cd34 + hematopoietic stem cells were successfully engrafted into balb / c rag-2/jak3/ mice ; however , the engraftment rate was far lower in c57/bl6 rag-2/jak3/ mice . transplantation of human peripheral blood mononuclear cells resulted in the same tendency . thus , a balb / c background offers superior engraftment capacity than a c57/bl6 background and provides an attractive model for human hematopoietic cell engraftment .

cardioembolic stroke ( ces ) , which causes 20% of all ischemic strokes each year , leads to severe neurological deficits . ces is associated with high mortality and is a common cause of its atrial fibrillation ( af ) , which has an increasing incidence with age [ 35 ] . proposed that af is an important and treatable cause of recurrent stroke and needs to be ruled - out by thorough evaluation before the diagnosis of cryptogenic stroke is assigned . ces is largely preventable through control of major primary cardioembolic risk factors , such as hyperlipidemia and high blood pressure . giralt et al . offered evidence of significant genetic involvement in ischemic stroke . in recent years , gene expression profiling of human disease tissues has provided insights into molecular mechanisms and eventually led to the identification of novel therapeutic targets . currently available high - throughput microarray experiments were developed to analyze genetic expression patterns with differentially expressed genes ( deg ) and dysregulated pathways . canonical reports claimed that gene expression patterns can identify biomarkers of ischemic stroke , which highlighted the relevance of the innate immune system through deg and signaling pathways [ 1113 ] . however , most methods did not consider regulatory cross - talk among pathways , and treated pathways as independent mechanisms . although it is intuitive that interacting pathways could influence each other , the presence of this frame and available technique have not been completely studied yet . antonio et al . developed an integrated approach to identify functional mirnas regulating pathway cross - talk in breast cancer with pairs of pathways . differential protein - protein interaction networks were constructed in ces with akaike information criterion ( aic ) method . to the best of our knowledge , there are few studies that constructed pathway networks correctly to discriminate controls versus ces . in this work we develop an integrated approach that is able to construct individual networks comprising pathways cross - talk to quantify differences between ces and controls . we used the individualized pathway aberrance score ( ipas ) to assess pathway statistics of every ingenuity pathways analysis ( ipa ) pathway . random forest ( rf ) classification was implemented to calculate the auc of every network . these procedures were tested by monte carlo cross - validation ( mccv ) for 50 bootstraps . . the novel approach may be the basis of individual medical treatment in ces , serving as therapy targeting markers . one biological dataset , e - geod-58294 , was derived from the gene expression omnibus ( geo ) database ( http://www.ncbi.nlm.nih.gov/geo/ ) . the platform was a - affy-44 affymetrix genechip human genome u133 plus 2.0 , which was used to read the gene chip % h0 . the linear models for microarray data ( limma ) was then used to preprocess data . after quantile data normalization performed by robust multi - array average ( rma ) , 20 544 genes were obtained . in order to identify a group of pathways significantly enriched in ces with respect to controls , we collected 589 biological pathways including 5169 genes from the ipa tool ( http://www.ingenuity.com/ ) . after genes of expression profile were enriched in ipa pathways , we focused on 4929 genes . raw p - values were adjusted by false - discovery rate ( fdr ) procedure for multiple testing corrections . a total of 23 accumulated normal samples ( ans ) were used to identify ipa pathways as reference . individual normal sample gene expression was standardized with the mean and standard deviation ( sd ) . for genes of every ces sample , as quantile normalization was performed . average z equation was recently proved to be a biologically valid modification of pathway analysis methods for ipas . z=(z1 , z2 , , zn ) represents the expression state of a pathway where zi denotes the standardized expression value of i - th gene and the number of genes existing in the pathway is n. gene statistics of each gene from every ces sample : each ipa pathway statistics : zi represents the standardized gene level statistics of 1-i gene and the number of genes existing in the pathway is n. z values of every pathway in ces samples were gathered after significance testing . differentially expressed pathways were selected with z<0.05 . the discriminating score ( ds ) was computed to quantify pathway cross - talk in each sample for the pair of pathways x and y. ds was defined as where mx and sx represent mean and standard deviation of expression levels of genes in a pathway x and my and sy in a pathway y . ds score indicates the relationships between pairs of pathway , with a larger value indicating relatively higher difference of activity between pathways . ds of normal samples was standardized using the mean and sd as reference . z values of every pathway pair in ces samples z values of differentially expressed pathways and pathway pairs were used to construct individual networks with cytoscape version 3.2.0 . it randomly selected expression data in proportion 6:4 to form the training and testing set . then the process was repeated in 50 bootstraps , randomly generating new training and test partitions each time . the number of main networks appearing in the 50 bootstraps was counted by ranking all networks with their auc values . one biological dataset , e - geod-58294 , was derived from the gene expression omnibus ( geo ) database ( http://www.ncbi.nlm.nih.gov/geo/ ) . the platform was a - affy-44 affymetrix genechip human genome u133 plus 2.0 , which was used to read the gene chip % h0 . the linear models for microarray data ( limma ) was then used to preprocess data . after quantile data normalization performed by robust multi - array average ( rma ) , 20 544 genes were obtained . in order to identify a group of pathways significantly enriched in ces with respect to controls , we collected 589 biological pathways including 5169 genes from the ipa tool ( http://www.ingenuity.com/ ) . after genes of expression profile were enriched in ipa pathways , we focused on 4929 genes . raw p - values were adjusted by false - discovery rate ( fdr ) procedure for multiple testing corrections . a total of 23 accumulated normal samples ( ans ) were used to identify ipa pathways as reference . individual normal sample gene expression was standardized with the mean and standard deviation ( sd ) . for genes of every ces sample , as quantile normalization was performed . average z equation was recently proved to be a biologically valid modification of pathway analysis methods for ipas . z=(z1 , z2 , , zn ) represents the expression state of a pathway where zi denotes the standardized expression value of i - th gene and the number of genes existing in the pathway is n. gene statistics of each gene from every ces sample : each ipa pathway statistics : zi represents the standardized gene level statistics of 1-i gene and the number of genes existing in the pathway is n. z values of every pathway in ces samples were gathered after significance testing . the discriminating score ( ds ) was computed to quantify pathway cross - talk in each sample for the pair of pathways x and y. ds was defined as where mx and sx represent mean and standard deviation of expression levels of genes in a pathway x and my and sy in a pathway y . ds score indicates the relationships between pairs of pathway , with a larger value indicating relatively higher difference of activity between pathways . ds of normal samples was standardized using the mean and sd as reference . z values of every pathway pair in ces samples z values of differentially expressed pathways and pathway pairs were used to construct individual networks with cytoscape version 3.2.0 . it randomly selected expression data in proportion 6:4 to form the training and testing set . then the process was repeated in 50 bootstraps , randomly generating new training and test partitions each time . the number of main networks appearing in the 50 bootstraps was counted by ranking all networks with their auc values . in the present study we developed an integrated approach that was sufficient to construct individual networks comprising pathways cross - talk to quantify differences between ces and controls . rf classification was implemented to calculate auc of every network , which was tested by mccv for 50 bootstraps . we obtained a heatmap in which pink squares indicate pathway pairs for classification in the training dataset for that bootstrap ( the frequency > 6 ) . there were 4 pairs of pathways in 46 bootstraps : cholesterol biosynthesis i and cholesterol biosynthesis ii , cholesterol biosynthesis i and cholesterol biosynthesis iii , cholesterol biosynthesis ii and cholesterol biosynthesis iii , uracil degradation ii and thymine degradation . individual networks were ordered with respect to their auc and 28 networks with auc > 0.9 were found between ces and controls . among them , 3 networks with auc=1.0 had the best performance for classification of ces and normal samples for all 50 bootstraps . as shown in figure 2 , the best individual networks were in 4 , 10 , and 23 bootstraps . therefore , the 3 pathway networks were able to significantly identify ces versus controls , which showed as biomarkers in the regulation and development of ces . then we found there were 22 pairs of pathways that commonly appeared in 3 networks ( table 1 ) , which revealed that the pathway pairs were important in regulating ces . given the substantial difference in the activities of main networks between ces and controls , we examined its effectiveness in classifying ces and normal samples based on their profiles . in the best 3 networks , we focused on pathways that had multi - cross - talk with others . the msp - ron signaling pathway had the most cross - talk , which played an important interaction role in the best networks . a previous study has reported that msp - ron signaling is important for the invasive growth of many types of cancers and appeared to have potential as a therapeutic target . pathway analysis has become the first choice for extracting and explaining the underlying pathology for high - throughput molecular measurements . personalized identification of altered pathway pairs is important for understanding disease mechanisms and for the future application of custom therapeutic decisions . existing pathway analysis methods are not suitable for identifying the pathway aberrance that may occur in an individual sample . therefore , we employed the ipas to analyze the personalized identification of networks , taking advantage of a vast number of normal samples . a key innovation of the method is ipas using ans in ces . proved that the average z equation can efficiently reveal noticeable aberrance in expression profiles and clinical significance , which sufficed to confirm the best averaged validation rate and distinguish a known survival - relevant pathway statistically . furthermore , ans data is expected to be available in more fields of medicine along with rapid advances in high - throughput databases . ds obtained lightly more improvement than the euclidean distance as a metric to quantify pathway cross - talk . in recent years , different validation technologies have been generally used to evaluate performance of pathways and networks in medical regression analysis . the mccv pays attention to a notable part of the sample at a time during network building and validation with multi - repeats . compared with conventional validation tests for capturing the best predictor variables , mccv showed superior performance , resulting from a form of cross - validation based on vast combinations of data sets . interestingly , mccv has not been utilized in individual networks comprising pathways cross - talk in ces patients . in this study we developed an integrated approach to quantify differences between ces and controls with the mccv test , which suggests that mccv worked better , based on strong predictive ability . screened networks were efficient in distinguishing differences among individual ces samples , and can provide broader carcinogenic insight in personalized medicine . the final purpose of our approach was to detect the best network able to discriminate ces versus controls . we found that the 3 best networks were similar and had 22 common pairs of pathways . we tended to select network 10 to differentiate ces disease from normal samples , with the fewest pairs of pathways ( figure 2b ) . our novel approach identified 3 networks able to accurately classify ces and normal samples in individuals . study participants were members of the cardiovascular health study ( chs ) , a prospective cohort consisting of 5,888 men and women aged 65 years who were randomly selected from medicare - eligibility lists in four u.s . communities ( forsyth county , nc ; washington county , md ; sacramento county , ca ; and pittsburgh , pa ) . a detailed description of methods and procedures in the chs has previously been published ( 26 ) . briefly , persons eligible to participate were not institutionalized or wheelchair dependent , did not require a proxy for consent , were not receiving treatment for cancer , and were expected to remain in their respective regions for 3 years . from 1989 to 1990 , 5,201 participants were recruited in the original cohort . between 1992 and 1993 , 687 subjects ( predominantly african american ) were also recruited . baseline evaluation of study participants included standardized questionnaires , physical examination , anthropometric measurements , resting electrocardiography , and laboratory examinations . from 1989 through 1999 , participants were followed up every 6 months , alternating between telephone calls and clinic visits ; biennial telephone calls have continued since then . the institutional review board at each center approved the study , and each participant gave informed consent . for this analysis , the 19921993 clinic visit was used as baseline . of the 5,553 participants alive at the 19921993 clinic visit , we excluded 773 people with prevalent diabetes , 780 people with missing data on fabp4 and nefa , 205 people with missing data on prevalent or incident type 2 diabetes , and 55 individuals with missing covariate information . plasma samples collected at the 19921993 examination were stored at 70c until analyzed at the central laboratory at the university of vermont . the interassay coefficient of variation ( cv ) was 2.615.32% ( detectable range 5250 ng / ml ) . this technique relies on the acylation of coa by the fatty acids in the presence of added acyl - coa synthetase . acyl - coa produced is oxidized by added acyl - coa oxidase with generation of hydrogen peroxide and in the presence of peroxidase permits the oxidative condensation of 3-methy - n - ethyl - n(-hydroxyethyl)-aniline with 4-aminoantipyrine to form a purple - colored adduct . we assessed medication use at baseline and annually by a validated medication inventory ( 27 ) through 2007 . in addition , fasting glucose was measured on blood specimens from the examinations in years 19921993 , 19961997 , 19981999 , and 20052006 ; nonfasting glucose was measured on blood specimens from 1994 to 1995 . a participant was classified as having type 2 diabetes if any of the following conditions were met : 1 ) use of insulin or oral hypoglycemic agents , 2 ) fasting glucose level of 7 mmol / l ( 126 mg / dl ) , or 3 ) a nonfasting glucose level of 11.1 mmol / l ( 200 mg / dl ) . age , sex , race , years of education , smoking status , and alcohol consumption were based on self - report . leisure - time activity ( kilocalories per week ) was assessed using a modified minnesota leisure - time activities questionnaire ( 28 ) . bmi was calculated as weight in kilograms divided by the square of height in meters . missing values for smoking and height to calculate bmi were carried forward from previous years if available . serum albumin was assessed using the kodak ektachem 700 analyzer ( eastman kodak , rochester , ny ) . plasma triglyceride was measured by enzymatic methods on an olympus demand system ( olympus , lake success , ny ) . hdl was measured by an enzymatic method after precipitation of apolipoprotein b containing lipoproteins with dextran sulfate / magnesium sulfate . interassay cvs using reference standards for triglycerides and hdl were 1.78 and 2.15% , respectively . cystatin c was measured by nephelometer using a particle - enhanced immunonephelometric assay ( n latex cystatin c ; dade behring , newark , de ) ( 30 ) with a interassay cv of 2.93.3% . baseline characteristics of study participants were summarized according to categories of fabp4 and nefa ; continuous variables were presented as means sd and categorical variables as percentages . cox proportional hazards regression was used to estimate the association of fabp4 and nefa with incident type 2 diabetes to allow adjustment for covariates . because the distribution of fabp4 differed appreciably between men and women , fabp4 was modeled as sex - specific tertiles with the lowest tertile as the referent category . nefa was similarly modeled as tertiles based on the overall distribution and also continuously per sd . individuals were censored for death , loss to follow - up , or end of diabetes ascertainment ( 20062007 ) . potential confounders included in adjusted models were age , race , and sex ( where appropriate ) ( model 1 ) with the addition of bmi ( included in nefa model only ) , education ( less than high school versus high school or more ) , cystatin c ( log transformed and included in fabp4 model only ) , serum albumin ( included in nefa model only ) , kilocalories of physical activity ( log transformed ) , alcohol intake ( none and < 0.5 , 0.51 , and > 1 drinks / day for women ; none and < 1 , 12 , and > 2 drinks / day for men ) , smoking status ( never , former , and current ) , self - reported health status ( fair / poor versus better ) , and estrogen use for women ( model 2 ) . given the production of fabp4 in adipocytes , we examined interaction between bmi and fabp4 . to evaluate intermediate pathways by which fabp4 might lead to type 2 diabetes , we adjusted for c - reactive protein ( log transformed ) , homa - ir , triglycerides ( log transformed ) , hdl , and ldl . to evaluate the joint impact of fabp4 and nefa on diabetes risk we also tested for an interaction between fabp4 and nefa on the risk of incident type 2 diabetes . in secondary analyses , we evaluated whether there were statistically significant interactions between log fabp4 and age or waist circumference or between nefa and sex or bmi . because measured weight may not always reflect adiposity in older adults , we excluded participants with unintentional weight loss ( defined as self - reported loss of 10 pounds not due to diet or exercise during the past 12 months ) , prevalent cardiovascular disease ( coronary heart disease or stroke ) , or cancer in a sensitivity analysis . since plasma nefa varies with dietary intake ( 31 ) and lipid metabolism and a single measure may not be a good estimate of average concentrations over the long term , we repeated the analysis while restricting follow - up to the first 5 years . schoenfeld residuals and plots of the residuals over time were used to evaluate proportional hazards assumptions ; there were no meaningful violations . stata , version 11.1 ( statacorp , college station , texas ) , was used for all analyses , and p values < 0.05 were considered statistically significant . plasma samples collected at the 19921993 examination were stored at 70c until analyzed at the central laboratory at the university of vermont . the interassay coefficient of variation ( cv ) was 2.615.32% ( detectable range 5250 ng / ml ) . this technique relies on the acylation of coa by the fatty acids in the presence of added acyl - coa synthetase . acyl - coa produced is oxidized by added acyl - coa oxidase with generation of hydrogen peroxide and in the presence of peroxidase permits the oxidative condensation of 3-methy - n - ethyl - n(-hydroxyethyl)-aniline with 4-aminoantipyrine to form a purple - colored adduct . we assessed medication use at baseline and annually by a validated medication inventory ( 27 ) through 2007 . in addition , fasting glucose was measured on blood specimens from the examinations in years 19921993 , 19961997 , 19981999 , and 20052006 ; nonfasting glucose was measured on blood specimens from 1994 to 1995 . a participant was classified as having type 2 diabetes if any of the following conditions were met : 1 ) use of insulin or oral hypoglycemic agents , 2 ) fasting glucose level of 7 mmol / l ( 126 mg / dl ) , or 3 ) a nonfasting glucose level of 11.1 mmol / l ( 200 mg / dl ) . age , sex , race , years of education , smoking status , and alcohol consumption were based on self - report . leisure - time activity ( kilocalories per week ) was assessed using a modified minnesota leisure - time activities questionnaire ( 28 ) . bmi was calculated as weight in kilograms divided by the square of height in meters . missing values for smoking and height to calculate bmi were carried forward from previous years if available . serum albumin was assessed using the kodak ektachem 700 analyzer ( eastman kodak , rochester , ny ) . plasma triglyceride was measured by enzymatic methods on an olympus demand system ( olympus , lake success , ny ) . hdl was measured by an enzymatic method after precipitation of apolipoprotein b containing lipoproteins with dextran sulfate / magnesium sulfate . interassay cvs using reference standards for triglycerides and hdl were 1.78 and 2.15% , respectively . cystatin c was measured by nephelometer using a particle - enhanced immunonephelometric assay ( n latex cystatin c ; dade behring , newark , de ) ( 30 ) with a interassay cv of 2.93.3% . baseline characteristics of study participants were summarized according to categories of fabp4 and nefa ; continuous variables were presented as means sd and categorical variables as percentages . cox proportional hazards regression was used to estimate the association of fabp4 and nefa with incident type 2 diabetes to allow adjustment for covariates . because the distribution of fabp4 differed appreciably between men and women , fabp4 was modeled as sex - specific tertiles with the lowest tertile as the referent category . nefa was similarly modeled as tertiles based on the overall distribution and also continuously per sd . individuals were censored for death , loss to follow - up , or end of diabetes ascertainment ( 20062007 ) . potential confounders included in adjusted models were age , race , and sex ( where appropriate ) ( model 1 ) with the addition of bmi ( included in nefa model only ) , education ( less than high school versus high school or more ) , cystatin c ( log transformed and included in fabp4 model only ) , serum albumin ( included in nefa model only ) , kilocalories of physical activity ( log transformed ) , alcohol intake ( none and < 0.5 , 0.51 , and > 1 drinks / day for women ; none and < 1 , 12 , and > 2 drinks / day for men ) , smoking status ( never , former , and current ) , self - reported health status ( fair / poor versus better ) , and estrogen use for women ( model 2 ) . given the production of fabp4 in adipocytes , we examined interaction between bmi and fabp4 . to evaluate intermediate pathways by which fabp4 might lead to type 2 diabetes , we adjusted for c - reactive protein ( log transformed ) , homa - ir , triglycerides ( log transformed ) , hdl , and ldl . to evaluate the joint impact of fabp4 and nefa on diabetes risk we also tested for an interaction between fabp4 and nefa on the risk of incident type 2 diabetes . in secondary analyses , we evaluated whether there were statistically significant interactions between log fabp4 and age or waist circumference or between nefa and sex or bmi . because measured weight may not always reflect adiposity in older adults , we excluded participants with unintentional weight loss ( defined as self - reported loss of 10 pounds not due to diet or exercise during the past 12 months ) , prevalent cardiovascular disease ( coronary heart disease or stroke ) , or cancer in a sensitivity analysis . since plasma nefa varies with dietary intake ( 31 ) and lipid metabolism and a single measure may not be a good estimate of average concentrations over the long term , we repeated the analysis while restricting follow - up to the first 5 years . schoenfeld residuals and plots of the residuals over time were used to evaluate proportional hazards assumptions ; there were no meaningful violations . stata , version 11.1 ( statacorp , college station , texas ) , was used for all analyses , and p values < 0.05 were considered statistically significant . among 3,740 participants , 2,259 were women ( 60% ) and the mean age at baseline was 74.8 years ( range 6598 ) . median plasma fabp4 was 29.5 ng / ml ( interquartile range 22.039.3 ) , and women had substantially higher concentrations of fabp4 ( median 34.4 ng / ml ) than men ( median 22.8 ng / ml ) . compared with the lowest tertile of fabp4 , those in the highest tertile had higher measures of adiposity and were more likely to have insulin resistance , higher prevalence of dyslipidemia , poorer health , higher levels of cystatin c , and lower physical activity . details on correlation coefficients between fabp4 and risk factors for type 2 diabetes are presented in supplementary table 1 . characteristics of 3,740 participants by sex - specific tertiles of fabp4 in the chs during a mean follow - up of 9.5 years , 305 cases of incident diabetes occurred . hazard ratios ( hrs ) associated with each sd increase in log fabp4 were 1.39 ( 95% ci 1.191.62 ) overall , 1.35 ( 1.101.65 ) for women , and 1.45 ( 1.131.85 ) for men after adjustment for age , race , sex , education , physical activity , cystatin c , alcohol intake , smoking , self - reported health status , and estrogen use for women ( table 2 ) , p for sex - by - fabp4 interaction 0.10 ) . there was evidence for an interaction between bmi and fabp4 on the risk of diabetes ( p value for interaction between continuous bmi and log fabp4 : p = 0.009 overall , 0.02 for women , and 0.135 for men ) ( table 3 ) . fabp4 was associated with diabetes in people with lower bmi in both sexes , but the only significantly elevated risk was among lean men ( table 3 ) . incidence rate and hrs of type 2 diabetes according to fabp4 tertiles or sd of log fabp4 ( 0.46 ) in the chs hr of diabetes per sd increase in log fabp4 stratified by bmi in the chs * as expected , additional adjustment for potential mediating factors ( c - reactive protein , homa - ir , ldl and hdl cholesterol , and triglycerides ) attenuated the observed association further ( relative risk [ rr ] per sd increase of log fabp4 of 1.25 [ 95% ci 1.011.55 ] ) for women and 1.17 [ 0.901.53 ] for men ) . relations between each potential mediator and type 2 diabetes when added individually to the full model are shown in supplementary table 2 . there was no statistically significant interaction between fabp4 and nefa ( p = 0.16 ) , and results were similar with the addition of nefa to these models ( data not shown ) . in a sensitivity analysis , exclusion of individuals with reported unintentional weight loss , prevalent cardiovascular disease , and cancer tended to strengthen the association ( hr per sd increase in log fabp4 from 1.39 [ 95% ci 1.191.62 ] before exclusion to 2.42 [ 1.563.73 ] after exclusion for men and women combined ) . participants with higher concentrations of nefa were older , were more likely to be female , had higher measures of adiposity , and had higher levels of total cholesterol , triglycerides , hdl , and c - reactive protein levels ( supplementary table 3 ) . in a multivariable model adjusted for age , sex , ethnicity , education , bmi , leisure - time physical activity ( kilocalories ) , smoking , alcohol use , health status , estrogen use ( in women ) , and serum albumin , the hr associated with each sd of nefa ( 0.20 meq / l ) was 1.08 ( 95% ci 0.961.23 ) ( table 4 ) . results were similar with the addition of fabp4 to these models ( data not shown ) . when stratified by bmi ( < 25 , 2529.9 , and 30 kg / m ) , the results did not change materially and the p value for bmi - by - nefa interaction was 0.13 . in a secondary analysis restricted to the first 5 years of follow up , each sd of higher nefa was associated with an hr of 1.15 ( 0.971.36 ) in a fully adjusted model , and there was evidence for increased risk of diabetes at higher levels of nefa when analyzed by tertiles of nefa ( 1.0 [ ref . ] , 1.68 [ 95% ci 1.122.53 ] , and 1.63 [ 1.072.50 ] ) ( table 4 ) . there was no evidence for sex - by - nefa interaction ( p = 0.97 ) . hr ( 95% ci ) of diabetes by category of and per sd ( 0.20 meq / l ) increase in nefas in the chs table 1 presents baseline characteristics of subjects according to tertiles of fabp4 by sex . compared with the lowest tertile of fabp4 , those in the highest tertile had higher measures of adiposity and were more likely to have insulin resistance , higher prevalence of dyslipidemia , poorer health , higher levels of cystatin c , and lower physical activity . details on correlation coefficients between fabp4 and risk factors for type 2 diabetes are presented in supplementary table 1 . characteristics of 3,740 participants by sex - specific tertiles of fabp4 in the chs during a mean follow - up of 9.5 years , 305 cases of incident diabetes occurred . hazard ratios ( hrs ) associated with each sd increase in log fabp4 were 1.39 ( 95% ci 1.191.62 ) overall , 1.35 ( 1.101.65 ) for women , and 1.45 ( 1.131.85 ) for men after adjustment for age , race , sex , education , physical activity , cystatin c , alcohol intake , smoking , self - reported health status , and estrogen use for women ( table 2 ) , p for sex - by - fabp4 interaction 0.10 ) . there was evidence for an interaction between bmi and fabp4 on the risk of diabetes ( p value for interaction between continuous bmi and log fabp4 : p = 0.009 overall , 0.02 for women , and 0.135 for men ) ( table 3 ) . fabp4 was associated with diabetes in people with lower bmi in both sexes , but the only significantly elevated risk was among lean men ( table 3 ) . incidence rate and hrs of type 2 diabetes according to fabp4 tertiles or sd of log fabp4 ( 0.46 ) in the chs hr of diabetes per sd increase in log fabp4 stratified by bmi in the chs * as expected , additional adjustment for potential mediating factors ( c - reactive protein , homa - ir , ldl and hdl cholesterol , and triglycerides ) attenuated the observed association further ( relative risk [ rr ] per sd increase of log fabp4 of 1.25 [ 95% ci 1.011.55 ] ) for women and 1.17 [ 0.901.53 ] for men ) . relations between each potential mediator and type 2 diabetes when added individually to the full model are shown in supplementary table 2 . there was no statistically significant interaction between fabp4 and nefa ( p = 0.16 ) , and results were similar with the addition of nefa to these models ( data not shown ) . in a sensitivity analysis , exclusion of individuals with reported unintentional weight loss , prevalent cardiovascular disease , and cancer tended to strengthen the association ( hr per sd increase in log fabp4 from 1.39 [ 95% ci 1.191.62 ] before exclusion to 2.42 [ 1.563.73 ] after exclusion for men and women combined ) . participants with higher concentrations of nefa were older , were more likely to be female , had higher measures of adiposity , and had higher levels of total cholesterol , triglycerides , hdl , and c - reactive protein levels ( supplementary table 3 ) . in a multivariable model adjusted for age , sex , ethnicity , education , bmi , leisure - time physical activity ( kilocalories ) , smoking , alcohol use , health status , estrogen use ( in women ) , and serum albumin , the hr associated with each sd of nefa ( 0.20 meq / l ) was 1.08 ( 95% ci 0.961.23 ) ( table 4 ) . results were similar with the addition of fabp4 to these models ( data not shown ) . when stratified by bmi ( < 25 , 2529.9 , and 30 kg / m ) , the results did not change materially and the p value for bmi - by - nefa interaction was 0.13 . in a secondary analysis restricted to the first 5 years of follow up , each sd of higher nefa was associated with an hr of 1.15 ( 0.971.36 ) in a fully adjusted model , and there was evidence for increased risk of diabetes at higher levels of nefa when analyzed by tertiles of nefa ( 1.0 [ ref . ] , 1.68 [ 95% ci 1.122.53 ] , and 1.63 [ 1.072.50 ] ) ( table 4 ) . there was no evidence for sex - by - nefa interaction ( p = 0.97 ) . hr ( 95% ci ) of diabetes by category of and per sd ( 0.20 meq / l ) increase in nefas in the chs in this prospective study of 3,740 people , we demonstrate a positive association between plasma fabp4 and incident type 2 diabetes in older adults and such association was modified by bmi in a way that a statistically significant higher risk of diabetes with fabp4 was observed in lean men only . we also observed a significant positive association between plasma nefa and incident type 2 diabetes during the first 5 years that was attenuated and not statistically significant when the total follow - up period was considered . this association was not materially altered with adjustments for fabp4 , and there was no interaction between fabp4 and nefa with incident type 2 diabetes . to the best of our knowledge , this is the first large prospective study to evaluate associations between plasma fabp4 and incident type 2 diabetes among older men and women in a community setting . while several animal studies have examined the effects of fabp4 expression on adiposity , insulin resistance , and type 2 diabetes risk ( 7,10,11,32 ) , there are only limited data available in humans . in a cross - sectional study of 98 patients with coronary artery disease , serum levels of fabp4 were positively correlated with the prevalence of metabolic syndrome ( mets ) ( p = 0.037 ) and the number of mets components ( p = 0.035 ) ( 33 ) . in another cross - sectional study of 806 middle - aged men and women , each sd of higher log fabp4 was associated with a 1.85-fold increased odds of mets ( 95% ci 1.532.23 ) controlling for age , sex , and race ( 5 ) . ( 8) ( n = 229 ) and stejskal and karpisek ( 6 ) ( n = 138 ) have reported positive associations between serum concentrations of fabp4 and mets . baseline fabp4 was also shown to be associated with incident mets after 5 years of follow - up in 356 chinese individuals ( 9 ) . only one study has evaluated the association of fabp4 levels with incident type 2 diabetes . in a 10-year prospective study of 544 chinese participants with a mean age of 50 years , fabp4 levels above the population median ( 15.3 ng / ml for men and 20.4 ng / ml for women ) were associated with a twofold increased risk of type 2 diabetes after controlling for bmi and other confounding factors ( rr 2.25 [ 95% ci 1.403.65 ] ) ( 16 ) . we found that the association of fabp4 with type 2 diabetes risk was most pronounced in persons with bmi < 25 kg / m . in light of this finding , it is notable that the above chinese study has a lower bmi ( mean sd 24.3 3.7 kg / m ) than the chs study ( 26.4 4.6 kg / m ) ( 16 ) . the interaction between bmi and fabp4 is consistent with the positive association seen in the leaner chinese cohort . our working hypothesis is that expression of fabp4 may be directly associated with the size of adipocytes ; furthermore , we are hypothesizing that overweight / obese subjects have larger adipocyte size than lean people . our findings of no overall significant association between nefa and type 2 diabetes in older adults are contrary to other reports . ( 19 ) reported a 63% higher risk of type 2 diabetes in the fourth relative to the first quartile of nefa ( rr 1.63 [ 1.04 2.57 ] ) in middle - aged adults . similar results were noted in the pima study ( 18 ) with a 2.3-fold greater risk of type 2 diabetes comparing the highest with the lowest decile of plasma nefa . the mean duration of follow - up in our study ( 9.5 years ) was at least twice as long as that reported in the pima study ( 18 ) and nearly five times longer than the 2 years reported in the paris prospective study ( 34 ) . furthermore , subjects in our study were much older ( mean age 74.8 years ) compared with a mean age of 26 years in the pima study ( 18 ) , 48.9 years for the paris study ( 34 ) , and 52.8 years for atherosclerosis risk in communities ( aric ) study ( 19 ) . the longer follow - up duration and older age of participants in our cohort might explain the lack of an association in our data . in particular , it is possible that a single nefa concentration may be less strongly associated with type 2 diabetes risk over long - term follow - up compared with in the short term . nefa may better predict diabetes risk within a shorter period of follow - up as observed in the paris and pima studies ( 18,34 ) . this hypothesis is consistent with the larger effect size ( hr 1.63 [ 95% ci 1.072.50 ] comparing the 3rd to the 1st tertile of nefa ) observed in our analysis when follow - up time was restricted to the first 5 years of follow - up . if this hypothesis is correct , it suggests that it might be necessary to update nefa measurements over time to fully characterize nefa exposure . nonetheless , our findings also differ from the 53% lower risk of type 2 diabetes ( 95% ci 1973 ) per unit of log nefa observed in the insulin resistance atherosclerosis study ( iras ) ( 20 ) . iras showed that 2-h glucose was a major confounder ; unfortunately , we did not conduct an oral glucose tolerance test in our cohort at the time of fabp4 and nefa measurement ( 20 ) . further , iras was multiethnic with younger average age ( 55 vs. 75 years in the chs ) . what potential biologic mechanisms could causally relate fabp4 and nefa to type 2 diabetes ? as a lipid chaperone , fabp4 is expressed in adipocytes and macrophages and plays an important role in lipid metabolism and perhaps glucose utilization ( 35 ) . as such , it is possible that fabp4 is a mediator of the obesity type 2 diabetes association . such a hypothesis would be consistent with the fact that compared with wild - type mice , fabp4 knockout mice do not develop insulin resistance despite extreme adiposity when fed a high - fat diet ( 10 ) . on the other hand , fabp4 might influence the size of fat cells through de novo lipogenesis and lead to obesity . at this point fabp4 also inhibits stearoyl - coa desaturase-1 , an enzyme that plays a key role in de novo lipogenesis ( 36 ) and could influence plasma nefa . during de novo lipogenesis , plasma glucose is metabolized to saturated fatty acids ( myristic acid [ c14 ] and palmitic acid [ c16 ] ) ( 37 ) . stearoyl - coa desaturase-1 catalyzes the conversion of palmitic to palmitoleic acid and stearic ( c18 ) to oleic acid ( c18 ) , a glucose - requiring process ( 36 ) . in fabp4-deficient mice , there is an increased ratio of short - chain fatty acids ( c14 ) to longer chain ( c18 ) in muscle and adipose tissues ; a more favorable ratio leads to enhanced insulin receptor signaling , amp - activated kinase activity , and insulin - stimulated glucose uptake ( 38 ) . in addition , fabp4 attenuates the inhibition of hepatic sensitive lipase through its binding of fatty acids ( 17 ) . hepatic sensitive lipase activity can lead to lipolysis with elevated triglycerides and release of free fatty acids , thus leading to insulin resistance . higher nefa concentrations can increase insulin resistance ( 21,22 ) , exert toxic effects on pancreatic -cells ( 23,24 ) , and increase glucose production ( 25 ) . in addition , treatment with pioglitazone ( which reduces insulin resistance ) has been associated with reduced nefa and lipotoxicity ( 39 ) . additional adjustment for fabp4 did not alter the point estimate for nefa in this study , suggesting that the contribution of fabp4 to nefa concentration may be negligible . hence , we were unable to account for change in these biomarkers resulting from change in weight or other factors over time . weight loss is associated with a reduction in serum fabp4 in humans ( 40 ) , and in our study , subjects in the highest tertile of fabp4 were more likely to report unintentional weight loss . the fact that we observed a stronger association between fabp4 and diabetes risk after exclusion of subjects who reported unintentional weight loss , cancer , or cvd suggests that repeated measures of fabp4 might lead to even stronger relationships . as an observational study , we can not exclude residual or unmeasured confounding as an alternative explanation of observed associations . our sample consisted of caucasian and , to a lesser degree , african american adults , all of whom were aged 65 years ; results may not generalize to younger individuals or other race / ethnicities . we did not have measures of fasting glucose in all years , and , hence , we likely missed type 2 diabetes cases in intervening years . we did not have 2-h glucose at the time of fabp4 and nefa measurement for further adjustment . we can not exclude the possibility that obese people were less likely to attend clinic and have their blood glucose measured ; missing data on glucose that was differential based on obesity may have led to an underestimate of type 2 diabetes events . however , the fact that a similar proportion of obese subjects attended the baseline ( 17.6% ) and 19961997 ( 18.1% ) examination is reassuring . despite these limitations , our study has numerous strengths including a large sample size , a representative sample of older adults , inclusion of both men and women , the use of a valid and reproducible method to assess fabp4 and nefa , availability of data on numerous potential confounders , and long - term and nearly complete follow - up . in summary , we observed a positive association between plasma fabp4 and incident type 2 diabetes that was statistically significant only among lean men . lastly , our data support a statistically significant association between a single measure of nefa and incident type 2 diabetes in older adults during a shorter but not a longer follow - up period .

backgroundcardioembolic stroke ( ces ) , which causes 20% cause of all ischemic strokes , is associated with high mortality . previous studies suggest that pathways play a critical role in the identification and pathogenesis of diseases . we aimed to develop an integrated approach that is able to construct individual networks of pathway cross - talk to quantify differences between patients with ces and controls.material/methodsone biological data set e - geod-58294 was used , including 23 normal controls and 59 ces samples . we used individualized pathway aberrance score ( ipas ) to assess pathway statistics of 589 ingenuity pathways analysis ( ipa ) pathways . random forest ( rf ) classification was implemented to calculate the auc of every network . these procedures were tested by monte carlo cross - validation for 50 bootstraps.resultsa total of 28 networks with auc > 0.9 were found between ces and controls . among them , 3 networks with auc=1.0 had the best performance for classification in 50 bootstraps . the 3 pathway networks were able to significantly identify ces versus controls , which showed as biomarkers in the regulation and development of ces.conclusionsthis novel approach could identify 3 networks able to accurately classify ces and normal samples in individuals . this integrated application needs to be validated in other diseases . objectiveto examine the relation of fatty acid binding protein ( fabp)4 and nonesterified fatty acids ( nefas ) to diabetes in older adults.research design and methodswe ascertained incident diabetes among 3,740 cardiovascular health study participants ( 19922007 ) based on the use of hypoglycemic medications , fasting glucose 126 mg / dl , or nonfasting glucose 200 mg / dl . fabp4 and nefa were measured on specimens collected between 1992 and 1993.resultsmean age of the 3,740 subjects studied was 74.8 years . for each sd increase in log fabp4 , hazard ratios ( hrs ) for diabetes were 1.35 ( 95% ci 1.101.65 ) for women and 1.45 ( 1.131.85 ) for men controlling for age , race , education , physical activity , cystatin c , alcohol intake , smoking , self - reported health status , and estrogen use for women ( p for sex - fabp4 interaction 0.10 ) . bmi modified the fabp4-diabetes relation ( p = 0.009 overall ; 0.02 for women and 0.135 for men ) , in that statistically significant higher risk of diabetes was mainly seen in men with bmi < 25 kg / m2 ( hr per sd : 1.78 [ 95% ci 1.132.81 ] ) . there was a modest and nonsignificant association of nefa with diabetes ( ptrend = 0.21 ) . however , when restricted to the first 5 years of follow - up , multivariable - adjusted hrs for diabetes were 1.0 ( ref . ) , 1.68 ( 95% ci 1.122.53 ) , and 1.63 ( 1.072.50 ) across consecutive tertiles of nefa ( ptrend = 0.03).conclusionsplasma fabp4 was positively associated with incident diabetes in older adults , and such association was statistically significant in lean men only . a significant positive association between plasma nefa and incident diabetes was observed during the first 5 years of follow - up .

diabetic retinopathy ( dr ) is a leading cause of blindness among working group in developed countries . it is estimated that the number of diabetes patients will achieve 366 million by 2030 , the majority of which will be type 2 diabetes mellitus ( t2 dm ) . after initial diagnosis , over 80% of t2 dm patients dr is a prototypical microvascular disorder characterized by microaneurysms , capillary nonperfusion , intraretinal hemorrhages , intraretinal microvascular abnormalities , and neovascularization [ 46 ] . however , recent studies have identified neuroretinal abnormalities in diabetics , even before the evidence of visible microvascular changes [ 710 ] . dr patients usually exhibit reduced responses in full - field and multifocal electroretinography , decreased blue - yellow color sensitivity , and contrast sensitivity before the microvascular lesions occurrence . large amount of neuronal cells is detected to be damaged in the very early stage of the disease , while microvascular lesions are usually difficult to be identified by fundus photography or ffa , which would cause easily clinical missed diagnosis followed by worsen progression . retinal neurodegeneration has also been identified in diabetic rodent models , which reproduces most aspects of the early stages of dr . lewis rats with streptozotocin - induced type 1 diabetes have shown the most accelerated loss of retinal ganglion cells ( rgcs ) at 8 months after the onset of diabetes . the spontaneous development of diabetes in ins2akita mice for 5 - 6 months also reveals the changes in neurosensory retina , including rgcs apoptosis as well as marked alterations to the morphology of surviving cells , reduction of cholinergic and dopaminergic amacrine cells , and a distinct thinning of the inner plexiform layer and the inner nuclear layer [ 1416 ] . furthermore , morphologic alterations of astrocytes and microglial cells in the inner retina and impaired glutamate metabolism by mller cells are found in other rodents [ 1719 ] . type 1 diabetic animal models were used in the majority of these researches , and to date retinal neural structural and functional studies of type 2 diabetic animal model are scarce . the db / db mouse ( bks / db/ ) is a typical model of type 2 diabetes that exhibits hyperglycemia associated with obesity a few 48 weeks after birth . physiological studies have revealed early inner retinal neuronal dysfunction including prolonged latencies of the oscillatory potentials and impaired b - wave at 24 weeks of age . an increased apoptosis of rgcs and other retinal neurons was observed in 60 weeks old db / db mice . however , the structural and functional features of inner retina on db / db mice during the early stage of diabetes remain unclear . this study was designed to follow the development of neurosensory retina damage from both structural and functional aspects in the early stages of diabetes in db / db mice . thirty - seven 8-week - old male db / db mice ( bks / db/ ) and 37 age - matched nondiabetic littermates ( bks / db+/+ ) were provided by the animal laboratory center of nanjing university and were kept in our spf - certified vivarium and exposed to a 12-hour light - dark cycle . all experiments conformed to standard environmental conditions ( at room temperature of 23c and humidity of 60% ) . to assess the chronological sequence of the retinal abnormalities associated with diabetes , 6 diabetes db / db mice were compared with 6 age - matched nondiabetic mice at difference ages ( 12 , 16 , 20 , 24 , and 28 weeks , resp . ) . visual functions were measured using pattern electroretinography ( perg ; roland consult , germany ) and in vivo retinal structures were visualized with spectral domain oct ( cirrus hd - oct , zeiss meditec , germany ) . at last , mice were anesthetized with intraperitoneal injection of a mixture containing 5% urethane and 10% chloralic hydras ( 1 : 1 ) ( 1020 mg kg body weight ) . all animal procedures were performed according to guide for the care and use of laboratory animals published by the institute for laboratory animal research . , mice ensured to natural light - adapted state were weighted and anesthetized as above mentioned . self - modified electrodes were made of acupuncture needles , welded to the signal - recorded wire . a recording electrode ( 0.25 mm diameter silver wire configured to a semicircular loop of 2 mm radius ) reference and ground electrodes were inserted under the skin of ipsilateral pars buccalis and tail , respectively . the pupils were not dilated , and eyes were not refracted for the viewing distance since the mouse eye has a large depth of focus . visual stimuli consisted of horizontal bars with a contrast of 99% at 0.05 cycles / degree spatial frequency , 1 hz temporal frequency . perg waveforms consisted of two main waves : a positive wave with a peak latency of 90 to 100 ms ( p1 ) followed by a broad negative with peak latency in the range of 200 to 300 ms ( n1 ) . after anesthetizing , mice were dilated with tropicamide 1% and underwent spectral domain oct ( cirrus hd - oct , zeiss meditec , germany ) as reported elsewhere [ 26 , 27 ] . in brief , mice were placed on the three - dimensional table . the mouse eyeball was coated with viscoelastic material and covered with a coverslip to form a plano - concave lens . then the 90 dpt noncontact slit lamp lens ( volk , superfield nc , volk optical inc . , macular cube scanning in the cirrus hd - oct was performed with the 512 128 scan pattern where a 6 6 mm area on the retina is scanned with 128 horizontal lines , each consisting of 512 a - scans per line with the scanning speed of 27000 a - scans per second . during the analysis procedure then retinal thicknesses are averaged in nine retinal subfields which are an area of a 6 mm diameter circle centered on the mouse optic disk . the eyes were dipped in ffa ( mixture of 95% ethyl alcohol , 10% neutral formalin , and glacial acetic acid , 17 : 2 : 1 ) for 1 min and fixed in 0.1 m buffer phosphate ( ph 7.4 ) containing 4% paraformaldehyde for 2 hours at 4c . then , the anterior segment of the eye was dissected away , the vitreous cleared , and the eyecup was left for further fixation overnight . serial sections 7 m thick were prepared for the assessment of retinal morphology , immunofluorescence staining , and tunel immunoreactivity . after deparaffinization and rehydration , the sections were stained with hematoxylin and eosin ( h&e ) . the measurements were taken at the regions of the retina ( at 480 m form ora serrata ) and were examined to ensure similar locations of measurements for all eyes . image analyses of ten sections of each eye were used to quantify total retinal thickness , the thickness of the inner nuclear layer ( inl ) and outer nuclear layer ( onl ) . images of h&e sections were captured with a microscope ( olympus ) using the program image j for quantification . the terminal deoxynucleotidyl transferase - mediated biotinylated utp nick - end labeling ( tunel ) assay was performed with dapi ( 4,6-diamidino-2-phenylindole ) staining . the prepared tissue sections were deparaffinized in xylene for 10 min and hydrated through a graded ethanol series . the tunel assay was then carried out following the protocol described by the manufacturers ( in situ cell death detection ; roche molecular biochemicals ) . after staining retinal sections were mounted with fluorescence mounting medium and images quantitative observations of retinal cells count from semithin section were defined as five hp fields ( 40 10 ) since 200 um up from the optic papilla . after deparaffinization and rehydration , the sections were incubated with ionized calcium binding adaptor molecule 1 ( iba-1 ; dilution 1 : 150 , wako , neuss , germany ) and f4/80 ( m-300 ; dilution 1 : 200 , santa cruz ) at 48c for 2 days and then rinsed in pbs ( 3 1 hr ) . then , the retina was incubated at 48c in sheep anti - mouse and anti - rabbit antibodies conjugated to fluorescein - isothiocyanate ( fitc ) ( boehringer mannheim gmbh , germany ) dilution 1 : 30 in 0.1% triton x-100 in 0.01 m pbs at ph 7.4 , for 3 days , and rinsed again . the retina was mounted on a slide in glycerol diluted with pbs at a ratio of 1 : 9 . whole retina thicknesses obtained from all oct devices were compared one to each other using unpaired student 's t - test . measurements of blood glucose ( vein blood of tail using contour ts ) and body weight were conducted in diabetic and nondiabetic mice before visual function testing or they were killed for other experiments at each time point . when the result of blood glucose value was beyond the upper limit of detection ( 33.3 mmol / l ) , we set 33.3 mmol / l as the statistical default . there were significant statistic differences of body weight and blood glucose level between the two groups , and hyperglycemia in diabetic mice runs in parallel with a significant increase of weight . p1 amplitudes , measured from the peak of the p1 wave to the trough of the n1 wave , were slightly reduced in diabetes mice at the age of 12 and 16 weeks , but they were significantly reduced in diabetes mice when compared with controls at the age of 20 , 24 , and 28 weeks ( figure 2(a ) ) . besides , a significant delay of p1 latency recorded in the diabetic mice at the ages of 24 and 28 weeks was observed when compared with the age - matched control mice ( figure 2(b ) ) . these data showed abnormal perg responses in diabetic db / db mice for 20 weeks , which also suggested that rgcs dysfunction might have occurred in early period of db / db mice . measurements of retinal thickness in diabetic and in nondiabetic mice at 20 , 24 , and 28 weeks are shown in figure 3 . total retinal thickness ( measured from inner limiting membrane to bruch 's membrane ) was significantly decreased in diabetic mice in comparison with nondiabetic mice at 28 weeks . furthermore , a reduction in onl was observed in diabetic mice in comparison with nondiabetic mice at 28 weeks . the retinal thickness was measured by oct noninvasively in 7 diabetic db / db mice and 7 nondiabetic mice at 28 weeks . one eye was selected at random in each mouse and only that eye was included in the analysis . as shown in table 1 , oct revealed a significant thinner in total retinal thickness of db / db mice than the control mice ( p < 0.01 ) . specifically , the differences were significant also for all quadrants . moreover , both the inner and outer sectors thicknesses of superior as well as nasal quadrants in db / db mice were significantly thinner than nondiabetic mice . additionally , significant thinning occurred in inner sector of the temporal quadrant in diabetic mice compared with control mice , despite the fact that no significant difference was detected in the temporal quadrant between the two groups . after examinations of perg , ffa were conducted which monitor the flow of a fluorescent dye through the retinal vasculature . all db / db as well as control mice did not show any alternations corresponding to relevant vascular abnormalities at each endpoint . figure 4 shows the images of ffa from db / db and db/+ mice at 28 weeks and there was no visible neovascularization and cellular capillaries in the retina . retinal sections were stained for dying photoreceptors by tunel staining to further determine the histological retina changes . a significant increase in tunel - positive immunofluorescence was observed in diabetic mice in comparison with retinas from nondiabetic mice at 20 , 24 , and 28 weeks ( figure 5 ) . tunel - positive cells were mainly localized in the rgcs layer , as well as the inl , implying the rgcs apoptosis which was also suggested by preg results . in addition , the number of apoptosis cells was increased from 1 - 2/hp at 20 weeks to 8 - 9/hp at 28 weeks ( figure 5 ) . to confirm the possible neuroinflammation in retinal neurodegeneration , iba-1-labeled microglia / macrophages and f4/80-labeled mature macrophages staining were performed . as shown in figure 6 , no positive expression of the two markers was observed in either 20-week - old diabetics or nondiabetics mice . however , db / db mice at 24 and 28 weeks showed obvious fluorescence of positive iba-1 and f4/80 expression in rgcs layer and inl , while no specific immunofluorescence exhibited in the retina of control mice ( figure 6(a ) ) . moreover , positive f4/80 was also expressed in the onl of db / db mice ( figure 6(b ) ) . these results confirmed the presence of iba-1 and f4/80 positive cells in rgcs layer and inl by the time of 24 weeks . when compared with normal heterozygous littermates , the db / db ( lepr ) mice can be affected with hyperglycemia , hyperlipidemia , and accelerated degeneration of the retinal capillaries and pericytes . it has been reported that 26-week - old db / db mice showed early features of dr , manifesting pericyte and endothelial cell loss , bruch 's membrane thickening , and increased blood flow in the retina [ 2931 ] . however , rodent models can reproduce most aspects of the early stages of dr but fail to develop retinal neovascularization or other advanced lesions ( retinal hemorrhages and microaneurysms ) of retinopathy . although cheung et al . had reported that 15-month - old db / db mice showed increased density of retinal capillaries in the inner nuclear layer , in current study , we did not found any obvious microvascular changes using ffa in db / db mice alive for 28 weeks . moreover , the reduction of retinal thickness in the db / db mice by using oct also supports that there was no retinal edema and/or retinal blood barrier damage . the relatively short lifespan of the mice and thus shorter duration of diabetes ( most studies are < 1 year ) were probable reason for failing to observe neovascularization in these models . therefore , the results of our study were supporting previous researches which implied that db / db mice developed capillary changes under microscope even in the early period of life span , but the lesion was not severe enough to cause the breakdown of retinal blood barrier . the results of perg presented in our study indicated that diabetic condition acts to be injurious events that lead to exacerbation of rgcs death . perg signal depends on the structural and functional integrity of rgcs and will greatly reduce when rgcs were selectively degenerated after optic nerve lesion . perg , therefore , is extensively used to probe rgc function in clinical and experimental models of diabetes and optic neuropathies [ 3436 ] and may be more sensitive to assess early damage of dr . previous studies showed that perg impairment could be detected in diabetic patients without or with minimal signs of retinopathy . this suggests that retina functional loss may occur in the early stage of this disease . therefore , we used perg to evaluate the changes in rgcs function and we found that the p1 amplitudes significantly reduced at the follow - up examinations for 20 weeks in db / db mice ; then a significantly increased latency was found later . all these results indicated that the deficit function of rgcs in db / db mice had certainly occurred . it was reported that rgcs die in a compartmentalized manner such that the mechanisms responsible for death of the rgcs body and dendrites may be different from those responsible for axonal injury ; both of them compromised the rgcs dysfunction . oct was performed as an indicator of progressive neural retinal pathology in animal models of retinal degeneration [ 38 , 39 ] . in the present study , we found a significant reduction in overall retinal thickness as well as all four quadrants , depending on the oct scanning , in db / db mice when compared with control mice at the age of 28 weeks . moreover , the results of h&e showed that total retinal thickness as well as onl ( at 480 m form ora serrata ) was significantly decreased in diabetic mice in comparison with nondiabetic mice at only 28 weeks , but there was no difference between the 2 groups at 20 and 24 weeks . these results are mainly due to the apoptosis of the ganglion cell layer and photoreceptors . in a recent study , bogdanov et al . reported that total retinal thickness in both central and peripheral retina was significantly decreased in db / db diabetic mice at 16 and 24 weeks , and a thinning in both onl and inl as well as a reduction in the number of cells in the gcl unfortunately , in our study only the db / db mice at 28 weeks were measured by oct , with the original intention to validate the reduced perg response . whether there existed oct alternations before 28 weeks so further studies are needed to determine the early changes of oct in db / db mice and evaluate the correlation between oct measured and histologically measured changes in retinal thickness following the life periods . recent studies have approached a common view that cell apoptosis is the base of diabetic neurodegeneration in cytopathogenesis . reportedly , various retinal neurocytes in early dr have been in apoptosis state , in particular the rgcs [ 4244 ] . accompanying cell apoptosis , inflammation has been considered to be an important pathogenesis in early stages of experimental dr , which has shown both of microglial and macrophages reaction represented during the alternations of functional and structural in retina [ 4547 ] . here , to further confirm the rgcs dysfunction determined by perg previous study reported that 15-month - old db / db mice had increased apoptosis of rgcs and other cells in the neural retina . in our study , apoptotic rgcs were observed in 20-week - old db / db mice , consistent with the reduced perg amplitudes . moreover , the number of apoptotic cells was increasing with age , accompanied with evident induction of iba-1 and f4/80 expression , both of which were considered as established marker for microglia / macrophages in the retina . additionally , iba-1 and f4/80 positive cells as well as apoptosis cells were also found in the inl , which was implied to be affected by db / db mice . all these findings indicated that db / db mice undergoing diabetes in 20 weeks or longer had developed neurodegeneration and neuroinflammation . in conclusion , we demonstrated that functional and structural retinal neurodegeneration exactly occurred in db / db mice with type 2 diabetes mellitus as early as 20 weeks , preceding any detectable alternations of blood vessel visibly . the retina lesions that develop in type 2 diabetes are similar to those that develop in type 1 diabetes , although the severity and/or incidence may be different . thus , it is prominently important to trace the degenerated phases and change rule of diabetic nerve cells in the early stage of dr and timely block and reverse these cells ' degeneration . these actions would play a significant role in prevention of early diabetic retinal neuropathy and nerve protection treatment , which are promising to be a potently effective target for the early intervention of diabetic retinopathy in future . infected dental plaque biofilm and the impaired host response remain the main causes of a wide range of gingival diseases . the ability of the patient to adhere to a proper oral care plan is also a critical factor that controls the tissue reaction and disease progression . the clinical manifestations of gingival inflammation differ according to the severity , distribution , and the response to treatment ranging between being localized and highly responsive to treatment to long standing lesions with persistent redness and massive bleeding on probing with variable degrees of swelling . the efficacy of treatment and the subsequent tissue response vary according to the etiology and idiopathic , local , or systemic cause . under healthy conditions , the neutrophils pass from the gingival connective tissue through the junctional epithelium into the gingival sulcus , providing a dynamic balance between continuously invading microorganisms and the immune response . the antioxidants , either enzymatic or nonenzymatic , play a crucial role in the tissue equilibrium via neutralizing such harmful agents ( ros ) [ 5 , 6 ] . under certain inflammatory conditions , the total amount of antioxidants decreases to its lowest levels , which necessities their supplementation by external source [ 7 , 8 ] . one of the most important antioxidant agents is vitamin c ( vit - c ) which has an important role in different body functions [ 7 , 8 ] . despite its mild acidity ( ph 4.2 ) , it requires an alkaline medium to perform its function as a reducing agent . to be activated , many changes in its structure occur in three successive cycles of oxidation ( figure 1 ) . vitamin c , being a strong reducing agent , can regulate the resolution of the inflammatory process and stimulate the tissue repair . it modulates the release of catabolic inflammatory cytokines [ 1012 ] , chemotaxis of the immune cells , and activation of the phagocytosis . the neutrophils and lymphocytes containing vitamin c show lower apoptotic potentiality and higher proliferation rate . furthermore , it enhances the synthesis of collagen type i , reduced by inflammatory process , keeps the balance between collagen i and collagen iii , and modifies the rate of fibroblast proliferation . it also reduces the potentiality of scaring via inhibiting cross - linking of collagen fibers and fibrosis . it acts as a cofactor in hydroxyproline synthesis to produce collagen type iv and improves endothelial cell vitality and function [ 1013 ] . the aim of the present study is to investigate the exact role and efficiency of the locally injected vitamin c in the treatment of persistent gingival inflammation after single or multiple dosing based on clinical and histopathological examination . each patient signed an informed detailed consent form before participation , explaining the benefits , steps , and side effects of the treatment protocol . the study was designed as a single arm , unblinded , and unrandomized trial as the trial did not depend on patient selection but it was rather based on an detection of a chronic persistent phenomenon . only patients with persistent inflammation patients were recruited among those diagnosed with plaque induced gingivitis in the postgraduate periodontology clinic at cairo university between the years 2013 and 2016 . an initial evaluation , including medical and dental history , clinical examination , and radiographic examination , was conducted by nmy and ara to determine patient eligibility for the study . fifty patients who met the following inclusion criteria were examined : ( 1 ) aged between 20 and 50 y , ( 2 ) medically free , and ( 3 ) generalized plaque induced gingivitis . all the reasons that could provoke an inflammatory reaction were excluded : ( 1 ) systemic diseases , ( 2 ) pregnant and lactating mothers , ( 3 ) treatment with antibiotic medication within 1 month before the trial , and ( 4 ) local causes ( smoking , mouth breathing , local trauma , and periodontitis ) ( figure 2 ) . each patient underwent full - mouth sessions of supragingival debridement using ultrasonic and hand instrumentation and received personalized oral hygiene instructions . about twenty patients achieved a minimal residual inflammation ( > 15% of plaque and bleeding scores according to o'leary 's scoring system ) and optimal soft tissue conditions during two weeks . combination antibiotic therapy ( amoxicillin 500 mg and metronidazole 500 mg/3 times per day for 7 days ) was administrated in order to control the amount of the condition . after 7 days of the complexion of the antibiotic course , reevaluation of the remaining 30 patients was undergone . at the end of the maintenance period ( 4 weeks ) , the included patients were 20 who suffered from persistent gingival inflammation in the esthetic anterior region ( figure 2 ) . the site of interest was anesthetized using lidocaine - epinephrine 1 : 100,000 . intraepidermal injection ( mesotherapy approach ) of 11.5 ml ( 200300 mg concentration ) of l - ascorbic acid was locally introduced in relation to the keratinized gingival tissues with prevalent extension to the whole target region , respectively , using insulin syringes . the current dose is equivalent to the treated region ( distance extends between six maxillary anteriors ) . during the first injection visit the same dose was repeated once per week until inflammation subsided . at the end of each session , patients were prescribed a rescue analgesic ( ibuprofen 200 mg ) to be used as needed . the patients were asked to abstain from mechanical oral hygiene procedures in relation to the target region for the day of procedure only . immediately before injection procedure , the following clinical measurements were performed by the same examiners ( nmy and ara ) : plaque and bleeding score using o'leary 's scoring system and local bleeding score using sulcus bleeding index ( sbi ) as a more specific index : score 0 : healthy looking and no bleeding on probingscore 1 : healthy looking and bleeding on probingscore 2 : bleeding on probing , change in color , and no edemascore 3 : bleeding on probing , change in color , and slight edemascore 4 : bleeding on probing , change in color , and obvious edemascore 5 : spontaneous bleeding , change in color , and marked edemathe latter index examined color , swelling , and bleeding tendency . at the completion of the injection visits , patient satisfaction was performed by using a 5- graded self - assessment analysis : excellent ( 4 ) , improved over 75% ; good ( 3 ) , improved 5075% ; moderate ( 2 ) , improved 2550% ; fair ( 1 ) , improved less than 25% ; no change or worse ( 0 ) , not improved or darkened . score 0 : healthy looking and no bleeding on probing score 1 : healthy looking and bleeding on probing score 2 : bleeding on probing , change in color , and no edema score 3 : bleeding on probing , change in color , and slight edema score 4 : bleeding on probing , change in color , and obvious edema score 5 : spontaneous bleeding , change in color , and marked edema preoperative and postoperative ( after 1 week of last injection ) gingival biopsies were excised , immediately fixed in 10% neutral buffered formalin , and then processed in the routine way for preparing a paraffin block . tissue sections were cut and stained with hematoxylin & eosin ( h&e ) for histopathological examination . finally , epidermal and dermal changes before and after ascorbic acid application were assessed using computer image analyzer software leica qwin 515 system ( england ) . the experimental period was between september 2013 and april 2016 ( last follow - up visit ) . twenty sites of localized chronic gingival inflammation were included with no definite cause ranging between marginal gingivitis and diffuse gingivitis . improvement was reported in all cases after maximum 2 injections except 2 cases that needed one more injection . measurement of the sbi was done following phase i therapy and after the completion of the treatment . preoperatively , it was noted that 12 ( 60% ) out of 20 patients scored 3 and 8 patients ( 40% ) scored 4 on sbi index . postoperatively , the range changed into 20 patients ( 100% ) scoring zero with variable degree of response to the applied treatment . seven patients ( 35% ) out of twenty treated patients were totally free of inflammation with zero sbi score after 1 injection visit . eleven ( 55% ) patients showed the same results after 2 injection visits . only 2 patients ( 10% ) out of twenty needed further injection to reach the zero score . all the enrolled patients showed great satisfaction about the treatment results , even the patients who were not fully treated . all patients ( 100% ) showed great satisfaction with the results ( score 4 ) ( figures 3 and 4 ) . the surface epithelium of parakeratinized stratified squamous type was hyperplastic with broad and elongated rete pegs , forming numerous epithelial arcades . obvious intracellular edema and extracellular edema of epithelial cells disturbing the intercellular desmosomal junctions were seen . an intense acute and chronic inflammatory cells infiltrate was observed dispersed in the whole thickness of connective tissue along with coarse blood capillaries ( table 1 ) . basement membrane was masked in some area by inflammatory cells ( figure 5 ) . microscopic examination revealed lower grade of inflammation . on the contrary to the preoperative sections , the connective tissue was formed of proliferating fibroblasts , collagen fiber formation , and few aggregates of chronic inflammatory cells . the characteristic phenomena were the presence of vacuolated epithelial cells especially in basal cell layer ( at the site of injection ) and the appearance of minute capillaries in subepithelial ct areas ( figure 5 ) . obvious reduction in the epithelial thickness was detected with minimal epithelial rete pegs and epithelial arcades . well - formed collagen fiber bundles with numerous and widely distributed blood vessels were detected in the connective tissue . fewer chronic inflammatory cells were detected but restricted to the deep connective tissue ( figure 5 ) . due to diversity of the gingival diseases , treatment plan should be variably designated based on the associated causative factors . however , one of the embarrassing problems is the presence of chronic or resistant inflammatory conditions with no definite aetiopathogenesis . they are usually characterized by persistent redness and bleeding on probing with different degrees of tissue edema which may be localized or diffused . in these cases , massive antibiotic course or corticosteroids regimens followed by surgical intervention if needed were the conventionally and commonly used protocols . despite the incredible results accompanying corticosteroids , they are usually avoided due to their adverse effects . therefore , there is a need for other safe nonsurgical therapeutic agents in order to control such conditions or , at least , improve the tissue healing and reduce inflammatory manifestations prior to the surgical removal of remaining pseudopocketing . dermal mesotherapeutic techniques using antioxidants such as vit - c showed promising results in the treatment of similar localized inflammatory conditions . the aim of the current study is the regulation of the overresponse of the inflammatory process , promoting healing , increasing the collagen content of the affected tissues , and improving the gingival circulation . in the current study , twenty patients were enrolled and evaluated at the baseline ( after 4 weeks following phase i therapy ) and postoperatively in the recall visit ( after 1 week after the last intraepidermal vitamin c injection ) . the long follow - up period preceding the therapy was essential in order to ensure that gingival inflammation is resistant to the conventional therapy . during inflammation , it was found that the tissue antioxidant level ( vit - c , vit - e , etc . ) decreases rapidly , indicating the need of its supplementation . on the other hand , the free radicals production increases at the site of inflammation [ 19 , 20 ] . extra doses of antioxidants , especially vitamin c , are essential . in localized inflammatory conditions , the administration of the needed higher doses ( higher than 500 mg ) can not be absorbed by the gastrointestinal tract which easily excreted through urine . moreover , in order to reach this dose at the site of inflammation , administration of very high systemic doses is needed , which could be harmful to the patient . , the incidence of vitamin c deficiency is nearly neglected due to its availability in a wide range of fruits and vegetables . due to the impaired financial status of our patients and the absence of the deficiency conditions , the used technique is much cheaper than providing a long term special food protocol or oral supplementations in order to improve their health status . the local injection of vit - c was preferred rather than the usage of topical vitamin c gel or dentifrice that was previously used by daniels and jefferies and shimabukuro et al . . many studies used different forms of lipid soluble topical vitamin c in order to overcome its limited absorption . these studies met several limitations such as very long term improvement time ( 12 weeks ) , easily dislodged from the oral tissues , limited absorption , instability when exposed to solutions , air , heat , or light , and localized enamel erosions [ 9 , 24 , 25 ] . total numerical evaluation of the gingival inflammation ( using sbi ) was determined , resulting in an average value per each patient . this analysis indicates 100% reduction in gingival inflammation from the average baseline value over the 721 days . intraepidermal vitamin c injection could be either single or double or triple with one week apart according to the severity of the condition . more cautious handling of the inflamed tissues during the first visit is quite important in order to avoid tearing or necrosis , especially in thin biotype tissues . improvement of the tissue color and form was clearly observed in the recall visits after 2 months . once inflammation subsided , representative tissue biopsies have been obtained 7 days following the last injection from the patients indicated for gingivoplasty or gingivectomy . clinically significant reduction of the pseudogingival enlargement was associated with return of the basal keratinocytes to their normal proliferative pattern which is the main role of vitamin c [ 26 , 27 ] . the anti - inflammatory action of vit - c was also evident and was detected in our specimens as a reduced intraepithelial edema and inflammatory cells . other characteristic changes which occurred after vit - c administration were in accordance with nusgens et al . . an increased number of fibroblasts were clearly detected forming more collagen fibers that showed more maturation and bundles formation following second injection . these features are consistent with its known essential role in the formation of new connective tissue in a healing wound . this is because it acts as a cofactor for enzymes critical in collagen formation , the main component of the connective tissue that forms the framework around which the new tissue is rebuilt . this is why , despite the increase in number of blood vessels , redness and bleeding tendency markedly decreased clinically . these vessels provide more nutritional and oxygen support to chronically irritated and continuously damaged mucosal areas , improving their healing . they detected that vitamin c promotes the development of an organization of the basement membrane and also restores the epidermal barrier within 2 - 3 weeks . furthermore , it promotes the wound closure and reduces its contraction which limits the incidence of scare formation . histologically , a characteristic cellular vacuolization was observed in all groups . in the preoperative specimens , vacuolated cells appeared as clear cells with small pyknotic dark nucleus representing signs of degeneration . in contrast , vit - c associated vacuolated cells were individually distributed along the epithelial layers with greatest aggregations in basal cells . a similar vacuolization was found in immediate and 15-minute biopsies excised after intradermal injection of local anesthesia described by kimura et al . . they attributed this phenomenon to the injection procedure itself rather than the used solution spatially with the presence of vacuolization in biopsies . however , the presence of these cells in after - week biopsies may be due to the ability of vitamin c to increase the cell resistance to death . furthermore , the basal localization of vacuolated cells , adjacent to injection , may indicate proper infusibility of vit - c . all these positive clinical changes were met by further patients ' cooperation and resulted in their end - treatment satisfaction . these data suggest a significant enhancement of the gingival health by the usage of the antioxidant approach . finally , we recommend the usage of the intraepidermal vitamin c injection as an adjunctive approach for the conventional nonsurgical treatment modality .

purpose . to describe both the functional and pathological alternations in neurosensory retina in a murine model of spontaneous type 2 diabetes ( db / db mouse ) . methods . db / db ( bks / db/ ) mice and heterozygous littermates ( as control group ) at various ages ( 12 , 16 , 20 , 24 , and 28 weeks ) were inspected with pattern electroretinogram ( perg ) , fundus fluorescein angiography ( ffa ) , and optical coherence tomography ( oct ) . histological markers of neuroinflammation ( iba-1 and f4/80 ) were evaluated by immunohistochemistry . in addition , levels of retinal ganglion cell death were measured by terminal dutp nick - end labeling ( tunel ) . results . significant alternations of perg responses and increased retinal ganglion cells ( rgcs ) apoptosis were observed in diabetic db / db mice for 20-week period when compared with control group . iba-1 and f4/80 expression in microglia / macrophages became evidently for 24-week period , thus supporting the perg findings . furthermore , obvious thinning of nasal and dorsal retina in 28-week - old db / db mice was also revealed by oct . no visible retinal microvascular changes were detected by ffa throughout the experiments on db / db mice . conclusions . diabetic retina underwent neurodegenerative changes in db / db mice , which happened at retinal ganglion cell layer and inner nuclear layer . but there was no obvious abnormality in retinal vasculature on db / db mice . objective . the purpose of this study is to investigate the role and efficiency of the locally injected vitamin c in the treatment of persistent gingival inflammation . design . twenty adult patients with persistent chronic gingival inflammation were included in this study . the same dose of sterile vitamin c was injected in gingival tissues after the completion of phase i therapy . gingival biopsies were taken after total resolution of inflammation . the specimens were examined histologically , using h&e stain . results . clinical evaluation revealed great improvement of the injected sites with recall visits . histopathological results revealed marked decrease in inflammatory cells and epithelial thickness and a higher number of newly formed subbasal capillaries . conclusions . vitamin c is an effective adjunctive treatment in reducing various degrees of chronic gingival inflammation .

the endoplasmic reticulum ( er ) fulfills multiple cellular functions . once er functions are perturbed by various pathological conditions , unfolded or misfolded proteins accumulate in the er lumen , resulting in er stress characterized by increasing er molecular chaperones and diminishing global protein synthesis . activation of the signaling network in response to er stress is known as unfolded protein response ( upr ) . there are three distinct upr signaling pathways triggered in response to er stress , which are mediated by ( 1 ) rna - dependent protein kinase - like endoplasmic reticulum kinase ( perk ) , ( 2 ) activating transcription factor 6 ( atf6 ) , and ( 3 ) inositol - requiring enzyme 1 ( ire1 ) , respectively , [ 2 , 3 ] . under physiological conditions , perk , atf6 , and ire1 are associated with the abundant luminal chaperone bip ( also known as glucose - regulated protein 78 , grp78 ) . when the er is overloaded with newly synthesized proteins or is stimulated by agents that cause unfolded proteins to accumulate , grp78/bip preferentially associates with the unfolded proteins , releasing perk , atf6 , and ire1 to activate downstream signaling molecules . in the perk - mediated pathway , activated perk phosphorylates eif-2. the phosphorylated eif-2 subsequently inhibits global protein synthesis , preventing further influx of nascent proteins into an already saturated er lumen . paradoxically , eif-2 phosphorylation enhances the translation of activating transcription factor-4 ( atf4 ) and subsequently upregulates the expression of atf4-target genes , including several protein chaperones . recent studies also demonstrated that eif-2 phosphorylation upregulates atf6 expression and enhances atf6 activity and therefore increases the expression of its target genes . although upr signaling pathways are activated classically by unfolded er proteins , increasing evidence demonstrates that lipids / lipoproteins can also trigger upr . for instance , a prior study reported that accumulation of free cholesterol in the er was able to activate upr signaling pathways in mouse peritoneal macrophages . also , studies from our laboratory demonstrated that treatment of mouse macrophages with lipoproteins carrying apolipoprotein ( apo ) b48 but lacking apoe ( e / b48 ) induced foam cell formation , and enhanced phosphorylation of perk and eif-2 , increased expression of atf4 and several molecular chaperons and reduced global protein synthesis [ 7 , 8 ] . furthermore , we observed that overexpression of dominant - negative mutants of perk or eif-2 , or treatment with the eif-2 inhibitor , 2-aminopurine ( 2-ap ) , suppressed foam cell formation induced by e / b48 lipoproteins [ 7 , 8 ] . these findings suggest that activation of the perk - eif2 signaling pathway is an underlying mechanism by which e / b48 lipoproteins induce foam cell formation . such a transformation of macrophages into a foam cells is an early step of atherogenesis . individuals with defective isoforms of apoe , such as apoe4 , develop postprandial hypercholesterolemia and atherosclerosis . similarly , apoe knockout ( apoe ) mice manifest an elevated plasma cholesterol and develop atherosclerosis in a manner that resembles the human disease [ 10 , 11 ] . the hypercholesterolemia in apoe mice results mainly from the increased levels of e / b48 lipoproteins . it is interesting to note that atherosclerotic lesions in the aorta sinus of apoe mice show upr , as reflected by increased perk phosphorylation in the lesions [ 12 , 13 ] . data from the present report demonstrated that treatment of apoe mice with the eif-2 inhibitor 2-ap reduced atherosclerotic lesions in their aortas compared to lesions observed in control mice . taken together , these findings suggest that activation of eif-2 signaling pathway is an underlying mechanism for the development of atherosclerosis in apoe mice and that inhibition of this upr pathway might provide a therapy strategy for treatment of atherosclerosis . male apoe mice were obtained from jackson laboratory ( bar harbor , me ) . these mice were generated using embryonic stem cells from the 129 strain of mice and were crossbred to c57bl/6 for over 10 generations . they were maintained under barrier conditions in a temperature - controlled environment and fed with a mouse chow containing approximately 5% fat and 19% protein by weight ( harlan teklad , madison , wi ) . at 6 weeks of age one group of the mice was gavage - fed 2-ap at a dose of 200 mg / kg body weight ( bw ) in 200 l water once every other day , and another group of mice was fed with same volume of water as a control . after 24 weeks of treatment , mice were fasted overnight and anesthetized with ketamine hydrochloride ( 80 mg / kg bw ) and xylazine hydrochloride ( 16 mg / kg bw ) . approximately 0.5 ml of blood was collected from the posterior vena cava of each mouse . thereafter , a 23-gauge needle was inserted into the left ventricle and 4% paraformaldehyde was delivered into the animal at pressure of 80 mm hg . a small incision was made into the liver to allow efflux of blood and fixative . after fixation , the heart and the proximal aorta were removed from the body and stored in 4% paraformaldehyde at 4c before sectioning . the distal aorta ( 2 mm from the heart to the iliac bifurcation ) was opened longitudinally using microscissors and pinned flat on a black wax surface in a dissecting pan under a dissecting microscope ( smz1000 , nikon instruments inc . , this en face preparation was fixed overnight and stained with oil - red - o . the photo - image of the aorta was captured with a coolsnap digital camera ( nikon instruments ) mounted on the smz1000 dissecting microscope . the atherosclerotic lesion area and the total aortic area were measured using a metamorph imaging system ( nikon instruments ) . the proximal aorta attached to the heart was used to prepare cross - sections , as described previously . briefly , the heart was sectioned transversely immediately below and parallel to a plane formed by the line between atrial leaflets . the portion of the heart with the attached aorta was embedded either in oct or in paraffin , and sectioned from the attached aorta towards the root of the aorta where the aorta valves were attached . sections ( 58 m ) were cut from the site where the aorta valve cups appear at the aorta root . melville , ny ) equipped with a cool snaps color digital camera and a metamorph computer image acquisition system . the average area ( m ) and morphological features ( foam cell deposits , cholesterol clefts , lipid cores and fibrous - caps ) of the lesions in 16 sections were determined for each mouse . immunostaining was performed with the use of the vectastain abc system ( vector laboratories , inc . , the endogenous peroxidase activity was blocked with 5% hydrogen peroxide for 10 min . after blocking with 5% normal goat or rabbit serum , the sections were incubated with primary antibodies against grp78 or phosphorylated eif-2 ( abcam inc . , cambridge , ma ) . after treatment with a secondary antibody produced from goats or rabbits , sections were stained with diaminobenzidine or nova red substrates and counterstained with hematoxylin . the immunostaining of the aortic sections were viewed using a microscope equipped with an hq2 coolsnap high - resolution camera ( nikon ) and the megamorth computer image acquisition system . the levels of plasma cholesterol and triglycerides were measured by spectrophotometric quantification using reagents obtained from sigma chemical co. ( st . cholesterol - reaction reagent was incubated at 37c for 30 min , and the absorbance was read at 530 nm with a dynex microplate reader ( thermo labsystems , franklin , ma ) . for measuring triglycerides , the mixture of plasma and triglyceride - reaction reagent was incubated at 37c for 10 min , and the absorbance was read at 530 nm . plasma concentrations of cholesterol and triglycerides were determined based on the absorbance obtained by incubation of the cholesterol and triglyceride standards provided by sigma . for determination of the cholesterol level in various lipoproteins , a 100 l plasma sample obtained from individual mice was fractionated using a fast performance liquid chromatography ( fplc ) ( kta fplc 900 , amersham biosciences , piscataway , nj ) in a buffer containing 0.15 m nacl , 0.01 m na2hpo4 , 0.1 mm edta , ph 7.5 , at a flow rate of 0.5 ml / min . it had already been established that fractions 1417 contain very low - density lipoprotein ( vldl ) and chylomicrons , fractions 1825 contain ldl , and fractions 2640 contain high - density lipoprotein ( hdl ) [ 14 , 15 ] . the cholesterol content in various lipoproteins was calculated from the concentration in the fplc fractions [ 14 , 15 ] . the antibodies against phosphorylated eif-2 , grp78 and lysosomal acid lipase ( lal ) were obtained from abcam inc . ( cambridge , ma ) , while the antibodies against eif-2 and -actin were obtained from santa cruz biotechnology inc . aortas from two mice were pooled , homogenized in 20 mm tris - cl and centrifuged at 14,000 rpm for 10 min at 4c . supernatants containing 15 g protein were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis on 10% gels and transferred to polyvinylidene difluoride membranes . membranes were blocked with 5% fat - free milk in tbs - t ( 2.5 mm tris , 15 mm nacl , 0.01% tween 20 ; ph 7.6 ) and then consequently incubated with primary antibodies against indicated proteins and horseradish peroxidase - conjugated secondary antibodies , as previously described . immunoreactive bands were visualized using ecl - plus chemiluminescence reagent ( ge healthcare healthcare - amersham ) and analyzed with a gs-700 imaging densitometer ( bio - rad , hercules , ca ) . the differences between 2-ap treatment and control groups were analyzed by student 's t - test . the pharmacological properties of 2-ap , such as effective dose 50 ( ed50 ) and effective half - life ( t1/2 ) , have not been tested in vivo . the material safety data sheet produced by invivogen ( san diego , ca ) indicates that the lethal dose 50 ( ld50 ) of 2-ap via oral administration and intraperitoneal injection of rats is 723 and 270 mg / kg body weight ( bw ) , respectively . a preliminary study in our laboratory demonstrated that gavage feeding of 2-ap to apoe mice at doses greater than 400 mg / kg bw once every other day reduced food intake and resulted in death ( data not show ) . in the present study , we observed that feeding 2-ap at a dose of 200 mg / kg bw once every other day did not reduce food intake and body weight , and did not increase mortality within 24 weeks as compared to untreated control apoe mice ( table 1 ) . specifically , the average of food intake of apoe mice at 2430 weeks of age with or without 2-ap treatment was 0.24 0.03 and 0.25 0.07 g chow / g bw / day , respectively . the body weight of these mice at 30 weeks of age was about 27 g. no significant difference was observed between the 2-ap treated and the untreated control mice . we previously reported that 2-ap inhibited e / b48 lipoprotein - induced transformation of macrophages into foam cells . figures 1(a ) and 1(b ) show examples of cross - sections and en face preparations of aortas obtained from apoe mice treated with or without 2-ap . data in figures 1(c ) and 1(d ) show that the mean size of atherosclerotic lesions in the aorta sinus and the surface area of the entire aorta of 2-ap - treated apoe mice were reduced by about 55% and 39% , respectively , compared to those in the untreated control apoe mice . consistent with the smaller lesion size , the advanced lesions were reduced markedly in 2-ap - treated mice . for instance , the number of lipid cores ( acellular areas ) in the aortic sinus in the 2-ap - treated and untreated control apoe mice were 1.2 0.6 and 0.4 0.1/section ( p < 0.05 ) , respectively . these results , together with our previous findings that 2-ap inhibits e / b48 lipoprotein - induced foam cell formation , suggest that 2-ap is able to inhibit atherogenesis in vitro and in vivo . an increase in plasma cholesterol and triglycerides has been suggested to be a risk factor for atherosclerosis . to determine whether the reduction in atherosclerotic lesions induced by 2-ap treatment was due to a change in plasma cholesterol and triglycerides , we measured the levels of plasma cholesterol and triglycerides in apoe mice with or without 2-ap treatment . as the data in table 1 show that the average plasma concentrations of total cholesterol and triglycerides were 513 and 146 , respectively , in apoe mice without 2-ap treatment and that 2-ap treatment did not significantly alter these plasma lipid levels . to investigate whether 2-ap feeding affected the distribution of cholesterol among the lipoproteins , we fractionated mouse plasma with an fplc system ( figure 2(a ) ) . as the data in figure 2(b ) show , the plasma cholesterol levels in vldl , ldl , and hdl fractions were comparable in 2-ap - treated and untreated control mice . these results suggest that the reduced atherosclerotic lesions in 2-ap treated mice were not due to altered levels of plasma lipids . we previously reported that transformation of macrophages into foam cells by e / b48 lipoproteins was associated with er stress , as reflected by increased phosphorylation of perk and eif-2. further that 2-ap inhibited e / b48 the present report studied the effect of 2-ap on the expression level of total and phosphorylated eif-2 in the aorta of apoe mice . data in figure 3(a ) show that the level of total eif-2 was comparable in aortas obtained from apoe mice treated with or without 2-ap treatment . in contrast , the level of phosphorylated eif-2 in the aorta was about 64% lower in 2-ap - treated apoe mice than in untreated control mice ( figure 3(b ) ) . the data in figure 3(c ) demonstrate that the protein level of chaperon grp78 was reduced by ~53% in 2-ap - treated apoe mice as compared to the untreated control littermates . we previously reported that intralysosomal accumulation of lipids / lipoproteins induced by e / b48 lipoproteins was associated with decreased lysosomal hydrolase lal , and that 2-ap treatment restored e / b48 lipoprotein - reduced expression of lal . therefore , the present report compared the protein levels of lal in the aorta of apoe mice with or without 2-ap treatment . data in figures 3(a ) and 3(c ) show that the protein level of aortic lal was ~2.6 fold higher in 2-ap - treated apoe mice than in their untreated littermates . having demonstrated the inhibitory activity of 2-ap on er stress in the aorta , we then compared the expression of grp78 and phosphorylated eif-2 in the lesional area in the aorta sinus . as the representative immunostaining sections in figure 4 show , the atherosclerotic lesions , especially the areas close to the plaque surface , accumulated cells positively stained by antibodies against grp78 or phosphorylated eif-2. the number of positively stained cells in 2-ap - treated apoe mice was much less than those in untreated control mice . these results are consistent with the western blot data shown in figure 3(a ) . these findings provide evidence that 2-ap inhibits er stress in the aorta of apoe mice . the most important finding of this report is that oral feeding of the eif-2 phosphorylation inhibitor 2-ap reduced atherosclerotic lesions in apoe mice . this finding suggests that induction of eif-2 phosphorylation is a causal mechanism for the development of atherosclerosis in apoe mice . four eif-2 kinases have been identified in mammalian cells , which each catalyze eif-2 phosphorylation in response to various stressful cellular events [ 18 , 19 ] . specifically , er stress activates perk , heme deprivation activates heme - regulated inhibitor ( hri ) , virus infection activates double - stranded rna - activated protein kinase ( pkr ) and amino acid deprivation activates general control nonderepressible-2 ( gcn2 ) . previous studies from our laboratory demonstrated that treatment of mouse macrophages with e / b48 lipoproteins induced phosphorylation of perk but no other eif-2 kinases and that overexpression of a nonphosphorylatable perk mutant attenuated e / b48 lipoprotein - induced eif-2 phosphorylation and foam cell formation [ 7 , 8 ] . these findings suggest that perk is responsible for the increased eif-2 phosphorylation observed in e / b48 lipoprotein - treated macrophages . a high plasma level of e / b48 lipoproteins is a hallmark feature of apoe mice [ 10 , 20 ] . data from the present report demonstrated that 2-ap treatment reduced eif-2 phosphorylation in the aorta apoe mice . it is highly likely that plasma e / b48 lipoproteins infiltrate into the arterial walls of apoe mice , where they activate perk and sequentially increase eif-2 phosphorylation in vascular cells , including resident macrophages . indeed , phosphorylation of eif-2 induced by e / b48 lipoproteins has been associated with increased expression of calreticulin and grp 78 . inhibition of eif-2 by a nonphosphorylatable eif-2 mutant or an 2-ap attenuated e / b48 lipoprotein - induced expression of these protein chaperons . data from the present report showed that 2-ap treatment reduced the expression of grp78 . reduction in global protein synthesis is another hallmark consequence of eif-2 phosphorylation . under physiological conditions , inhibition of global protein synthesis and upregulation of protein chaperones reduces the accumulation of newly synthesized proteins in the er , thereby relieving er stress [ 21 , 22 ] . however , constant phosphorylation of eif-2 and inhibition of global protein synthesis might overpower normal cellular functions , causing pathological conditions [ 21 , 22 ] . for example , we previously observed that e / b48 lipoprotein - induced eif-2 phosphorylation was associated with reduced global protein synthesis and reduced expression of lysosomal hydrolases , such as lysosomal acid lipase ( lal ) . in addition , we previously observed that inhibition of eif-2 phosphorylation by 2-ap , or a nonphosphorylatable eif-2 mutant , attenuated the reduced expression of lal and inhibited foam cell formation induced by e / b48 lipoproteins [ 7 , 8 ] . lal is the sole hydrolase responsible for cleavage of cholesteryl esters delivered to lysosomes [ 24 , 25 ] . taken together , our previous studies suggest that reduction in lysosomal hydrolases could be a fundamental mechanism by which e / b48 lipoproteins trigger intracellular lipid / lipoprotein accumulation and transform macrophages into foam cells . correspondingly , the present report demonstrated that treatment of apoe mice with 2-ap significantly elevated the protein level of lal in the apoe mouse aorta . it is possible that the e / b48 lipoproteins deposited in the arterial wall induce eif-2 phosphorylation , which downregulates lysosomal hydrolases , such as lal , in the resident macrophages , and therefore reduces the degradation of e / b48 lipoproteins leading to intralysosomal e / b48 lipoprotein accumulation and resulting in foam cell formation and atherosclerosis development . this postulation is supported by the evidence showing that patients with a deficiency of lal manifest both an accumulation of lipids in cells and develop premature atherosclerosis . further more , overexpression of lal reduces atherosclerotic lesions in mice deficient in low - density lipoprotein receptor . this report demonstrated that er stress occurred in the aorta of apoe mice , as reflected by increased phosphorylation of perk and eif-2 and by increased expression of chaperon grp78 . we also observed that inhibition of eif-2 phosphorylation by 2-ap reduced atherosclerotic lesions in the aorta of apoe mice . these observations suggest that induction of er stress , that is , activation of the perk - eif2 signaling cascade , is a causal mechanism for development of atherosclerosis in apoe mice . such insights will inform novel strategies for using er stress inhibitors such as 2-ap to prevent or treat atherosclerosis . the tsc lines used in this study were b6ts4 and egfp - ts3.5 , which were derived from a blastocyst of the c57bl/6 and icr mouse strain , respectively . tscs were cultured as described previously . in brief , cells were cultured on mitomycin - c ( sigma - aldrich , st . louis , mo , usa)-treated primary mouse embryonic fibroblasts in rpmi1640 medium ( thermo fisher scientific , san jose , ca , usa ) with 20% fetal bovine serum ( thermo fisher scientific ) , 25 ng / ml human recombinant fgf4 ( wako pure chemicals , osaka , japan ) , 1 g / ml heparin ( sigma - aldrich ) , 100 m 2-mercaptoethanol ( sigma - aldrich ) , 1% glutamax ( thermo fisher scientific ) , and 1 mm sodium pyruvate ( thermo fisher scientific ) . representative colony morphologies are shown in fig . 1 , together with descriptions of their morphological characters . there was an additional type ( type 5 ) , but this was not analyzed here because it rarely emerged under normal tsc culture conditions . at 96 h after passaging , colonies were picked up using a glass capillary pipette under a dissecting microscope and used for rt - qpcr analysis . total rna for rt - qpcr was extracted with rneasy micro kits ( qiagen , venlo , netherlands ) from tsc colonies . following extraction , the first strand cdna was synthesized with a superscript iii reverse transcriptase reagent set ( thermo fisher scientific ) . gene expression was assessed by qpcr on a steponeplus instrument ( thermo fisher scientific ) using quantitect sybr green pcr kits ( qiagen ) according to the manufacturer s instructions . samples were denatured at 95c for 10 min , followed by 40 amplification cycles consisting of denaturation at 95c for 15 sec , and an annealing and extension step at 60c for 1 min . raw cq ( ct ) values ( pcr cycles at which the fluorescence signal crosses threshold ) were calculated using stepone software ( v. 2.1 ; thermo fisher scientific ) setting baseline and appropriate threshold values . all runs were performed in triplicate and an identical sample was used in each different runs as an inter - run calibration sample to correct for the technical variance between the runs and thus compare results from different plates [ 4 , 6 ] . the primer sets used for tsc marker genes were the following : cdx2 , 5gcagtccctaggaagccaag3 and 5gcagccagctcacttttcct3 ; elf5 , 5gtggcatcctggaatgggaa3 and 5cactaacctccggtcaaccc3. for quantification of reference genes ( atp5b , canx , cyc1 , gapdh , ubc , rn18s ) , the primer sets from the mouse genorm kit ( primerdesign ltd . , southampton , uk ) were used . actb was analyzed as an additional reference gene using the primer set 5ctgtcgagtcgcgtcca3 and 5acccattcccaccatcacac3. the baseline cycles and cycle thresholds were established manually for each gene . to assess the stability of the reference genes , the raw cq values of each reference gene were analyzed with the genorm algorithmic tool using qbase software ( biogazelle ) . these values were converted into normalized relative quantities ( nrqs ) with multiple reference genes based on the modified classic --ct method or a single reference gene . nrqs were scaled in relation to the mean across all samples for each target or to reference genes . we ranked the stability of the reference genes based on the mean expression stability ( genorm m value ) provided by the genorm program . to determine the optimal number of reference genes across different colony types , qbase was also used to calculate pairwise variation ( v value ) . normalization of the expression levels of tsc marker genes ( cdx2 and elf5 ) was performed by qbase software using reference gene(s ) ranked as described above . to compare the gene expression levels between colony types within the tsc lines , normalized relative mrna levels were analyzed with kruskal - wallis tests followed by dunnett s multiple comparison tests ; p < 0.05 was considered statistically significant . the tsc lines used in this study were b6ts4 and egfp - ts3.5 , which were derived from a blastocyst of the c57bl/6 and icr mouse strain , respectively . tscs were cultured as described previously . in brief , cells were cultured on mitomycin - c ( sigma - aldrich , st . louis , mo , usa)-treated primary mouse embryonic fibroblasts in rpmi1640 medium ( thermo fisher scientific , san jose , ca , usa ) with 20% fetal bovine serum ( thermo fisher scientific ) , 25 ng / ml human recombinant fgf4 ( wako pure chemicals , osaka , japan ) , 1 g / ml heparin ( sigma - aldrich ) , 100 m 2-mercaptoethanol ( sigma - aldrich ) , 1% glutamax ( thermo fisher scientific ) , and 1 mm sodium pyruvate ( thermo fisher scientific ) . representative colony morphologies are shown in fig . 1 , together with descriptions of their morphological characters . there was an additional type ( type 5 ) , but this was not analyzed here because it rarely emerged under normal tsc culture conditions . at 96 h after passaging , colonies were picked up using a glass capillary pipette under a dissecting microscope and used for rt - qpcr analysis . total rna for rt - qpcr was extracted with rneasy micro kits ( qiagen , venlo , netherlands ) from tsc colonies . following extraction , the first strand cdna was synthesized with a superscript iii reverse transcriptase reagent set ( thermo fisher scientific ) . gene expression was assessed by qpcr on a steponeplus instrument ( thermo fisher scientific ) using quantitect sybr green pcr kits ( qiagen ) according to the manufacturer s instructions . samples were denatured at 95c for 10 min , followed by 40 amplification cycles consisting of denaturation at 95c for 15 sec , and an annealing and extension step at 60c for 1 min . raw cq ( ct ) values ( pcr cycles at which the fluorescence signal crosses threshold ) were calculated using stepone software ( v. 2.1 ; thermo fisher scientific ) setting baseline and appropriate threshold values . all runs were performed in triplicate and an identical sample was used in each different runs as an inter - run calibration sample to correct for the technical variance between the runs and thus compare results from different plates [ 4 , 6 ] . the primer sets used for tsc marker genes were the following : cdx2 , 5gcagtccctaggaagccaag3 and 5gcagccagctcacttttcct3 ; elf5 , 5gtggcatcctggaatgggaa3 and 5cactaacctccggtcaaccc3. for quantification of reference genes ( atp5b , canx , cyc1 , gapdh , ubc , rn18s ) , the primer sets from the mouse genorm kit ( primerdesign ltd . , southampton , uk ) were used . actb was analyzed as an additional reference gene using the primer set 5ctgtcgagtcgcgtcca3 and 5acccattcccaccatcacac3. the baseline cycles and cycle thresholds were established manually for each gene . to assess the stability of the reference genes , the raw cq values of each reference gene were analyzed with the genorm algorithmic tool using qbase software ( biogazelle ) . these values were converted into normalized relative quantities ( nrqs ) with multiple reference genes based on the modified classic --ct method or a single reference gene . nrqs were scaled in relation to the mean across all samples for each target or to reference genes . we ranked the stability of the reference genes based on the mean expression stability ( genorm m value ) provided by the genorm program . to determine the optimal number of reference genes across different colony types , qbase was also used to calculate pairwise variation ( v value ) . normalization of the expression levels of tsc marker genes ( cdx2 and elf5 ) was performed by qbase software using reference gene(s ) ranked as described above . to compare the gene expression levels between colony types within the tsc lines , normalized relative mrna levels were analyzed with kruskal - wallis tests followed by dunnett s multiple comparison tests ; p < 0.05 was considered statistically significant .

we previously reported that the apolipoprotein ( apo ) b48-carrying lipoproteins obtained from apoe knockout ( apoe/ ) mice , so called e/b48 lipoproteins , transformed mouse macrophages into foam cells and enhanced the phosphorylation of eukaryotic translation initiation factor 2 ( eif-2 ) . furthermore , the eif-2 phosphorylation inhibitor , 2-aminopurine ( 2-ap ) , attenuated e/b48 lipoprotein - induced foam cell formation . the present report studied the effect of 2-ap on atherosclerosis in apoe/ mice . our results showed that the level of food intake , bodyweight , plasma cholesterol , and triglycerides was comparable in apoe/ mice treated with or without 2-ap . however , the mean size of atherosclerotic lesions in the aorta sinus as well as the surface area of the entire aorta of 2-ap - treated apoe/ mice were reduced by about 55% and 39% , respectively , compared to samples from untreated control apoe/ mice . in addition , the 2-ap - treated apoe/ mice showed a significant decrease in glucose - regulated protein 78 ( grp78 ) and phosphorylated eif-2 in their aortic samples as compared to levels in untreated control apoe/ mice . these observations suggest that endoplasmic reticulum stress is a causal mechanism for the development of atherosclerosis in apoe/ mice and that therapeutic strategies can be developed for using eif-2 phosphorylation inhibitors , such as 2-ap , to prevent or treat atherosclerosis . mouse trophoblast stem cells ( tscs ) form colonies of different sizes and morphologies , which might reflect their degrees of differentiation . therefore , each colony type can have a characteristic gene expression profile ; however , the expression levels of internal reference genes may also change , causing fluctuations in their estimated gene expression levels . in this study , we validated seven housekeeping genes by using a geometric averaging method and identified gapdh as the most stable gene across different colony types . indeed , when gapdh was used as the reference , expression levels of elf5 , a tsc marker gene , stringently classified tsc colonies into two groups : a high expression groups consisting of type 1 and 2 colonies , and a lower expression group consisting of type 3 and 4 colonies . this clustering was consistent with our putative classification of undifferentiated / differentiated colonies based on their time - dependent colony transitions . by contrast , use of an unstable reference gene ( rn18s ) allowed no such clear classification . cdx2 , another tsc marker , did not show any significant colony type - specific expression pattern irrespective of the reference gene . selection of stable reference genes for quantitative gene expression analysis might be critical , especially when cell lines consisting of heterogeneous cell populations are used .

epilepsy surgery is an established treatment in selected patients with drug - resistant epilepsy resulting in seizure freedom or a significant reduction of seizure frequency in the majority of operated patients.13 a detailed clinical assessment is critical before a decision about surgery can be made in order to localise the seizure onset zone . for many patients , however , especially in mri - negative patients , invasive seizure monitoring is required for localising the seizure onset zone and also for delineating the area of planned resection against eloquent cortex.4 despite the potential benefits of the invasive workup , they have to outweigh the risks related to the neurosurgical procedure . the rates of reported complications related to invasive monitoring differ widely between studies , from no complications at all5 up to 26.3%.6 the reasons for these varying proportions may be related to the definitions of complications and to study methodology ( eg , if data are obtained retrospectively or prospectively ) . many reports use the terms transient and permanent , surgical and neurological complications.7 8 additionally , severity scales from 1 to 4,9 or from 1 to 5 have been used.4 in the swedish national epilepsy surgery register all complications related to diagnostic or therapeutic epilepsy surgery procedures are reported and are graded as major or minor.10 a few studies have evaluated the usefulness and safety versus the risks associated with implantation of intracranial electrodes , and the benefits of invasive workup for epilepsy surgery in general seem to outweigh the risks.11 12 increased number of electrodes and longer duration of monitoring has been shown to be associated with increased frequency of adverse events.13 other aspects not explored include age and medication . higher age was shown to be associated with an increased risk for complications at epilepsy surgery in an earlier swedish national study10 and , therefore , it would be of interest to investigate whether the risk of complications related to invasive monitoring is also age related . valproate treatment may induce thrombocytopenia which seems to be dose dependent with a negative correlation between valproate trough levels and platelet counts.14 although several studies have found no indication that patients on valproate treatment are at an increased risk of surgical bleeding complications,15 16 there is a worry about the possible risk , and some epilepsy surgeons do not operate patients who are on valproate treatment . the aim of the current study was to analyse data from the prospective population - based swedish national epilepsy surgery register 19962010 , in order to describe the complications related to preoperative invasive investigations with intracranial electrodes . secondary aims were to explore if the risk of complications is age related , if there was a higher risk of haemorrhagic complications in patients who were treated with valproate , and whether complications occurring during invasive monitoring is a risk factor for complications at therapeutic surgery . every epilepsy surgery procedure in sweden is reported to the swedish national epilepsy surgery register . the register includes data on all patients who have been operated in sweden since 1990 . it was initiated by the swedish board of health and welfare as a quality control register , and is controlled by the swedish data inspection board . the validity of the data collected from the centres is regularly checked by intrinsic checkpoints within the database and by external revision . two epilepsy nurses visit the six centres regularly and compare the data entered into the central database with the original data from the patient files for a random yearly sample of around 25% of the patients operated at each centre . the items controlled in this external revision include side and site of operation , complications , main histopathological findings and seizure outcome at the two - year follow - up . so far , there have been no mismatches in the reporting on any of these central items . the regional board of medical ethics at the university of gothenburg considered this study a quality control study not necessitating informed consent for research . in this study , we have analysed complications after invasive eeg monitoring in all patients who , prior to surgery , underwent investigations with subdural strips or grids , intracerebral depth electrodes , foramen ovale electrodes or epidural electrodes from january 1996 to december 2010 . ( during this time period , stereo - eeg ( seeg ) was not performed in sweden , the depth electrodes used were freely placed most often in conjunction with grid electrodes ) . two or more electrode types were often combined , and when reporting complications in relation to electrode type the most complication - prone electrode type has been accounted for . the register does , however , not provide information on the number of electrodes and the duration of monitoring . complications are reported as major or minor to the register , and the type of complication is commented upon . minor complications are defined as those that resolve within 3 months , while major complications affect activities of daily living and last longer than 3 months . major complications also include any significant neurological deficits , even if they do not affect activities of daily living.10 the antiepileptic drug treatment at the presurgical investigation is reported , but not the dosage . the swedish national epilepsy surgery register does not contain information on laboratory variables , such as trough levels of antiepileptic drugs or platelet counts . between 1 january 1996 and 31 december 2010 , 865 patients underwent therapeutic surgical procedures . seven hundred and ninety - eight of the operations were resections : 523 were temporal lobe resections ( tlr ) and 142 were frontal lobe resections ( flr ) . there were 44 parietal lobe resections ( plr ) , 27 occipital lobe resections ( olr ) , 19 multilobar resections ( mlr ) and 43 hemispherectomies . two hundred and seventy - six of the 865 patients ( 32% ) underwent diagnostic intracranial monitoring with invasive electrodes prior to the therapeutic surgical procedure . two hundred and sixty - four of them had a resective procedure later , and there were eight multiple subpial trans - sections , three callosotomies and one exploration . forty three per cent ( n=61 ) of the patients who eventually had an flr were invasively monitored , versus 30% ( n=156 ) of the patients who had a tlr , 57% of the patients who had a plr , 30% of the patients who had an olr , 63% of the patients who had an mlr and 5% of the patients who had a hemispherectomy . patient age at surgery ranged from two years to 58 years ( median 26 years ) , 134 ( 48.6% ) were female and 142 ( 51.4% ) were male . for five patients , data on complications to the invasive investigation were missing , which is why complication data are reported for 271 patients . for another five patients , data on electrode type was missing , hence , the analysis of electrode type related to surgical complications is limited to 271 patients . minor complications occurred in 13 of 271 cases ( 4.8% ) ( table 1 ) . complications related to intracranial monitoring haematomas were most common , associated with a 3.7% risk ( n=10 ) . of these , seven ( 2.6% ) were subdural haematomas and three were epidural haematomas ( 1.1% ) . two patients suffered from wound infections and one from electrode dislocation , representing a complication rate of 0.7 and 0.4% . individual patient data for all complications occurring during intracranial monitoring are given in table 2 . individual complications occurring during intracranial monitoring lflr , left frontal lobe resection ; ltlr , left temporal lobe resection mst , multiple subpial trans - section ; rflr , right frontal lobe resection rtlr , right temporal lobe resection . ten per cent ( n=6 ) of the 61 patients who were investigated for an flr and 4% ( n=6 ) of the 156 patients investigated for a tlr suffered from complications from the invasive electrode monitoring . in the group of patients who had complications , five out of the six who had frontally located electrodes had a subdural haematoma . among the patients with complications from temporally located electrodes , there was a slight , non - significant difference in complication rate between female ( 3.9% , n=5 ) and male patients ( 5.7% , n=8 ) . among the 108 patients who were monitored with subdural grids , eight ( 7.4% ) suffered from complications , mostly haematomas . as shown in table 3 , grids had the highest complication rate , followed by intracerebral depth electrodes . two patients in the study had epidural electrodes , and one of them got a subdural haematoma . electrode types and complications electrode type alone or in combination , most complication prone first . d , electrode dislocation ; eh , epidural haematoma ; i , infection ; sh , subdural haematoma . when patients were divided into age categories ( table 4 ) there was a difference between the age groups . children ( age 518 years ) and young adults ( age 1835 years ) had complications in 3.2 and 3.6% , respectively , while 8.3% of adults 35 years old had complications . age and complications sixty of the 271 patients ( 22% ) in who reports on complications or not had been registered were treated with valproate at the time of presurgical investigations , alone or in combination with other antiepileptic drugs . three of the 60 patients on valproate had a bleeding during invasive monitoring ( 5% ) compared with seven of the 211 patients who were not treated with valproate ( 3.3% ) . treatment with valproate and having a bleeding was associated with an or of 1.53 ( 95% ci 0.38 to 6.12 ) compared to having a bleeding and not being treated with valproate . this association was , however , not significant as shown by the wide ci including 1 . in the whole swedish series of 865 therapeutic surgeries 19962010 , there were 26 major complications ( 3%).17 a post hoc analysis of a possible relationship between having a complication during invasive monitoring and at surgery was performed . two of 13 patients ( 15% ) who had a complication at the invasive monitoring in addition also suffered a major complication related to the therapeutic procedure , compared to 24/852 ( 3% ) patients who had a complication only at therapeutic surgery . having a complication during invasive monitoring was associated with a significant or of 6.27 ( ci 1.32 to 29.9 ) of also having a complication at therapeutic surgery compared to the risk of having a complication only at surgery . there was no relationship between the kind of complication ( bleeding or other ) at the one or the other procedure . in this series , the rate of haemorrhagic complications ( 3.6% of the patients ) was comparable with that reported in a recent systematic review concerning adverse events related to invasive eeg monitoring with subdural grid electrodes which identified 21 studies including 2542 patients.13 in this review , the mean pooled prevalence of haematomas was 4% . the development of subdural haematoma was the most common type of complication ( 2.6% ) in our series , more than half the complications were in this category ( n=7 ) . the prevalence of subdural haematomas varies widely in the literature and is reported to be common in some studies , whereas , other groups report no subdural haematomas at all in their series . in the abovementioned review , subdural haematomas were documented in 41 of 2356 patients ( pooled prevalence 1.7%).13 three ( 1.1% ) of our patients developed epidural haematomas during the invasive eeg monitoring . this is slightly higher than the 0.6% reported by arya et al.13 even higher rates have been reported by hamer et al ( 2.5%),6 while several studies report no epidural haematomas at all.1820 two ( 0.7% ) of the patients in our study had wound infections , whereas , there were no intracranial infections . this is a low number compared to other studies . in the review by arya et al , infections emerged as the most common type of complication with a pooled prevalence of 5.3% ( 2.3% neurologic infections and 3.0% superficial infections).13 some series also have significantly higher infection rates.9 21 in one prospective study , the infection rate was shown to increase if more than 100 electrode contacts were used , if more than ten electrode cables were present , or if electrodes remained implanted for more than 14 days.22 since the swedish national epilepsy surgery register does not contain data on number of electrode contacts or days , we were not able to analyse the impact of these factors in our series . we hypothesise that one reason we do not have any bone flap or deep infections may be attributed to short surgical time23 since we , in general , use fewer grids than reported from many centres in complication series . in our series , grid electrodes caused more complications than other types of electrodes , as eight of 13 affected patients had grid electrodes alone or in combination with other electrode types . this correlation between subdural grids and complications has also been shown by other groups.4 24 the reasons for this increased risk are unclear but could be mechanical or haemodynamic ( the grid disturbing the csf circulation ) . whether the addition of strips to a subdural grid recording increases the risk of complications can not be excluded , although strips per se have a low complication rate . patients who are investigated for frontal lobe seizure onset tend to have more grid electrodes than other patients . this group of patients also suffered from complications more often than other groups in our series , nearly 10% ( 6 out of 61 ) , and all were haematomas . next in line were the patients investigated for temporal lobe epilepsy , almost 4% ( 6 out of 156 ) had complications . we assume that the high rate of haemorrhagic complication in patients with frontal lobe seizures was related to the more common use of grids . there was a ( non - significant ) trend towards higher risk for complications in patients who were 35 years or more in our series . the association between complications and age has previously been documented in relation to invasive monitoring , resective epilepsy surgery procedures and other neurosurgical procedures , and has been discussed in terms of increasing tissue fragility and affection of the cerebrovascular system with increasing age.6 10 25 on the other hand , blauwblomme et al studied adverse events related to invasive eeg monitoring in children and found a very high complication rate , almost half the patients suffered from some kind of complication . this can partly be explained by the fact that in this series a wide range of adverse events was considered as complications , for example , cerebrospinal leakage.9 minor adverse events such as headache , nausea , light fever and tiredness during the first postoperative days can be associated with the implantation of subdural electrodes , but most surgeons consider them as expected effects of the implantation and they are rarely documented . in the post hoc exploration of a possible relationship between complications during invasive monitoring and at surgery it was notable that two of the 13 patients with complications in our study also suffered from major complications after the therapeutic resection.17 since this association was not explained by bleedings , some other vulnerability may be at play , for which we presently have no explanation . in order to verify and elucidate this finding , prospective multicentre studies of complications during invasive monitoring and in relation to resective surgery , collecting more detailed patient data is needed . there was no significant association between valproate treatment and bleeding complications during invasive monitoring and , therefore , there is no support for the notion that valproate treatment is an important risk factor for bleedings . however , the or of 1.53 implies that there is a small increase in risk and it would be valuable if this could be further studied in larger samples preferably with data on valproate dosage , trough levels and platelet counts . in one large single centre study , the risk of bleedings requiring acute decompression was shown not to be related to the surgical learning curve , and so , not to diminish over time.4 the importance of minimising such potentially life - threatening complications during presurgical investigations can not be overstated . five deaths directly related to subdural grid implantation were documented in the systematic review.13 this does not , however , cover complications related to depth electrodes . in general , complications have been reported to be less common with depth electrodes than with subdural grids,4 5 7 2629 but at least two fatalities have been reported with seeg recordings.30 31 also , engel et al early on reported two deaths from intracerebral haemorrhage caused by depth electrodes.32 an important issue when striving to lower the risk of serious complications related to invasive monitoring is surveillance . it is clear from the literature that some reported deaths have been related to lack of 24 h professional surveillance.33 while serious complications can not totally be avoided , their effects may be minimised with rapid intervention such as acute decompression . in our series , the four swedish centres in which subdural grids are used all have personalised 24 h surveillance of those patients and have , therefore , managed to act rapidly in case of complications . the spectrum of definitions and scales makes it difficult to compare studies and , therefore , reporting only percentages is not very meaningful . the main issues are study methodology on the one hand , and the definitions of complications on the other . most studies are single - centre retrospective studies , while a few studies analyse prospectively recorded data.9 22 24 it would be valuable if an agreement could be reached on how to report complications associated with invasive eeg monitoring , and agree about what is to be considered a complication and what could rather be described as a more or less expected adverse event . in the present study , we have not considered transient csf leakage , fever and headache as complications , as all these commonly occur after implantation of intracranial electrodes . neither have we had as a routine to report small asymptomatic subdural haematomas . we acknowledge that this is a limitation of the study , and are aware that it leads more to a top of the iceberg reporting , but we capture symptomatic complications and especially all those necessitating interventions . another limitation of the study is that the register does not contain information on the number of electrodes and duration of invasive monitoring , and these variables could , therefore , not be related to the risk of complications . the main strength of the study is the prospective collection of population - based data in a large national series . also , our external quality control ensures that the data has been correctly entered from the patient files into the register . in this prospective population - based epilepsy surgery series , the most common complications were haematomas , and subdural grids carried the highest risk of complications . close supervision and rapid interventions led to avoidance of permanent morbidity . whether the slightly increased risk of haematomas with valproate treatment is clinically relevant needs further investigation , as does the finding of an increased risk for complications at epilepsy surgery for patients who had a complication during invasive monitoring . there is a need for consensus on how to report complications related to invasive monitoring in order to provide patients with adequate information on risks . scoliosis , newly developed in a skeletally mature patient , is termed degenerative scoliosis or de novo scoliosis . prevalence and incidence of degenerative scoliosis increase with age . an overall increase is seen due to the demographic shift towards an aging society [ 17 , 33 , 39 ] . generally it is assumed that degenerative scoliosis is caused by asymmetric disc degeneration and facet joint degeneration [ 1 , 3 , 4 , 7 , 27 , 30 , 36 ] and that the onset is marked by disc degeneration [ 17 , 25 ] . this etiology sets degenerative scoliosis apart from other types of scoliosis , such as adolescent idiopathic scoliosis and scoliosis secondary to neuromuscular disease . in addition , degenerative scoliosis develops most frequently in the lumbar spine , where degenerative changes are most prevalent , whereas in neuromuscular and idiopathic scoliosis the major curve is usually in the thoracic or thoracolumbar spine . . showed that the normal spine has a specific pattern of vertebral rotation , with a predominant rotation to the right at the mid - thoracic level . most prevalent curve types of adolescent idiopathic scoliosis were found to demonstrate comparable patterns [ 8 , 11 , 12 , 14 , 21 , 23 , 32 , 35 , 40 ] , which correspond to the predominant rotational pattern in the normal spine . this implies that once the spine starts to decompensate , due to a still unknown cause , it follows this already built - in rotational tendency . in degenerative scoliosis factors causing asymmetrical degeneration and the eventual disruption of the spinal equilibrium whether a pre - existing rotational pattern also plays a role in degenerative scoliosis is unknown . an argument against this is that the normal lumbar spine does not show an obvious rotation deviating from the midline . we hypothesize that spinal decompensation based on degenerative changes is purely coincidental and that left and right curves in lumbar degenerative scoliosis are equally distributed . however , if the distribution of curve direction is unequal , this may point to a pre - existing rotational tendency that has so far not been demonstrated . symptoms of degenerative scoliosis are most frequently progressive back pain , radiculopathy and neurogenic claudication [ 1 , 3 , 7 , 9 , 13 , 15 ] . aging progressively affects all structures of the spinal units , eventually leading to degenerative instabilities such as spondylolisthesis , spinal stenosis and scoliosis . relationships between scoliotic pattern and patient symptoms are unclear , although speculations on such relationships are frequently made [ 1 , 3 , 7 , 30 , 42 ] . some authors suggest that pain at the convexity is caused by muscle fatigue of the paraspinal muscles [ 3 , 30 , 42 ] . other authors claim that pain at the convexity can also be caused by facet joints . pain at the concavity of the scoliotic curve is thought to be caused by destruction of facet joints and degenerative changes in disc spaces . radicular pain at the concavity can arise from narrowed foramen [ 1 , 3 , 42 ] or ruptured discs causing radiculopathy [ 1 , 3 ] . dynamic overstretch of a nerve root could also cause radicular pain at the convex side of the scoliosis . the first objective of this study is to assess whether a dominant direction of compensation exists in degenerative scoliosis . our second objective is to investigate whether asymmetric symptoms are related to the curve direction , i.e. whether pain and radicular symptoms in degenerative lumbar scoliosis are predominantly present at one side relative to the curve direction . in this retrospective study we filtered the outpatient records of our institution for patients who presented between 1996 and 2007 and were diagnosed with degenerative scoliosis . patients aged 50 and over at the initial presentation of degenerative scoliosis and had a scoliosis of at least 10 in the coronal plain were included . patients were excluded if they had a previous history of idiopathic scoliosis , neuromuscular scoliosis , or scoliosis secondary to an underlying pathology . the patient records were reviewed in order to obtain patient data about age , gender , length , height , weight , symptoms , physical examination and history . for each patient , curve direction , apical level , apical vertebra , cobb angle in the coronal plane , and length of the curve were determined . the apex of the curve was determined in the coronal plane and the apical level was determined . the apical vertebra was defined according to the scoliosis research society ( srs ) as the vertebra with the greatest distance from the midline with the most rotation . according to the srs classification curves with an apex at the 12th thoracic vertebral body or the first lumbar vertebral body are thoracolumbar curves . curves with an apex between the first lumbar disc and the fourth lumbar vertebral body are lumbar curves . the cobb angle is the angle between the two most tilted vertebrae within a scoliotic curve . the length of the curves was taken as the number of vertebrae between these two most tilted vertebrae . patient symptoms recorded were low back pain , pain in the buttocks , leg pain , hypo- or hyperesthesia and weakness affecting the legs . all statistical analyses were performed with spss 15.0 for windows ( spss , inc , chicago , il ) . the binominal test was used to compare the observed frequencies of right and left curves to an equal distribution . in addition , it was tested whether curve direction depended on apex level using the chi - square test . whether the observed frequencies of ipsilateral and contralateral pain and/or radicular symptoms relative to the scoliotic curve direction differs from an equal distribution was tested using a binominal test . all statistical analyses were performed with spss 15.0 for windows ( spss , inc , chicago , il ) . the binominal test was used to compare the observed frequencies of right and left curves to an equal distribution . the tests were performed for all curves combined and also separately per apical level . in addition , it was tested whether curve direction depended on apex level using the chi - square test . whether the observed frequencies of ipsilateral and contralateral pain and/or radicular symptoms relative to the scoliotic curve direction differs from an equal distribution the group consisted of 71 women and 17 men with a mean age of 70 ( 70.1 10.3 ; range 5093 ) . of these patients 50 all patients had a single curve in the thoracolumbar range , except one patient who had a double curve . for this double curve , the curve with the largest cobb angle was considered in the analyses . of the total group , 39 curves ( 44% ) were to the right and 49 ( 56% ) were to the left ( p = 0.337 ) . apical levels ranged from the 12th thoracic disc to the 4th lumbar vertebra ( median apex , 2nd lumbar disc ) . the mean cobb angle was 25 ( 25.4 13.0 ; range 1083 ) . eighty - three patients had lumbar curves and five patients had thoracolumbar curves according to the srs classification . fifty - eight percent ( 49 ) of the lumbar curves were convex to the left ( p = 0.156 ) . eighty - three percent ( 5 ) of the thoracolumbar curves were convex to the right ( p = 0.375 ) . the apical vertebrae of the curves varied from l1 to l4 ( table 1 ) , with l3 the median apical vertebra.table 1the distribution of curve direction for each apical vertebraapical vertebra of curvennumber of curves convex to the rightnumber of curves convex to the leftp ( binominal test)l165 ( 83%)1 ( 17%)0.219l23520 ( 57%)15 ( 43%)0.500l33912 ( 31%)27 ( 69%)0.024*l482 ( 25%)6 ( 75%)0.289the p level indicates whether the distribution deviates significantly from an equal left right distribution*statistically significant ( p < 0.05 ) the distribution of curve direction for each apical vertebra the p level indicates whether the distribution deviates significantly from an equal left right distribution * statistically significant ( p < 0.05 ) left and right distributions were also tested per apical vertebra ( table 1 ) . for apical level l1 an inclination for convexity to the right eighty percent ( 4 ) of patients with the apex of the curve at l1 had a convexity to the right ( p = 0.219 ) . in 35 a small majority , 57% ( 20 ) of curves were convex to the right ( p = 0.500 ) . curves with an apex at l3 ( n = 39 ) , however , showed a significant inclination to the left . in this group 69% ( 27 ) had a curve convexity to the left ( p = 0.024 ) . also most ( 75% , n = 6 ) of the curves with the apex at l4 were convex to the left ( p = 0.289 ) . the curve direction depended significantly on the apical level ( p = 0.011 ) and on the level of the apical vertebra ( p = 0.017 ) ( fig . 1 ) . the direction of the curve depends significantly on the apical vertebra ( chi - square , p = 0.017 ) . when tested separately per level , the left right distribution for curves with an apex at l3 differed significantly from a random distribution ( binominal , p = 0.024 ) distribution of left and right convex curves per apical vertebra . the direction of the curve depends significantly on the apical vertebra ( chi - square , p = 0.017 ) . when tested separately per level , the left right distribution for curves with an apex at l3 differed significantly from a random distribution ( binominal , p = 0.024 ) all patients had low back pain . only four patients ( 5% ) had unilateral back pain . this was not related to the curve direction , since two patients had pain at the same , and two at the contralateral side of curve convexity . of these patients 57% ( 41 ) had unilateral leg pain , the others had pain in both legs . the side at which unilateral radicular leg pain occurred was unrelated to the curve direction ; 21 patients ( 51% ) had symptoms ipsilateral to the curve convexity and 20 ( 49% ) patients contralateral to the scoliotic curve ( p = 1.000 ) . seventy - two patients had symptoms of the lower extremities of which 22 had weakness , 1 hyperesthesia , 6 hypoesthesia and 11 showed neurological symptoms during physical examination . the group consisted of 71 women and 17 men with a mean age of 70 ( 70.1 10.3 ; range 5093 ) . of these patients 50 all patients had a single curve in the thoracolumbar range , except one patient who had a double curve . for this double curve , the curve with the largest cobb angle was considered in the analyses . of the total group , 39 curves ( 44% ) were to the right and 49 ( 56% ) were to the left ( p = 0.337 ) . apical levels ranged from the 12th thoracic disc to the 4th lumbar vertebra ( median apex , 2nd lumbar disc ) . the mean cobb angle was 25 ( 25.4 13.0 ; range 1083 ) . eighty - three patients had lumbar curves and five patients had thoracolumbar curves according to the srs classification . fifty - eight percent ( 49 ) of the lumbar curves were convex to the left ( p = 0.156 ) . eighty - three percent ( 5 ) of the thoracolumbar curves were convex to the right ( p = 0.375 ) . the apical vertebrae of the curves varied from l1 to l4 ( table 1 ) , with l3 the median apical vertebra.table 1the distribution of curve direction for each apical vertebraapical vertebra of curvennumber of curves convex to the rightnumber of curves convex to the leftp ( binominal test)l165 ( 83%)1 ( 17%)0.219l23520 ( 57%)15 ( 43%)0.500l33912 ( 31%)27 ( 69%)0.024*l482 ( 25%)6 ( 75%)0.289the p level indicates whether the distribution deviates significantly from an equal left right distribution*statistically significant ( p < 0.05 ) the distribution of curve direction for each apical vertebra the p level indicates whether the distribution deviates significantly from an equal left right distribution * statistically significant ( p < 0.05 ) left and right distributions were also tested per apical vertebra ( table 1 ) . for apical level l1 an inclination for convexity to the right eighty percent ( 4 ) of patients with the apex of the curve at l1 had a convexity to the right ( p = 0.219 ) . in 35 a small majority , 57% ( 20 ) of curves were convex to the right ( p = 0.500 ) . curves with an apex at l3 ( n = 39 ) , however , showed a significant inclination to the left . in this group 69% ( 27 ) had a curve convexity to the left ( p = 0.024 ) . also most ( 75% , n = 6 ) of the curves with the apex at l4 were convex to the left ( p = 0.289 ) . the curve direction depended significantly on the apical level ( p = 0.011 ) and on the level of the apical vertebra ( p = 0.017 ) ( fig . 1 ) . the direction of the curve depends significantly on the apical vertebra ( chi - square , p = 0.017 ) . when tested separately per level , the left right distribution for curves with an apex at l3 differed significantly from a random distribution ( binominal , p = 0.024 ) distribution of left and right convex curves per apical vertebra . the direction of the curve depends significantly on the apical vertebra ( chi - square , p = 0.017 ) . when tested separately per level , the left right distribution for curves with an apex at l3 differed significantly from a random distribution ( binominal , p = 0.024 ) all patients had low back pain . only four patients ( 5% ) had unilateral back pain . this was not related to the curve direction , since two patients had pain at the same , and two at the contralateral side of curve convexity . of these patients 57% ( 41 ) had unilateral leg pain , the others had pain in both legs . the side at which unilateral radicular leg pain occurred was unrelated to the curve direction ; 21 patients ( 51% ) had symptoms ipsilateral to the curve convexity and 20 ( 49% ) patients contralateral to the scoliotic curve ( p = 1.000 ) . seventy - two patients had symptoms of the lower extremities of which 22 had weakness , 1 hyperesthesia , 6 hypoesthesia and 11 showed neurological symptoms during physical examination . in this retrospective radiographic study we have assessed the direction of curve decompensation in a group of 88 patients with degenerative lumbar scoliosis . it was found that curve direction depended significantly on the apical level of the curve . interestingly , the majority of curves with an apex above l2 were convex to the right , whereas curves with an apex below l2 were more frequently convex to the left . the srs classification does not differentiate in this respect , since the transition from left to right - sided curves falls within the class of lumbar curves . hence , when looking at all lumbar curves combined , no predominant curve direction is found . vertebral rotation and lateral deviation of the spine are coupled phenomena , with the rotation of vertebral bodies directed into the convexity of the curve . it was previously demonstrated that in the normal non - scoliotic spine a predominant left - sided rotation exists in the high thoracic vertebrae , whereas the mid- and lower thoracic vertebrae are predominantly rotated to the right . this prevalent rotational pattern in the normal spine corresponds with the predominance of right - sided thoracic and thoracolumbar curves in idiopathic and neuromuscular scoliosis [ 19 , 38 ] . however , in idiopathic and neuromuscular scoliosis often a left - sided compensatory curve is seen at the lumbar level [ 19 , 38 ] . although in lumbar degenerative scoliosis , the scoliotic curve is essentially at the lumbar level , it apparently does show the same predominant direction as the compensatory lumbar curve in idiopathic or neuromuscular scoliosis . the strong relationship between apical level and curve direction does indicate that also in degenerative scoliosis the innate curvature of the spine plays a role in the direction of the curve . in this study we have analyzed curve directions in the coronal plane , although scoliosis is essentially a complex 3d deformation . nevertheless , a good impression can be obtained from 2d images because lateral deviation is coupled to rotational deviation [ 20 , 38 ] . in idiopathic scoliosis , it is assumed that biomechanical factors play a role in the development and progression of the curvature . it is thought that a spine with scoliosis experiences greater loading on the concave side and that this asymmetrical loading causes asymmetrical growth and progression of deformity . similar processes may play a role in degenerative scoliosis , only in this case , the greater loads at the concave side may induce degenerative changes which could result in a further progression of the scoliosis . these degenerative changes , however , can be diverse , ranging from degenerative changes in the intervertebral discs to spondylolysis or spondylolisthesis , rotatory dislocations and destruction of facet joints , depending on the weakest link . it is known that disc degeneration temporarily induces segmental instability [ 16 , 24 ] , making the spinal construct more vulnerable to forces that can increase a slight pre - existing rotatory pattern , such as dorsally directed shear loads ( ddsl s ) [ 5 , 18 ] . the fact that degenerative scoliosis occurs most often in the lumbar spine , where most vertebrae are subject to these ddsl s supports this assumption . in our patient population , we found that all patients presented with back pain and most patients experienced radicular leg pain . several possible mechanisms have been put forward to explain the causes of radicular pain in degenerative scoliosis . radicular pain may result from impingement of the nerve root due to decreased foraminal width or overstretching of the neural elements . it decreases during extension , ipsilateral lateral side bending , and ipsilateral axial rotation and increases during flexion , contralateral lateral side bending and contralateral axial rotation . based on this , in scoliosis a decreased foraminal width can be expected at the concavity of the curve . although this was corroborated in an mri study of ploumis et al . , they found that the foraminal width at the concave side was still within the normal range . they concluded that ligamentum flavum hypertrophy , posterior disc bulging , and bony overgrowth are more likely to contribute to stenosis irrespective of scoliosis . this would also explain why in our study radicular leg pain is not related to the direction of the curve . hence , for a better understanding of the symptoms related to degenerative scoliosis the underlying local deformations should be studied in more detail . one of the potential effects of severe degeneration is scoliosis . in 88 patients with degenerative lumbar scoliosis we found a significant relationship between curve direction and the apex of the curve . curves with an apex above l2 were mostly convex to the right , whereas curves with an apex below l2 were more frequently convex to the left . this indicates that in degenerative scoliosis the innate curvature or rotatory pattern of the spine is an important factor for the direction of spinal decompensation . although the normal spine does not show a pre - existing rotation at the lumbar level , the asymmetry of the whole spine is presumably directive for the decompensatory changes . this supports the concept that symptoms are the result of specific degenerative changes irrespective of the scoliotic curve .

objectivein some patients who undergo presurgical workup for drug - resistant epilepsy invasive seizure monitoring is needed to define the seizure onset zone and delineate eloquent cortex . such procedures carry risks for complications causing permanent morbidity and even mortality . in this study , prospective data on complications in a national population - based sample were analysed.designcomplication data from the prospective swedish national epilepsy surgery register were analysed for 271 patients in whom therapeutic surgery was preceded by invasive monitoring 19962010.resultscomplications occurred in 13/271 patients ( 4.8% ) . subdural grids carried the highest risk of complications ( 7.4% ) . there was no surgical mortality or permanent morbidity . subdural haematomas were most common ( n=7 ) followed by epidural haematomas ( n=3 ) . valproate treatment and having a haematoma was associated with an or of 1.53 ( ci 0.38 to 6.12 ) compared to having a haematoma without valproate treatment . having a complication during invasive monitoring was associated with a significant or of 6.27 ( ci 1.32 to 29.9 ) of also having a complication at therapeutic surgery compared to the risk of having a complication only at surgery.conclusionsin this prospective population - based epilepsy surgery series , the most common complications were haematomas , and subdural grids carried the highest risk . close supervision and rapid interventions led to avoidance of permanent morbidity . the clinical implications of the slightly increased risk of haematomas with valproate treatment needs further investigation as does the finding of an increased risk for complications at epilepsy surgery for patients who had a complication during invasive monitoring . due to the aging population , degenerative scoliosis is a growing clinical problem . it is associated with back pain and radicular symptoms . the pathogenesis of degenerative scoliosis lies in degenerative changes of the spinal structures , such as the intervertebral disc , the facet joints and the vertebrae itself . possibly muscle weakness also plays a role . however , it is not clear what exactly causes the decompensation to occur and what determines the direction of the curve . it is known that in the normal spine a pre - existing rotation exists at the thoracic level , but not at the lumbar level . in this retrospective study we have investigated if a predominant curve pattern can be found in degenerative scoliosis and whether symptoms are predominantly present at one side relative to the curve direction . the lumbar curves of 88 patients with degenerative scoliosis were analyzed and symptoms were recorded . it was found that curve direction depended significantly on the apical level of the curve . the majority of curves with an apex above l2 were convex to the right , whereas curves with an apex below l2 were more frequently convex to the left . this would indicate that also in degenerative scoliosis the innate curvature and rotational pattern of the spine plays a role in the direction of the curve . unilateral symptoms were not coupled to the curve direction . it is believed that the symptoms are related to local and more specific degenerative changes besides the scoliotic curve itself .

teneligliptin is a novel dipeptidyl peptidase4 ( dpp4 ) inhibitor belonging to the relatively novel pharmacological class of antihyperglycaemic agents that are now recommended as second or firstline agents in specific situations 1 , 2 , 3 . in a phase ii clinical trial , a 4week course of teneligliptin ( 20 mg ) monotherapy produced significant least squares ( ls ) mean reductions of 2.78 0.43 , 1.93 0.51 , and 2.08 0.42 mmol / l in 2h postprandial glucose level after breakfast , lunch and dinner , respectively , in japanese patients with type 2 diabetes ( t2 dm ) 4 . we therefore conducted the present phase iii , randomized , doubleblind , placebocontrolled study to assess the clinical efficacy and safety of teneligliptin in korean patients with t2 dm that was inadequately controlled with diet and exercise . the present study was designed to confirm the efficacy and safety of teneligliptin compared with placebo . the study was conducted in accordance with the principles of good clinical practice and was approved by the appropriate institutional review boards and regulatory agencies . participants considered eligible for the study were patients aged 18 years with t2 dm inadequately controlled [ glycated haemoglobin ( hba1c ) 7.0% and < 10.0% ] through diet and exercise for 8 weeks , who had not taken an oral antihyperglycemic agent ( oha ) for 8 weeks . exclusion criteria included current or history of significant comorbidities such as cardiovascular , hepatic or renal disease . patients with adequate compliance ( 80% ) after a 2week singleblind placebo runin period underwent baseline evaluation and were randomized according to hba1c ( using an 8.0% threshold ) into two parallel groups ( teneligliptin and placebo ; 2 : 1 matching ) for 24 weeks . ( hba1c < 7 and < 6.5% ) , changes in fasting plasma glucose ( fpg ) , and homeostatic model assessment of insulin resistance ( homair ) and cell function ( homa ) at week 24 . subjects who did not meet progressively stricter glycaemic goals immediately stopped the study without rescue therapy and underwent the evaluation planned for the final visit . these subjects were included in our analysis . threeway analysis of variance ( anova ) was used to compare the primary endpoint between the treatment groups with baseline hba1c and previous antihyperglycaemic agents as fixed effects . for other endpoints ( fpg , homa and homair ) , analysis of covariance was used with previous antihyperglycaemic agents as fixed effects and baseline hba1c as a covariate . the differences between the parallel groups were calculated as ls means standard error ( s.e . ) . responder rate ( hba1c < 6.5% , hba1c < 7.0% ) was analysed using multiple logistic regression analysis with the same factors as used in the threeway anova . of the 222 subjects screened , 142 eligible subjects were randomized to treatment as follows : 99 received teneligliptin and 43 received placebo . in total , our analysis set consisted of 141 subjects for whom baseline and postbaseline values of the primary efficacy endpoint were available ( figure s1 , file s1 ) . the treatment groups were not significantly different with respect to demographic or clinical characteristics ( table s1 , file s1 ) . the mean baseline hba1c value was 7.63% in the teneligliptin group and 7.77% in the placebo group . the changes in hba1c from baseline to week 24 in the teneligliptin and placebo groups were ls mean s.e . 0.09% and 0.03 0.12% , respectively ( p < 0.001 ; table 1 ) . a greater decrease in hba1c was observed with teneligliptin compared with placebo at week 8 , which was sustained throughout the randomized treatment period ( figure 1 ) . significantly more subjects in the teneligliptin group achieved an hba1c level of < 7% at week 24 than in the placebo group ( 69.39% in the teneligliptin group vs 20.93% in the placebo group ; p < 0.001 ; figure s2 , file s1 ) . approximately 34.69% of all subjects in the teneligliptin group achieved an hba1c level < 6.5% at week 24 , whereas only 4.65% achieved this level in the placebo group ( p = 0.0016 ; figure s2 , file s1 ) . in subgroup analysis of subjects with high or low hba1c level ( 8.0 or < 8.0% ) at baseline , the effect of teneligliptin was greater in the hba1c 8.0% group ( 1.15 0.99% , n = 25 ) than in the hba1c < 8.0% group ( 0.69 0.70% , n = 73 ; p = 0.0019 ) . effects of teneligliptin and placebo on glucose metabolism between baseline and week 24 . ci , confidence interval ; fpg , fasting plasma glucose ; hba1c , glycated haemoglobin ; homa , homeostatic model assessment of cell function ; homair , homeostatic model assessment of insulin resistance ; s.d . , standard deviation ; s.e . , * according to threeway analysis of variance ( anova ) . * * p < 0.0001 versus placebo according to threeway anova . teneligliptin decreased the fpg level from baseline to week 24 , while the fpg level did not change in the placebo group . at week 24 , the difference in change in fpg between the two groups was 1.21 mmol / l [ 95% confidence interval ( ci ) 1.72 , 0.70 ; p < 0.0001 ( table 1 ) ] . cell function , as assessed by homa , was improved in the teneligliptin group at week 24 but not in the placebo group . at week 24 , the difference in homa change between the groups was 12.23% [ 95% ci 5.78 , 18.67 ; p = 0.0003 ( table 1 ) ] . the teneligliptin group exhibited an improved homa score compared with the placebo group at week 8 and throughout the randomized treatment period ( figure s3 , file s1 ) . no significant difference in homair was observed between the two treatment groups . during this study , 81 adverse events ( aes ) occurred in 49 ( 34.5% ) of the 142 randomized subjects . among the 81 aes , 43 occurred in 29 ( 29.59% ) teneligliptintreated subjects , and 38 occurred in 20 ( 45.5% ) placebotreated subjects . the incidences of aes were not significantly different between the two groups at week 24 ( p = 0.0660 ) . hypoglycaemia occurred in one case in the placebo group but in no cases in the teneligliptin group . the results of the present study showed that 24 weeks of teneligliptin as a monotherapy was well tolerated and significantly decreased the hba1c level in korean patients with t2 dm . this is the first 24week , phase iii , doubleblind , randomized , placebocontrolled study on the efficacy and safety of teneligliptin . at present , more than five competitive reversible inhibitors are currently on the market , and each has different characteristics . a cochrane review that included 11 trials with sitagliptin and 14 trials with vildagliptin reported reductions in hba1c level of 0.7% and 0.6% , respectively , compared with placebo 5 . another dpp4 inhibitor , saxagliptin , was also shown to reduce hba1c by 0.450.63% compared with the placebo group 6 . in a recent phase ii study with 12 weeks of teneligliptin treatment ( n = 324 ) , significant ls mean reductions in hba1c for 10 , 20 and 40mg groups vs a placebo group were 0.9% ( 95% ci 1.0 , 0.7 ) , 0.9% ( 95% ci 1.1 , 0.7 ) , and 1.0% ( 95% ci 1.2 , 0.9 ) , respectively ( all p < 0.001 ) 7 . another recent study assessed the efficacy of teneligliptin when added to an ongoing metformin treatment at week 16 , and the mean reductions in hba1c and fasting plasma glucose level compared with placebo were 0.78% and 1.24 mmol / l , respectively 8 . the results of the present study in week 8 are consistent with these previous reports . specifically , the differences in changes ( baseline to week 8) in hba1c and fpg between the teneligliptin 20mg group and the placebo group were 0.80% ( 95% ci 1.02 , 0.59 ) and 0.95 mmol / l ( 95% ci 1.37 , 0.53 ) , respectively ( all p < 0.001 ) . moreover , the differences in hba1c and fpg changes between the teneligliptin 20mg group and the placebo group were maintained until week 24 [ 0.94% ( 95% ci 1.22 , 0.65 ) and 1.21 mmol / l ( 95% ci 1.72 , 0.70 ) , respectively ( all p < 0.001 ) ] , and a large proportion ( 69.39% ) of patients achieved an hba1c level < 7% compared with those using other dpp4 inhibitors ( 40% ) 9 , 10 , 11 . according to our results , teneligliptin has similar efficacy in the treatment of diabetes to other orally administered dpp4 inhibitors . cell destruction is key to the pathophysiology of t2 dm , and cell conservation delays disease progression 12 . improvements in cell function have been observed in clinical trials with the dpp4 inhibitors sitagliptin , saxagliptin , vildagliptin , alogliptin and linagliptin 13 , 14 , 15 , 16 , 17 . teneligliptin also resulted in increased homa index relative to placebo in the present study , suggesting that , like other dpp4 inhibitors , teneligliptin modestly improves pancreatic function . in the present study , the incidences of aes and adverse drug reactions were not significantly different between the teneligliptin and placebo groups at week 24 . together , these results indicate that teneligliptin is well tolerated and safe in korean patients with diabetes . in conclusion , this study showed that 20 mg of teneligliptin exhibits efficacy and safety after 24 weeks when administered once daily as a monotherapy to korean patients with t2 dm who are adhering to a diet and exercise control programme . k. a. , c. h. , b. j. , h. c. , c. w. , m.k . baseline characteristics . table s2 effects of teneligliptin and placebo on weight , and lipid profiles between baseline and week 24 . percentage of responders with hba1c < 7.0 or < 6.5% from baseline to week 24 . * p < 0.0001 vs. placebo by multiple logistic regression analysis with adjustment for hba1c ( < 8.0 or 8.0 ) and for antidiabetic medication ( prior to the study ) . * * p < 0.05 vs. placebo by multiple logistic regression analysis with adjustment for hba1c ( < 8.0 or 8.0 ) and for antidiabetic medication ( prior to the study ) . * * * p < 0.005 vs. placebo by multiple logistic regression analysis with adjustment for hba1c ( < 8.0 or 8.0 ) and for antidiabetic medication ( prior to the study ) . changes in mean homa score throughout the randomized treatment period . * by ancova . * the binding of a drug - like molecule to a protein leads to displacement of water molecules from the protein s binding pocket , and the thermodynamics of this displacement process is thought to contribute significantly to the overall thermodynamics of protein protein hydrogen bonds may incur a large energetic penalty , whereas displacement of water from hydrophobic parts of the binding pocket may help drive ligand - binding . intuitively , one may view different parts of the protein s surface as imposing different surface energies on the nearby water , with correspondingly different thermodynamic consequences for water displacement by various ligands . the use of molecular distribution functions to analyze molecular dynamics ( md ) simulations has led to important advances in the study of binding site water and its role in molecular recognition ; parallel progress with the 3d rism approach also deserves mention but is not considered here . key early contributions include development of watermap ( schrdinger llc ) , stow , and other approaches , which have provided new insight and shown promise as tools to help discover small molecules that will bind a targeted binding pocket . such methods frequently define spherical sites , where water is present at high density , to represent the distribution of water in the binding site . this hydration site approach ( hsa ) is motivated in part by the practical consideration that , in regions where water is present at lower density , it becomes more difficult to obtain converged values of the local orientational entropy of water . this is a simple consequence of the lower number of water samples available from the simulation in such low - density locations . the hsa strategy of limiting attention to hydration sites where water is present at high density maximizes the chances for good numerical convergence of the orientational entropy . however , as previously discussed , the regions in a binding site where water is present at high density can have a complex shape , which is not easily represented by a collection of spheres . this limitation has been addressed in a grid - based implementation of inhomogeneous solvation theory ( ist ) , termed gist . instead of constructing hydration sites , gist discretizes the smooth distributions of water density and other properties onto a fine , three - dimensional grid . the problem of converging the local orientational entropy of water is overcome through the use of a highly efficient nearest - neighbor ( nn ) method , as opposed to histogram methods , which require more sampling to reach adequate convergence . gist can also take advantage of the fact that regions of lower density contribute proportionately less than regions of higher density regions to the overall orientational entropy of the displaced water . this density - weighting means that , if one is interested in the integral of the orientational entropy over a volume containing both high and low density regions , one can converge the overall integral to an acceptable tolerance , so long as the high - density regions are well converged . alternatively , the grid approach makes it straightforward to focus on regions where water is present at high density , as done in hsa , without simplifying their shapes . here protein system . in order to establish a clear basis for comparing methods , we study coagulation factor xa ( fxa ) with a set of small molecule inhibitors used in early studies of the watermap method , and we derive scoring functions based on both gist and hsa methods . for this initial test of gist s applicability to protein ligand modeling , we do not seek to establish a full - fledged protein ligand scoring function , suitable for virtual screening or lead optimization . instead , as previously done , we ask how well the gist treatment of hydration can capture affinity differences between closely related congeneric pairs of ligands , where differences in binding affinity that result from contributions other than solvation such as configurational entropy and protein our results support the applicability of gist and , in addition , provide an unexpected outcome regarding the role of energetically versus entropically unfavorable water . we ran explicit - water md simulations of fxa and used both gist and our local hsa implementation to extract information about the structure and thermodynamics of the water in the binding site . we then considered the displacement of this binding site water by various fxa inhibitors , whose binding site poses are known or could be inferred from the known poses of very similar compounds . candidate scoring functions , based on the computed properties of the water displaced by each ligand , were trained on subsets of the experimental affinity data and then tested on separate sets , in order to assess the utility of the hydration data to resolve the relative binding affinities of pairs of congeneric ligands . details of the computational methods are presented in the following subsections . as previously detailed , gist uses a three - dimensional rectangular grid of cubic voxels in the region of interest and processes the snapshots of an md trajectory to compute the following thermodynamic quantities for each voxel , k , centered at location rk : ek , swnorm , the mean solute water interaction energy of a water molecule in voxel k. ek , wwnorm , one - half the mean interaction energy of the water in voxel k with all other waters . the factor of 1/2 , which was not included in the definition of the corresponding water water interaction energy in our initial presentation of gist , is customary in liquid - state theory ; it allows the total energy of neat water to be written as the sum of the individual water energies . tsk , swtrans , norm , the single - body ( one - water ) translational entropy of water in voxel k , relative to bulk , normalized to the mean number of waters in the voxel . tsk , sworient , norm , the single - body ( one - water ) orientational entropy of water in voxel k , relative to bulk , normalized to the mean number of waters in the voxel . note that we previously used a somewhat different notation for these quantities ; for example , ek , swnorm was eswnorm(rk ) . here , a 20.5 20.5 22.5 grid was centered on the active site of fxa . the grid spacing of 0.5 provides voxels large enough to give statistically meaningful data but small enough to still give a high resolution description of the density distribution functions . it is worth noting that the volume of each voxel is 33.5 times less than that of a hydration site ( see below ) , as the latter represents a sphere of radius 1 . the gist lennard - jones and electrostatic energies were computed from stored md frames , using the minimum image convention and no cutoff , and the reference value of the bulk water the main gist calculations presented here used 100 000 frames saved at 1 ps intervals during a 100 ns production md run , but shorter durations were also examined , to study convergence , as detailed below . when a ligand binds a protein , it displaces water from the protein s binding site . if the displaced water was unfavorable relative to bulk , then water displacement should make a favorable contribution to the ligand s binding affinity . with this in mind , we initially looked for a correlation between measured ligand binding affinities and regional hydration free energies ( eq 25 of ref ( 28 ) ) , where the region was defined as those voxels covered by each ligand in a bound pose . however , finding little correlation ( data not shown ) , we conjectured that any underlying correlation had been obscured by noise , due to sharp variations in the hydration energies with even small changes in ligand position . this sharpness traces , at least in part , to our use of a single pose for each ligand , and a restrained protein structure in the water simulations . we addressed this issue by constructing scoring functions which are based on the gist data but are less sensitive to the details of local water properties , due to the use of cutoffs in local water density , energy , and entropy . the use of cutoffs to construct a well - behaved scoring function from local hydration data was first introduced in the context of a hydration site model . we tested three such scoring functions based on the gist hydration data available from the grid described above . in all three cases , voxel k can contribute to a ligand s score only if the voxel s center , rk , lies within the van der waals radius of any atom of the ligand . for a given ligand , then , each voxel is assigned a binary displacement indicator , dk , which equals 1 if the center of the voxel lies within the van der waals radius of any ligand atom and 0 if it does not . the van der waals radii are drawn from the software package crystal maker ( crystalmaker software ltd . ) , which in turn relies on bondi . we also allowed the scoring function to focus on voxels where water is present at high density by setting up an additional binary indicator , gk , which is set to 1 if the water density in voxel k exceeds a cutoff gco , and 0 if it does not . this cutoff is one of the trained parameters , so it will be greater than zero only if imposing a density cutoff actually improves the accuracy of the scoring function . finally , we set up similar cutoffs for the total energy and entropy , eknorm and tsk , swnorm , associated with each voxel k , and used these to define additional binary masks based on energy and entropy thresholds . thus , the binary mask , ek , equals 1 if eknorm exceeds the cutoff , eco , and 0 otherwise ; and the binary mask , sk , equals 1 if tsk , swnorm exceeds the cutoff , sco , and 0 otherwise . like the density cutoff , gco , the values of eco and sco are fitted parameters and hence are free to go to zero if imposing these cutoffs does not improve the accuracy of the scoring function . the total energy and entropy were computed as eknorm ek , swnorm + 2ek , wwnorm 2eww , the quantity eknorm is the mean interaction energy of the water in voxel k with the protein and all other waters , relative to what its interactions would be in bulk , 2eww , bulknorm , computed in the same convention as the other gist quantities . with the voxels binary masks in place for density , energy , and entropy , we now define the three candidate scoring functions , one using both the energy and entropy data from gist , the second using only the energy data , and the third using only the entropy data:1 here , eaff and saff are additional fitted parameters , which specify the affinity increments provided by voxels surpassing the energy and entropy cutoffs , respectively , and also meeting the criteria dk = gk = 1 . note that in this study , each of these scoring functions was trained separately and has its own fitted values of gco and c , as well as eco and eaff and/or sco and saff . we adjusted the scoring functions described above to fit the measured relative binding free energies , gexpt , of 28 different congeneric pairs of fxa inhibitors ( see below ) . separate training and test sets were used , in order to avoid overfitting of the parameters . thus , we used a random number generator to split the 28 pairs into two arbitrarily selected sets of 14 apiece . ten such random splits were carried out , creating 10 distinct training and test sets . parameters were optimized for each training set and then tested on the corresponding test set . we report means and standard deviations over these 10 splits for the resulting fitted parameters and accuracy metrics . we further assess the significance of these results by comparing them with results obtained after a shuffling operation , which used the gsl_permutation function in the gnu scientific library , to randomly exchange the entropy and enthalpy values among pairs of voxels . for each training set , the parameters were adjusted as follows , using ges as an example . we scanned values of eco and sco from 0 to 4 kcal / mol and values of gco from 0 to 4 ( in units of neat water density ) , each in increments of 0.1 . this scan yields 41 41 41 = 68 921 combinations of the three cutoff values . for each combination , linear regression was then used to obtain values of eaff and saff that provide the highest correlation coefficient ( r ) of the relative scores for the congeneric pairs , ges , to the corresponding experimental values , gexpt , for the training set . the optimized values of the five fitted parameters were then used to compute ges for the congeneric pairs in the training set , and the reliability of the scoring function was evaluated based on the resulting value of r for the test set . analogous procedures were used for the other two scoring functions , ge and gs . these require scanning only 41 41 = 1681 cutoff combinations and yield only one of the two affinity parameters , eaff or saff , rather than both as for ges . hydration sites in the fxa binding site were defined and analyzed thermodynamically based on the same md simulation used for the gist calculations , using the first 10 ns ( 10 000 frames ) , in accord with the common practice of using approximately 210 ns simulations for hsa calculations . we first collected all instances , in these 1000 frames , of water molecules within 5 of any heavy atom of any bound ligand ( see below ) . for each water molecule in this set , we counted the number of neighboring waters from the same set , using the criterion of an oxygen , a water molecule can count as its own neighbor , if two instances of it in different frames meet the distance criterion . the location of the first hydration site was then set to the coordinates of the water oxygen with the most neighbors . this water molecule and all of its neighbors were then removed from consideration as potential hydration sites , and the location of the next hydration site was set to the coordinates of the remaining water oxygen with the most neighbors , based on the initial counts . this removal process was iterated until the number of neighbors of all remaining waters was less than twice that expected for a 1000 frame simulation of bulk water ( i.e. , < 280 from 1000 frames ) . each hydration site then was associated with all water instances , from the full 10 000 md frames ( above ) , whose oxygens lay within 1 of the site . each hydration site i was associated with a mean energy ei and a one - body entropy si . the energy of a water molecule in a given hydration site was calculated as half the difference between the total energy of the water a script invoking the program amber , with settings matched to those of the md simulation , was used to compute these energies . the mean energy of the hydration site then is the average of these energies for all water molecules that populate the site , minus the average energy of a water molecule in neat water from matched calculations . the water entropy si associated with hydration site i is the sum of its one - body translational and orientational entropies , si , swtrans k g(r ) ln g(r ) dr and si , sworient ( ( kni)/ ) g( ) ln g( ) d , where r is position in the protein frame of reference , k is boltzmann s constant , is bulk water density , g(r ) is the local water density referenced to bulk , ni is the number of water molecules associated with hydration site i , 8 , vhs indicates an integral restricted to the spherical hydration site , and defines orientational coordinates in the protein frame of reference . the translational entropy was computed by the histogram method , where spherical coordinates r , , centered on the hydration site were divided into uniformly spaced bins in r , cos , to generate 512 three - dimensional bins of equal volume , with r [ 0 , 1 ] , [ 0 , ] , and [ 0 , 2 ] . the orientational entropy associated with a hydration site was computed via the same nearest neighbor method used by gist for individual voxels . we used the hydration sites described above as the basis for three cutoff - based scoring functions , whose functional forms build on prior work . like the three gist scoring functions ( above ) , the free hsa - based scoring functions are based on , respectively , both energy and entropy , energy alone , and entropy alone:2 here , the sums range over hydration sites i ; ei and si equal 1 if ei and tsi are greater than cutoff values eco and sco , respectively , and 0 otherwise ; di is a displacement function , defined below , which accounts for the overlap of the ligand , in a given pose , with hydration site i ; eaff and saff are fitted constants ; and c is a constant offset . note that the hsa scoring parameters in eq 2 are set independently of the gist scoring parameters in eq 1 , despite the use of some equivalent symbols . one is identical to that used in the previous paper , while the second , as discussed below , applies a physically motivated cap to this quantity:3 here , (x ) , the heaviside step function , equals 0 if x 0 and 1 otherwise ; rco is a distance cutoff ; rij is the distance between hydration site i and atom j of the ligand being scored ; and the sum runs over all atoms belonging to the ligand being scored , i. for dinocap , which is modeled on the prior hydration site scoring function , each ligand atom within rco of the hydration site makes a contribution that scales between 0 and 1 as it approaches the center of the site , so the displacement function accounts for the degree to which a ligand displaces the water in the site . however , this approach is nonphysical in the sense that , if rij < rco for multiple ligand atoms j and a single site i , then the displacement of solvent from the site may be multiply counted . that is , for the energy / entropy scoring function , a site might contribute more than eaff + saff ; for the energy - only scoring function , it might contribute more than eaff ; and for the entropy - only scoring function , it might contribute more than saff . indeed , in the present study , we found values of dinocap up to 3.2 , implying that a single site might contribute over three times . we therefore also considered the alternative displacement function , dicap , which is capped at a value of 1 , so that no hydration site may contribute more than one - fold to a ligand s score , in accordance with the fact that the site can not be displaced more than once . the parameters of the hsa - based scoring functions were adjusted in the same manner as those of the gist - based scoring functions , except that rco took the place of gco . thus , values of rco were scanned from 2 to 3 , in steps of 0.1 and , as for gist , values of eco and sco were scanned from 0 to 4 kcal / mol in increments of 0.1 kcal / mol . ( note that no hydration sites had energies greater than 3.7 kcal / mol . ) the sums in eq 2 were evaluated for each ligand and with each combination of rco , eco , and sco . for each set of cutoffs scanned , values of eaff and saff were obtained by linear regression against the differences in measured binding free energies for a training set of congeneric ligand pairs , for each scoring function in eq 2 . the parameters that yielded the highest correlation coefficients were chosen and were tested for their ability to reproduce the difference in binding affinities for the congeneric pairs in the test set . this procedure was applied to the same 10 training and test sets used for the gist scoring function . as done for gist , we assessed the significance of the hsa results by comparing them with results obtained after a shuffling operation , which randomly exchanges the entropy and enthalpy values across pairs of hydration sites . both gist and the hsa methods take as input a boltzmann sample of water configurations for a given configuration of the protein . here , we used molecular dynamics ( md ) simulations to generate this sample from the canonical ensemble ( nvt ) , as follows . we used the structure of fxa from protein data bank entry 1fjs , as previously done , and our assignment of protonation states was also consistent with this prior study . we removed the ligand from the binding site and used tleap and other amber tools to assign protein parameters from the amber99sb force field and solvate the protein with 8557 tip3p water molecules . the simulations used a periodic box with dimensions 66.5 72.2 60.9 , which afforded at least 10 between any protein atom and the edge of the periodic box . four disulfide bonds were set up to join cysteine pairs 7/12 , 27/43 , 156/170 , and 181/209 , and two ions observed in the crystal structure ( ca and cl ) were restrained to their original positions . the resulting simulation system had 29 338 atoms , comprising the protein , the two ions , and the water molecules . energy minimization , followed by md simulation , was carried out with the amber 12 software using pmemd.cuda on a single gpu . first , the energy of the system was minimized in two rounds ; both used 1500 steps of the steepest descents algorithm followed by the conjugate gradient method for a maximum of 2000 steps . in the first round , all protein atoms were harmonically restrained to their initial positions with a force constant of 100 kcal / mol / . in the second round , the system was further relaxed keeping only non - hydrogen protein atoms restrained , with the same force constant . the energy minimized system was then heated with a series of 20 ps constant - volume and -temperature md simulations with the first simulation at 50 k and the temperature incremented by 50 k every 20 ps until 300 k was reached . the system was then equilibrated for 10 ns at 300 k at a constant pressure of 1 atm . at the final volume , the system was then equilibrated for an additional 5 ns at constant volume . the final md production run of 100 ns was at constant number of particles , volume , and temperature ( nvt ) , and system configurations were stored every 1 ps , for a total of 100 000 stored configurations . during all md simulations , all protein atoms were harmonically restrained to their positions following the energy minimization step , with a force constant of 100 kcal / mol / . the shake algorithm was used to constrain the lengths of all bonds involving hydrogen atoms . particle mesh ewald was implemented to account for long - range electrostatic interactions , and the leapfrog algorithm was used to propagate the trajectory . for the constant pressure simulations , isotropic position scaling was implemented with a pressure relaxation time of 0.5 ps . the main gist and hsa solvation maps however , in order to study the convergence properties of gist , we performed two additional 20 ns nvt production runs storing configurations every 0.05 ps ; one was begun identically with the 100 ns run , and the other was begun with the last md configuration of the 100 ns run . we trained and tested the scoring functions with a set of 28 congeneric ligand pairs ( see supporting information ) , where the members of each pair differ only by small , localized chemical changes in rigid moieties , leading to differential displacement of solvent . as previously discussed , this approach minimizes the contributions of free energy terms other than hydration and thus allows a focus on the quantity of central interest in this study . the 28 pairs used here are a subset of 31 drawn from several experimental series for use in a previous computational study : we eliminated three pairs ( matter:25/matter:28 ; matter:28/2bmg : i1h ; mueller:3/mueller:2 ) , because we were not confident of the conformation of at least one member of the pair , and hence of the location of the displaced solvent . in particular , mueller:3 differs from mueller:2 by a phenyl group whose orientation is not clear , because it is attached by a rotatable bond ; and for the other two pairs , an aromatic ring changes to a nonaromatic ring , whose conformation is uncertain . it is worth noting that , for this set of ligands , there is essentially no correlation between binding free energy and molecular weight ( r = 0.12 ) . ligand poses were drawn from available cocrystal structures or generated from a cocrystal structure of a closely related ligand by a small chemical adjustment . in all cases , the cocrystal structure was aligned with the simulated protein ( above ) to generate an initial pose in the binding site for which the hydration structure was computed . final poses for modeling solvent displacement were generated by protonating the ligands , then minimizing the initial poses in the simulated protein structure while allowing the ligand and protein hydrogen atoms to relax . the atomic partial charges for each ligand were obtained from the restrained electrostatic potential ( resp ) method , using quantum - mechanically derived electrostatic potentials with the 6 - 31 g * basis set . note that these parameters were used only to generate the ligand poses studied with the gist and hsa scoring functions . the hsa hydration sites are informative about water density but do not capture the level of detail available from gist s grid - based method . the two approaches are compared in figure 1 , which displays the hsa sites ( blue spheres ) computed for the binding pocket of factor xa , along with gist s gridded representation of water density , contoured at three different levels . contours of water density at 6 times that of bulk ( g = 6 ) appear as discrete , mostly convex droplets ( yellow contours , left panel ) , although a few of these high - density regions are elongated , rather than round . every high - density droplet is matched by a spherical hsa site , but there are many hsa sites in the binding pocket that do not enclose one of these high - density droplets . contours at 4 times bulk density ( g = 4 ) appear as more and larger droplets and match the hsa sites rather well ( gold contours , middle panel ) . contours at twice bulk density ( g = 2 ) form long , curved strands , which follow the contours of the protein surface ( orange contours , right panel ) , and are not well represented by the hsa sites . these begin to delineate the first hydration shell of the protein . finally , contours at still lower density ( e.g. , g = 1.5 ) include parts of the second hydration shell ( not shown ) . overall , the hsa representation captures the droplet - like distribution of the highest water densities but does not distinguish between high and medium density regions and does not capture the complex distributions of water density that become apparent at densities roughly twice that of bulk . this observation has practical relevance , because the scoring functions developed here include regions where water density is of this order , as detailed in the next subsection . comparison of the gist and hsa representations of water density in the factor xa binding pocket . hsa sites ( blue spheres ) are the same in all three panels . from left to right , the gist contour levels are at g = 6 , 4 , and 2 . the gist water densities are based on the occupancy of grid voxels by water oxygens , and the boundaries of the grid box may be discerned in the right - hand panel . it is also worth remarking that a hydration site should not be directly equated with a single bound water , as the sites occupancies are typically well below one . thus , for the hsa sites within 5 of the bound ligands , we find a mean occupancy of 0.58 waters molecules , with a standard deviation of 0.22 . these values are similar to those reported previously for factor xa in an early implementation of watermap : the occupancies reported in table 1 of the prior report correspond to a mean of 0.51 water molecules per hydration site , with a standard deviation of 0.22 . a gist - based scoring function based on both the local energy and one - body entropy of displaced water yields good correlations with the measured binding affinity differences for the 28 congeneric ligand pairs ( table 1 , top row ) . the mean r value is 0.94 across 10 training sets drawn at random from the full set of 28 pairs , and a high correlation ( r = 0.84 ) is preserved when the optimized parameters are applied to the respective test sets . the best results are obtained when voxels are excluded if their water density is less than about twice the bulk density ( gco = 1.9 ) . each voxel which furthermore meets the energy cutoff ( about 0.6 kcal / mol / water above bulk ) contributes about 0.2 kcal / mol of affinity . the entropy terms are somewhat puzzling : the entropy cutoff of 3.3 kcal / mol / water is much higher than the energy cutoff of 0.6 kcal / mol / water , and the positive value of saff seemingly indicates that displacing low entropy water disfavors binding , rather than favoring it , as might be anticipated . these results suggest that the energy term may be more meaningful ; this topic is further discussed below . the quality of each scoring function is reported in terms of coefficients of determination ( r ) . the test - set results ( test r ) are the most meaningful ; the training - set results ( train r ) are included for completeness . all quantities are averages across the 10 splits , with associated standard deviations . results are presented for the actual hydration data and for hydration data shuffled among voxels ( gist ) or hydration sites ( hsa ) , and hsa results are presented for capped and uncapped displacement functions ( see methods ) . scoring functions were constructed based on both energy and entropy data ( e / s ) , energy data only ( e ) , and entropy data only ( s ) ; the parameters are defined in the methods section . it is of interest to visualize the energy and entropy scoring regions ; i.e. , those voxels which meet either both the density and energy cutoffs or both the density and entropy cutoffs , respectively . as shown in figure 2 ( left ) , the energy scoring region tends to localize at extended regions of the nonpolar surface . these are places where water molecules lose more energy due to desolvation by the protein than they gain by making new interactions with the protein . ( the projection from three to two dimensions makes some scoring regions appear deceptively close to polar protein atoms . ) the analogous visualization for the energy - only scoring function , which is discussed below , is very similar ( data not shown ) . the locations of the entropy scoring regions ( figure 2 , middle ) are more complicated . some ( white arrows ) lie at the surface of polar atoms ; others ( pink arrows ) lie at hydrophobic locations . the frequent localization of entropy scoring regions at polar surfaces , and the unexpectedly positive value of saff , likely reflects the fact that polar surfaces can tightly bind waters , leading to unfavorable entropies but favorable energies ( figure 2 , right ) typical of traditional entropy enthalpy compensation . displacement of water from such regions may be net unfavorable , and this might help account for the positive value of saff . on the other hand , the displacement of entropically disfavored water from subregions where energy is not particularly favorable should favor ligand binding . thus , the energetically mixed nature of the entropic scoring regions further suggests that it may not give a clear signal in the overall scoring function . gist contours in binding site of fxa , in molecular surface representation . left : energy scoring region , which meets both density and energy cutoff criteria for the combined energy middle : entropy scoring region , which meets both density and entropy cutoff criteria for the combined energy entropy scoring function . right : normalized total water energy , contoured at 2.6 kcal / mol / water . graphic generated with vmd . in order to study the significance of the energy and entropy terms in more detail , we also considered a scoring function based only on density and energy , and another based only on density and entropy . as shown in table 1 ( second and third rows ) , the energy - only scoring function performs just as well ( r = 0.85 for the test sets ) as the original one based on both energy and entropy . in addition , the fitted parameters are similar in magnitude and sign to those of the original energy term . in contrast , the entropy - only scoring function yielded a poor correlation with experimental data ( r = 0.35 for the test sets ) , and the sign of saff is reversed relative to that in the original scoring function . thus , the hydration energy alone carries all of the predictive power of the gist - based scoring function , at least for fxa with this ansatz . this result is consistent with the analysis of the combined energy / entropy scoring function , above . as a further check of the statistical significance of the present results , we shuffled the gist data among voxels and then refitted all three gist - based scoring functions using the shuffled data . although correlations as high as r = 0.82 are obtained for the training sets , the test - set results are all poor ( r 0.4 0.2 ) . this result supports the significance of the high correlations obtained with the true ( unshuffled ) data and indicates that the low correlations observed for the entropy - only scoring function should be viewed as statistically insignificant . finally , we examined the amount of md simulation data required to generate the high correlations observed above . first , we reanalyzed the original set of md frames , which had been saved at 1 ps intervals . for increasing numbers of frames from this set , we reran the 10 training and test calculations and computed the mean and standard deviation of the resulting 10 values of r. as shown in figure 3 ( left ) , the value of r and its standard deviation ( error bars ) appear to plateau at about 60 ns for the combined energy entropy scoring function ( top left ) . interestingly , the plateau starts much earlier , at about 30 ns , for the energy - alone scoring function ( lower left ) . it appears that the more slowly convergent entropy term delays convergence of the energy entropy scoring function , so that leaving out the entropy term in the energy - only scoring function speeds convergence . this result is , again , consistent with the irrelevance of the entropy term in the gist scoring functions . we then asked whether shorter md simulations might give better convergence if frames were saved at shorter time intervals . first , we reprocessed the first 20 ns of the same simulation , now processing frames saved at 0.05 ps . as shown in figure 3 ( top middle ) , the combined energy / entropy scoring function now converges somewhat more quickly , especially as measured by the reduction in the standard deviation of r across the 10 train / test calculations . the improvement is more marked for the energy - only scoring function , as well - converged results are now available after only 10 ns of simulation time , although the standard deviation of r remains slightly higher than that from the 100 ns simulation . we then extended the 100 ns simulation by 20 ns , saving frames at 0.05 ps intervals , and examined convergence over this short simulation . the results are further improved , with good convergence and tight error bars achieved within about 5 ns for the energy - only scoring function , and 10 ns for the combined energy / entropy one . the improvement in these results , relative to those from the early 20 ns segment , suggests that the water structure continued to equilibrate somewhat during the 100 ns run , so it would have been appropriate to use a somewhat longer equilibration period in the md protocol . in summary , at least for fxa , a simulation of 10 ns or less suffices to gain all the benefit of these gist scoring functions . convergence of r values for gist scoring functions , as a function of simulation duration . bottom row : energy - only scoring function . left : 100 ns simulation , frames saved at 1 ps intervals . middle : the first 20 ns of the same 100 ns simulation , frames saved every 0.05 ps . right : 20 ns simulation initiated from the last frame of the 100 ns simulation , frames saved every 0.05 ps . as discussed in the methods section , a prior hsa scoring function was constructed in such a way that the thermodynamic contribution of each hydration could be counted multiple times , if more than one ligand atom lay within a cutoff distance . this function gave good results but is arguably nonphysical , because once the water in a hydration site has been displaced , it can not be displaced again . here , we present results for a set of similarly constructed hsa - based scoring functions in which , as before , a hydration site can be counted multiple times ; as well as a second set , in which the contribution of each hydration site is capped , so that it can only contribute once . for both the uncapped and capped models , we examine scoring functions based on energy and entropy , energy only , and entropy only , as also done for gist . with the original uncapped approach , where a hydration site can contribute multiple times , the combined energy / entropy scoring function provides high correlation coefficients for both the training ( r = 0.88 ) and test ( r = 0.80 ) sets ( table 1 , fourth row , marked e / s ) . these values of r are slightly lower than those for gist , but the difference is not statistically significant . interestingly , the fitted value of eaff ( 1.70 kcal / mol ) is much greater than that of saff , which in fact is assigned a positive sign ( 0.09 kcal / mol ) . in addition , the training procedure puts much sharper constraints on the energy terms than the entropy terms , as indicated by the fact that the standard deviations of eo and eaff ( 0.08 and 0.31 kcal / mol , respectively ) are lower than those for so and saff ( 0.50 and 0.60 kcal / mol ) . thus , the training results suggest that the entropy term is of lower importance than the energy term , much as observed in the case of gist . accordingly , a scoring function based entirely on energy ( table 1 , row 5 , marked e ) performs as well as the one with both energy and entropy , and furthermore yields values of rco , eco , and eaff very similar to those of the combined energy entropy scoring function . on the other hand , an entropy - only scoring function ( table 1 , row 6 , marked s ) also performs fairly well , with a value of r = 0.66 for the test sets , and the fitted value of saff , 1.52 kcal / mol , is essentially the same as the fitted value of eaff for the energy - only scoring function , 1.50 kcal / mol . thus , the entropy - only scoring function appears to largely replicate the energy - only scoring function , with some drop in the correlation with experimental data . these results are similar to those for gist , as in both cases , the energy - only scoring function performed as well as the energy / entropy one , while the entropy - only one is worse . however , the decline in performance on going to entropy - only is much greater for gist than for hsa . in the second variant of the hsa - based scoring functions , no hydration site can contribute more than one - fold to the difference between two ligands affinities . imposing this physically reasonable cap on the contribution of each site reduced the experimental correlation of the scoring function with experimental data to 0.66 for the test sets with the combined energy entropy scoring function but had essentially no effect on the correlations for the energy - only and entropy - only test - set results . thus , the results remain consistent with a conclusion that the energy term alone is enough to gain all the benefit of the scoring functions . the only other major change , relative to the uncapped version of the scoring function , is that the fitted values of eaff and saff changed from about 1.5 kcal / mol to about 2.3 kcal / mol , except that saff for the combined energy / entropy scoring function remained small . these increases presumably have the effect of compensating for the reduced values of the displacement function , i.e. , for the fact that dicap dinocap in eqs 2 and 3 . in order to further test the statistical significance of the hsa scoring results , we shuffled the energies and entropies among hydration sites and then refitted all six hsa - based scoring functions with the shuffled data . as summarized in the last six rows of table 1 , the uncapped scoring functions now yield poor correlations with experimental data ( r 0.4 ) , just as observed for gist , and the correlations for the capped scoring functions fall even lower . these results support the significance of the correlations obtained with the actual ( unshuffled ) data . the fact that the shuffled results are worse for the capped hsa scoring functions suggests , but does not prove , that applying the physically reasonable cap might reduce spurious correlations . it is of interest to examine the locations of hsa scoring sites relative to the gist scoring regions . we focus here on the energy - only scoring functions , since it is not clear that including entropy adds useful information , at least for fxa within the present functional form . as shown in figure 4 ( left ) , the hsa sites do not capture the complex shapes of the gist scoring regions , and substantial parts of the gist scoring region are entirely missed by the hsa scoring sites . these regions the spatial relationship of the scoring sites to the ligands studied here may be appreciated from figure 4 ( right ) , which shows the van der waals surface of a representative ligand . comparison of gist and hsa scoring locations , in the context of the fxa binding site ( gray ) . left : scoring sites from the capped hsa energy - only scoring function ( blue spheres ) and energy scoring regions from the gist energy - only scoring function ( orange contours ) . gist results are present only within the limits of the gist grid ( orange lines ) . right : a representative ligand , pdb het i d 4qc , in van der waals representation ( pink ) . ligand modeling is that the detailed representation of water structure and thermodynamics it affords works at least as well in a simple scoring function as the prior and present site - based hsa implementations . in addition , the gist results converge well within 510 ns of md simulation time , depending upon whether one uses the energy - only model or the energy / entropy model . these simulation times are commensurate with those normally used for the hsa approach ; see the methods . additional considerations include the fact that site - based approaches may require less data storage and that they paint a simple picture of water structure in a protein binding site . on the other hand , the gist grid files produced are still small ( < 1 mb ) , and we anticipate that the more detailed rendering of hydration structure and thermodynamics afforded by the grid approach will be useful for insight and prediction . note , in particular , that there are hsa sites which are only partly occupied by the more refined gist scoring regions , as well as gist scoring regions that are not identified by the hsa model , as evident from figure 4 . more generally , the fact that gist is a more direct representation of the common underlying inhomogeneous solvation theory facilitates the interpretation of its results and provides clear pathways to future enhancements , such as the incorporation of higher - order correlations , as touched on below . a fast implementation of gist , based upon focused grand canonical monte carlo sampling , may be of particular interest in the near term for high - throughput applications . another novel and striking result of the present study is that both the gist and hsa models provide clear signals that ligands can gain affinity by displacing energetically unfavorable binding site water , whereas the displacement of entropically unfavorable water seems to play a negligible role . the energetically unfavorable water highlighted by this study localizes at hydrophobic patches of the protein surface , perhaps especially in concave regions where water molecules are expected to lose hydrogen bonds . the concept that energy may outweigh entropy in cases of strong hydrophobic binding has been raised before , in both experimental and computational contexts . nonetheless , water entropy is typically thought to play a central role in hydrophobic binding . here , interestingly , neither the gist or hsa models made a compelling case that the displacement of entropically unfavorable water consistently enhances affinity . we conjecture that the lack of a clear correlation of water entropy with affinity may reflect the fact that low water entropy often results from energetically favorable water protein interactions , so that water may actually be quite stable in many locations where its entropy is low . this view is consistent with the experimental observation that the entropy of hydration of small ions is strongly negative , although the free energy is also strongly negative . it may be possible in the future to devise a more sophisticated scoring function that would account for the enthalpy entropy compensation between stabilizing energy and destabilizing entropy and focus on regions where this compensation breaks down , such as in the binding cavity of the synthetic host molecule cucurbituril . it is also worth mentioning that different protein binding sites affect water differently , so a different result might be obtained for a different protein . finally , it may be that capturing the entropic aspect of the hydrophobic effect requires accounting for water water correlations , which are absent from the one - body entropy considered here . if so , the entropy term may become more important once pairwise correlations have been incorporated into gist s entropy calculations . our observation that the displacement of high energy water plays a greater role in ligand scoring than displacement of low entropy water appears to contrast with a prior hsa - based study of the same system , where the fitted scoring function placed approximately equal weight on water energy and entropy . however , the range of fitted values for saff in our hsa models nearly spans the value of 0.66 kcal / mol for the corresponding parameter in the prior study , srwd . in addition , the prior study did not examine the uncertainty in its fitted energy and entropy parameters or evaluate a scoring function based purely on water energy . therefore , the results of these two studies should not be regarded as inconsistent . in summary , the grid - based gist method of extracting information about hydration thermodynamics from md simulations with explicit water has provided encouraging results in its first application to protein ligand binding . it thus appears to hold significant promise as a broadly applicable method of understanding the role of binding site water in protein ligand binding , and as a tool to improve the accuracy of methods for discovering high affinity targeted ligands . it is anticipated that the detailed representation of water distributions and thermodynamics which gist affords will make it particularly informative . we are currently working to develop such applications and to release an open - source implementation of gist within the ambertools software package for others to study and use .

we assessed the 24week efficacy and safety of teneligliptin , a novel dipeptidyl peptidase4 inhibitor , in korean patients with type 2 diabetes mellitus ( t2 dm ) that was inadequately controlled with diet and exercise . the present study was designed as a multicentre , randomized , doubleblind , placebocontrolled , parallelgroup , phase iii study . patients ( n = 142 ) were randomized 2 : 1 into two different treatment groups as follows : 99 received teneligliptin ( 20 mg ) and 43 received placebo . the primary endpoint was change in glycated haemoglobin ( hba1c ) level from baseline to week 24 . teneligliptin significantly reduced the hba1c level from baseline compared with placebo after 24 weeks . at week 24 , the differences between changes in hba1c and fasting plasma glucose ( fbg ) in the teneligliptin and placebo groups were 0.94% [ leastsquares ( ls ) mean 1.22 , 0.65 ] and 1.21 mmol / l ( 1.72 , 0.70 ) , respectively ( all p < 0.001 ) . the incidence of hypoglycaemia and adverse events were not significantly different between the two groups . this phase iii , randomized , placebocontrolled study provides evidence of the safety and efficacy of 24 weeks of treatment with teneligliptin as a monotherapy in korean patients with t2 dm . water molecules in the active site of an enzyme occupy a complex , heterogeneous environment , and the thermodynamic properties of active - site water are functions of position . as a consequence , it is thought that an enzyme inhibitor can gain affinity by extending into a region occupied by unfavorable water or lose affinity by displacing water from a region where it was relatively stable . recent advances in the characterization of binding - site water , based on the analysis of molecular simulations with explicit water molecules , have focused largely on simplified representations of water as occupying well - defined hydration sites . our grid - based treatment of hydration , gist , offers a more complete picture of the complex distributions of water properties , but it has not yet been applied to proteins . this first application of gist to protein ligand modeling , for the case of coagulation factor xa , shows that ligand scoring functions based on gist perform at least as well as scoring functions based on a hydration - site approach ( hsa ) , when applied to exactly the same simulation data . interestingly , the displacement of energetically unfavorable water emerges as the dominant factor in the fitted scoring functions , for both gist and hsa methods , while water entropy plays a secondary role , at least in the present context .

yeast cultures were grown in nutrient - rich ypd media ( 1% yeast extract , 2% peptone , and 2% dextrose ) at 30 on a rotary shaker unless otherwise noted . for induction of autophagy , cells were grown to mid - log phase ( a600 = 0.7 ) in ypd , collected by centrifugation ( 3000 rpm 5 min ) , washed with water , then resuspended in nitrogen starvation media ( 1.7 g / liter yeast nitrogen base without amino acids and without ammonium sulfate , plus 2% dextrose ) . for northern blot analysis of temperature - sensitive mutants , cultures were grown at 30 to an od600 of 0.7 and then incubated at 37 for 1 hr before rna extraction . spei cut atg31ses1 intergenic fragment ( pcr amplified with oligos ddo1281/-1282 , which added an artificial xhoi site ) into bluescript sk+ ( all oligos used are listed in supporting information , table s2 ) . two - step pcr mutagenesis was performed using pdd1232 as template and t7 and t3 primers with mutagenic primers ( ddo184/-1284 ; ddo183/-1285 , respectively ) to amplify the fragment containing the tdna deletion ; this fragment was then cloned into bluescript sk+ as above to create pdd1233 . direct - site - directed mutagenesis was performed on pdd1232 to create pdd1248 ( tdna b - box ) , pdd1249 ( tdna b - box mutant ) , pdd1261 ( tdna a - box mutant ) , and pdd1260 ( tdna::ecori bamhi linker ) , using ddo1391/-1392 , ddo1393/-1394 , ddo1474/-1475 , and ddo1466/-1467 primer sets , respectively . the b - box mutant had the invariant cytosine and following guanine bases changed to gc , and the a - box mutant scrambled the entire consensus . plasmids pdd1262 ( flipped orientation of the tdna ) and pdd1272 ( tdna::etc9 ) were created by cloning ecori bamhi - digested pcr - amplified fragments using ddo1468/-1469 ( flip ) , or ddo1534/-1535 ( etc9 ) , respectively , into ecori yeast genomic dna was used as pcr template . plasmid pdd1263 ( tdna::etc4 ) was constructed by directly ligating complementary oligonucleotides ( ddo1489/-1490 ) containing ecori yeast strains were generated from wild - type s. cerevisiae w303 - 1a ; genotypes of all strains used and generated in this study are given in table 1 . parent tdna::ura3 ( ddy46054607 ) strains were created by amplifying ura3 with primers ddo1279/-1280 containing homology to the flanking region of tv(uac)d , and then this dna was transformed into wild - type ddy3 followed by selection of ura+ colonies and pcr identification of homologous recombinants . linearized tdna mutant plasmids were digested with xhoi and spei , individually transformed into a tdna::ura3 strain , and 5- fluoroorotic acid ( 5-foa ) resistant colonies were isolated . yeast strains for chromatin immunoprecipitation ( chip ) were created by crossing existing brf13x - flag ( ddy1495 ) and tfc13x - flag ( ddy3860 ) strains to ddy4607 , and then flag - tagged ura+ progeny were backcrossed to each tdna mutant . all strains are isogenic to s. cerevisiae w303 - 1a except ddy5010 , which is s288c . the 9x - myc epitope tag was amplified from a trp1 marked cassette ( knop et al . atg319x - myc trp+ homologous recombinants were identified by pcr ( ddo1419/-1464 ) and confirmed by western blotting . atg31 and atg8 knockout strains were constructed by standard yeast homologous recombination . for autophagy induction alkaline phosphatase assays , pho13 pho860 strains were created by crossing a pho13::ura3pho860::his3 strain ( kindly provided by daniel klionsky ) to wild - type and tdna mutants . most northern results were verified with three ( but at least two ) independently isolated mutant strains ; table 1 lists only the specific strains shown in the figures . 5-race analysis was performed on rna isolated from the ddy420 brf1 ii.6 mutant using the first choice rlm - race kit ( ambion / life technologies , am1700 ) . individual clones were sequenced by standard sanger sequencing and mapped to the s. cerevisiae genome on the saccharomyces genome database at http://www.yeastgenome.org ( cherry et al . 2012 ) . quantitative rt pcr was performed as follows : first - strand cdna was synthesized from 0.5 g of total rna after dnase treatment ( rq1 dnase , promega m6101 ) . synthesis was extended from long transcript specific primer ddo1284 using protoscript m - mulv first - strand cdna synthesis kit ( neb e6300s ) . quantitative reverse transcription pcr ( qrt pcr ) was performed on 1:4 diluted cdna using primers ddo1606/-1555 and sybr green super mix ( bio - rad 170 - 8882 ) with 60 annealing temperature . reactions were run and analyzed using a bio - rad myiq as described ( kim et al . 2011 ) and examined by agarose gel electrophoresis to verify that only the predicted pcr products were amplified . the primers were designed to specifically amplify readthrough transcripts ; the strategy is described and illustrated in figure s2 . primers ddo1527/-1555 or ddo1576/-1577 were used to amplify desired regions surrounding tv(uac)d . for western analysis , yeast minilysates were prepared by glass bead lysis of log - phase cultures directly in lysis buffer ( 50 mm tris ph 7.5 , 1% sds , 5 mm edta , 14.3 mm -mercaptoethanol , 1 mm pmsf , 2 g / ml leupeptin and pepstatin ) . pellets from 5 ml ypd culture at a600 1.0 were resuspended in 200 l lysis buffer and then vortexed with glass beads at 4 for 10 min . lysis buffer , 100 l , was added , and the mixture was boiled for 3 min , cooled on ice , and centrifuged at 4 to remove cell debris . to 100 l of clarified minilysate , an equal amount of 2 sds page loading buffer was added , and after boiling , 15 l was loaded on 12% acrylamide protein gel . proteins were transferred to millipore immobilon membrane by semidry transfer and incubated in blotto ( 10 tbs/10% sds/5% dry milk ) for 1 hr . primary myc antibody ( c - myc 9e10 , santa cruz biotechnology ) , anti - mouse ig - horseradish peroxidase secondary antibody ( ge healthcare ) were used for western analysis . immuno - star western chemiluminescent kit ( bio - rad ) yeast strains were grown in ypd rich media to a600 = 1.0 ( 10 cells / ml ) . cells were harvested by centrifugation , washed once with distilled water , and resuspended at 10 cells / ml in media lacking nitrogen . cells were incubated for 6 days at 30 on rotatory shaker , and every other day 200 l of culture dilutions was plated on ypd plates in triplicate . plates were incubated at 30 for 48 hr and survival rate was obtained by counting resulting colonies . figure 1a depicts the atg31ses1 locus , showing the location of the tv(uac)d tdna , and the extent of transcripts normally produced from the region . to test the initial hypothesis that tv(uac)d might act as an insulator by preventing promiscuous activation of atg31 by regulatory elements associated with the strong promoter of the neighboring divergent ses1 gene , we created tv(uac)d deleted ( tdna ) mutant strains . mutation of the tv(uac)d trna gene upstream of atg31 results in readthrough of the intergenic sut467 transcript . ( b ) northern blot analysis of atg31 expression in wild - type and tdna strains reveals the extended transcript . the atg31 coding sequence probe hybridized to rna of 800 bp in wild - type strains ( black arrow ) and to rna of 1200 bp after tdna deletion ( red arrow ) . the sut467 probe hybridized to the predicted 300-bp transcript in wild - type cells ( blue arrow ) and to the same 1200-bp extended transcript in tdna strains . each pair of lanes contained total rna from independent wild - type and mutant strains . ( c ) b - box deletion ( b - box ) or mutation of the invariant cytosine in the b - box ( b - box mut ) also resulted in extended readthrough transcription . strains used were : ( b ) ddy4625 and ddy3 ( wild - type ) ; ddy4653 and 4624 ( tdna ) ; ( c ) ddy3 ( wt ) ; ddy4652 ( tdna ) ; ddy4769 ( b - box ) ; and ddy4925 ( b - box mut ) . northern blot analysis using a probe homologous to the atg31 coding sequence ( figure 1b , left ) showed not only an apparent slight increase in the level of atg31 mrna in the tdna strains ( compared to act1 controls ) , but also an increase in the length of the transcript by 400 nucleotides ( shifting from 800 bases in wild - type strains to 1200 bases ) , with apparent absence of the normal length mrna . recent tiling array and rna - seq studies have identified widespread pervasive and intergenic transcription in eukaryotic cells , and in yeast this often appears to occur as bidirectional transcription from strong promoters ( neil et al . inspection of data from these studies indicated that the sut467 intergenic transcript initiates upstream of the ses1 promoter and terminates near the tdna ( figure 1a ) ; therefore , we hypothesized that the extended atg31 transcript in the tdna strain was a readthrough sut467 transcript that interferes with the production of the normal atg31 transcript . to confirm that the extended transcript in tdna strains was due to readthrough of sut467 and not a 3 extension , we repeated the northern analysis using a probe specific for the transcribed sut467 rna sequence ( figure 1b , right ) . the results showed that this probe hybridized to rna of 300 bases in wild - type strains , consistent with previous annotations of sut467 . the tdna strains showed a longer 1200-base transcript , the same length as when using the atg31 coding - sequence probe . we concluded that in the absence of the tdna at this region , sut467 readthrough occurs and interferes with normal atg31 transcription initiation , producing only the observed extended rna . since our gross deletion of the tdna sequence removed 90 bp of chromosome iv , we confirmed this readthrough effect by creating strains that either had only the b - box sequence of the tdna deleted or contained a mutation in the invariant cytosine residue in the b - box . both of these mutations of the tdna were expected to result in loss of tfiiic binding and inhibition of pol iii complex assembly . northern blot analysis with either probe shown in figure 1c confirmed that in each of these mutant backgrounds , complete readthrough of sut467 occurred as in the tdna strains . the slightly shorter transcript observed in the tdna strain compared to the b - box and point mutant strains is also consistent with the long transcript being a readthrough from upstream of the tdna , as this reflects the 90-bp deletion . also observed were shorter rnas hybridizing to only the sut467 probe , which appear to terminate between the tdna and atg31 . our previous studies of heterochromatin barrier and insulator function of tdnas have shown that assembled tfiiic alone can block the spread of silencing from the hmr locus and can insulate a uas from a promoter ( simms et al . 2008 ) . to determine which components of the pol iii complex are required to block sut467 progression , we analyzed atg31 transcripts in various temperature - sensitive strains compromised for pol iii complex function and formation when pulsed at the nonpermissive temperature before rna extraction . temperature - sensitive mutations in rna polymerase iii subunit genes rpc31 and rpc160 affect transcription initiation and elongation , respectively ( dieci et al . 1995 ; thuillier et al . 1995 ) . these mutants had relatively little effect on the ability of the tdna to block progression of sut467 ( lanes 2 and 3 compared to wild type in lane 1 ) as evidenced by a minimal alteration of the ratio of normal to extended transcripts . in contrast , mutations in the tfiiib subunit encoding brf1 gene ( brf1-ii.6 and -ii.9 ) that impair interactions of brf1p with tbp ( andrau et al . 1999 ) showed a major shift to the longer extended transcript ( lanes 4 and 5 ) , with relatively little normal length rna . interestingly , this mutant also showed an intermediate length atg31 transcript that initiates just upstream of the tdna coding sequence ( see 5-race analysis below ) . ( a ) northern analysis of temperature - sensitive mutants of the pol iii complex was performed as in figure 1 , except that each culture was shifted from 30 to 37 for 1 hr prior to rna extraction . extended atg31 transcripts are most prominent in tfiiib and tfiiic subunit mutants , which also express an intermediate length atg31 transcript . strains used in lanes 18 were ddy3 ( wt ) ; ddy232 ( rpc31 - 236 ) ; ddy246 ( rpc162112 ) ; ddy416 ( brf1 ii.9 ) ; ddy420 ( brf1 ii.6 ) ; ddy261 ( tfc3 g349e ) ; ddy3 ( wt ) ; and ddy4300 ( tfc6 promoter mutant ) . 5-race was performed to map transcriptional start sites ( tss ) for the various transcripts observed in the northern blot analysis of the brf1 ii.6 mutant . colored solid boxes represent the range of alternative tss , which were observed in three distinct clusters . the exact saccharomyces genome database coordinates of all mapped tss are given in figure s1 . rna isolated from the temperature - sensitive , dna - binding defective tfc3 g349e mutant strain ( lefebvre et al . 1994 ) showed apparent equal amounts of both normal and long atg31 transcripts , with a small relative amount of the intermediate transcript ( figure 2a , lane 6 ) . a strain harboring a mutation in the tfc6 promoter that results in reduced expression of tfc6p and slow growth ( kleinschmidt et al . 2011 ) showed a similar pattern ( lane 8) , shifted a bit more to the long and intermediate transcripts . these results demonstrate that loss of tfiiic function also results in readthrough sut467 transcription , but this could be due to loss of tfiiib assembly in the absence of full tfiiic activity . to verify that the transcription start site of our readthrough transcript initiates in the region of the annotated sut467 transcriptional start site ( tss ) and to map the tss of the observed intermediate transcript , we performed 5-race analysis on rna isolated from the brf1-ii.6 mutant , because it contains all three transcripts as detected by northern blotting . as shown schematically in figure 2b , 5-race ends that correspond to the annotated atg31 mrna , and within a 94-nucleotide range that overlaps the annotated sut467 tss , were mapped . the intermediate transcript was found to begin very close to the beginning of the trna coding sequence . the exact saccharomyces genome database chromosome iv coordinates corresponding to each individually mapped 5-race end are listed in figure s1 . to further assess the mechanistic requirements of each pol iii transcription factor in preventing pol ii readthrough transcription , we constructed yeast strains specifically modified at the atg31 upstream tdna locus and analyzed the long vs. short rna phenotypes . inverting the orientation of the tdna had no effect , as no extended atg31 mrna was detected ( figure 3a , lane 1 ) . mutation of the a - box within the tdna or replacement of the tdna with the etc4 site resulted in only the extended transcript being produced ( figure 3a , lanes 2 and 3 ) . each of these replacements was expected to bind tfiiic , but not be able to efficiently recruit tfiiib or pol iii . interestingly , replacing the tdna with the tdna remnant upstream of the tim21 gene , recently referred to as etc9 ( nagarajavel et al . 2013 ) , was sufficient to block readthrough transcription ( figure 3a , lane 4 ) . this tdna remnant has previously been shown to bind both tfiiic and tfiiib , but not the pol iii enzymatic complex ( guffanti et al . strains were constructed to recruit the entire pol iii complex , tfiiib and tfiiic , or tfiiic alone to the atg31ses1 intergenic region . each construct was tested for the ability to block readthrough and for binding of pol iii transcription factor complexes to the ectopic locations . ( a ) schematic of the modified atg31 loci and northern blot of each strain using the atg31 probe . lane 1 , ddy4816 ( tdna flip ) ; lane 2 , ddy4817 ( a - box mut ) ; lane 3 , ddy4819 ( etc4 replacement ) ; lane 4 , ddy4970 ( etc9 replacement ) ; and lane 5 , ddy4925 ( b - box mut ) . replacement of the tdna by etc4 , or mutating the a - box or b - box , resulted in the presence of the extended transcript ( red labels ) . however , inversion of the tdna sequence or replacement with the etc9 sequence still blocked readthrough ( black labels ) . ( b and c ) confirmation of expected pol iii transcription factor binding in the above mutants by chromatin immunoprecipitation . each tdna mutant strain was crossed to strains containing either brf13x - flag or tfc13x - flag alleles , and then subjected to chip analysis using anti - flag antibody . ( b ) the absence of tfiiib upstream of atg31 in the a - box mutant , etc4 replacement , and b - box mutant correlates with the presence of the extended transcript , suggesting that tfiiib binding is required to block readthrough . strains used ( left to right ) were ddy4935 , -4943 , -4949 , -5003 , and -4946 . ( c ) chip analysis of brf13xflag and tfc13xflag strains demonstrates that tfiiic but not tfiiib is bound in etc4 replacement strains , indicating that tfiiic binding alone can not block readthrough transcription . strains used ( left to right ) were ddy4938 , -5003 , -4949 , -3860 , -5006 , and -4917 . these results suggest that recruitment of tfiiib is the critical step that prevents readthrough transcription of sut467 , as binding of tfiiic alone at the etc4 site is not sufficient to block pol ii progression . mutation of the a - box has been demonstrated to impair tfiiib assembly ( huibregtse and engelke 1989 ) , and these interpretations assume that each of these sequences used to replace the tdna have the same in vivo binding characteristics at the atg31 locus as they do in their native chromosomal locations . to confirm such assumptions regarding the presence or absence of each transcription factor complex at these sequences when moved to the atg31 locus , we crossed a 3x - flag - epitope - tagged brf1 allele into each of these mutants . chip results using anti - flag antibody shown in figure 3b demonstrate that the tdna flip and etc9 alleles are strongly enriched for tfiiib at levels comparable to wild - type tdnas , while insertions unable to block readthrough transcription ( a - box mut , etc4 , and b - box mut ) had significantly reduced tfiiib chip signals , comparable to background signals observed in the no antibody control panels . primers amplifying a separate control tdna on chromosome iii showed similar enrichment in each of the samples , indicating that equivalent amounts of chip dna were added to each pcr reaction . we also created strains containing a 3x - flag - epitope - tagged tfc1 allele to assess the binding of tfiiic at modified atg31 loci . the results in figure 3c show that tfiiic but not tfiiib is associated with the etc4 insertion , and both tfiiic and tfiiib are bound at the etc9 insertion and at the wild - type tdna locus . importantly , and contrary to results seen in our earlier tdna heterochromatin blocking studies ( simms et al . 2008 ) , tfiiic binding alone to etc4 is not sufficient to block cryptic transcript readthrough . previous studies on the heterochromatin barrier activity of tdnas revealed the involvement of other chromatin - associated proteins in this extratranscriptional function ( donze et al . 2007 ) . to assess the potential role of these tdna associated proteins in blocking readthrough transcription , we performed northern blot analysis ( using the atg31 coding sequence probe ) on rna isolated from a number of these mutants . the results in figure 4a showed that each of these mutants contain mostly normal - length atg31 transcripts ; however , low levels of readthrough are apparent in some strains , most obvious in nhp6 ( lane 3 ) and smc3 ( lane 12 ) mutants in the particular blot shown . however , the intensity of these signals was relatively weak and was often difficult to consistently distinguish from background in different blots . genetic factors involved in tdna chromatin boundary function have minimal effects on blocking of pol ii progression through tv(uac)d . ( a ) rna from strains containing mutations that weaken tdna boundary function were analyzed by northern blotting using the atg31 probe . strains in lanes 19 were ddy3 , -947 , -1376 , -2236 , -2058 , -2509 , -1631 , -1676 , and -5010 ; lane 10 , sg154.2 ; lanes 1113 , roy1032 , -1060 , and -1063 ; lane 14 , ddy4925 . ( b ) rt pcr analysis of readthrough transcription also shows only minimal effects . to confirm these apparent low levels of readthrough , we used readthrough transcript - specific primers to develop an rt pcr assay to measure differences in the relative levels of the long transcript compared to a wild - type strain . the inset in figure 4b shows an inverted ethidium - stained gel image that verifies that the primers specifically amplified the readthrough cdna , as the b - box mutant strain showed significantly higher levels of rt pcr product than the wild - type strain . there also appears to be a low level of readthrough in the wild - type strain , which is consistent with a genome - wide transcriptome analysis that identified a single readthrough clone overlapping this locus ( miura et al . quantitative rt pcr was performed on the same rna samples shown in the northern blot in figure 4a , and those that showed a significant increase in the long atg31 transcript relative to the wild - type parent are shown in figure 4b . the b - box mutant strain measured 80-fold more readthrough transcript than wild type in this assay , while other mutants were confirmed to have modest ( ranging from 2- to 15-fold ) yet detectable increased levels of the long transcript as suggested by the northern analysis . given the extended 5-utr present on the long atg31 transcript , we next asked to what extent translation of the atg31 protein was affected by readthrough sut467 transcription . we created atg319x - myc epitope - tagged strains in wild - type and tdna mutant backgrounds and then analyzed atg31 protein expression by western blotting . in each strain producing the long transcript , we observed a drastic reduction in atg31p levels ( figure 5a ) . autophagy is a conserved cellular response that recycles cellular components upon nutrient limitation and during normal regulated molecular turnover and involves the formation of autophagosome vesicles that capture and degrade macromolecules after fusion with other membrane bound vesicles ( reggiori and klionsky 2013 ; stanley et al . ( a ) western blot analysis was performed on wild - type and tdna mutants containing an atg319x - myc tag allele to determine whether the extended transcript affects translation of atg31p . no other bands were observed on the western blot , demonstrating that the extended transcript does not lead to production of an extended polypeptide . strains used were ddy5012 ( wt ) , ddy5014 ( tdna ) , ddy5018 ( b - box ) , and ddy5020 ( b - box mut ) . ( b ) inhibition of autophagy induction in yeast expressing the extended transcript as measured by the pho860 alkaline phosphatase assay . strains used were : ddy5051 ( wt control ) , ddy5072 ( tdna ) , ddy5078 ( b - box ) , ddy5081 ( b - box mut ) , ddy5044 ( atg8 ) , and ddy5046 ( atg31 ) . ( c ) production of the extended transcript is associated with reduced survival under nitrogen starvation conditions . strains used were : ddy5012 ( wt ) , ddy5081 ( b - box mut ) , and ddy4764 ( atg31 ) . since atg31p is required for the formation of autophagosomes upon nitrogen starvation ( kabeya et al . 2007 ) , we tested the efficiency of this response using two well - characterized assays to measure autophagy induction . we first created a series of strains that produce the readthrough transcript and contain the pho860 and pho13 alleles . these mutations reduce background alkaline phosphatase levels , and the phosphatase activity of the precursor pho860 protein can be activated only if it is proteolytically processed during autophagy ( klionsky 2007 ) . since atg31 is required for these events , we reasoned that the reduction in atg31p levels due to the extended 5-utr would result in reduced processing of pho860p upon induction of autophagy and therefore reduced levels of alkaline phosphatase activity upon shifting cells to nitrogen starvation conditions . figure 5b shows this to be the case , as strains producing the readthrough transcript showed significantly reduced induction of phosphatase activity compared to a pho13 pho860 strain producing only normal atg31 mrna ( wt in figure 5b ) . complete deletion of atg31 or atg8 in control strains severely reduced starvation - induced phosphatase activity as expected . these results confirm that induction of autophagy is compromised in strains that predominately produce the long atg31 transcript . as a second assay for the efficiency of autophagy induction , we tested the viability of yeast cells producing the long transcript when placed under autophagy - inducing conditions . previous studies have shown that complete deletion of atg31 results in reduced survival of cells undergoing nitrogen starvation due to inhibition of autophagy ( kabeya et al . when we tested a strain containing the b - box mutation in the tdna ( producing the long transcript ) , we found an intermediate level of survival compared with wild - type and atg31 strains ( figure 5c ) . this loss of survival is not due to effects on the neighboring ses1 gene , as wild - type and tdna mutant strains show equivalent expression levels of ses1 when analyzed by northern blotting ( figure s3 ) . this result suggests that readthrough of the cryptic transcript reduces atg31p translation to a level that compromises fitness of the cells during nitrogen starvation . figure 1a depicts the atg31ses1 locus , showing the location of the tv(uac)d tdna , and the extent of transcripts normally produced from the region . to test the initial hypothesis that tv(uac)d might act as an insulator by preventing promiscuous activation of atg31 by regulatory elements associated with the strong promoter of the neighboring divergent ses1 gene , we created tv(uac)d deleted ( tdna ) mutant strains . mutation of the tv(uac)d trna gene upstream of atg31 results in readthrough of the intergenic sut467 transcript . ( b ) northern blot analysis of atg31 expression in wild - type and tdna strains reveals the extended transcript . the atg31 coding sequence probe hybridized to rna of 800 bp in wild - type strains ( black arrow ) and to rna of 1200 bp after tdna deletion ( red arrow ) . the sut467 probe hybridized to the predicted 300-bp transcript in wild - type cells ( blue arrow ) and to the same 1200-bp extended transcript in tdna strains . each pair of lanes contained total rna from independent wild - type and mutant strains . ( c ) b - box deletion ( b - box ) or mutation of the invariant cytosine in the b - box ( b - box mut ) also resulted in extended readthrough transcription . strains used were : ( b ) ddy4625 and ddy3 ( wild - type ) ; ddy4653 and 4624 ( tdna ) ; ( c ) ddy3 ( wt ) ; ddy4652 ( tdna ) ; ddy4769 ( b - box ) ; and ddy4925 ( b - box mut ) . northern blot analysis using a probe homologous to the atg31 coding sequence ( figure 1b , left ) showed not only an apparent slight increase in the level of atg31 mrna in the tdna strains ( compared to act1 controls ) , but also an increase in the length of the transcript by 400 nucleotides ( shifting from 800 bases in wild - type strains to 1200 bases ) , with apparent absence of the normal length mrna . recent tiling array and rna - seq studies have identified widespread pervasive and intergenic transcription in eukaryotic cells , and in yeast this often appears to occur as bidirectional transcription from strong promoters ( neil et al . inspection of data from these studies indicated that the sut467 intergenic transcript initiates upstream of the ses1 promoter and terminates near the tdna ( figure 1a ) ; therefore , we hypothesized that the extended atg31 transcript in the tdna strain was a readthrough sut467 transcript that interferes with the production of the normal atg31 transcript . to confirm that the extended transcript in tdna strains was due to readthrough of sut467 and not a 3 extension , we repeated the northern analysis using a probe specific for the transcribed sut467 rna sequence ( figure 1b , right ) . the results showed that this probe hybridized to rna of 300 bases in wild - type strains , consistent with previous annotations of sut467 . the tdna strains showed a longer 1200-base transcript , the same length as when using the atg31 coding - sequence probe . we concluded that in the absence of the tdna at this region , sut467 readthrough occurs and interferes with normal atg31 transcription initiation , producing only the observed extended rna . since our gross deletion of the tdna sequence removed 90 bp of chromosome iv , we confirmed this readthrough effect by creating strains that either had only the b - box sequence of the tdna deleted or contained a mutation in the invariant cytosine residue in the b - box . both of these mutations of the tdna were expected to result in loss of tfiiic binding and inhibition of pol iii complex assembly . northern blot analysis with either probe shown in figure 1c confirmed that in each of these mutant backgrounds , complete readthrough of sut467 occurred as in the tdna strains . the slightly shorter transcript observed in the tdna strain compared to the b - box and point mutant strains is also consistent with the long transcript being a readthrough from upstream of the tdna , as this reflects the 90-bp deletion . also observed were shorter rnas hybridizing to only the sut467 probe , which appear to terminate between the tdna and atg31 . our previous studies of heterochromatin barrier and insulator function of tdnas have shown that assembled tfiiic alone can block the spread of silencing from the hmr locus and can insulate a uas from a promoter ( simms et al . 2008 ) . to determine which components of the pol iii complex are required to block sut467 progression , we analyzed atg31 transcripts in various temperature - sensitive strains compromised for pol iii complex function and formation when pulsed at the nonpermissive temperature before rna extraction temperature - sensitive mutations in rna polymerase iii subunit genes rpc31 and rpc160 affect transcription initiation and elongation , respectively ( dieci et al . 1995 ; thuillier et al . 1995 ) . these mutants had relatively little effect on the ability of the tdna to block progression of sut467 ( lanes 2 and 3 compared to wild type in lane 1 ) as evidenced by a minimal alteration of the ratio of normal to extended transcripts . in contrast , mutations in the tfiiib subunit encoding brf1 gene ( brf1-ii.6 and -ii.9 ) that impair interactions of brf1p with tbp ( andrau et al . 1999 ) showed a major shift to the longer extended transcript ( lanes 4 and 5 ) , with relatively little normal length rna . interestingly , this mutant also showed an intermediate length atg31 transcript that initiates just upstream of the tdna coding sequence ( see 5-race analysis below ) . ( a ) northern analysis of temperature - sensitive mutants of the pol iii complex was performed as in figure 1 , except that each culture was shifted from 30 to 37 for 1 hr prior to rna extraction . extended atg31 transcripts are most prominent in tfiiib and tfiiic subunit mutants , which also express an intermediate length atg31 transcript . strains used in lanes 18 were ddy3 ( wt ) ; ddy232 ( rpc31 - 236 ) ; ddy246 ( rpc162112 ) ; ddy416 ( brf1 ii.9 ) ; ddy420 ( brf1 ii.6 ) ; ddy261 ( tfc3 g349e ) ; ddy3 ( wt ) ; and ddy4300 ( tfc6 promoter mutant ) . 5-race was performed to map transcriptional start sites ( tss ) for the various transcripts observed in the northern blot analysis of the brf1 ii.6 mutant . colored solid boxes represent the range of alternative tss , which were observed in three distinct clusters . the exact saccharomyces genome database coordinates of all mapped tss are given in figure s1 . rna isolated from the temperature - sensitive , dna - binding defective tfc3 g349e mutant strain ( lefebvre et al . 1994 ) showed apparent equal amounts of both normal and long atg31 transcripts , with a small relative amount of the intermediate transcript ( figure 2a , lane 6 ) . a strain harboring a mutation in the tfc6 promoter that results in reduced expression of tfc6p and slow growth ( kleinschmidt et al . 2011 ) showed a similar pattern ( lane 8) , shifted a bit more to the long and intermediate transcripts . these results demonstrate that loss of tfiiic function also results in readthrough sut467 transcription , but this could be due to loss of tfiiib assembly in the absence of full tfiiic activity . to verify that the transcription start site of our readthrough transcript initiates in the region of the annotated sut467 transcriptional start site ( tss ) and to map the tss of the observed intermediate transcript , we performed 5-race analysis on rna isolated from the brf1-ii.6 mutant , because it contains all three transcripts as detected by northern blotting . as shown schematically in figure 2b , 5-race ends that correspond to the annotated atg31 mrna , and within a 94-nucleotide range that overlaps the annotated sut467 tss , were mapped . the intermediate transcript was found to begin very close to the beginning of the trna coding sequence . the exact saccharomyces genome database chromosome iv coordinates corresponding to each individually mapped 5-race end are listed in figure s1 . to further assess the mechanistic requirements of each pol iii transcription factor in preventing pol ii readthrough transcription , we constructed yeast strains specifically modified at the atg31 upstream tdna locus and analyzed the long vs. short rna phenotypes . inverting the orientation of the tdna had no effect , as no extended atg31 mrna was detected ( figure 3a , lane 1 ) . mutation of the a - box within the tdna or replacement of the tdna with the etc4 site resulted in only the extended transcript being produced ( figure 3a , lanes 2 and 3 ) . each of these replacements was expected to bind tfiiic , but not be able to efficiently recruit tfiiib or pol iii . interestingly , replacing the tdna with the tdna remnant upstream of the tim21 gene , recently referred to as etc9 ( nagarajavel et al . 2013 ) , was sufficient to block readthrough transcription ( figure 3a , lane 4 ) . this tdna remnant has previously been shown to bind both tfiiic and tfiiib , but not the pol iii enzymatic complex ( guffanti et al . 2006 ) . strains were constructed to recruit the entire pol iii complex , tfiiib and tfiiic , or tfiiic alone to the atg31ses1 intergenic region . each construct was tested for the ability to block readthrough and for binding of pol iii transcription factor complexes to the ectopic locations . ( a ) schematic of the modified atg31 loci and northern blot of each strain using the atg31 probe . lane 1 , ddy4816 ( tdna flip ) ; lane 2 , ddy4817 ( a - box mut ) ; lane 3 , ddy4819 ( etc4 replacement ) ; lane 4 , ddy4970 ( etc9 replacement ) ; and lane 5 , ddy4925 ( b - box mut ) . replacement of the tdna by etc4 , or mutating the a - box or b - box , resulted in the presence of the extended transcript ( red labels ) . however , inversion of the tdna sequence or replacement with the etc9 sequence still blocked readthrough ( black labels ) . ( b and c ) confirmation of expected pol iii transcription factor binding in the above mutants by chromatin immunoprecipitation . each tdna mutant strain was crossed to strains containing either brf13x - flag or tfc13x - flag alleles , and then subjected to chip analysis using anti - flag antibody . ( b ) the absence of tfiiib upstream of atg31 in the a - box mutant , etc4 replacement , and b - box mutant correlates with the presence of the extended transcript , suggesting that tfiiib binding is required to block readthrough . strains used ( left to right ) were ddy4935 , -4943 , -4949 , -5003 , and -4946 . ( c ) chip analysis of brf13xflag and tfc13xflag strains demonstrates that tfiiic but not tfiiib is bound in etc4 replacement strains , indicating that tfiiic binding alone can not block readthrough transcription . strains used ( left to right ) were ddy4938 , -5003 , -4949 , -3860 , -5006 , and -4917 . these results suggest that recruitment of tfiiib is the critical step that prevents readthrough transcription of sut467 , as binding of tfiiic alone at the etc4 site is not sufficient to block pol ii progression . mutation of the a - box has been demonstrated to impair tfiiib assembly ( huibregtse and engelke 1989 ) , and this mutation also allows readthrough . these interpretations assume that each of these sequences used to replace the tdna have the same in vivo binding characteristics at the atg31 locus as they do in their native chromosomal locations . to confirm such assumptions regarding the presence or absence of each transcription factor complex at these sequences when moved to the atg31 locus , we crossed a 3x - flag - epitope - tagged brf1 allele into each of these mutants . chip results using anti - flag antibody shown in figure 3b demonstrate that the tdna flip and etc9 alleles are strongly enriched for tfiiib at levels comparable to wild - type tdnas , while insertions unable to block readthrough transcription ( a - box mut , etc4 , and b - box mut ) had significantly reduced tfiiib chip signals , comparable to background signals observed in the no antibody control panels . primers amplifying a separate control tdna on chromosome iii showed similar enrichment in each of the samples , indicating that equivalent amounts of chip dna were added to each pcr reaction . we also created strains containing a 3x - flag - epitope - tagged tfc1 allele to assess the binding of tfiiic at modified atg31 loci . the results in figure 3c show that tfiiic but not tfiiib is associated with the etc4 insertion , and both tfiiic and tfiiib are bound at the etc9 insertion and at the wild - type tdna locus . importantly , and contrary to results seen in our earlier tdna heterochromatin blocking studies ( simms et al . 2008 ) , tfiiic binding alone to etc4 is not sufficient to block cryptic transcript readthrough . previous studies on the heterochromatin barrier activity of tdnas revealed the involvement of other chromatin - associated proteins in this extratranscriptional function ( donze et al . 2007 ) . to assess the potential role of these tdna associated proteins in blocking readthrough transcription , we performed northern blot analysis ( using the atg31 coding sequence probe ) on rna isolated from a number of these mutants . the results in figure 4a showed that each of these mutants contain mostly normal - length atg31 transcripts ; however , low levels of readthrough are apparent in some strains , most obvious in nhp6 ( lane 3 ) and smc3 ( lane 12 ) mutants in the particular blot shown . however , the intensity of these signals was relatively weak and was often difficult to consistently distinguish from background in different blots . genetic factors involved in tdna chromatin boundary function have minimal effects on blocking of pol ii progression through tv(uac)d . ( a ) rna from strains containing mutations that weaken tdna boundary function were analyzed by northern blotting using the atg31 probe . strains in lanes 19 were ddy3 , -947 , -1376 , -2236 , -2058 , -2509 , -1631 , -1676 , and -5010 ; lane 10 , sg154.2 ; lanes 1113 , roy1032 , -1060 , and -1063 ; lane 14 , ddy4925 . ( b ) rt pcr analysis of readthrough transcription also shows only minimal effects . to confirm these apparent low levels of readthrough , we used readthrough transcript - specific primers to develop an rt pcr assay to measure differences in the relative levels of the long transcript compared to a wild - type strain . the inset in figure 4b shows an inverted ethidium - stained gel image that verifies that the primers specifically amplified the readthrough cdna , as the b - box mutant strain showed significantly higher levels of rt pcr product than the wild - type strain . there also appears to be a low level of readthrough in the wild - type strain , which is consistent with a genome - wide transcriptome analysis that identified a single readthrough clone overlapping this locus ( miura et al . quantitative rt pcr was performed on the same rna samples shown in the northern blot in figure 4a , and those that showed a significant increase in the long atg31 transcript relative to the wild - type parent are shown in figure 4b . the b - box mutant strain measured 80-fold more readthrough transcript than wild type in this assay , while other mutants were confirmed to have modest ( ranging from 2- to 15-fold ) yet detectable increased levels of the long transcript as suggested by the northern analysis . given the extended 5-utr present on the long atg31 transcript , we next asked to what extent translation of the atg31 protein was affected by readthrough sut467 transcription . we created atg319x - myc epitope - tagged strains in wild - type and tdna mutant backgrounds and then analyzed atg31 protein expression by western blotting . in each strain producing the long transcript , we observed a drastic reduction in atg31p levels ( figure 5a ) . autophagy is a conserved cellular response that recycles cellular components upon nutrient limitation and during normal regulated molecular turnover and involves the formation of autophagosome vesicles that capture and degrade macromolecules after fusion with other membrane bound vesicles ( reggiori and klionsky 2013 ; stanley et al . ( a ) western blot analysis was performed on wild - type and tdna mutants containing an atg319x - myc tag allele to determine whether the extended transcript affects translation of atg31p . no other bands were observed on the western blot , demonstrating that the extended transcript does not lead to production of an extended polypeptide . strains used were ddy5012 ( wt ) , ddy5014 ( tdna ) , ddy5018 ( b - box ) , and ddy5020 ( b - box mut ) . ( b ) inhibition of autophagy induction in yeast expressing the extended transcript as measured by the pho860 alkaline phosphatase assay . strains used were : ddy5051 ( wt control ) , ddy5072 ( tdna ) , ddy5078 ( b - box ) , ddy5081 ( b - box mut ) , ddy5044 ( atg8 ) , and ddy5046 ( atg31 ) . ( c ) production of the extended transcript is associated with reduced survival under nitrogen starvation conditions . strains used were : ddy5012 ( wt ) , ddy5081 ( b - box mut ) , and ddy4764 ( atg31 ) . since atg31p is required for the formation of autophagosomes upon nitrogen starvation ( kabeya et al . 2007 ) , we tested the efficiency of this response using two well - characterized assays to measure autophagy induction . we first created a series of strains that produce the readthrough transcript and contain the pho860 and pho13 alleles . these mutations reduce background alkaline phosphatase levels , and the phosphatase activity of the precursor pho860 protein can be activated only if it is proteolytically processed during autophagy ( klionsky 2007 ) . since atg31 is required for these events , we reasoned that the reduction in atg31p levels due to the extended 5-utr would result in reduced processing of pho860p upon induction of autophagy and therefore reduced levels of alkaline phosphatase activity upon shifting cells to nitrogen starvation conditions . figure 5b shows this to be the case , as strains producing the readthrough transcript showed significantly reduced induction of phosphatase activity compared to a pho13 pho860 strain producing only normal atg31 mrna ( wt in figure 5b ) . complete deletion of atg31 or atg8 in control strains severely reduced starvation - induced phosphatase activity as expected . these results confirm that induction of autophagy is compromised in strains that predominately produce the long atg31 transcript . as a second assay for the efficiency of autophagy induction , we tested the viability of yeast cells producing the long transcript when placed under autophagy - inducing conditions . previous studies have shown that complete deletion of atg31 results in reduced survival of cells undergoing nitrogen starvation due to inhibition of autophagy ( kabeya et al . when we tested a strain containing the b - box mutation in the tdna ( producing the long transcript ) , we found an intermediate level of survival compared with wild - type and atg31 strains ( figure 5c ) . this loss of survival is not due to effects on the neighboring ses1 gene , as wild - type and tdna mutant strains show equivalent expression levels of ses1 when analyzed by northern blotting ( figure s3 ) . this result suggests that readthrough of the cryptic transcript reduces atg31p translation to a level that compromises fitness of the cells during nitrogen starvation . the results described in this study demonstrate that stable rna polymerase iii transcription factor complexes containing tfiiib assembled at tdnas have the capacity to block the progression of intergenic transcription by rna polymerase ii . high - throughput microarray and sequencing technologies have led to the identification of much more diversity in transcriptomes , from prokaryotes to humans , than was previously appreciated ( core et al . such pervasive transcripts include the cryptic unstable transcripts ( cuts ) , stable unannotated transcripts ( suts ) , xrn1-sensitive unstable transcripts ( xuts ) , and meiotic unannotated transcripts ( muts ) ( wyers et al . additionally , alterations in the prevalence of intergenic transcripts and transcript start and end sites have been observed under different growth and stress conditions ( xu et al . since the vast majority of these cryptic transcripts and transcript isoforms have unknown functions , it has been speculated that they may represent inherent sloppiness of the transcriptional process , referred to as transcriptional noise ( struhl 2007 ) . where functions of such pervasive transcription have been identified in s. cerevisiae , it appears that it is not necessarily the rna produced but the act of transcription itself that leads to the observed function . the short noncoding srg1 transcript inhibits ser3 expression by transcriptional interference and promoter occlusion mechanisms , as the path of the srg1 transcript overlaps transcription factor binding sites within the ser3 promoter ( martens et al . , the srg1 transcript terminates near the beginning of ser3 , while mutation of the tdna at sut467 results in uncontrolled readthrough all the way to the end of atg31 . similar cis - linked mechanisms may be at work at other yeast loci where noncoding transcription appears to block initiation or elongation of adh1 ( bird et al . 2006 ) , imd2 ( kuehner and brow 2008 ) , ura2 ( thiebaut et al . 2008 ) , flo11 ( bumgarner et al . 2009 ) , pho84 ( camblong et al . 2007 ) , and ime4 ( hongay et al . 2006 ; gelfand et al . there is evidence that there are also trans - effects of the noncoding rna product regulating the pho84 locus ( camblong et al . additionally , full repression of yeast gal genes ( houseley et al . 2008 ) , ime1 ( van werven et al . 2012 ) , and again pho84 ( camblong et al . 2007 ) requires chromatin modifications associated with ongoing noncoding transcription . while more instances are likely yet to be identified , this handful of yeast genes has incorporated intergenic transcription into their regulatory programs and generally appears to use it as a means of repression . however , we show in this study that if left unchecked , progression of noncoding transcription can have negative consequences on neighboring gene expression , resulting in reduced fitness of cells . this result demonstrating a cryptic transcript - blocking activity of bound pol iii complexes can be added to the list of extratranscriptional effects of the rna polymerase iii system . our results presented here demonstrate that the tdna upstream of atg31 protects against such repressive transcriptional interference effects . our data are consistent with a model in which tfiiib , as part of the pol iii complex associated with the tv(uac)d tdna , serves as a physical impediment to elongating rna pol ii initiating at the sut467 transcriptional start site . in the absence of tfiiib , nearly complete readthrough by pol ii occurs to produce an extended sut467atg31 rna transcript . this transcript is not efficiently , if at all , translated into atg31 protein , as scanning ribosomes ( kozak 2005 ) attaching at the 5 end of the extended transcript would encounter start and stop codons before reaching the atg31 start codon . this readthrough transcript appears to be both capped and polyadenylated , as the 5-race protocol includes a phosphatase treatment before decapping and adaptor ligation , ensuring that only capped 5 ends are mapped , and the long transcript is enriched in northern analysis of poly(a)-purified rna ( a. korde , unpublished data ) . the small amount of atg31p we detect in our western blots likely results from low levels of normal atg31 transcripts that are undetectable in northern blots from cells grown in rich media . extracts from nitrogen - starved tdna mutants show a slight increase in protein levels , along with detectable normal atg31 transcripts in northern blots of rna isolated from the same cultures ( a. korde , unpublished data ) . this suggests that under conditions that induce atg31 , limited normal initiation is slightly enhanced , but protein levels are still lower than in wild - type cells . previous work from our lab and others has shown that certain extratranscriptional effects associated with tdnas can be mediated by binding of the tfiiic complex alone . propagation of silencing at the hmr mating locus can be blocked by replacing the tdna downstream of the hmr - i silencer with an etc site , and insertion of an etc site between uasg and gal10 insulates the promoter from gal4p activation ( simms et al . heterochromatin boundary activity of tfiiic - only containing complexes is also observed in schizosaccharomyces pombe ( noma et al . additionally , the etc6 site within the tfc6 promoter may modulate transcription by an insulator - like mechanism ( kleinschmidt et al . in this case of preventing readthrough of intergenic transcription , the binding of tfiiic alone is clearly not sufficient . while tfiiic binds to b - box sequences in vitro with extremely high affinity ( lefebvre et al . 2003 ) , this binding is somehow tempered by passage of the pol iii enzymatic complex during transcription of the internal control element regions . on the other hand , after recruitment of tfiiib by tfiiic , the tightly bound tfiiib complex appears to be fixed , as in vitro experiments have shown that tfiiib dna complexes are resistant to high salt and heparin treatments ( kassavetis et al . 1990 ; kassavetis et al . 1995 ) . the fully assembled tfiiib complex also is thought to be kinetically trapped ( cloutier et al . 2001 ) , with a half - life on the order of a full yeast cell cycle , and fully assembled tfiiib likely persists at tdnas until regulated release during mitosis or stationary phase ( fairley et al . 2003 ; roberts et al . such characteristics of tfiiib are consistent with our results that suggest that formation of this complex is the major impediment to cryptic transcript readthrough by sut467 . these results are also compatible with earlier in vitro studies that demonstrated the ability of pol iii to transcribe through assembled tfiiic but not assembled tfiiib ( bardeleben et al . our results suggest that tfiiic yields to pol ii in a similar manner as it does to pol iii , since replacing the tdna with etc4 allowed readthrough of pol ii even though chromatin immunoprecipitation analysis revealed that tfiiic was bound to the ectopic etc4 site ( figure 3c ) . while we have not mapped the exact 3 end of the sut467 transcript , the annotated end mapped by tiling array analysis ( xu et al . 2009 ) places it within 20 bp of the 5 extent of the expected tfiiib footprint at this tdna ( schematically depicted in figure s1 ) . estimation of this 5 end of the tfiiib footprint is based on earlier in vitro footprinting studies ( kassavetis et al . 1989 ) and a recent global bootprinting analysis of in vivo bound pol iii transcription factors ( nagarajavel et al . the location of the 3 end of sut467 is consistent with tfiiib being a transcriptional roadblock that is resistant to displacement by transcribing pol ii . a curious sidelight to this study is the appearance of the intermediate length transcript in tfiiib and tfiiic mutants , but not in tdna mutants . this is most likely initiated by pol ii , as in brf1 , tfc3 , and tfc6 mutants , the tdna terminator sequence is still present , so it is unlikely that this is a pol iii transcript . we speculate that in these mutants , tfiiib binding still occurs , but is unstable , and perhaps dissociation of the brf1p and bdp1p subunits occurs before loss of tbp at the site . such a lingering tbp might then recruit factors necessary to then subsequently recruit pol ii immediately upstream of the tdna . alternatively , the pol iii complex may mask a cryptic pol ii promoter , which is revealed in a subset of cells containing mutations in the pol iii transcription factors . there is mounting evidence that a much larger fraction of genomes is transcribed than was previously appreciated . while rna degradation pathways generally keep most of these transcripts at low levels ( wolin et al . 2012 ) , it has become clear that the act of intergenic transcription can have significant effects on neighboring genes . due to such observations , one must consider how mutation of a specific genomic locus may affect expression of nearby genes in addition to the targeted gene when assigning the actual cause of observed phenotypes ( wei et al . iii extratranscriptional effects , we are conducting rna - seq analysis of wild - type vs. tfc6p underexpressing mutant strains . previous studies have been conducted to determine the global effects of pol iii deficiencies ( conesa et al . 2005 ) , but the rna was analyzed by coding sequence microarray , which could not detect effects involving intergenic transcription . inspection of preliminary rna - seq results suggests that when the pol iii complex is globally compromised , several tdna proximal genes may be affected as described here , and in other possibly unique ways ( q. wang , a. korde , and c. nowak , unpublished results ) . this type of result also raises the question of how to interpret phenotypes due to mutations that may globally affect intergenic transcription ( which may be relevant in mutants of other dna and chromatin binding proteins ) , or as shown here for mutation at a specific locus , as unchecked cryptic transcription can lead to unexpected and even detrimental misexpression of downstream genes . while a subset of pervasive transcription products themselves may be noise neglected bilateral anterior shoulder dislocation is a very rare condition , often related to seizures or major trauma . open reduction is recommended whenever hill - sachs lesion is > 25% of the joint and the dislocation is elder than 3 weeks . we describe a case report of a 28-year - old man left handed jehovah s witness laborer assessed 12 weeks after bilateral anterior shoulder dislocation . the patient was evaluated with clinical examination , and it was observed an asymptomatic intrarotation of both shoulders with a mild left circumflex nerve deficit . he was able to perform flexion and abduction of both arms up to 60 and 10 of extrarotation . pre - operative constant scores were 49 in left and 55 in right shoulder , pre - operative disabilities of the arm , shoulder , and hand ( dash ) scores were 57 in left and 53 in right shoulder , and visual analogue scales ( vas ) was 2 . the surgeon treated both shoulder ( not simultaneously ) by open reduction and bristow - latarjet coracoids transfer procedure . a 1 year after operations , left flexion was 180 while right was 160 , bilateral abduction was 180 . he was able to return to his pre - injury activities , the constant score was 89 left and 83 right , dash score was 17 left and 13 right and vas was 0 . atraumatic bilateral neglected anterior shoulder dislocation can be treated with open bristow - latarjet procedure to provide a stable glenohumeral joint in laborer patient and permit a return to the pre - injury activities , to create a greater extension of the glenoid arc and to avoid future dislocation . bilateral anterior shoulder dislocation is rare , because almost always one extremity takes the brunt of the impact during trauma incidence . usually happen almost always secondary to major trauma ( 50% ) , affect mainly young men ( 70% ) and it is associated with other injuries ( greater tuberosity fractures in 15% , rotator cuff tears , neurological associated lesions ) . muscle contractions due to neurological or psychiatric spasm or electrocution were identified like alternative cause . neglected bilateral anterior shoulder dislocation is very rare condition , often related to seizures or major trauma . for anterior dislocation , open reconstructive options include infraspinatus tendon transfer , disimpaction grafting or allograft repair , bankart repair and shoulder replacement . the goal of surgical treatment is repair of articular defects and maintenance of shoulder stability to allow an early rehabilitation . a 28-year - old male jehovah s witness laborer , affected by iron - deficiency anemia , was evaluated in orthopedic office for restricted mobility and pain ( during daily activities ) in both shoulders from about 12 weeks . he reported that previously these inabilities were interpreted as paraspinal cervical muscle contracture and treated only with muscle relaxant drugs . at the time of evaluation , patient showed mild pain and inabilities in shoulders external rotation and abduction . physical examination revealed : bilateral normal internal rotation ; bilateral 10 external rotation , abduction and flexion about 60 and axillary nerve numbness . anteriorposterior x - ray images evidenced bilateral anterior shoulder dislocation and computer tomography - scan showed bilateral hill - sachs lesion ( < 25% ) ( fig . 1 ) . the surgeon suggested a step - by - step surgical treatment conscious about patient s medical history and refusal of whatever blood transfusion for religious reasons . the first step was surgical treatment of dominant limb and then right side after 1 month . surgical procedures were both performed under general anesthesia , beach chair position and fluoroscan aid . the deltopectoral approach was used and for both cases were evidenced a glenoid fossa deficiency with hypoplasia of scapular neck and glenoid . bleeding was controlled and intraoperative blood salvage was used to accommodate wishes of the patient to refuse homologous and autologous transfusions , as required his confession of faith . through post - operatively management , shoulders were supported by a sling with 15 of abduction and maintained for 3 weeks . a 4 weeks later , clinical and radiographic evaluation were performed , and the patient was introduced to passive shoulder motion , while 2 weeks later he started an active controlled shoulder motion . he was followed for next 6 months by upper extremities therapist team . the 1 year after last operations , the patient was clinical and radiographical evaluated and he was able to perform left flexion up to 180 and 160 in right , bilateral abduction was 180 ( fig . the constant score was 89 left and 83 right , disabilities of the arm , shoulder and hand ( dash ) score was 17 left and 13 right and visual analogue scales were 0 . anterioposterior and axillary x - ray images evidenced bilateral anterior shoulder dislocation and computer tomography - scan showed bilateral hill - sachs lesion . clinical evaluation and x - ray images 1-year as expressed by other authors , this type of lesion can be present in 4% of all shoulder dislocation , even if the most of cases were related to medical conditions including myasthenia gravis , cerebral palsy and scapular myopathy . neglected anterior shoulder dislocation is an exceptional condition whose treatment has not guidelines mutually agreed . in our opinion , treatment of this type of lesion depends on many factors : time of dislocation , age of patient , functional limitation , success of reduction , presence and amount of the hill - sachs lesion and glenoid fossa defect and surgeon s experience . conservative treatment should be considered in patient without shoulder pain or low functional demands and in case of surgical or anesthesiological contraindications . however , in young , active , high demands patients , conservative choice without stabilization should be not the proper procedure due to possible concurrent hill - sachs lesion , to possible concurrent glenoid defect and resulting high risk of instability . solomon et al . suggested closed reduction only up to 6 weeks post - injury due to high risk of iatrogenic fracture or neurovascular damage . open reduction is the recommended procedure with hill - sachs lesion more than 25% of articular surface and after 3 weeks . for anterior dislocation , open reconstructive options include infraspinatus tendon transfer , disimpaction grafting or allograft repair , bankart repair and shoulder replacement . the goal of surgical treatment is repair of articular defects and maintenance of shoulder stability to allow an early rehabilitation . many surgical solutions were proposed like eden - hybinette , arthroscopical bristow - latarjet procedure but recently , longo et al . showed how eden - hybinette procedure has clinical outcomes very similar to the bristow - latarjet with a higher rate of post - operative osteoarthritis and recurrence . authors treated the young patient with an open bristow - latarjet procedure primarily to repair the old dislocation and remedy the glenoid fossa defect . hill - sachs lesions were not treated during surgery because involved < 25% and intraoperatively glenohumeral joint was stable after reduction . author s opinion is to refer surgical treatment of hill - sachs lesion in case of lesion involving over 25% of articular surface to maintain glenohumeral stability by open remplissage with the infraspinatus tendon ( in this technique the head defect was filled with the infraspinatus tendon , by tying the sutures from the anchor , so bringing the tendon down to the defect ) or shoulder arthroplasty if the lesion is over 50% of humeral head . currently , however , literature reports only few studies about results of operative treatment of chronic anterior shoulder dislocation with arthroplasty and this type of surgery should be considered in selected cases : elderly patients ; intraoperatively instability due to severe humeral head cortical depression and/or glenoid defect and degenerative arthropathy . gavriilidis recommended this surgical procedure with large head defects and experienced good mid - term results with benefits for a range of motion , pain and patient satisfaction . this case report showed that anterior shoulder dislocation could also occur without a trauma mechanism due to glenoid fossa dysplasia . despite ethical and religious positions of patient about blood transfusions , open bristow - latarjet procedure may be performed with recovery of good upper extremity function ( constant score and dash score ) , low of recurrence and good clinical outcomes . atraumatic bilateral neglected anterior shoulder dislocation can be treated with an open bristow - latarjet procedure to provide a stable glenohumeral joint in laborer patient . this open surgical procedure is reliable , secure and burdened with fewer complications over time , especially even in special cases ( jehovah s witness ) to create a greater extension of the glenoid arc and to avoid future dislocation . though very rare , atraumatic bilateral neglected anterior shoulder dislocation does occur and may present to orthopedic evaluation .

the major function of eukaryotic rna polymerase iii is to transcribe transfer rna , 5s ribosomal rna , and other small non - protein - coding rna molecules . assembly of the rna polymerase iii complex on chromosomal dna requires the sequential binding of transcription factor complexes tfiiic and tfiiib . recent evidence has suggested that in addition to producing rna transcripts , chromatin - assembled rna polymerase iii complexes may mediate additional nuclear functions that include chromatin boundary , nucleosome phasing , and general genome organization activities . this study provides evidence of another such extratranscriptional activity of assembled rna polymerase iii complexes , which is the ability to block progression of intergenic rna polymerase ii transcription . we demonstrate that the rna polymerase iii complex bound to the trna gene upstream of the saccharomyces cerevisiae atg31 gene protects the atg31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic sut467 transcription unit . this protection is predominately mediated by binding of the tfiiib complex . when tfiiib binding to this trna gene is weakened , an extended sut467atg31 readthrough transcript is produced , resulting in compromised atg31 translation . since the atg31 gene product is required for autophagy , strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy - inducing nitrogen starvation conditions . given the recent discovery of widespread pervasive transcription in all forms of life , protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled rna polymerase iii complexes and possibly other dna binding proteins . introduction : neglected bilateral anterior shoulder dislocation is a very rare condition , often related to seizures or major trauma . open reduction is recommended whenever hill - sachs lesion is > 25% of the joint and the dislocation is elder than 3 weeks.case report : we describe a case report of a 28-year - old man left handed jehovah s witness laborer assessed 12 weeks after bilateral anterior shoulder dislocation . the patient was evaluated with clinical examination , and it was observed an asymptomatic intrarotation of both shoulders with a mild left circumflex nerve deficit . he was able to perform flexion and abduction of both arms up to 60 and 10 of extrarotation . pre - operative constant scores were 49 in left and 55 in right shoulder , pre - operative disabilities of the arm , shoulder , and hand ( dash ) scores were 57 in left and 53 in right shoulder , and visual analogue scales ( vas ) was 2 . radiological examination were bilateral anteroposterior shoulder x - rays and computer tomography scan . the surgeon treated both shoulder ( not simultaneously ) by open reduction and bristow - latarjet coracoids transfer procedure . a 1 year after operations , left flexion was 180 while right was 160 , bilateral abduction was 180 . he was able to return to his pre - injury activities , the constant score was 89 left and 83 right , dash score was 17 left and 13 right and vas was 0.conclusion:atraumatic bilateral neglected anterior shoulder dislocation can be treated with open bristow - latarjet procedure to provide a stable glenohumeral joint in laborer patient and permit a return to the pre - injury activities , to create a greater extension of the glenoid arc and to avoid future dislocation .

atrophic gastritis ( ag ) is a histopathologic entity characterized by chronic inflammation of the gastric mucosa with loss of gastric glandular cells and replacement by intestinal - type epithelium , pyloric - type glands , and fibrous tissue . atrophy of the gastric mucosa is the endpoint of chronic processes , such as chronic gastritis associated with helicobacter pylori ( h. pylori ) infection , other unidentified environmental factors , and autoimmunity directed against gastric glandular cells . pepsinogen ( pg ) is precursors of pepsin , and consists of two biochemically and immunologically distinct types , namely , pg i and pg ii . serum pg levels are related to gastritis , gastric mucosal lesion , with a particular relationship to ag . decreased serum pg i levels and the pg i / ii ratio can be used to assess gastric atrophy . miki et al . reported that the pg i / ii ratio of more than 3 has a sensitivity of 93.3% and specificity of 87.7% for the diagnosis of normal fundic gland mucosa . h. pylori infection is the most common cause of ag , and at least 50% of the world s population is infected with h. pylori . h. pylori infection can lead to variety of upper gastrointestinal disorders , including peptic ulcer disease , gastric mucosa - associated lymphoid tissue lymphoma , and gastric cancer . in addition , h. pylori infection has been linked to several extra - gastric disorders , such as atherosclerosis and coronary artery disease ( cad ) . some studies have shown a positive association between h. pylori infection and cad , while others have shown no significant association . recently , two studies have suggested that ag irrespective of h. pylori status was associated with atherosclerosis in general population . to the best of our knowledge , however , no previous reports have investigated the effects of ag on the prevalence of cad in general population . therefore , we investigated the relationship between ag , which is diagnosed based on serum pg levels , and cad in general japanese individuals . the human dry dock , is one of the most popular medical services in japan , for the purpose of the medical health checkup promoting public health through early detection of chronic diseases and their risk factors . a standard human dry dock features anamnesis and a survey of lifestyle , a physical examination , serum and urine examination , a chest x - ray , abdominal ultrasonography , and other tests . the fee is paid by participants or supported ( fully or partially ) by their employers or medical insurers . this study was designed to cross - sectionally evaluate the relationship between ag and cad in japanese population . the study included 1,758 male and 875 female subjects , aged 2190 years , who attended the human dry dock of oike clinic in kyoto , japan in 2009 . medical history and lifestyle factors were obtained from a self - administered questionnaire completed by all the subjects , which included medication use , family history of diseases , and habits of smoking and drinking . in addition , all participants underwent physical examinations , routine biochemical screening tests obtained by venipuncture after an overnight fast . all participants gave their informed consent , and the study was approved by the ethics committee of oike clinic . body mass index ( bmi ) was calculated as body weight in kilogram divided by the square of the participant s height in meters . systolic blood pressure and diastolic blood pressure were measured in the right upper arm of participants in a sedentary position using an automatic oscillometric blood pressure recorder . hypertension was defined as systolic blood pressure / diastolic blood pressure 140/90 mmhg or pharmacological treatment for hypertension . dyslipidemia was defined as total cholesterol 5.7 mmol / l , triglyceride 1.7 mmol / l , or pharmacological treatment for hyperlipidemia . hyperuricemia was defined as serum uric acid 416.3 mol / l , or pharmacological treatment for hyperuricemia . atrophic gastritis was defined as pg i 70 ng / ml and pg i / ii ratio 3.0 . coronary artery disease was defined as a previous myocardial infarction based on the clinical history or electrocardiogram . triglyceride value was presented as median ( interquartile range ) due to skewed distribution , and other continuous variables were presented as mean standard deviation ( sd ) . unpaired student s t tests , mann - whitney s u test , or test were conducted as appropriate to assess statistical significance of differences between groups , using stat view software ( ver . 5.0 ; sas institute , cary , nc ) . multiple logistic regression analysis was performed to assess the combined influence of variables on the prevalence of cad . to examine the effects of various factors on the prevalence of cad , the following factors were considered as independent variables : ag , age , sex , obesity , hypertension , diabetes mellitus , hyperlipidemia , hyperuricemia , and habits of smoking and drinking . the human dry dock , is one of the most popular medical services in japan , for the purpose of the medical health checkup promoting public health through early detection of chronic diseases and their risk factors . a standard human dry dock features anamnesis and a survey of lifestyle , a physical examination , serum and urine examination , a chest x - ray , abdominal ultrasonography , and other tests . the fee is paid by participants or supported ( fully or partially ) by their employers or medical insurers . this study was designed to cross - sectionally evaluate the relationship between ag and cad in japanese population . the study included 1,758 male and 875 female subjects , aged 2190 years , who attended the human dry dock of oike clinic in kyoto , japan in 2009 . medical history and lifestyle factors were obtained from a self - administered questionnaire completed by all the subjects , which included medication use , family history of diseases , and habits of smoking and drinking . in addition , all participants underwent physical examinations , routine biochemical screening tests obtained by venipuncture after an overnight fast . all participants gave their informed consent , and the study was approved by the ethics committee of oike clinic . body mass index ( bmi ) was calculated as body weight in kilogram divided by the square of the participant s height in meters . systolic blood pressure and diastolic blood pressure were measured in the right upper arm of participants in a sedentary position using an automatic oscillometric blood pressure recorder . hypertension was defined as systolic blood pressure / diastolic blood pressure 140/90 mmhg or pharmacological treatment for hypertension . diabetes mellitus was defined as fasting blood glucose 7.0 mmol / l or pharmacological treatment for diabetes mellitus . dyslipidemia was defined as total cholesterol 5.7 mmol / l , triglyceride 1.7 mmol / l , or pharmacological treatment for hyperlipidemia . hyperuricemia was defined as serum uric acid 416.3 mol / l , or pharmacological treatment for hyperuricemia . atrophic gastritis was defined as pg i 70 ng / ml and pg i / ii ratio 3.0 . coronary artery disease was defined as a previous myocardial infarction based on the clinical history or electrocardiogram . triglyceride value was presented as median ( interquartile range ) due to skewed distribution , and other continuous variables were presented as mean standard deviation ( sd ) . unpaired student s t tests , mann - whitney s u test , or test were conducted as appropriate to assess statistical significance of differences between groups , using stat view software ( ver . multiple logistic regression analysis was performed to assess the combined influence of variables on the prevalence of cad . to examine the effects of various factors on the prevalence of cad , the following factors were considered as independent variables : ag , age , sex , obesity , hypertension , diabetes mellitus , hyperlipidemia , hyperuricemia , and habits of smoking and drinking . characteristics of the 2,633 subjects enrolled in this study are shown in table 1 . among 2,633 study subjects , 531 subjects ( 20.2% ) mean age was significantly higher in the ag - positive group than that in the ag - negative group ( 64.5 9.4 vs 56.2 11.6 years , p<0.0001 ) . serum uric acid concentration was lower in ag - positive group than that in ag - negative group ( 327.1 83.3 vs 345.0 83.3 mol / l , p<0.0001 ) . proportions of drinker were significantly fewer in the ag - positive group than those in the ag - negative group ( 36.0% vs 40.9% , p<0.0001 ) . the prevalence of hyperuricemia was significantly lower in the ag - positive group than that in the ag - negative group ( 19.2% vs 25.0% , p = 0.0051 ) . the prevalence of cad was significantly higher in the ag - positive group than that in the ag - negative group ( 5.8% vs 2.8% , p = 0.0005 ) ( table 2 ) . multiple logistic regression analysis demonstrated that age ( odds ratio [ or ] , 1.07 ; 95% confidence interval [ ci ] , 1.041.09 ) , hypertension ( or , 2.75 ; 95% ci , 1.674.53 ) , habits of smoking ( or , 2.53 ; 95% ci , 1.544.15 ) , and ag ( or , 1.67 ; 95% ci , 1.032.72 ) were significantly associated with cad ( table 3 ) . we have shown that ag determined by serum pg levels is significantly associated with cad . this significant association remained unchanged even after adjustment for age , sex , obesity , hypertension , diabetes mellitus , dyslipidemia , hyperuricemis , and habits of smoking and drinking . atrophic gastritis represents the end stage of chronic inflammation of gastric mucosa , and h. pylori infection of the stomach is by far the most common cause of ag . h. pylori infection can eventually lead to loss of the normal gastric mucosal architecture , with destruction of gastric glands and replacement by fibrosis and intestinal - type epithelium . this process of ag and intestinal metaplasia are involved in the induction of an inflammatory response characterized by an influx of neutrophils , mononuclear cells , and t - helper 1 cells , typically aimed at clearing intracellular infections . however , h. pylori is not an intracellular pathogen , and thus the t - helper 1 response results in epithelial cell damage rather than in the removal of h. pylori . the ongoing infection with h. pylori thus causes a lifelong proinflammatory response coupled to cellular damage and initiates the cancer cascade . the underlying hypothetical mechanisms include chronic low - grade activation of the coagulation cascade and accelerating atherosclerosis due to the vascular endothelial damage resembling the gastric epithelial damage through the induction of inflammatory response . on the other hand , several studies demonstrated that h. pylori infection was associated with atherogenic lipid profiles including increased serum triglyceride and total cholesterol concentrations , and decreased hdl cholesterol concentrations . however , some studies have shown no significant association between h. pylori infection and cad . this issue is still controversial , thus further studies are needed to confirm the relationship between h. pylori infection and cad . torisu et al . reported that pulse wave velocity , which is an early preclinical marker of atherosclerosis , was significantly higher in healthy subjects with ag diagnosed by serum pg test method than in those without ag . they reported that serum ghrelin levels which might be protective against atherosclerosis , were significantly lower in ag - positive subjects than in ag - negative subjects . reported that ag diagnosed by histologic method might cause hyperhomocysteinemia , which is an independent risk factor for atherosclerosis , in general population . in their report , carotid intima - media thickness in subjects with ag was found to be thicker than that in controls , although it did not reach statistically significant levels . those two reports have suggested that ag irrespective of h. pylori status was associated with atherosclerosis . our study based on a large number of subjects suggested that ag was an independent risk factor for cad . taken together these findings , it seems plausible that ag influences the development of atherosclerotic changes in coronary arteries . second , it was not clear which extent of ag was related to h. pylori infection , because the serum immunoglobulin g antibody to h. pylori was not measured . subjects with autoimmune gastritis and h. pylori - induced gastritis were included in ag - positive group . however , the prevalence of autoimmune gastritis is low in japan , therefore the relationship between ag and cad could be regarded as the relationship between h. pylori - induced ag and cad . third , we did not measure proinflammatory cytokines such as c - reactive protein , tumor necrosis factor - alpha , and interleukin-6 , although they might not be markedly increased in the end stage of chronic inflammation . finally , we did not measure homocysteine , ghrelin , and others , each of which has been reported to be related to atherosclerosis . in conclusion reduction of health inequalities , including those between social groups and geographical areas , has been a major health policy goal in the united states ( us ) for the past 4 decades.[1 - 5 ] cardiovascular diseases ( cvd ) , including heart disease and stroke , have been the number one cause of death in the united states for the past eight decades , and contribute greatly to overall health inequalities for the nation . while cvd mortality rates are widely reported by age , sex , and race / ethnicity , geographical disparities in cvd mortality are mostly limited to reporting differences by rural - urban or state of residence.[7 - 9 ] analyses of geographical disparities in cvd mortality over time , especially by region or county of residence , and their socioeconomic and behavioral determinants are less common , although a few recent us studies have examined county - level variations in cvd mortality as a function of area - based deprivation or socioeconomic characteristics.[5,10 - 14 ] although us data have identified higher rates of cvd morbidity and mortality in several southern states and the southeastern region , research on whether the magnitude and patterns of geographical disparities in cvd mortality rates at various levels of geography ( such as region , state , and county ) have changed over time is either limited or lacking.[5,12 - 15 ] while national - level analyses are important in understanding overall social - group disparities in cvd , it is crucial to know from a policy standpoint as to how specific regions , states , or geographical areas are performing in reducing their cvd mortality rates and associated risk factors relative to each other or nation as a whole . in the us , states and local communities such as counties are generally responsible for development and implementation of public policies to tackle public health problems , for collecting social , environmental , and health data , and for providing a broad range of social and health services to their residents . documenting disparities between geographical areas with the lowest and highest cvd rates moreover , a spatial - temporal analysis should help identify geographical areas or regions which not only have high rates of cvd mortality but have also experienced slower mortality reductions , indicating the need for urgent action for cvd prevention and control . the aim of our study is to examine changes in the extent of geographical disparities in cvd mortality among 9 census regions , 50 states and the district of columbia , and 3,141 counties of the united states between 1969 and 2011 . using small - area national vital statistics mortality and census data , we model variations in county - level cvd mortality rates as a function of area deprivation , urbanization , racial / ethnic composition , smoking , obesity , physical inactivity , diabetes , and health care access . additionally , we use the national longitudinal mortality study ( nlms ) to model regional and state - level disparities in cvd mortality risks after adjusting for individual - level socioeconomic and demographic characteristics . to analyze geographical disparities in cvd mortality over time , we used the national vital statistics mortality database , which has been the cornerstone of health and disease monitoring among sociodemograhic groups and geographical areas in the us for over a century.[3 - 9,17 ] the national mortality database is based on information from death certificates of every death occurring in the united states each year . while the national mortality database provides the number of deaths ( numerator data ) by year , age , sex , race , geographic area , and cause of death , the corresponding population statistics developed by the us census bureau serve as the denominator for computing mortality rates.[6 - 9,17,18 ] the mainland united states consists of 50 states and the district of columbia , which are grouped into 9 census regions as shown in figure 1 . states are divided into counties , and the number of counties varies by state . in all , there are 3,143 counties in the united states . in our study , cvd mortality rates were computed annually for all 9 regions between 1969 and 2011 . for smaller geographical areas such as states and counties , mortality trends are presented for three time periods due to data availability and space constraints . state - specific cvd mortality rates were computed for 1969 , 1990 , and 2011 . cvd mortality rates were computed for 3,141 counties for the time periods : 1969 - 1974 , 1990 - 1999 , and 2003 - 2007 . mortality rates for all geographic areas were age - adjusted by the direct method using the age - composition of the 2000 us population as the standard.[4 - 9 ] trends in cardiovascular disease ( cvd ) mortality by geographic region , united states , 1969 - 2011 new england = maine + new hampshire + vermont + massachusetts + rhode island + connecticut middle atlantic = new york + new jersey + pennsylvania east north central = ohio + indiana + illinois + michigan + wisconsin west north central = minnesota + iowa + missouri + north dakota + south dakota + nebraska + kansas south atlantic = delaware + maryland + district of columbia + virginia + west virginia + north carolina + south carolina + georgia + florida east south central = kentucky + tennessee + alabama + mississippi west south central = arkansas + louisiana + oklahoma + texas mountain = montana + idaho + wyoming + colorado + new mexico + arizona + utah + nevada pacific = washington + oregon + california + alaska + hawaii log - linear regression models were used to estimate annual rates of decrease in cvd mortality for each census region . specifically , the logarithm of region - specific mortality rates were modeled as a linear function of time ( calendar year ) , which yielded annual exponential rates of change in mortality rates . in order to summarize state- and county - level disparities in mortality , we used various disparity measures such as the coefficient of variation ( cv ) , interquartile range , quintile and percentile ratios , and absolute and relative mean deviation indices . moreover , disparities in mortality were described by rate ratios ( relative risks ) and rate differences ( absolute inequalities ) , which were tested for statistical significance at the 0.05 level . we used weighted least squares regression to model county - level variations in age - adjusted cvd mortality rates as a function of area deprivation , urbanization , racial / ethnic composition , smoking , obesity , physical inactivity , diabetes , and health uninsurance rates , and availability of primary care physicians . the data on county - level covariates were obtained from several sources such as the census , behavioral risk factor surveillance system , and area resource file.[19 - 22 ] for area deprivation , we used a factor - based deprivation index from the 2000 decennial us census . the deprivation index consisted of 22 socioeconomic indicators , which are viewed as broadly representing educational opportunities , labor force skills , economic , and housing conditions in a given county . selected indicators of education , occupation , wealth , income distribution , unemployment rate , poverty rate , and housing quality were used to construct the 2000 index . effects of both continuous and categorical measures of the deprivation index and smoking , obesity , and diabetes prevalence rates were estimated in the regression models . cardiovascular deaths in each county were used as weights in the weighted regression models because the number of deaths is proportional to the inverse of the variance of mortality rates . to examine regional and state - level variations in cvd mortality , we also used the 1979 - 2002 nlms , that allowed us to examine geographical differences in mortality after adjusting for individual - level socioeconomic and demographic characteristics . the nlms is a longitudinal dataset for examining socioeconomic , occupational , and demographic factors associated with all - cause and cause - specific mortality in the united states.[25 - 28 ] the nlms is conducted by the national heart , lung , and blood institute ( national institutes of health [ nih ] ) in collaboration with the us census bureau , the national cancer institute ( nih ) , the national institute on aging ( nih ) , and the national center for health statistics ( centers for disease control and prevention).[25 - 28 ] the nlms consists of 30 current population survey ( cps ) and census cohorts between 1973 and 2002 whose survival ( mortality ) experiences were studied between 1979 and 2002 . the cps is a sample household and telephone interview survey of the civilian non - institutionalized population in the united states and is conducted by the us census bureau to produce monthly national statistics on unemployment and the labor force . data from death certificates on the fact of death and the cause of death are combined with the socioeconomic and demographic characteristics of the nlms cohorts by means of the national death index.[25 - 28 ] detailed descriptions of the nlms have been provided elsewhere.[25 - 27 ] the full nlms consists of approximately 3 million individuals drawn from 30 cps and census cohorts whose mortality experience has been followed from 1979 through 2002 , with the total number of deaths during the 23-year follow - up being 341,343 . however , our study uses the public - use micro - data sample that contains only selected population cohorts between 1979 and 1991 , with a maximum mortality follow - up of 11 years . state- and region - level differentials in mortality risks were adjusted by multivariate cox proportional hazards regression for age and for additional covariates such as sex , race / ethnicity , marital status , metropolitan / non - metropolitan residence , education , income / poverty level , and occupation . the public - use nlms sample for 1979 - 2002 included 780,461 individuals aged 25 at the baseline and 50,430 cvd deaths during the 11-year mortality follow - up . in estimating the mortality risk , all those surviving beyond the 11-year follow - up ( i.e. , 4,018 days of follow - up ) and those dying from causes other than cvd during the follow - up period were treated as right - censored observations . to analyze geographical disparities in cvd mortality over time , we used the national vital statistics mortality database , which has been the cornerstone of health and disease monitoring among sociodemograhic groups and geographical areas in the us for over a century.[3 - 9,17 ] the national mortality database is based on information from death certificates of every death occurring in the united states each year . while the national mortality database provides the number of deaths ( numerator data ) by year , age , sex , race , geographic area , and cause of death , the corresponding population statistics developed by the us census bureau serve as the denominator for computing mortality rates.[6 - 9,17,18 ] the mainland united states consists of 50 states and the district of columbia , which are grouped into 9 census regions as shown in figure 1 . states are divided into counties , and the number of counties varies by state . in all , there are 3,143 counties in the united states . in our study , cvd mortality rates were computed annually for all 9 regions between 1969 and 2011 . for smaller geographical areas such as states and counties , mortality trends are presented for three time periods due to data availability and space constraints . state - specific cvd mortality rates were computed for 1969 , 1990 , and 2011 . cvd mortality rates were computed for 3,141 counties for the time periods : 1969 - 1974 , 1990 - 1999 , and 2003 - 2007 . mortality rates for all geographic areas were age - adjusted by the direct method using the age - composition of the 2000 us population as the standard.[4 - 9 ] trends in cardiovascular disease ( cvd ) mortality by geographic region , united states , 1969 - 2011 new england = maine + new hampshire + vermont + massachusetts + rhode island + connecticut middle atlantic = new york + new jersey + pennsylvania east north central = ohio + indiana + illinois + michigan + wisconsin west north central = minnesota + iowa + missouri + north dakota + south dakota + nebraska + kansas south atlantic = delaware + maryland + district of columbia + virginia + west virginia + north carolina + south carolina + georgia + florida east south central = kentucky + tennessee + alabama + mississippi west south central = arkansas + louisiana + oklahoma + texas mountain = montana + idaho + wyoming + colorado + new mexico + arizona + utah + nevada pacific = washington + oregon + california + alaska + hawaii log - linear regression models were used to estimate annual rates of decrease in cvd mortality for each census region . specifically , the logarithm of region - specific mortality rates were modeled as a linear function of time ( calendar year ) , which yielded annual exponential rates of change in mortality rates . in order to summarize state- and county - level disparities in mortality , we used various disparity measures such as the coefficient of variation ( cv ) , interquartile range , quintile and percentile ratios , and absolute and relative mean deviation indices . moreover , disparities in mortality were described by rate ratios ( relative risks ) and rate differences ( absolute inequalities ) , which were tested for statistical significance at the 0.05 level . we used weighted least squares regression to model county - level variations in age - adjusted cvd mortality rates as a function of area deprivation , urbanization , racial / ethnic composition , smoking , obesity , physical inactivity , diabetes , and health uninsurance rates , and availability of primary care physicians . the data on county - level covariates were obtained from several sources such as the census , behavioral risk factor surveillance system , and area resource file.[19 - 22 ] for area deprivation , we used a factor - based deprivation index from the 2000 decennial us census . the deprivation index consisted of 22 socioeconomic indicators , which are viewed as broadly representing educational opportunities , labor force skills , economic , and housing conditions in a given county . selected indicators of education , occupation , wealth , income distribution , unemployment rate , poverty rate , and housing quality were used to construct the 2000 index . effects of both continuous and categorical measures of the deprivation index and smoking , obesity , and diabetes prevalence rates were estimated in the regression models . cardiovascular deaths in each county were used as weights in the weighted regression models because the number of deaths is proportional to the inverse of the variance of mortality rates . to examine regional and state - level variations in cvd mortality , we also used the 1979 - 2002 nlms , that allowed us to examine geographical differences in mortality after adjusting for individual - level socioeconomic and demographic characteristics . the nlms is a longitudinal dataset for examining socioeconomic , occupational , and demographic factors associated with all - cause and cause - specific mortality in the united states.[25 - 28 ] the nlms is conducted by the national heart , lung , and blood institute ( national institutes of health [ nih ] ) in collaboration with the us census bureau , the national cancer institute ( nih ) , the national institute on aging ( nih ) , and the national center for health statistics ( centers for disease control and prevention).[25 - 28 ] the nlms consists of 30 current population survey ( cps ) and census cohorts between 1973 and 2002 whose survival ( mortality ) experiences were studied between 1979 and 2002 . the cps is a sample household and telephone interview survey of the civilian non - institutionalized population in the united states and is conducted by the us census bureau to produce monthly national statistics on unemployment and the labor force . data from death certificates on the fact of death and the cause of death are combined with the socioeconomic and demographic characteristics of the nlms cohorts by means of the national death index.[25 - 28 ] detailed descriptions of the nlms have been provided elsewhere.[25 - 27 ] the full nlms consists of approximately 3 million individuals drawn from 30 cps and census cohorts whose mortality experience has been followed from 1979 through 2002 , with the total number of deaths during the 23-year follow - up being 341,343 . however , our study uses the public - use micro - data sample that contains only selected population cohorts between 1979 and 1991 , with a maximum mortality follow - up of 11 years . state- and region - level differentials in mortality risks were adjusted by multivariate cox proportional hazards regression for age and for additional covariates such as sex , race / ethnicity , marital status , metropolitan / non - metropolitan residence , education , income / poverty level , and occupation . the public - use nlms sample for 1979 - 2002 included 780,461 individuals aged 25 at the baseline and 50,430 cvd deaths during the 11-year mortality follow - up . in estimating the mortality risk , all those surviving beyond the 11-year follow - up ( i.e. , 4,018 days of follow - up ) and those dying from causes other than cvd during the follow - up period were treated as right - censored observations . figure 1 shows annual trends in cvd mortality among 9 census regions . during 1969 - 2011 , cvd mortality rates declined at the fastest pace in new england and mid - atlantic regions and at the slowest rate in the southeast and southwestern regions of the united states . the average annual rates of decline in mortality during 1969 - 2011 were 2.94% for new england , 2.7% for mid - atlantic , 2.23% for southwest , and 2.12% for southeast . in 1969 , the mortality rate was 9% higher in the southeast than in new england , but this differential increased to 22% in 1990 and 48% in 2011 . a similar increase in relative risk of cvd mortality was seen over time for the southeast and southwest regions when compared to new england and mountain regions ( figure 1 ) . even after adjusting for individual - level socioeconomic and demographic characteristics in the nlms , those in the southeast and east northcentral regions maintained 18 - 19% higher cvd mortality risks than their counterparts in the mountain region ( table 1 ) . the adjusted effects of other individual - level covariates on cvd mortality risks in the nlms are worth noting ( table 1 ) . individuals with low education and incomes had 32 - 40% higher cvd mortality risks than their counterparts with high education and income levels . service workers and manual laborers had 17 - 19% higher cvd mortality risks than those employed in professional and managerial occupations . divorced / separated and never married individuals had 29 - 32% higher cvd mortality risks than married individuals . hispanics and asian / pacific islanders had 35 - 41% lower cvd mortality risks than their non - hispanic counterparts of equivalent socioeconomic backgrounds . age- and covariate - adjusted relative risks of cardiovascular disease ( cvd ) mortality among us adults aged 25+years according to baseline socioedemographic characteristics and region of residence : the us national longitudinal mortaliy study , 19792002 ( n=780,461 ) notes : relative risks ( hazard ratios ) were derived from multivariate cox proportional hazards regression models . adjusted for age , sex , race / ethnicity , marital status , metropolitan / nonmetropolitan residence , educational attainment , occupation , income / poverty level , and geographic region in 2011 , southeastern states such as mississippi and alabama had the highest cvd mortality rates , nearly two times higher than the rates for minnesota and hawaii ( table 2 ) . state patterns were similar in 1969 and 1990 , with substantially increased risks of cvd mortality for most southern states . in 1990 , mississippi and louisiana had the highest mortality rates , 51% , and 42% higher than the rate for hawaii . in 1969 , south carolina had the highest mortality rate , 52% higher than the rate for alaska ( table 2 ) . controlling for individual - level sociodemographic characteristics in the nlms reduced state differentials ; however , individuals in indiana , michigan , louisiana , and kentucky maintained 30 - 35% higher cvd mortality risks than their counterparts in new mexico ( table 3 ) . age - adjusted cardiovascular disease mortality rates by state : united states , 1969 , 1990 , and 2011 notes : rates are per 100,000 population and are directly age - adjusted to the 2000 us standard population . relative risks of cardiovascular disease ( cvd ) mortality among us adults aged 25 years , according to state of residence : the us national longitudinal mortaliy study , 1979 - 2002 ( n=780,461 ) notes : estimated relative risks ( hazard ratios ) were derived from multivariate cox proportional hazards regression models . adjusted for age only . adjusted for age , sex , race / ethnicity , marital status , metro / non - metro residence , educational attainment , occupation , and income / poverty level absolute disparities in state - level cvd mortality , as measured by interquartile range and absolute mean deviation , however , relative disparities in state - level cvd mortality rates , as measured by cv , relative mean deviation index , and quintile and percentile ratios , widened over time . the coefficient of variation in state - level cvd mortality increased by 48% from 10.0 in 1969 to 14.8 in 2011 . the relative mean deviation index indicated a 43% increase in state - level disparity in cvd mortality between 1969 and 2011 ( table 4 ) . summary measures of geographical disparities in cardiovascular disease ( cvd ) mortality , united states , 1969 - 2011 ( 50 states and district of columbia ; 3,141 counties ) interquartile range=3 quartile - 1 quartile ; q1=first quintile ; q4=fourth quintile . p10=10 percentile ; p90=90 percentile county - level variations in area deprivation and cvd mortality rates were closely related , with the weighted correlation being -0.53 ( figure 2 and table 5 ) . consistent with high deprivation levels in the southeast , individuals in this region had the highest cvd mortality rates ( figure 2 ) . area deprivation , smoking , obesity , physical inactivity , diabetes prevalence , urbanization , racial / ethnic composition , lack of health insurance , and lower access to primary medical care were all significant predictors of county - level cvd mortality rates ( table 5 ) . in the multivariate models , these covariates ( excluding health insurance and physician availability because of multicollinearity ) accounted for 52.7% of the county variance . a 10-percentage - point increase in obesity prevalence similarly , a 10-percentage - point increase in diabetes prevalence was associated with a 57.7-point increase in the cvd mortality rate . in multivariate categorical models , consistent gradients in cvd mortality were found by area deprivation and smoking , obesity and diabetes prevalence . even after adjusting for behavioral risk factors , those in the most deprived counties had 15% higher cvd mortality than those in the most affluent counties . cvd mortality rates were 18% higher in areas with smoking rates 36% , compared with areas with smoking rates < 12% . counties with obesity rates 40% had 54% higher cvd mortality than counties with an obesity rate < 15% . counties with a diabetes prevalence 14% had 19% higher cvd mortality than counties with a diabetes prevalence < 6% ( table 5 ) . area ( county ) socioeconomic deprivation index and age - adjusted cardiovascular disease ( cvd ) mortality rates per 100,000 population for the united states ( 2000 us population used as standard ; 3,141 counties ) weighted least squares regression models showing the impacts of the continuous and categorical socioeconomic deprivation index , smoking , obesity , physical activity , diabetes prevalence , ruralurban continuum , and racial / ethnic composition on countylevel ageadjusted cardiovascular disease ( cvd ) mortality rates : united states , 20032007 ( n=3,141 ) notes : b = unstandardized regression coefficient ; =standardized regression coefficient ; r = percentage variance explained . health uninsurance and primary care physician availability rates were not used as covariates in the multivariate model because of estimation problems due to multicollinearity . the 2000 census socioeconomic deprivation index is a continuous variable with a mean of 100 and a standard deviation of 20 . current smoking prevalence among adults aged 18+in 2000 - 2003 . obesity or diabetes prevalence among adults aged 18+in 2006 - 2008 . percentage of physically active adults aged 18+in 2007 , where phyically active = at least 150 minutes of moderate physical activity per week , or 75 minutes of vigorous activity per week , or an equivalent comination of moderate and vigorous physical activity . the 2003 rural - urban continuum is used a continuous variable , with code 1 being the most urbanized county and code 9 being the most rural county . percentage of black , american indian / alaska native , asian / pacific islander , and hispanic populations in 2000 . number of primary care doctors per 100,000 population in 2005 . adjusted for socioeconomic deprivation , smoking , obesity , and pa prevalence , rural - urban continuum , and minority concentration . source : based on the us national vital statistcs system , behavioral risk factor surveillance system , us census , and area resource file county - level differentials in cvd mortality rates , as measured by relative disparity indices , widened over time ; the relative mean deviation index and coefficient of variation indicated , respectively , a 52% and 61% increase in county - level disparity in cvd mortality rates between 1969 and 2007 . absolute county - level disparities in cvd mortality , however , declined over time ( table 4 ) . figure 1 shows annual trends in cvd mortality among 9 census regions . during 1969 - 2011 , cvd mortality rates declined at the fastest pace in new england and mid - atlantic regions and at the slowest rate in the southeast and southwestern regions of the united states . the average annual rates of decline in mortality during 1969 - 2011 were 2.94% for new england , 2.7% for mid - atlantic , 2.23% for southwest , and 2.12% for southeast . in 1969 , the mortality rate was 9% higher in the southeast than in new england , but this differential increased to 22% in 1990 and 48% in 2011 . a similar increase in relative risk of cvd mortality was seen over time for the southeast and southwest regions when compared to new england and mountain regions ( figure 1 ) . even after adjusting for individual - level socioeconomic and demographic characteristics in the nlms , those in the southeast and east northcentral regions maintained 18 - 19% higher cvd mortality risks than their counterparts in the mountain region ( table 1 ) . the adjusted effects of other individual - level covariates on cvd mortality risks in the nlms are worth noting ( table 1 ) . individuals with low education and incomes had 32 - 40% higher cvd mortality risks than their counterparts with high education and income levels . service workers and manual laborers had 17 - 19% higher cvd mortality risks than those employed in professional and managerial occupations . divorced / separated and never married individuals had 29 - 32% higher cvd mortality risks than married individuals . hispanics and asian / pacific islanders had 35 - 41% lower cvd mortality risks than their non - hispanic counterparts of equivalent socioeconomic backgrounds . age- and covariate - adjusted relative risks of cardiovascular disease ( cvd ) mortality among us adults aged 25+years according to baseline socioedemographic characteristics and region of residence : the us national longitudinal mortaliy study , 19792002 ( n=780,461 ) notes : relative risks ( hazard ratios ) were derived from multivariate cox proportional hazards regression models . adjusted for age , sex , race / ethnicity , marital status , metropolitan / nonmetropolitan residence , educational attainment , occupation , income / poverty level , and geographic region in 2011 , southeastern states such as mississippi and alabama had the highest cvd mortality rates , nearly two times higher than the rates for minnesota and hawaii ( table 2 ) . state patterns were similar in 1969 and 1990 , with substantially increased risks of cvd mortality for most southern states . in 1990 , mississippi and louisiana had the highest mortality rates , 51% , and 42% higher than the rate for hawaii . in 1969 , south carolina had the highest mortality rate , 52% higher than the rate for alaska ( table 2 ) . controlling for individual - level sociodemographic characteristics in the nlms reduced state differentials ; however , individuals in indiana , michigan , louisiana , and kentucky maintained 30 - 35% higher cvd mortality risks than their counterparts in new mexico ( table 3 ) . age - adjusted cardiovascular disease mortality rates by state : united states , 1969 , 1990 , and 2011 notes : rates are per 100,000 population and are directly age - adjusted to the 2000 us standard population . relative risks of cardiovascular disease ( cvd ) mortality among us adults aged 25 years , according to state of residence : the us national longitudinal mortaliy study , 1979 - 2002 ( n=780,461 ) notes : estimated relative risks ( hazard ratios ) were derived from multivariate cox proportional hazards regression models . adjusted for age only . adjusted for age , sex , race / ethnicity , marital status , metro / non - metro residence , educational attainment , occupation , and income / poverty level absolute disparities in state - level cvd mortality , as measured by interquartile range and absolute mean deviation , decreased over time . however , relative disparities in state - level cvd mortality rates , as measured by cv , relative mean deviation index , and quintile and percentile ratios , widened over time . the coefficient of variation in state - level cvd mortality increased by 48% from 10.0 in 1969 to 14.8 in 2011 . the relative mean deviation index indicated a 43% increase in state - level disparity in cvd mortality between 1969 and 2011 ( table 4 ) . summary measures of geographical disparities in cardiovascular disease ( cvd ) mortality , united states , 1969 - 2011 ( 50 states and district of columbia ; 3,141 counties ) interquartile range=3 quartile - 1 quartile ; q1=first quintile ; q4=fourth quintile . county - level variations in area deprivation and cvd mortality rates were closely related , with the weighted correlation being -0.53 ( figure 2 and table 5 ) . consistent with high deprivation levels in the southeast , individuals in this region had the highest cvd mortality rates ( figure 2 ) . area deprivation , smoking , obesity , physical inactivity , diabetes prevalence , urbanization , racial / ethnic composition , lack of health insurance , and lower access to primary medical care were all significant predictors of county - level cvd mortality rates ( table 5 ) . in the multivariate models , these covariates ( excluding health insurance and physician availability because of multicollinearity ) accounted for 52.7% of the county variance . a 10-percentage - point increase in obesity prevalence similarly , a 10-percentage - point increase in diabetes prevalence was associated with a 57.7-point increase in the cvd mortality rate . in multivariate categorical models , consistent gradients in cvd mortality were found by area deprivation and smoking , obesity and diabetes prevalence . even after adjusting for behavioral risk factors , those in the most deprived counties had 15% higher cvd mortality than those in the most affluent counties . cvd mortality rates were 18% higher in areas with smoking rates 36% , compared with areas with smoking rates < 12% . counties with obesity rates 40% had 54% higher cvd mortality than counties with an obesity rate < 15% . counties with a diabetes prevalence 14% had 19% higher cvd mortality than counties with a diabetes prevalence < 6% ( table 5 ) . area ( county ) socioeconomic deprivation index and age - adjusted cardiovascular disease ( cvd ) mortality rates per 100,000 population for the united states ( 2000 us population used as standard ; 3,141 counties ) weighted least squares regression models showing the impacts of the continuous and categorical socioeconomic deprivation index , smoking , obesity , physical activity , diabetes prevalence , ruralurban continuum , and racial / ethnic composition on countylevel ageadjusted cardiovascular disease ( cvd ) mortality rates : united states , 20032007 ( n=3,141 ) notes : b = unstandardized regression coefficient ; =standardized regression coefficient ; r = percentage variance explained . health uninsurance and primary care physician availability rates were not used as covariates in the multivariate model because of estimation problems due to multicollinearity . the 2000 census socioeconomic deprivation index is a continuous variable with a mean of 100 and a standard deviation of 20 . percentage of physically active adults aged 18+in 2007 , where phyically active = at least 150 minutes of moderate physical activity per week , or 75 minutes of vigorous activity per week , or an equivalent comination of moderate and vigorous physical activity . the 2003 rural - urban continuum is used a continuous variable , with code 1 being the most urbanized county and code 9 being the most rural county . percentage of black , american indian / alaska native , asian / pacific islander , and hispanic populations in 2000 . number of primary care doctors per 100,000 population in 2005 . adjusted for socioeconomic deprivation , smoking , obesity , and pa prevalence , rural - urban continuum , and minority concentration . source : based on the us national vital statistcs system , behavioral risk factor surveillance system , us census , and area resource file county - level differentials in cvd mortality rates , as measured by relative disparity indices , widened over time ; the relative mean deviation index and coefficient of variation indicated , respectively , a 52% and 61% increase in county - level disparity in cvd mortality rates between 1969 and 2007 . absolute county - level disparities in cvd mortality , however , declined over time ( table 4 ) . cardiovascular disease mortality rates have decreased for all regions and states in the united states . yet , geographical disparities in mortality , in relative terms , have widened over time as several areas in the south experienced slower mortality declines than those in the northeast and western regions of the country . geographical disparities are very marked , with several southern states having nearly twice the risk of cvd mortality than states in the northeastern and western united states . existence of such marked and growing geographical disparities in cvd mortality appears contrary to the goals of the national health initiative that calls for further reductions in cardiovascular disease inequalities in the united states by 2020 . our results are consistent with the previous studies that have shown historically higher rates of cvd mortality in the southern region of the united states.[7,12 - 15 ] because of the persistence of this geographical pattern , the south is often referred to as the stroke or heart disease belt of the united states . since behavioral risk factors such as smoking , unhealthy diet , physical inactivity , and obesity are known to account for about 80% of cvd deaths , geographical disparities in cvd mortality may be understood in terms of geographical distribution of these risk factors . our analysis confirms the significance of geographical distribution of smoking , obesity , physical inactivity , and diabetes prevalence in explaining county - level disparities in cvd mortality rates . nearly 40% of the variance in cvd mortality , and geographical differences in smoking explain about 28% of the variance . smoking , obesity , and physical inactivity rates are highest in the south , and increases in obesity rates have been more marked in the southern states . moreover , smoking rates have declined more slowly in the south than elsewhere in the united states . patterns and increasing geographical disparities in cvd mortality shown here are consistent with those observed previously for the united states and europe.[5,13,33 - 35 ] a recent study showed widening rural - urban disparities in cvd mortality rates in the united states , with those in rural areas experiencing 16% and 26% higher mortality in 1990 and 2009 respectively than their urban counterparts . disparities in cvd mortality between most deprived non - metropolitan areas and most affluent metropolitan areas of the united states also increased markedly between 1990 and 2009 in both absolute and relative terms . coronary heart disease mortality rates have been found to be higher in inner - city areas and in local authority areas in the north of england than those in the south . another study showed a substantial , widening gap in coronary heart disease mortality between the worst health and best health areas of britain over a 10-year period . with the prevalence of many chronic disease risk factors rising in the developing world due to urbanization , development , and globalization , the global burden of cardiovascular diseases is expected to increase further , especially in low- and middle - income countries which account for more than 80% of cvd deaths globally . [ 30,36 - 38 ] cardiovascular disease is the leading cause of death not only in the industrialized world , but also in low- and middle - income countries.[30,36 - 38 ] globally , a major shift has been occurring in the distribution of disease burden as a number of low- and middle - income countries are experiencing an increasing proportion of deaths and years of life lost due to non - communicable diseases such as heart disease , stroke , and copd.[30,36 - 38 ] most of the cvd deaths are preventable through policy measures that are aimed at reducing behavioral risk factors such as smoking , physical inactivity , unhealthy diet , and heavy drinking that account for about 80% of cardiovascular diseases globally . cardiovascular disease burden varies greatly across the world regions , with india and china accounting for > 30% of all global cvd deaths.[30,36 - 38 ] similar analyses of geographical disparities in cardiovascular disease prevalence and mortality rates in developing countries can highlight rural - urban , province- , or district - level disparities , thus indicating the need for targeted action and population - wide interventions to reduce cardiovascular disease incidence and associated behavioral risks . the following countries have the highest disease burden ( in terms of number of heart disease deaths ) : us and germany among high - income countries ; china and indonesia in the east asia and pacific region ; russia and ukraine in europe and central asia ; brazil and mexico in latin america and the caribbean ; india and pakistan in south asia ; and nigeria and ethiopia in sub - saharan africa.[30,36 - 38 ] because of macro - societal forces , such as globalization and urbanization , people in developing countries are increasingly being exposed to such cvd risk factors as smoking , drinking , physical inactivity , and unhealthy diet . at the same time , they do not have similar access to public health education and prevention programs and access to primary care as their counterparts in the industrialized world . geographical inequalities in the united states remain quite marked despite the impressive overall decline in cvd mortality over the past several decades . the growing geographical disparities in cvd mortality are a major public health concern . because cardiovascular diseases are the leading cause of death and account for nearly one - third of all us deaths , the widening inequalities in cvd mortality contribute greatly to overall health and mortality inequalities in the united states . these disparities in mortality may indicate significant geographical inequities in cvd prevention and control efforts . population - wide interventions such as comprehensive tobacco control policies , smoking cessation programs , increased access to primary medical care , physical activity campaigns , and anti - obesity programs can be implemented to reduce cvd risks in the entire population while targeting those in the more disadvantaged areas of the country such as the south . a broad course of policy action related to the wider social determinants can be a particularly effective strategy in reducing cvd inequalities . health and social policy interventions such as improved access to health services , and reductions in inequalities in education , poverty , unemployment , occupation , housing , and access to health - promoting physical or built environments are essential for tackling long - term inequalities in cvd mortality between geographical areas in the united states . previous research has identified higher risks of cvd mortality in the southern region of the united states . our study shows that , despite the substantial decline in overall mortality , regions , states , and counties in the southeastern united states continue to show substantially increased risks of cvd mortality.county- and state - level inequalities in cardiovascular - disease mortality increased consistently between 1969 and 2011 as geographical areas in new england and mid - atlantic regions of the us experienced faster mortality declines than those in the south.both area- and individual - level socioeconomic characteristics influence geographical disparities in cvd mortality in the united states . additionally , preventable or modifiable risk factors such as smoking , obesity , physical inactivity , diabetes , and healthcare access account for much of the geographical inequality in cvd mortality.widening geographical in us cardiovascular - disease mortality may be related to increasing temporal differences in material living conditions and health - risk behaviors such as smoking , obesity , physical inactivity , and unhealthy diet between geographical areas.from a policy standpoint , narrowing the socioeconomic gap and inequalities in smoking , obesity , and physical inactivity between affluent and disadvantaged areas has the greatest potential to reduce cvd and overall mortality rates in the united states.with the prevalence of many chronic disease risk factors rising in the developing world due to globalization and development , the global burden of cardiovascular diseases will likely increase further , especially in low- and middle - income countries , which account for more than 80% of cvd deaths globally . previous research has identified higher risks of cvd mortality in the southern region of the united states . our study shows that , despite the substantial decline in overall mortality , regions , states , and counties in the southeastern united states continue to show substantially increased risks of cvd mortality . county- and state - level inequalities in cardiovascular - disease mortality increased consistently between 1969 and 2011 as geographical areas in new england and mid - atlantic regions of the us experienced faster mortality declines than those in the south . both area- and individual - level socioeconomic characteristics influence geographical disparities in cvd mortality in the united states . additionally , preventable or modifiable risk factors such as smoking , obesity , physical inactivity , diabetes , and healthcare access account for much of the geographical inequality in cvd mortality . widening geographical in us cardiovascular - disease mortality may be related to increasing temporal differences in material living conditions and health - risk behaviors such as smoking , obesity , physical inactivity , and unhealthy diet between geographical areas . from a policy standpoint , narrowing the socioeconomic gap and inequalities in smoking , obesity , and physical inactivity between affluent and disadvantaged areas has the greatest potential to reduce cvd and overall mortality rates in the united states . with the prevalence of many chronic disease risk factors rising in the developing world due to globalization and development , the global burden of cardiovascular diseases will likely increase further , especially in low- and middle - income countries , which account for more than 80% of cvd deaths globally .

atrophic gastritis is characterized by chronic inflammation of gastric mucosa by helicobacter pylori infection and other factors . helicobacter pylori infection has been linked to coronary artery disease . to our knowledge , however , no reports are available on the relationship between atrophic gastritis and coronary artery disease . in this study , we investigated the relationship between atrophic gastritis , which is diagnosed based on serum pepsinogen levels ( pepsinogen i 70 ng / ml and pepsinogen i / ii ratio 3.0 ) , and the prevalence of coronary artery disease in general japanese population . among 2,633 study subjects , 531 subjects ( 20.2% ) were diagnosed as atrophic gastritis . the prevalence of coronary artery disease was higher in the atrophic gastritis - positive group than that in the atrophic gastritis - negative group ( 5.8% vs 2.8% , p = 0.0005 ) . multiple logistic regression analysis demonstrated that atrophic gastritis was independently associated with coronary artery disease ( odds ratio , 1.67 ; 95% confidence interval , 1.032.72 ) , after adjustment for age , sex , obesity , hypertension , diabetes mellitus , dyslipidemia , hyperuricemia , and habits of smoking and drinking . these results suggest that atrophic gastritis is an independent risk factor for coronary artery disease . chronic inflammation of gastric mucosa may be associated with the prevalence of coronary artery disease . objectives : this study examined trends in geographical disparities in cardiovascular - disease ( cvd ) mortality in the united states between 1969 and 2011.methods:national vital statistics data and the national longitudinal mortality study were used to estimate regional , state , and county - level disparities in cvd mortality over time . log - linear , weighted least squares , and cox regression were used to analyze mortality trends and differentials.results:during 1969 - 2011 , cvd mortality rates declined fastest in new england and mid - atlantic regions and slowest in the southeast and southwestern regions . in 1969 , the mortality rate was 9% higher in the southeast than in new england , but the differential increased to 48% in 2011 . in 2011 , southeastern states , mississippi and alabama , had the highest cvd mortality rates , nearly twice the rates for minnesota and hawaii . controlling for individual - level covariates reduced state differentials . state- and county - level differentials in cvd mortality rates widened over time as geographical disparity in cvd mortality increased by 50% between 1969 and 2011 . area deprivation , smoking , obesity , physical inactivity , diabetes prevalence , urbanization , lack of health insurance , and lower access to primary medical care were all significant predictors of county - level cvd mortality rates and accounted for 52.7% of the county variance.conclusions and global health implications : although cvd mortality has declined for all geographical areas in the united states , geographical disparity has widened over time as certain regions and states , particularly those in the south , have lagged behind in mortality reduction . geographical disparities in cvd mortality reflect inequalities in socioeconomic conditions and behavioral risk factors . with the global cvd burden on the rise , monitoring geographical disparities , particularly in low- and middle - income countries , could indicate the extent to which reductions in cvd mortality are achievable and may help identify effective policy strategies for cvd prevention and control .

type 1 and type 2 diabetes account for more than 95% of all diabetes cases worldwide . although clinical characteristics show clear differences between type 1 and type 2 diabetes , recent developments indicate that the pathogenesis of the two forms of the disease share a number of fundamental features . comparative analyses particularly revealed chronic , low - grade inflammatory processes such as systemically elevated levels of proinflammatory immune mediators in both types of diabetes [ 3 , 4 ] . in type 1 diabetes , systemically increased concentrations of inflammatory mediators may promote immune - mediated destruction of autologous pancreatic beta cells [ 57 ] , whereas in type 2 diabetes , increased systemic levels of inflammatory mediators are supposed to contribute to insulin resistance , a major pathogenetic feature of the disease [ 811 ] . previous studies identified the adipose tissue as a prominent source of proinflammatory cytokines and chemokines [ 12 , 13 ] . this finding gains special importance in view of the observations that an increased adipose tissue mass is associated with an elevated risk for type 2 diabetes [ 14 , 15 ] and an accelerated progression of type 1 diabetes [ 16 , 17 ] . further analyses identified the adipocyte population as an important cellular source of proinflammatory mediators within the adipose tissue and implicate that adipocyte - derived mediators promote local inflammatory processes as well as systemic low - grade inflammation [ 1820 ] . recently , we demonstrated that the ( pro-)inflammatory activity of adipocytes is under control of heat shock protein ( hsp ) 60 [ 2123 ] , a prominent member of the hsp family . the capacity to specifically bind to adipocytes and to induce the release of proinflammatory cytokines and chemokines qualifies hsp60 as a candidate for the induction and progression of inflammatory processes associated with the development of diabetes . initial evidence for a role of hsp60 in the progression of insulin - deficient / type 1 diabetes came from observations in the nonobese diabetic ( nod ) mouse , an animal model of type 1 diabetes which shows aberrant hsp60 expression in pancreatic beta cells already in the prediabetic phase . in this model , moreover , patients with type 1 diabetes have increased hsp60 levels and show delayed disease progression after treatment with the hsp60-derived peptide p277 [ 28 , 29 ] . in obese individuals , hsp60 plasma concentrations were increased and positively correlated with body mass index and insulin resistance . these observations indicate that hsp60 stimulates the proinflammatory activity of adipocytes which then might contribute to the ( pro-)inflammatory processes associated with diabetes . however , the increased proinflammatory activity observed in ( pre-)diabetic individuals may not only be determined by their elevated hsp60 levels but also by an increased responsiveness of their adipocyte population to the stress protein . currently , it is not known whether adipocytes from diabetes - prone individuals exhibit an altered hsp60 responsiveness . we therefore hypothesized that adipocytes of diabetes - predisposed individuals exhibit an increased responsiveness to the ( pro-)inflammatory effects of the stress protein hsp60 . to test the hypothesis , we compared the hsp60 responsiveness of adipocytes from the nod mouse , as preferred animal model of type 1 diabetes [ 32 , 33 ] , and from the new zealand obese ( nzo ) mouse , a model of obesity and the metabolic syndrome . using adipocytes from c57bl/6j mice as reference mouse strain without diabetes risk , we investigated the decisive events assumed to be involved in hsp60-mediated adipocyte activation : ( 1 ) the binding of hsp60 , ( 2 ) the activation of signalling pathways , and ( 3 ) the release of inflammatory mediators . normoglycemic mice ( blood glucose concentration < 10 mmol / l ) of the strains c57bl/6j ( female ) , nod ( female ) , and nzo ( male ) at an age of 70 days were obtained from the breeding colonies at the german diabetes center . animal experimentation was performed in accordance with the principles of laboratory animal care and was approved by the local state animal welfare committee . preadipocytes were isolated from the visceral adipose tissue depot of the various mouse strains , cultivated , and differentiated to mature adipocytes as previously described . the purity of the cell populations and their differentiation state were confirmed by lipid - specific oil red o staining and facs analyses as described . antibodies directed against -actin and signalling proteins ( phospho - erk1/2 mapk thr202/tyr204 , phospho - p38 mapk , phospho - sapk / jnk thr183/tyr185 , phospho - nfb p65 ser536 ) were purchased from cell signalling ( danvers , ma , usa ) . murine anti - human hsp60 abs were from santa cruz ( heidelberg , germany ; clones h-1 , h-300 , k-19 , lk1 ) , bd biosciences ( san diego , ca , usa ; clone 24/hsp60 ) , novus ( littleton , co , usa ; clone mab11 - 13 ) , lifespan ( seattle , wa , usa ; clone hspd1 ) , and thermo scientific ( rockford , il , usa ; clone 4b9/89 ) . isotype controls were from cell signalling , recombinant human hsp60 from loke aps diagnostics ( risskov , denmark ) , recombinant mouse and rat hsp60 from stressgen biotechnologies ( victoria , bc , canada ) , and recombinant hamster hsp60 from immpact biotechnologies gmbh ( hamburg , germany ) . for inhibition studies , the specific erk1/2 inhibitor ( pd98059 ) and the nfb inhibitor sn50 ( calbiochem , darmstadt , germany ) were used . for hsp60 binding studies , 0.5 10 c57bl/6j , nod , or nzo mouse - derived ( pre-)adipocytes were either directly incubated with fluorescent - labelled hsp60 ( 100 nm , hsp60 * ) ( 45 min , 4c ) or preincubated with a tenfold molar excess of unlabelled hsp60 from different species or ovalbumin ( ova , sigma - aldrich , steinheim , germany ) as described . for identification of hsp60 binding epitopes , hsp60 * ( 100 nm ) was preincubated with 025 g / ml of anti - hsp60 antibodies ( 45 min , 4c ) before application to ( pre-)adipocytes . after washing and fixation of the cells in 2% paraformaldehyde , hsp60 * binding was calculated from the geometric mean fluorescence after subtracting the autofluorescence as determined by analyses in a facs calibur flow cytometer ( bd biosciences ) . c57bl/6j , nod , and nzo mouse - derived ( pre-)adipocytes were exposed to medium or 120 g / ml recombinant human hsp60 ( enzo life sciences , lrrach , germany ) . after 24 h , the concentrations of the inflammatory mediators interleukin-6 ( il-6 ) , mouse chemokine cxcl-1 ( kc ) , and monocyte chemoattractant protein-1 ( mcp-1 ) were determined in the culture supernatants by multiplex - beads - systems ( luminex corp . , austin , tx , usa ) . to identify hsp60-activated signaling pathways , c57bl/6j , nod , and nzo mouse - derived ( pre-)adipocytes were seeded in 6 cm petri dishes ( 2 10 cells/5 ml medium ) . cells were treated with medium or hsp60 ( 10 g / ml ) ( 15 min , 37c ) and subsequently washed and treated with lysis - buffer ( 50 mm tris - hcl , 150 mm nacl , 1% np-40 , 0.25% na - desoxycholat ) including protease and phosphatase inhibitors ( roche , mannheim , germany ) ( 20 min , 4c ) . after centrifugation ( 15 min , 10,000 g , 4c ) cell lysates were subjected to sds - page ( 10% ) and appropriate antibodies for detection of activated signal proteins were applied for immunoblot analysis . signals were visualized by the lumi - imager system ( roche applied science , mannheim , germany ) . to analyse the effect of specific signal protein inhibitors on the hsp60-mediated secretion of inflammatory mediators , 1 10 c57bl/6j , nod , and nzo mouse - derived ( pre-)adipocytes were seeded in 1 ml per well of a 48 well plate and incubated ( 1 h , 37c ) either with medium ( control ) , erk1/2 inhibitor pd98059 ( 100 g / ml ) , or nfb inhibitor sn50 ( 50 g / ml ) . afterwards , cells remained unstimulated ( medium ) or were stimulated with hsp60 ( 10 g / ml ) for 24 h. supernatants were collected to determine the concentrations of il-6 , kc , and mcp-1 by multiplex - beads - assays . to characterize the interaction between hsp60 and primary ( pre-)adipocytes from c57bl/6j , nod , and nzo mice , we investigated the binding of fluorescent - labeled hsp60 ( hsp60 * , 100 nm ) to preadipocytes and in vitro differentiated , mature adipocytes of the three mouse strains . facs analyses demonstrated substantial binding of hsp60 to adipocyte populations of all mouse strains ( figures 1(a)1(f ) ) . comparison of the mean fluorescence signals revealed hsp60 binding intensities of preadipocytes from c57bl/6j , nod , and nzo mice within a range of geo means from 19 to 30 ( figure 1(g ) ) . among the adipocyte populations , nzo mouse - derived adipocytes revealed maximum hsp60 binding ( geo mean 38.0 10.7 ) . the specificity of hsp60-(pre-)adipocyte interaction was proven by inhibition of hsp60 * binding to cells preincubated with excess unlabelled hsp60 ( 1000 nm ) to 23.1 2.7% ( c57bl/6j ) , 20.4 1.9% ( nod ) , and 28.7 2.5% ( nzo ) ( p < 0.05 ) ( figure 1(h ) ) . the extent of inhibition of hsp60 binding to mature adipocytes was only marginally lower than that observed for preadipocytes . based on our previous observation that different eukaryotic hsp60 species recognize the same receptor structure(s ) on cells of the murine adipocyte line 3t3-l1 , we investigated the effect of eukaryotic ( human , mouse , rat , and hamster ) and prokaryotic hsp60 species ( e. coli , m. bovis ) on the binding of human hsp60 to primary c57bl/6j , nod , and nzo mouse - derived ( pre-)adipocytes . strong inhibition of hsp60 * binding to preadipocytes was observed after preincubation of hsp60 * with the mammalian hsp60 species ( figure 2 ) . preincubation with human hsp60 induced maximum inhibitory effects by reducing hsp60 * binding to preadipocytes from the three mouse strains to 28.9 14.5% ( c57bl/6j ) , 33.9 18.4% ( nod ) , and 34.1 17.4% ( nzo ) . except for groel - mediated inhibition of hsp60 * binding to c57bl/6j mouse - derived preadipocytes , preincubation with prokaryotic hsp60 species did not significantly affect hsp60 * binding to preadipocyte populations ( figure 2 ) . for primary , in vitro differentiated adipocytes comparable results were obtained ( data not shown ) . our further experiments focused on the identification of the hsp60 epitope(s ) potentially involved in the interaction of the stress protein with ( pre-)adipocytes . in an initial approach , we investigated the effect of antibodies against distinct regions of the hsp60 molecule , on the binding of hsp60 to c57bl/6j , and nod mouse - derived preadipocytes . facs analyses revealed largely comparable patterns of antibody - mediated inhibition of hsp60 binding to preadipocyte populations of both mouse strains ( table 1 ) . preincubation of the cells with antibodies against the n - terminal ( aa1 - 50 ( clone h1 ) , aa1 - 200 ( clone 24/hsp60 ) ) , and c - terminal ( aa523 - 573 ( clone k19 ) ) regions of the hsp60 molecule resulted in strongest reduction of hsp60 binding to 25.5 0.6% . subsequently , we preincubated fluorescent - labeled hsp60 with increasing concentrations ( 025 g / ml ) of the antibodies initially found to inhibit hsp60 binding ( clones h1 , 24/hsp60 , k-19 ( table 1 ) ) prior addition to ( pre-)adipocytes from c57bl/6j , nod , or nzo mice ( figure 3 ) . in c57bl/6j mouse - derived preadipocytes , hsp60 binding was reduced dose - dependently to 51.2 9.1% by the application of the antibody clone h-1 ( aa1 - 50 , 25 g / ml ) and to 28.0 2.6% by applying antibody clone 24/hsp60 ( aa1 - 200 , 25 g / ml ) . in contrast , clone k-19 ( aa523 - 573 ) did not induce clear dose - dependent inhibition of hsp60 binding . comparable inhibitory effects were obtained for preadipocytes from nod and nzo mice . clone h-1 ( aa1 - 50 ) reduced hsp60 binding to 38.2 12.5% ( nod ) and to 47.0 2.9% ( nzo ) ; clone 24/hsp60 ( aa1 - 200 ) reduced hsp60 binding to 23.3 4.9% and to 18.6 3.5% in nod and nzo mouse - derived cells , respectively . as in c57bl/6j preadipocytes , no clear dose - dependent effects were observed for nod and nzo mouse - derived preadipocytes preincubated with the antibody against the c - terminal region of hsp60 ( clone k-19 , aa523 - 573 ) . in binding studies with mature adipocytes , to compare adipocyte populations from different mouse models of diabetes for their ability to release inflammatory mediators , we investigated the accumulation of kc , il-6 , and mcp-1 in cultures of ( pre-)adipocytes derived from c57bl/6j , nod , and nzo mice ( figure 4 ) . unstimulated cells of the three mouse strains spontaneously accumulated substantial amounts of most of the mediators in their supernatants ( figures 4(a ) and 4(b ) ) . independent of the mouse strain , preadipocyte and adipocyte populations released kc in a concentration range of 4.312.9 ng / ml and mcp-1 in a range of 13.327.1 ng / ml . the amounts of il-6 released from c57bl/6j and nod mouse - derived ( pre-)adipocytes were in a range of 0.79.8 ng / ml , whereas ( pre-)adipocytes from nzo mice released exceptionally low levels of the cytokine ( 0.7 0.3 ng / ml and 0.9 0.3 ng / ml il-6 , resp . ) exposure of the ( pre-)adipocyte populations to rising hsp60 concentrations ( 120 g / ml ) for 24 h caused a dose - dependent increase of the secretion of kc , il-6 , and mcp-1 in a cell type- and mouse strain - specific manner ( figures 4(c)4(h ) ) . however , whereas the cytokine release from nod mouse - derived cell populations largely resembled the pattern of c57bl/6j mouse - derived cells , adipocyte populations from nzo mice showed an enhanced release of kc and il-6 . in particular , a strong , 3.9 0.2-fold increase of kc release from nzo mouse - derived adipocytes was observed when compared to adipocytes from c57bl/6j ( 1.7 0.2-fold increase , p < 0.05 ) ) and from nod mice ( 2.4 0.7-fold increase ) ( figure 4(d ) ) . moreover , the release of il-6 induced by hsp60 ( 20 g / ml ) was strikingly increased from nzo mouse - derived preadipocytes ( 7.7 1.0-fold ) and adipocytes ( 19.1 4.0-fold ) when compared to the corresponding cell populations of c57bl/6j and nod mice ( p < 0.05 ) ( figures 4(e ) and 4(f ) ) . we further investigated the effect of hsp60 on the stimulation of proinflammatory signaling pathways by analyzing the activation ( phosphorylation ) of members of the map kinase family ( erk1/2 , jnk(p46 ) , p38 ) and of the transcription factor nfb in ( pre-)adipocytes of c57bl/6j , nod , and nzo mice ( figure 5 ) . hsp60 treatment did not activate erk1/2 , jnk(p46 ) , and p38 in preadipocytes of the three mouse strains ( figure 5(a ) ) . on the other hand , nfb activation was increased to a similar degree in nod and nzo mouse - derived preadipocytes ( 2.6 0.2-fold and 2.6 0.5-fold , resp . ) ( p < 0.05 ) but not in c57bl/6j mouse - derived cells . in contrast to the hsp60 effects observed in preadipocytes , hsp60 exposure of mature adipocytes increased the activation of erk1/2 ( 2.1 0.2-fold , p < 0.01 ) and nfb ( 3.5 0.2-fold , p < 0.01 ) in c57bl/6j mouse - derived cells , whereas in cells from the diabetes predisposed mouse strains , hsp60-induced activation of the investigated pathways was only moderate or low ( < 2-fold ) ( figure 5(b ) ) . these results show that hsp60 preferentially controls erk1/2- and nfb - dependent pathways in a differential manner depending on the maturation state of the adipocytes and on the donor mouse strain . we therefore focused our further analyses on the involvement of erk1/2 and nfb in hsp60-induced cytokine release by the use of specific inhibitors of the signaling molecules . exposure of adipocytes of c57bl/6j and nod mice to the erk1/2-inhibitor pd98059 reduced the hsp60-induced release of il-6 , kc , and mcp-1 to 16 to 67% ( p < 0.05 ) of the levels detectable in cultures treated with hsp60 alone ( figures 6(a ) , 6(c ) , and 6(e ) ) . however , in nzo mouse - derived adipocytes , the hsp60-induced cytokine pattern showed a differential response to erk1/2 inhibition , whereas in the presence of pd98059 , the formation of il-6 remained largely unaffected ( relative il-6 secretion 89.7 10.9% ) ( figure 6(a ) ) , the residual kc formation was in the range of the levels detected in c57bl/6j and nod mouse derived cells ( 48.7 3.8% , p < 0.001 ) ( figure 6(c ) ) and the release of mcp-1 was strongly reduced to 28.7 9.1% ( p < 0.001 ) ( figure 6(e ) ) . inhibition of nfb by sn50 resulted in the reduction of cytokine release in a similar range in cultures of ( pre-)adipocytes from c57bl/6j and nod mice ( 2970% ) ( p < 0.01 ) ( figures 6(b ) , 6(d ) , and 6(f ) ) . the strongest inhibition of hsp60-induced il-6 , kc , and mcp-1 release was found in sn50-exposed ( pre-)adipocytes from nzo mice . in nzo mouse - derived preadipocytes , the nfb inhibitor significantly reduced il-6 release to 3.0 2.1% , kc release to 15.0 4.7% , and mcp-1 release to 5.1 3.6% ( p < 0.001 ) of the cytokine levels measured in the absence of the inhibitor . in adipocytes , nfb inhibition reduced il-6 release to less than 0.1% , kc release to 19.7 9.8% , and mcp-1 release to 26.3 9.1% ( p < 0.001 ) . previous observations suggest an essential contribution of obesity - associated inflammatory processes to the development of type 2 diabetes [ 10 , 11 ] as well as type 1 diabetes . further studies , based on the finding of the strong immunomodulatory capacity of adipocytes , identified the stress protein hsp60 as a potent inductor of proinflammatory mediators from murine and human adipocytes [ 21 , 31 ] . these findings implicate that hsp60-induced adipocyte mediators promote the development of obesity- and diabetes - associated inflammatory processes and further raise the question whether adipocytes from diabetes - prone subjects exhibit an aberrant reactivity to the stress protein . to address this issue , we examined the effect of hsp60 on adipocyte populations derived from the currently best characterized animal models of the two prevailing forms of human diabetes . diabetes observed in the nod mouse reflects major features of human type 1 diabetes [ 32 , 33 ] , whereas the metabolic abnormalities developing in the nzo mouse largely resemble the dysregulations of energy metabolism and glucose homeostasis associated with human type 2 diabetes . as reference , adipocytes from the metabolically healthy mouse strain c57bl/6j were used . for our studies , we selected adipocyte populations isolated from the visceral fat depot as adipose tissue from this anatomical location had been identified as a major source of mediators responsible for diabetes - associated metabolic and immunologic disorders [ 36 , 37 ] . as previous reports indicate that the functional properties of adipocytes depend on their maturation state [ 38 , 39 ] , we investigated hsp60 effects on preadipocytes as well as terminally differentiated adipocytes . our approach focused on the decisive steps assumed to be involved in hsp60-mediated inflammatory adipocyte activation : ( 1 ) the binding of hsp60 to adipocytes regarded as the primary event in hsp60-adipocyte interaction , ( 2 ) the activation of intracellular signaling pathways , and ( 3 ) the resulting release of inflammatory mediators which may act as signaling molecules in an autocrine manner and/or in the attraction and stimulation of immune cells . initial comparative facs studies with fluorescent - labeled hsp60 revealed that preadipocytes and mature adipocytes of all investigated mouse strains are able to bind hsp60 . binding of the stress protein was highly specific as demonstrated by efficient inhibition after preincubation of the cells with unlabelled hsp60 . interestingly , the observed characteristics of hsp60-(pre-)adipocyte interaction showed striking similarities with hsp60 binding by macrophages which was investigated in more detail in previous studies [ 40 , 41 ] . further studies revealed that the receptor structure for hsp60 acts in a stereospecific manner , includes at least two functionally different components engaged in binding and signal transduction , differs from receptors for other heat shock proteins , and triggers endocytosis of its bound ligand . considering the observed similarities in hsp60 binding to ( pre-)adipocytes and macrophages and in view of the close relationship between cells of the adipocyte and macrophage lineages , it may be concluded that hsp60 binding to adipocytes is mediated by similar receptor - mediated mechanisms as the above mentioned mechanisms described for macrophages . interestingly , although efficient hsp60 binding to adipocytes of all three mouse strains was observed , we found strongly enhanced binding of the stress protein to mature nzo mouse - derived adipocytes when compared to cells from c57bl/6j ( and nod ) mice of the same maturation state . this finding may be of special importance in view of the fact that nzo mice are characterized by an increased body fat mass which most likely includes a large proportion of terminally differentiated adipocytes . in more detailed studies , we attempted to identify hsp60 epitopes involved in the interaction of the stress protein with ( pre-)adipocytes . competition experiments with various hsp60 species and with inhibitory antibodies directed against defined hsp60 epitopes revealed that eukaryotic hsp60 molecules bind to ( pre-)adipocytes via highly conserved aminoacid sequences within the n - terminal region . collectively , our studies on hsp60-(pre-)adipocyte interaction demonstrate efficient hsp60-binding to primary nod mouse - derived adipocytes , thereby extending our previous observations of specific hsp60 interaction with adipocytes from nzo mice and with cells of the murine adipocyte line 3t3-l1 . hence , our findings provide further evidence for the assumption that the ability to bind hsp60 represents a general property of murine adipocytes irrespective of their origin and differentiation state . nevertheless , further extensive biochemical studies will be necessary to gain further insight into the structural and functional properties of the hsp60 receptor structure on ( pre-)adipocytes . previous studies identified stress proteins as potent inducers of intracellular signaling pathways . we therefore investigated the effects of hsp60 on the activation of the map kinases erk1/2 , jnk , and p38 and of the transcription factor nfb which are preferentially activated by stress signals but are also essential for the coordination of adipocyte differentiation [ 4446 ] . due to the marked interdependence of signaling and differentiation pathways , we hypothesized that the response of the adipocyte populations depends on the maturation state of the cells . in fact , hsp60 exposure induced increased erk1/2 activation in ( mature ) adipocytes from normal control c57bl/6j mice and enhanced activation of erk1/2 and p38 in adipocytes from nod mice . with regard to nfb , we found an elevated activation of the transcription factor in preadipocytes from diabetes - prone nod and nzo mice but reduced activation in mature adipocytes of the two mouse strains , when compared to the reactivity of cell populations from normal control c57bl/6j mice . as nfb represents an important transcriptional regulator of inflammatory mediators , our findings implicate that preadipocytes of nod and nzo mice exhibit an increased hsp60 responsiveness that might contribute to diabetes - promoting proinflammatory processes in these animals which are genetically predisposed to develop diabetes . collectively , our findings implicate that the hsp60-induced activation of intracellular signaling pathways in adipocytes not only depends on the differentiation state of the cells but also on the donor mouse strain . our observations particularly suggest that aberrant intracellular signaling and disturbed transcriptional regulation in adipocytes enhance diabetes - promoting inflammatory reactivity in animals with a genetic predisposition to develop diabetes . in fact , previous studies demonstrated aberrant activation of map kinases , including p38 , [ 48 , 49 ] and a dysregulation of transcription factors , including nfb [ 5052 ] in diabetes - prone mouse strains . although these findings are mainly derived from macrophages , they obviously reflect intrinsic disturbances of signaling and transcriptional processes in diabetes prone nod and nzo mice that may contribute to the aberrant activation of map kinases and nfb observed in hsp60-exposed ( pre-)adipocytes of these animals . further studies on the elucidation of the intracellular signaling cascade triggered by stress signals ( will ) have to consider the complex situation that most components of the stress response pathways are also critical for the control of adipocyte differentiation [ 45 , 5052 ] . the release of proinflammatory adipocyte mediators induced by hsp60 and the intracellular signaling pathways engaged in this process were further analyzed by focusing on the formation of kc , il-6 , and mcp-1 which had been identified as prominent factors released by adipocytes and are supposed to contribute to obesity - associated inflammatory processes [ 5355 ] . unstimulated preadipocytes and adipocytes of c57bl/6j , nod , and nzo mice released substantial amounts of these mediators , with the exception of nzo mouse - derived adipocyte populations which released markedly low amounts of il-6 . hsp60 , applied at concentrations that largely correspond to the levels found in individuals suffering from cardiovascular and arthritic disorders [ 56 , 57 ] , dose - dependently increased the release of kc , il-6 , and mcp-1 by preadipocytes and adipocytes from the three mouse strains . these results extend our initial observations on the pronounced hsp60 responsiveness of adipocytes of a murine cell line and of nzo mice [ 21 , 22 ] and further support the view that the capacity to release proinflammatory mediators in response to hsp60 reflects a common feature of adipocytes . however , with regard to the release of il-6 , inducibility by hsp60 was strikingly higher in nzo mouse - derived adipocyte populations than in cells from c57bl/6j and nod mice . the potential impact of the donor mouse strain on map kinase - dependent signaling and transcriptional regulation in the hsp60-induced formation of inflammatory adipocyte mediators was further elucidated by selective inhibition of the signaling molecules . blocking of signaling pathways by specific inhibitors [ 58 , 59 ] confirmed the involvement of erk1/2 and nfb in the hsp60-induced release of adipocyte mediators . unexpectedly , the experiments revealed strong differences between the reactivities of adipocyte populations from the two diabetes - predisposed mouse strains . after erk1/2 or nfb inhibition , the reactivity pattern of nod mouse - derived cells largely resembled the pattern of cells from normal control c57bl/6j mice . however , in adipocyte populations of nzo mice , erk1/2 dependency was low for il-6 production , intermediate for kc production , and high for mcp-1 production when compared to c57bl/6j and nod mouse - derived cells . moreover , nfb suppression caused strongest inhibition of 7598% in cultures of nzo mouse - derived cells pointing to a dominant role of nfb in the formation of proinflammatory mediators from hsp60-exposed adipocytes in this mouse strain . this finding appears to be in discrepancy with our above - mentioned results demonstrating that nod and nzo mouse - derived adipocytes show largely comparable activation levels of map kinases and nfb after hsp60 exposure . these contrasting observations obviously reflect strain - specific differences in the preferential usage of intracellular pathways of signaling and transcriptional regulation ( other than nfb - dependent ) to induce the formation of inflammatory mediators in response to hsp60 . this assumption is supported by the finding of aberrant activation of intracellular signaling pathways in diabetes - prone mouse strains [ 49 , 51 , 52 ] . furthermore , the promoter regions of many genes encoding inflammatory mediators ( e.g , il-6 , mcp-1 ) also have binding sites for other transcription factors including activator protein 1 and members of the ccaat - enhancer binding protein family [ 6062 ] . it might be speculated that these factors could be used alternatively to nfb in the induction of proinflammatory adipocyte mediators in different mouse strains , depending , for example , on their genetic predisposition to develop a specific form of diabetes . taken together , our observations in c57bl/6j , nod , and nzo mouse - derived adipocyte populations indicate that the basic structural requirements , such as receptor structure(s ) and hsp60 binding epitopes , involved in the initial recognition of the stress protein , share a high degree of similarity , and irrespective of the donor and of the differentiation state of the cells . however , clear differences between the mouse strains were found regarding the efficiency of hsp60 binding , the patterns of intracellular signaling pathways activated by hsp60 , and the resulting release of inflammatory mediators . these findings warrant further extensive studies for the detailed elucidation of mouse strain dependency and the roles of intracellular signaling pathways in hsp60-induced adipocyte stimulation . nod mice typically exhibit a lean constitution and , accordingly , a low number of ( mature ) adipocytes . moreover , the hsp60 responsiveness of nod mouse - derived adipocytes largely corresponds to the reactivity of adipocytes of normal healthy c57bl/6j mice . based on these considerations , it may be concluded that in nod mice , hsp60-induced proinflammatory adipocyte activities do not play a decisive role in the progression of ( insulin deficient ) diabetes . in nzo mice , however , improved hsp60 binding and dysregulation of il-6 formation reflect an increased proinflammatory reactivity , preferably of mature adipocytes . in addition , the pathological increase of the adipose tissue mass by adipocyte - hyperplasia and proliferation inevitably leads to an elevated number of ( mature ) adipocytes in these mice . as a result , the enhanced release of chemotactic and stimulatory adipocyte mediators may contribute not only to the acceleration of immune cell infiltration into the adipose tissue but also to an elevation of systemic inflammatory mediators which may aggravate peripheral insulin resistance , thereby promoting the progression of metabolic dysregulation and diabetes . moreover , during the development of obesity , the expanding adipose tissue itself may create an increasingly stressful environment in which infiltrating immune cells ( e.g , macrophages ) and adipocytes themselves may become relevant sources of hsp60 and other stress signals . the human cornea is the main refractive surface focusing light into the eye , so its transparency is critical for vision . globally , approximately 4.9 million individuals have bilateral cornea blindness , while 23 million are unilaterally cornea blind.1 donor tissue transplantation is the only widely accepted treatment but the need for donor corneas exceeds the supply and the gap only increases as the population ages . recent advances in corneal transplantation techniques with a shift from penetrating to safer lamellar procedures and wide application of limbal epithelial stem cell transplantation , have resulted in improved outcomes , and have expanded the number of cases of corneal blindness that can now be treated successfully.2 however , patients with severe pathologies ( e.g. , chemical burns , previously rejected grafts , autoimmune disease , and infections ) still have a high risk of rejection and failure , often needing multiple surgeries.38 corneal prostheses are available , but they remain mainly for end - stage disease and even if implanted as primary procedure they often are accompanied by vision - threatening complications,1,9 leaving an unmet need for a de novo solution for high - risk patients . in ukraine , as in many countries , there is a severe shortage of donor corneas . the annual need in 2010 was 4000 grafts , but only 511 corneal transplantations were performed due to the lack of donated tissues . fifty - four percent of these transplantations were performed for tectonic purposes due to the increasing number of infections and injuries.10 to solve the problems of lack of donor tissues as well as the high risk of rejection of allografted corneas , we had developed corneal implants made from interpenetrating networks of cross - linked recombinant human collagen type iii ( rhciii ) and 2-methacryloyloxyethyl phosphorylcholine ( mpc ) , a synthetic phospholipid11 ( figure 1 ) . mpc - polymer has antifouling properties and has been fda approved as a polymer coating in vascular stents.12 here , it was incorporated into rhciii to prevent neovascularization . schematic diagram showing the combination of recombinant human collagen type iii ( rhciii ) and 2-methacryloyloxyethyl phosphorylcholine ( mpc ) to form interpenetrating networks of rhciii - mpc . the rhciii and mpc were mixed together in a syringe , while the final hydrogel was molded into an implant . an image of vladimir filatov , pioneer of human donor cornea grafting , can be seen through the transparent implant . we previously tested implants comprising rhciii alone as substitutes for donor corneas in a phase 1 clinical study , where they promoted good tissue integration with stable regeneration of corneal epithelium , stroma and nerves , restoration of corneal reflex better than allograft ( p = 0.04 ) , maintenance of transparency in cornea and no tissue rejection reported on 4-year follow - up , in the absence of long - term steroid immunosuppression beyond week 7 postimplantation , in patients with keratoconus or central scarring , i.e. , low - risk patients,13 like donor corneas , however , rhciii only implants became neovascularized when grafted into a model of severe pathology , the alkali - burned rabbit cornea.14 incorporation of mpc into rhciii , on the other hand , yielded implants that repelled blood vessels14 while allowing regeneration.15,16 here , we report our initial experience with rhciii - mpc implants into three patients for whom donor cornea grafting carried a high risk of rejection . the primary aim of this early investigation was to assess the safety of such an approach . the secondary aim was to test the feasibility of restoring the integrity of the cornea . this study was performed in accordance to the declaration of helsinki convention of the council of europe on human rights and biomedicine , relevant laws of ukraine , and after approval by the bioethics commission of the filatov institute of eye diseases and tissue therapy of the national academy of medical sciences of ukraine and trial registration ( registered eudract no . 2013 - 002442 - 37 ) . after providing written informed consent , each patient was grafted with a rhciii - mpc implant consisting of rhciii ( 8% wt / vol ; from fibrogen , inc , san francisco , usa),17 mpc ( 4% wt / vol ) and poly(ethylene glycol ) diacrylate ( 1.37% wt / vol)11 by anterior lamellar corneal transplantation . patient 1 , an 80-year - old male , suffered an alkali burn to his right eye resulting in a persistent corneal ulcer resistant to conventional medical treatment and bandage contact lens wear . he suffered from pain , tearing , and photophobia due to the inability of the corneal epithelium to stably adhere to the underlying damaged and vascularized stroma . patient 1 s best corrected visual acuity ( bcva ) was 6/600 , i.e. , near blindness . patient 2 was a 72-year - old female with a previously rejected penetrating human cornea graft in her left eye , which was combined with cataract extraction and intraocular lens ( iol ) implantation . she suffered from corneal ulceration , which was unresponsive to medical treatment and bandage contact lens wear , and had the same symptoms as patient 1 , with only light perception , i.e. , effectively blind . patient 3 was a 52-year - old male who suffered from recurrent corneal erosions following an acid burn . his symptoms were similar to those of the other two patients . during the chronic stage of the burn , he received excimer laser phototherapeutic keratectomy and a human amniotic membrane ( ham ) graft for ocular surface healing . he has also had a cataract phacoemulsification and was implanted with an iol one year after the injury . all three patients needed surgery to treat the ulceration , restore corneal integrity , alleviate the associated pain and discomfort , and to improve vision . based on literature review all were considered high - risk patients for limbal epithelial graft rejection as well as penetrating or lamellar cornea graft rejection and failure . each patient s pathologic cornea was initially cut with a 45-mm - diameter trephine ( depending on lesion diameter ) to remove the lesioned area with a small epithelialized margin around it . the incision was then deepened with a diamond knife set to a depth of 250 or 350 m ( depending on lesion depth ) . a trephine of the same diameter was used to cut the biosynthetic implant of corresponding thickness . once in place , the implant was anchored with three to four overlying 100 nylon mattress sutures to avoid puncturing the implant material . alcon - couvreur n.v . , puurs , belgium ) , a short - term mydriatic ( cyclopentolate 1% , sentiss pharma pvt . ltd . , gurgaon , india ) and a nonsteroidal anti - inflammatory drug ( indomethacin 0.1% , bausch + lomb gmbh , dr . this was followed by a topical antiseptic ( chlorhexidine bigluconate 0.02% , farmacia , lugansk , ukraine ) and a steroid ( dexamethasone 0.1% , s.a . the patients wore 14-mm bandage contact lenses containing 36% water ( bausch & lomb purevision ) until epithelial regeneration was complete . patients were assessed at 1 , 3 , 6 , 9 , and 12 months postoperatively or as determined required by the physician . clinical assessment performed included slit - lamp biomicroscopy to check for signs of inflammation or infection , a fluorescein staining test to confirm epithelial integrity , corneal surface sensitivity assessment ( cochet bonnet esthesiometer , luneau ophthalmologie , france ) , best corrected visual acuity measurement , transpalpebral tonometry to measure intraocular pressure without damaging the epithelium ( diaton , ryazan state instrument - making enterprise , russia ) , ultrasound pachymetry ( sp-100 , tomey , japan ) and in vivo confocal microscopy ( confoscan4 , nidek , japan ) . all implants had similar optical properties to healthy human corneas . while they were mechanically weaker than human donor corneas , they were nevertheless robust enough to withstand the handling and grafting procedure . properties of rhciii - mpc hydrogels used as corneal implants doutch j , quantock aj , smith va , meek km . light transmission in the human cornea as a function of position across the ocular surface : theoretical and experimental aspects . zeng y , yang j , huang k , lee z , lee x. a comparison of biomechanical properties between human and porcine cornea . j biomech . crabb ra , chau ep , evans mc , barocas vh , hubel a. biomechanical and microstructural characteristics of a collagen film - based corneal stroma equivalent . merrett k , fagerholm p , mclaughlin cr , dravida s , lagali n , shinozaki n , watsky ma , munger r , kato y , li f , marmo cj , griffith m. tissue - engineered recombinant human collagen - based corneal substitutes for implantation : performance of type i versus type iii collagen . invest ophthalmol vis sci . 2008 ; 49 : 38873894 . epithelial coverage of the initially cell - free implants took on average 6 weeks ( table2 , figure 2 ) . all three implants remained free of neovascularization or epithelial erosions over the 912 months postoperation follow - up period . however , conjunctival epithelium invaded the implant surface in patient 3 , due to limbal stem cell deficiency ( figure 2 ) . there was no stromal edema , no prolonged inflammation nor any infection in any patient . touch sensitivity increased after surgery in all patients although not to the level of normal human corneas ( figure 3 ) . after the mild deturgescence of the edema that always accompanies corneal ulceration , the corneal thickness remained stable throughout the follow - ups . signs of punctate precipitate and haze appeared at the posterior surface of the implant during the third postoperative week in all cases , but resolved after two weeks of treatment with topical steroids . visual acuity improved in patients 1 and 2 from near blindness ( 6/600 and lp ) to moderate ( 6/38 ) and severe ( 6/75 ) vision loss , respectively , allowing for restricted function with enhancing aids . patient 3 , who had conjunctivalization of corneal surface , showed no change in vision but no longer had recurrent painful erosions seen preoperatively , suggesting that rhciii - mpc implants could stabilize the corneal surface pending further treatment , e.g. , limbal stem cell grafting to improve vision . implants remained stably incorporated without immunosuppression beyond the 6-week course of prophylactic anti - inflammatory medication . details of implanted patients showing their biometrics , diagnosis , treatment , and results cu = corneal ulcer ; rce = recurrent corneal erosion ; acb = acid burn ; alkb = alkali burn ; ptk = phototherapeutic keratectomy ; ce = cataract extraction ; iol = intraocular lens implantation ; phaco = cataract phacoemulsification ; ham = human amniotic membrane transplantation ; pk = penetrating keratoplasty ; lp = light perception . bcva = best corrected visual acuity ( lp , 6/6006/375 : near blindness ; 6/3006/150 : profound vision loss ; 6/1206/60 : severe vision loss : 6/486/24 : moderate vision loss ; 6/196/9.5 : mild vision loss ; 6/7.56/3.8 : normal range of vision ) . corneas of all three patients before and after implantation with tectonic grafts of rhciii - mpc . the green fluorescein staining delineates the large area of eroded epithelium . at 12 months postoperation , patient 2 had a failed 8.5-mm - diameter graft with a persistent ulcer and dense stromal opacification in the visual zone prior to surgery . a small 4-mm implant was grafted into the ulcerated area of the failed graft ( arrowhead ) and has remained relatively clear after 12 months . patient 3 had an opacity with an unstable epithelial surface and vascularized stroma prior to surgery . at 9 months postoperation , while the implant remained clear , the ingrowing conjunctiva has left the surface hazy . restoration of touch sensitivity after grafting with rhciii - mpc implants as an indication of nerve function restoration . central corneal touch sensitivity was assessed by contact esthesiometry in the patients corneas before and after implantation , with the nonoperated , contralateral eyes serving as controls . measurements were obtained using a cochet bonnet esthesiometer with a monofilament , where an increase in filament length ( mm ) corresponds to an increase in touch sensitivity ( n = 3 patients ) . in vivo confocal microscopy of the regenerated neo - cornea of patient 1 showed a regenerated epithelium ( figure 4a ) , which resembled that of a healthy cornea ( figure 4b ) . stromal cells were not clearly seen due to corneal haze , but they appear to have started populating the initially cell - free implant ( figure 4c ) but have not reached the steady state seen in a healthy cornea ( figure 4d ) . confocal microscopy through an implanted cornea at 12 months postoperation showing regenerated patient epithelium ( a ) that resembles that of a healthy cornea ( b ) . stromal cells have grown into the initially cell - free graft , although partially obscured by haze ( c ) , unlike the healthy cornea where the cells are clearly visible ( d ) . the preserved patient endothelium ( e ) has a number of larger sized cells but also endothelial cells that resemble those of a healthy cornea ( f ) . in many countries , such as ukraine , china , south east asia , and india , corneal blindness is a huge problem . in addition to the severe shortage in donated corneas,10 many patients suffer from severe pathologies that have a high risk of graft rejection or failure . poor outcomes of corneal transplantation are likely due to graft rejection and stem cell deficiency . graft rejection is directed at allogenic cells from donor human corneas.18 this is circumvented by cell - free rhciii - based corneal implants . the rhciii replaces the largely collagenous stroma of the native cornea allowing for repopulation by endogenous host corneal cells , as we had previously reported in low - risk patients.13 we have previously shown that the rhciii - mpc implants are composed of lamellae - like layers that are interconnected with fiber - like structures that crudely mimics the structure of the human cornea.11 furthermore , the coefficients of glucose and albumin diffusion through these hydrogels were comparable to that of the human cornea.15 additionally , mpc polymer has reported anti - inflammatory properties,19,20 possibly accounting for the capacity of rhciii - mpc implants to quiesce the immunopathologic corneas , allowing stable restoration of the ocular surface . corneal epithelial limbal stem cell transplantation , which is available in large urban centers in the developed world , is too cost prohibitive for the healthcare systems in many countries such as ukraine , as it requires specialized , certified clean rooms and staffing to expand the cultures , plus time for harvesting through cultivation of cells . in addition , where the damage to the cornea extends below the epithelium , the patients require a subsequent donor graft . in this case , a grafted rhciii - mpc implant can allow for healing of the epithelium and stroma , providing relief from pain and discomfort for patients and allowing for a future limbal epithelial graft as the second step in the treatment . in this initial observational study , small grafts of 45 mm were used to replace the lesioned area with a minimal margin of healthy tissue . the rationale for this was patient safety , i.e. , in case of a serious adverse device effect , the implant can be removed and replaced with a larger human donor cornea graft by deep lamellar keratoplasty , the current conventional treatment . following corneal wounding , overexpression of unaligned , mainly type iii collagen occurs to form a scar.21 bridging the wound gap with rhciii - mpc implants that have a regular lamellar structure,11 however , appears to provide a template for more controlled in - growth of stromal cells that in turn provides for an optically clear regenerated cornea . nonetheless , in patients with limbal epithelial stem cell deficiency , transplantation with corneal epithelial stem cells in conjunction with an implant will be needed to prevent conjunctival in - growth that decreases optical clarity . we have shown that under conditions that render standard of care suboptimal , e.g. , ukraine due to its on - going crisis , making the situation approach that in much of the developing world,22 rhciii implants have the potential to overcome the cornea supply shortage in various regions in the world , and avoid social and religious stigma some may have with allograft corneas . furthermore , rhciii - mpc implants may be an alternative to donor corneas and possibly a superior substitute in high - risk cases for restoring corneal integrity . despite of the limitations of this preliminary case study such as the very small sample population and relatively short followup of 912 months , our initial results nevertheless suggest that bioengineered rhciii - mpc implants may potentially be more efficacious alternatives to donor tissues in repairing corneas with severe pathologies . therefore , further clinical evaluation in form of a clinical study with high - risk patients is merited to determine their safety and full potential as alternatives to donor human cornea transplantation .

adipocytes release immune mediators that contribute to diabetes - associated inflammatory processes . as the stress protein heat shock protein 60 ( hsp60 ) induces proinflammatory adipocyte activities , we hypothesized that adipocytes of diabetes - predisposed mice exhibit an increased proinflammatory reactivity to hsp60 . preadipocytes and mature adipocytes from nonobese diabetic ( nod ) , new zealand obese ( nzo ) , and c57bl/6j mice were analyzed for hsp60 binding , hsp60-activated signaling pathways , and hsp60-induced release of the chemokine cxcl-1 ( kc ) , interleukin 6 ( il-6 ) , and macrophage chemoattractant protein-1 ( mcp-1 ) . hsp60 showed specific binding to ( pre-)adipocytes of nod , nzo , and c57bl/6j mice . hsp60 binding involved conserved binding structure(s ) and hsp60 epitopes and was strongest to nzo mouse - derived mature adipocytes . hsp60 exposure induced kc , il-6 , and mcp-1 release from ( pre-)adipocytes of all mouse strains with a pronounced increase of il-6 release from nzo mouse - derived adipocytes . compared to nod and c57bl/6j mouse derived cells , hsp60-induced formation of il-6 , kc , and mcp-1 from nzo mouse - derived ( pre-)adipocytes strongly depended on nfb - activation . increased hsp60 binding and hsp60-induced il-6 release by mature adipocytes of nzo mice suggest that enhanced adipocyte reactivity to the stress signal hsp60 contributes to inflammatory processes underlying diabetes associated with obesity and insulin resistance . corneas with severe pathologies have a high risk of rejection when conventionally grafted with human donor tissues . in this early observational study , we grafted bioengineered corneal implants made from recombinant human collagen and synthetic phosphorylcholine polymer into three patients for whom donor cornea transplantation carried a high risk of transplant failure . these patients suffered from corneal ulcers and recurrent erosions preoperatively . the implants provided relief from pain and discomfort , restored corneal integrity by promoting endogenous regeneration of corneal tissues , and improved vision in two of three patients . such implants could in the future be alternatives to donor corneas for high - risk patients , and therefore , merits further testing in a clinical trial .

transthyretin familial amyloid polyneuropathy ( ttrfap ) is an autosomaldominant , adultonset disorder associated with over 100 different mutations in the transthyretin ( ttr ) gene that cause transthyretin protein to deposit as amyloid in peripheral and autonomic nerves , heart , gastrointestinal ( gi ) tract , kidneys , eyes , and connective tissue of the transversal carpal ligament ( ando et al . , 2013 ; rowczenio et al . , 2014 ; sekijima , 2015 ) . this results in progressive organ dysfunction and death within an average of 10 years ( ando et al . , 2013 ) . ttrfap is a highly heterogeneous disease associated with a wide range of clinical manifestations that may present in varying degrees and combinations ( ando et al . , 2013 ; sekijima , 2015 ) . the disease can be difficult to recognize due to its variable clinical presentation and nonspecific symptoms . the age of onset ranges from the second to ninth decade of life ( ando et al . , 2013 ) , and incomplete penetrance of clinical disease can lead to sporadic cases without known affected relatives . accurate diagnosis of ttrfap is often delayed for years ( plantbordeneuve et al . , 2007 ; koike et al . , 2011 ; an accurate diagnosis is important to permit effective disease management , as tissue damage is largely irreversible , and available treatment options are most beneficial in early disease stages ( coelho et al . , 2013 ; plantbordeneuve , 2014 ; ericzon et al . , 2015 a particular challenge in making a diagnosis is that clinical manifestations are not necessarily uniform among carriers of the same ttr mutation and can vary even within the same family ( ando et al . , 2013 ) . the clinical phenotype is also influenced by genetic , epigenetic , or environmental factors other than the ttr mutation ( see table 1 ) . gi , gastrointestinal ; ttrfap , transthyretin familial amyloid polyneuropathy ; sd , standard deviation . urinary retention might be underdiagnosed as patients are usually not aware or cognisant of symptoms . lengthdependent peripheral sensorymotor neuropathy is a hallmark feature of ttrfap . in classical earlyonset ( < 50 years of age ) val30met ttrfap ( andrade , 1952 ) , distal small myelinated and unmyelinated nerve fibers associated with pain and temperature sensation become damaged first , which may manifest as paresthesia , dysesthesia , allodynia , hyperalgesia , or spontaneous pain in the feet . axonal degeneration then progresses relentlessly in a distal to proximal pattern reaching upper limbs usually 4 to 5 years after first symptoms . within a few years , larger myelinated sensory and motor nerve fibers become affected and impairment of light touch , vibration , and position sensation , and motor deficit appear in the distal lower limbs . lateonset cases are characterized by relative preservation of unmyelinated nerve fibers and conspicuous presence of axonal sprouting ( koike et al . , 2004 ) . these characteristics are responsible for impaired superficial and deep sensation , severe neuropathic pain , early distal motor involvement , and relatively mild autonomic symptoms ( conceio and de carvalho , 2007 ; koike et al . , the lengthdependent pattern of symmetric sensorymotor and autonomic polyneuropathy in ttrfap is not unique , and similar neurologic impairments can be observed in many conditions , all of which can mislead clinical diagnosis . the most common neuropathic misdiagnosis for sporadic ttrfap is chronic inflammatory demyelinating polyneuropathy ( cidp ) ( plantbordeneuve , 2014 ) . for instance , 20% of 90 french patients without a family history of ttrfap ( plantbordeneuve et al . , 2007 ) and 53% of 15 japanese patients with sporadic v30 m ttrfap ( koike et al . , 2011 ) although cidp is generally characterized by a demyelinating sensorymotor neuropathy , once extensive axonal lengthdependent damage is present , electrophysiological characteristics of ttrfap can resemble those of cidp due to secondary demyelination . furthermore , protein levels in cerebrospinal fluid can be elevated in patients with ttrfap , albeit less markedly than in cidp . in some cases , given the frequent misdiagnosis , there should be a high suspicion index for ttrfap in patients diagnosed with cidp that do not respond to immunomodulatory treatment . there have been numerous cases where ttrrelated amyloidosis was initially misdiagnosed as amyloid lightchain ( al ) amyloidosis ( cowan et al . , 2011 ; briani et al . , misdiagnosis can be due to occurrence of monoclonal gammopathy in elderly patients or false immunolabeling of amyloid deposits , leading to inappropriate , potentially harmful chemotherapeutic treatment . ttrfap is unlikely to be mistaken for other forms of hereditary amyloid neuropathy caused by mutation of the apolipoprotein ai , gelsolin , or 2microglobulin genes . however , although very rare and less rapidly progressive , hereditary neuropathy due to truncation mutations of the prion protein can closely mimic ttrfap ( mead and reilly , 2015 ) and should be considered in the differential diagnosis . consequently , it is important to obtain a complete clinical history with details of symptoms of systemic disease and a complete and detailed family history ( ando et al . , 2013 ; plantbordeneuve , 2014 ) . the occurrence of progressive peripheral sensorymotor polyneuropathy and at least one of the following is suggestive of ttrfap : family history of neuropathy , early autonomic dysfunction , cardiac involvement , diarrhea , constipation , alternating episodes of constipation and diarrhea , inexplicable weight loss , carpal tunnel syndrome , renal impairment , or vitreous opacity ( fig . symptom clusters that may warn of a diagnosis of transthyretin familial amyloid polyneuropathy ( ttrfap ) . of these combinations , peripheral neuropathy with early autonomic signs , such as erectile dysfunction or gi symptoms , and peripheral neuropathy with cardiac manifestations are the most common and important combinations in sporadic patients . rapid disease progression ( dohrn et al . , 2013 ) and failure to respond to immunomodulatory treatment are additional signs . the clusters of clinical symptoms mentioned above should raise suspicion of ttrfap , particularly if there is a positive family history . in patients with suspected ttrfap , the entire coding region of the ttr gene , that is all four exons , should be sequenced . tissue biopsy analysis and neurologic , cardiac , autonomic , and ophthalmologic evaluation can lend further credence to the correct diagnosis . once a single word is presented , its orthographically similar words are also partially activated . coltheart first introduced the concept of orthographic neighborhood of a target word , defined as all words of the same length that can be generated by changing just one letter while preserving letter positions . for example , cheap , chest , cleat , and wheat are all neighbors of cheat . grainger and his colleagues pointed out that the printed frequency of a word 's orthographic neighbors played an important role in identification process of this target word , which is termed as neighborhood frequency effect . the authors indicated that if the frequency of a target word was not the highest among its neighbors , those higher - frequency neighbors ( hfns ) would compete with the target word and , consequently , slow down its processing . this inhibition was reported in several studies of lexical decision [ 28 ] . in naming tasks , no effect or a facilitatory trend of neighborhood frequency grainger gave an explanation to the absence of neighborhood interference based on the analogy theory of word naming [ 10 , 11 ] . the pronunciations in alphabetic orthographic neighborhoods were of high consistency , a word usually sounded similar with its orthographic neighbors , and then the neighbors with higher frequency would provide support for the component phonology of the target word . however , there is a close relationship between visual forms and pronunciations in alphabetic words , thus it is still needed to determine whether the hfns affect the target naming during phonological processing or visual / orthographic analysis . as opposed to alphabetic languages , chinese with a logographic writing system lacks the grapheme - to - phoneme correspondence ( gpc ) rules , in which visual words map onto speech sounds through an addressed way [ 12 , 13 ] . considering different language characteristics , the definition of orthographic neighbors for alphabetic words is inapplicable for chinese characters . however , about 85% of chinese characters are semantic - phonetic compounds [ 14 , 15 ] with the phonetic and semantic radicals informing to some extent about the character 's pronunciation and meaning , respectively . by the medium of phonetic or semantic radicals , orthographic neighborhood of chinese characters can be formed . for instance , characters ( pronounced / ji1/ , meaning garbage ; the number here refers to chinese tone ; the same below ) , ( /ji2/ , draw ) , ( /ji2/ , unmitigated ) , ( /ji2/ , book ) , ( /ji2/ , danger ) , ( /ji2/ , class ) , ( /xi1/ , absorb ) , and ( /sa3/ , shoes ) have the same phonetic radical ( ji2 , and ) and are considered as a phonetic - radical orthographic neighborhood . moreover , because phonological consistency is much lower in chinese orthographic neighborhoods [ 16 , 17 ] , the higher - frequency neighbors were expected to inhibit the target naming according to analogy theory of word naming , and this has been proved . li et al . examined the neighborhood frequency effect in chinese character naming , in which neighbors with the highest frequency sounded differently from target characters in the with - hfn condition . results showed a significantly interference effect of neighborhood frequency , and participants spent longer time and made more errors in with - hfn condition than in without - hfn condition . and their follow - up fmri study found that naming with - hfn characters induced greater activations than naming without - hfn characters in bilateral inferior frontal gyrus ( bi - ifg ) which was related with the phonological competition and inhibition of extraneous phonological activation . the above findings revealed that higher - frequency neighbors exerted an inhibitory influence on chinese - character naming , and this inhibition might be linked to the phonological processing . but whether the hfn effect was special to the phonological level was still unclear because of the low temporal resolution of brain imaging , and the event - related potential ( erp ) technique is undoubtedly a valuable way to dissociate the phonological processing from other stages ( e.g. , visual stage ) . the bilaterally posterior n1 is usually considered as a visual - orthographic component , with amplitude peak at around 130 ms to 170 ms [ 2026 ] . the p2 component occurs around 200 ms after stimulus at centrofrontal sites and indexes lexical phonology in chinese reading [ 2729 ] . a previous study of neighborhood frequency effect using lexical decision found that english words with hfns reliably induced larger amplitude than that without hfns in the 180240 ms time window , while there was no difference between the two conditions at earlier stages , revealing the possible phonological influence of hfns . however , to our knowledge , there were no erp researches on neighborhood frequency effect in naming tasks . so , the present study intended to use erps to examine the mechanism of neighborhood frequency effect in chinese - character naming . according to the analogy theory of word naming , it could be expected that the higher - frequency neighbors would only affect the target processing at phonological stage , exhibiting an effect in p2 component but not n1 component . all the participants were right - handed native mandarin speakers with normal or corrected - to - normal vision . there were 48 characters as targets , 24 for with - hfn and without - hfn conditions , respectively ( see table 1 ) . for the without - hfn characters , a target character had the highest frequency within its neighborhood . for the with - hfn characters , at least one of its neighbors was of 3 per million higher frequency referred to as a previous study ; meanwhile , the target and its neighbor(s ) with the highest frequency sounded differently from each other . for example , ( pronounced /dan4/ , meaning birth , character frequency being 7.09 occurrences per million ; the same below ) , ( /xian2/ , saliva , 0.74 ) , ( /yan2/ , wriggly , 0.85 ) , and ( /yan2/ , feast , 0.65 ) form an orthographic neighborhood . the target character is , and its orthographic neighbors all have much lower character frequency , so this target is considered as a without - hfn character . ( /di2/ , barbarians , 5.41 ) , ( /qiu1/ , autumn , 24.03 ) , ( /huo3/ , partner , 28.72 ) , and ( /huo3/ , holmium , 0.02 ) form another neighborhood , and the target character is . the neighbor has the highest frequency in this neighborhood , and the difference in frequency between ( 5.41 ) and ( 24.03 ) is greater than 3 per million ; meanwhile the two characters sound different from each other , and thus the target is regarded as a with - hfn character . the mean number of hfns in one neighborhood was 2 ( range , 1 to 6 ; standard deviation = 1.47 ) . the irregular character indicates the character whose pronunciation is different from that of its phonetic radical ; for example , ( sa3 , shoes ) is an irregular character , because of its phonetic radical ( ji2 , and ) with a different pronunciation . the consistency value ( c ) can be calculated from the relative ratio of the number of orthographic neighbors with the same pronunciation ( n ) to the whole neighborhood size ( n ) , c = n / n . for example in the phonetic - radical neighborhood of ( ji2 , and ) , its neighborhood size is eight , and there are six neighbors with the same pronunciation / ji/ , ( /ji1/ , garbage ) , ( /ji2/ , draw ) , ( /ji2/ , unmitigated ) , ( /ji2/ , book ) , ( /ji2/ , danger ) , and ( /ji2/ , class ) , producing a consistency level of /ji / of 0.75 ( c = 6/8 ) . in the present study , the consistency of each target character was below 0.4 . the target characters were all left - right structure with the phonetic radical on the right side . moreover , the number of neighbors , character frequency , stroke , and consistency were balanced between conditions ( table 2 ) . character frequency information was collected from the chinese text computing database ( http://lingua.mtsu.edu/chinese-computing/ ) , and the neighborhood was defined on the basis of li and kang 's statistics . participants sat in a sound - attenuated room , at a viewing distance of 50 cm from the screen , with a visual angle of 5.7 5.7. they were instructed to sit comfortably and concentrate on the stimulus avoiding head movements or any other unnecessary movements . figure 1 displays the presentation format within each trial . a trial consisted of a 500 ms cross - fixation , a 500 ms blank screen , and a 500 ms target character . the participants were asked to silently name the target . to ensure that the phonology of the target character was accessed , the covert naming was followed with a homophonic decision , in which participants should make a decision on whether the target and the following probe were homophonic and click mouse buttons with their thumbs , with the left button for different pronunciation and the right button for homophony . for the compound - character probe , its phonetic radical was always different from that of the paired target . half of the probes sounded the same with the targets , and the other half sounded different . the list of the target characters was presented twice so that the number of trials in each condition rose to 48 . one target was paired up with different probes between the two presentations to reduce practice effects . the stimuli were programmed with the stim software and randomly shown on a lenovo monitor . electroencephalographic ( eeg ) activity was recorded from a 64-channel neuroscan version 4.3 system with a common vertex reference and rereferenced to the average of the left and right mastoids in the offline analysis . vertical eye movements ( veog ) were recorded by a pair of electrodes placed on the supraorbit and infraorbit of the left eye , and horizontal eye movements ( heog ) were recorded by a pair of electrodes placed beside the outer canthus of both eyes . eeg signals were recorded and digitized at a bandpass filter of 0.05100 hz , with amplifying at a sample rate of 500 hz . resistances across all the electrodes were kept below 5 k. the acquired data were corrected for eye movements and blinks and segmented to epochs of 800 ms posttarget interval and 100 ms pretarget baseline . epochs containing incorrect behavioral responses or with peak - to - peak differences larger than 100 v were rejected , and this led to a rejection rate of 8% over all trials , without statistical difference in the number of rejections between conditions ( p > 0.1 ) . the remaining epochs were filtered with a low - pass filter of 30 hz ( zero - phase shift mode , 12 db ) and averaged for each condition and participant . figure 2 displays the erp grand averages time locked to the onset of target characters for neighborhood frequency comparison . as can be seen from the figure , the erps show a negative polarity peaked at around 150 ms over posterior regions and a positive polarity peaked at about 170 ms over frontocentral regions . the 40 ms time windows were selected centering the n1 and p2 peaks ( n1 : 130170 ms ; p2 : 150190 ms ) . according to topographic mapping in figure 2 , the n1 amplitude was output at channels of po5-po6 , po7-po8 , o1-o2 , and the p2 amplitude was recorded at channels of frontal ( f1-f2 , f3-f4 ) , frontocentral ( fc1-fc2 , fc3-fc4 ) , central ( c1-c2 , c3-c4 ) , and centroparietal ( cp1-cp2 , cp3-cp4 ) lobes . mean erp amplitudes from corresponding time windows were computed for each participant in both conditions . the n1 amplitude was submitted to a repeated - measure anova with neighborhood frequency ( nf , with / without hfns ) and laterality ( left / right hemisphere ) . the p2 amplitude was submitted to a repeated - measure anova , with three within - subject variables : neighborhood frequency ( with-/without hfns ) , laterality ( left / right hemispheres ) , and lobe ( frontal / frontocentral / central / centroparietal lobes ) . the anova on n1 amplitude showed no significant main effect or interaction ( all ps > 0.1 ) . the anova on p2 amplitude showed a significant main effect of neighborhood frequency ( f(1 , 12 ) = 12.88 , p < 0.01 ) , and erp response to target characters was larger in without - hfn condition than in with - hfn condition ; a significant main effect was also observed for lobe ( f(1 , 12 ) = 8.73 , p < 0.01 ) , and post hoc analysis showed the smallest amplitude in the centroparietal lobe but no differences across other three lobes ; no significant effect was found for laterality ( f(1 , 12 ) < 1 , p = 0.68 ) or any interactions ( all ps > 0.1 ) . in the present study , there was no effect of neighborhood frequency on bilateral posterior n1 amplitude ; as to the p2 amplitude , naming chinese characters without hfns induced more positive erps than that with hfns . the current results uncovered the time course of early influence of orthographic neighbors with higher frequency in chinese - character naming . the absence of neighborhood frequency effect in n1 amplitude revealed that higher - frequency neighbors did not affect visual / orthographic analysis of the target characters , since n1 is usually associated with the visual form processing of written words / characters [ 22 , 26 ] . the finding was consistent with a previous study using english words , in which there was not hfn effect in the early time window before 180 ms . moreover , it was reported that the n1 originates predominately from the occipitotemporal regions ; thus the present result was identical with the nonsignificant hfn effect in the occipital areas reported by li et al . . the current amplitude difference between with- and without - hfn conditions in the p2 time - window indicated that higher - frequency neighbors exerted an influence on phonological processing of chinese characters . debruille also reported an hfn effect in english words with the erps response in the posttarget interval ranging from 180 ms to 240 ms . these findings suggested the universal role of hfns in phonological retrieval of target words / characters across language systems . however , the current hfn effect was greater p2 amplitude in without - hfn condition than that in with - hfn condition , while the neighborhood frequency effect in debruille was opposite . the difference might be ascribed to the language characteristics in phonological consistency . considering the high phonological consistency in alphabetic writing systems , higher - frequency neighbors would provide support for the target phonology , which might correspond with increased brain activity of larger erp amplitude . whereas the phonological consistency was low in chinese orthographic neighborhoods , different pronunciations of the hfns would be more easily activated because of the lower threshold of activation for these neighbors and compete with the phonological activation of the target characters , resulting in the interference on phonological processing of chinese characters , which may inhibit the relevant brain activity of p2 component . additionally , the distribution of p2 effect was mainly located in the frontal and centroparietal areas . neuroimaging studies reported that the left middle frontal gyrus was responsible for the addressed phonology , and the dorsal aspect of the left inferior parietal lobule was thought to be the region specific for phonological storage in chinese ; meanwhile , the right frontal regions were linked to phonological competition and inhibition [ 12 , 19 , 35 , 36 ] . these results indicated that the higher - frequency neighbors might exhibit a disruptive effect in the lexical route of chinese phonological access . the present results were consistent with our prediction based on the analogy theory of word naming , revealing that orthographic neighbors with higher frequency might impact on target naming at the phonological level . proposed that the orthographic neighborhood effect in chinese - character naming may be generated in two phases : orthographic facilitation from visually similar neighbors and phonological inhibition of higher - frequency neighbors . the current findings provided evidence for the phonological inhibition , suggesting negative influence of higher - frequency neighbors at the phonological retrieval in chinese - character naming . the present study used erps to separately examine the effect of higher - frequency neighbors at the early orthographic and phonological stages . results showed amplitude difference between with- and without - hfn conditions in p2 component but not in n1 component , suggesting a special role for the hfns in addressed phonology of chinese characters . the reduced amplitude of p2 for with - hfn characters might reveal the phonological inhibition from the higher - frequency neighbors due to their different phonological representations from the target characters .

abstracttransthyretin familial amyloid polyneuropathy ( ttrfap ) is a rare , progressive , lifethreatening , hereditary disorder caused by mutations in the transthyretin gene and characterized by extracellular deposition of transthyretinderived amyloid fibrils in peripheral and autonomic nerves , heart , and other organs . ttrfap is frequently diagnosed late because the disease is difficult to recognize due to phenotypic heterogeneity . based on published literature and expert opinion , symptom clusters suggesting ttrfap are reviewed , and practical guidance to facilitate earlier diagnosis is provided . ttrfap should be suspected if progressive peripheral sensorymotor neuropathy is observed in combination with one or more of the following : family history of a neuropathy , autonomic dysfunction , cardiac hypertrophy , gastrointestinal problems , inexplicable weight loss , carpal tunnel syndrome , renal impairment , or ocular involvement . if ttrfap is suspected , transthyretin genotyping , confirmation of amyloid in tissue biopsy , large and smallfiber assessment by nerve conduction studies and autonomic system evaluations , and cardiac testing should be performed . the present study explored the time course of neighborhood frequency effect at the early processing stages , examining whether orthographic neighbors with higher frequency exerted an influence on target processing especially at the phonological stage by using the event - related potential ( erp ) . thirteen undergraduate students were recruited in this study , and they were required to covertly name chinese characters with or without higher - frequency neighbors ( hfns ) ; meanwhile , their brain activity was recorded . results showed that the effect of neighborhood frequency was significant in frontocentral p2 amplitude , with a reduction for naming characters with hfns compared to those without hfns ; while there was no effect in posterior n1 amplitude . the only neighborhood frequency effect in p2 component suggested a special role for the hfns in phonological access of chinese characters . the decrease in amplitude for naming with - hfn characters might be associated with the phonological interference of higher - frequency neighbors due to their different pronunciations from the target characters .

a cataract increases lens opacity and reduces visual acuity ( va ) , thus impairing the patient 's quality of life . various techniques for cataract surgery have been developed since h. ridley introduced intraocular lenses ( iols ) composed of polymethylmethacrylate in 1949 . the stability of iols allows cataract surgery to be commonly performed worldwide , and technological advancements such as multifocal and toric iols have increased the procedure 's popularity [ 6 , 7 ] . to improve quality of life , cataract surgery postoperative va , contrast sensitivity , and optical aberrations were measured as objective indices of surgical success [ 1116 ] . after cataract surgery , improvement in va is typically tested with a high - contrast ( 100% ) chart under photopic and mesopic conditions . for example , visual inspection of human faces of the utmost importance in daily life involves a target of large size and low contrast . the change in va after cataract surgery has not been studied under various levels of contrast . the aim of this study was to assess the visual performance of pseudophakic eyes after cataract surgery at different contrast levels by using the oqas ( optical quality analysis system , visiometrics , terrassa , spain ) . this research was approved by the institutional review board of the catholic medical center at the catholic university of korea and conducted in accordance with ethical research guidelines . the present study adhered to the declaration of helsinki and was approved by the institutional review board of the catholic medical center at the catholic university of korea ( approval number : kc12risi0023 ) . patients completed an informed consent form approved by the institutional review board after the purpose of the study was explained to them . no patients had a history of ocular surgery , ocular disease , or general disorders affecting vision ( e.g. , diabetic retinopathy ) . vision of patients greater than 0.1 logmar was measured using the snellen test and classified as age - matched ( 50s , 60s , and 70s ) between normal and pseudophakic eyes . the patients with pseudophakic eyes had undergone phacoemulsification and received one of five iol types in the posterior chamber 26 months before the study . patients were stratified into the following age groups : 5059 years , 6069 years , and 7079 years . patients with failed iol implantation into the lens capsule , severe posterior lens capsule opacification or history of laser capsulotomy due to opacification , or any other eye complication were excluded . those with poor cooperation were also excluded . to prevent uncorrected refractive error from limiting contrast sensitivity or va [ 20 , 21 ] , va was measured using a snellen chart at 6 m. altered contrast sensitivity was simulated with the oqas by using the double - pass technique . with this approach , the retinal image , degree of haze inside the eye , and condition of visual function are analyzed in terms of objective scattering index ( osi ) , modulation transfer function ( mtf ) cut - off value , and strehl ratio , respectively [ 2225 ] . simulated va by oqas was evaluated at contrast of 100% , 20% , and 9% . testing at each contrast level was performed for all five types of iols : hoya pc-60ad ( hoya , corp , tokyo , japan ) , ec-1pal ( aaren scientific , ontario , canada ) , akreos mi-60 ( bausch & lomb , rochester , ny , usa ) , ny-60 ( hoya , corp , tokyo , japan ) , and xl stabi zo ( carl zeiss meditec , jena , germany ) . analysis of variance ( anova ) and independent - sample t - tests were performed using spss version 18.0 software ( ibm corporation , armonk , ny ) . the subjects ' characteristics are shown in table 1 . mean visual acuities for the normal eyes were 0.16 0.18 logmar , 0.30 0.18 logmar , and 0.52 0.17 logmar at 100% , 20% , and 9% contrast , respectively ( table 2 ) . in normal eyes from all age groups , simulated va decreased significantly when contrast was reduced ( 5059 , p = 0.000 ; 6069 , p = 0.000 ; 7079 , p = 0.020 ) . however , simulated va was highest among those aged 5059 and lowest among those aged 7079 . simulated va at 100% and 9% contrast decreased with increasing age ( 100% , p = 0.045 ; 9% , p = 0.010 ) . no significant differences were noted among the age groups at 20% contrast ( p = 0.070 ) . for pseudophakic eyes , mean visual acuities were 0.16 0.12 logmar , 0.33 0.20 logmar , and 0.56 0.21 logmar at 100% , 20% , and 9% contrast , respectively ( table 3 ) . as in the normal eyes , va decreased significantly when contrast was reduced ( p = 0.000 ) . however , nova was similar in all age groups ( p > 0.05 ) . figure 1 shows simulated va for the normal and pseudophakic eyes in each age group at all contrast levels . however , among those aged 7079 , the pseudophakic eyes exhibited significantly higher va ( p = 0.000 ) . table 4 shows the simulated mean visual acuities of the pseudophakic eyes according to iol type . subjects implanted with ec-1pal and ny-60 iols showed the lowest and the highest va , respectively , although no significant differences were noted among iols at any contrast level . regardless of iol type , va decreased significantly when contrast was reduced ( p = 0.000 ) . osi value , mtf cut - off value , and strehl ratio values are presented in table 5 . the mean values were 2.21 1.38 , 23.29 10.72 , and 0.18 0.47 , respectively , in the pseudophakic group and 1.99 1.41 , 23.41 9.72 , and 0.13 0.07 , respectively , in the normal group . the only significant finding was elevated osi values in normal eyes from the 7079 age group ( p = 0.008 ) . figure 2 shows representative oqas results obtained from a 69-year - old patient with normal eyes and a 72-year - old patient with pseudophakic eyes . in addition to questionnaires , va and contrast sensitivity assessments are used to evaluate ocular health after cataract surgery . recent technological advancements have allowed for the measurement of higher - order aberrations including spherical aberration and coma aberration as well as the optical analysis of light spread within the eye ( point spread function ) [ 2729 ] . however , such tests are useful only in characterizing symptoms . . used the oqas to measure the osi , mtf cut - off value , and strehl ratio after cataract and refractive surgeries and to assess visual function objectively . , we investigated vision at three contrast levels simulated using the oqas and stratified subjects with normal and pseudophakic eyes by age . among patients in their 50s and 60s , normal eyes exhibited sim va superior or similar to that of pseudophakic eyes however , among those in their 70s , pseudophakic eyes had significantly superior va ( p = 0.000 ) . alternatively , the normal eyes in this age group may have been free of any lens opacity that could be detected by slit - lamp examination but may have had fine opacities in the lens and/or vitreous chamber that scattered light , thereby reducing va . another significant finding was the lower osi value of the pseudophakic eyes in this group ( p = 0.008 ) , which is in line with the report by saad et al . that osi values increase with age in normal eyes . in both the normal and pseudophakic groups , va was directly related to contrast level regardless of age . furthermore , va decreased as age increased ; as reported by mathai et al . sim va was similar in normal and pseudophakic eyes of subjects aged 5069 but superior in the pseudophakic eyes of subjects aged 7079 years . therefore , visual function must have returned to normal levels in all patients with iols at 26 months after cataract surgery . the ny-60 and ec-1pal iols were associated with the highest and lowest visual acuities , respectively , regardless of contrast level . interestingly , use of the ny-60 iol is reportedly associated with fewer capsular folds than use of a three - piece iol . ny-60 iol users also report the absence of entoptic phenomena 1 year after surgery . in an eye with an iol , the presence of entoptic phenomena correlates directly with the osi value . this relationship may explain the superior va of the ny-60 group , although this improvement in va was associated with use of the acrysof sn60wf and tecnis zcb00 iols in previous reports . in this study , reduced contrast was associated with decreased acuity across groups , but no significant difference in simulated va among iol types was noted at any contrast level ( p = 0.413 ) . a possible limitation of our study is that the oqas 's he - ne diode laser may have introduced error to the osi readings , for example , in patients with dry eye symptoms or cloudy vitreous , which scatters light . in summary , va at 26 months after cataract surgery in pseudophakic eyes is similar to that of normal eyes and older patients with pseudophakic eyes have superior simulated va to age - matched controls , contrary to previous reports that the level of contrast and age affect visual function . these findings suggest that iols ensure simulated va similar to that of the normal eye even in very low - contrast conditions , as encountered when driving at night . therefore , iol implantation should have a beneficial impact on the patient 's quality of life . the oqas seems to be a useful instrument for the objective evaluation of visual quality under contrast after various surgeries such as cataract surgery , laser - assisted in situ keratomileusis ( lasik ) , laser - assisted subepithelial keratectomy ( lasek ) , and keratoconus surgery . the process involves the removal of pulp cavity content and the consequent reduction of microorganism counts , especially in non - vital teeth with periapical lesion . with proper instrumentation , the cavity is enlarged and smoothened to fittingly receive the filling . as tooth and canal anatomies are equally important during canal preparation , the appropriate use of the endodontic instruments and the close contact with canal walls for pulp extirpation and cleaning purposes is likewise crucial to the treatment . several techniques and instruments have been suggested to achieve the desired efficiency in root canal preparation2,6,11,15,19,23 . the concomitant use of chemicals that promote the removal of microorganisms is widely accepted . as regards the cleaning and disinfection of root canals , current literature sets forward conflicting results for manual , mechanical and ultrasonic instrumentation techniques1,3,810,12,13,16,23 . therefore , considering that in the treatment of non - vital teeth with chronic periapical reaction the presence of bacteria inside the root canal system accounts for the development and persistence of periapical lesions , this study was designed to investigate quantitatively the in vitro ability to remove dye adhered to proximally flattened root canals . three techniques were investigated : crown - down technique with rotary instrumentation using nickel - titanium files ( k3 and protaper systems ) ; ultrasonic crown - down technique ; and progressive manual technique without apical pressure - modified oregon technique . a set of 40 permanent human teeth freshly extracted from patients at the university of marlia for periodontal or prosthetic purposes and stored in 0.01% thymol solution was used in this study . teeth were mandibular left and right central incisors and lateral incisors , and mandibular right and left premolars , evenly distributed the groups . all teeth presented proximally flattened root canals . , so paulo , sp , brazil ) using an insulin syringe ( becton dickinson , curitiba , pr , brazil ) , upon the foramen . a fine kerr file was then repetitively pumped in the canal to prevent the formation of bubbles , whereupon teeth were left to rest at room temperature for 48 h to ensure complete curing of red dye . group i was treated with the crown - down technique using the k3 system ( kerr , sybron endo , california , usa ) ( table 1 ) and group ii with the crown - down technique using the protaper system ( dentsply maillefer , ballaigues , switzerland ) ( table 2 ) ; group iii was treated with the progressive manual technique without apical pressure ( modified oregon technique ) ; and group iv was treated with the ultrasonic crown - down technique . the root canal walls of groups i and ii were instrumented using an electric endodontic engine ( endo plus k ; vk driller equipamentos eltricos , so paulo , sp , brazil ) . the device was adjusted to a constant clockwise 250-rpm speed and to a # 3 torque . group iii was instrumented using the modified oregon technique as defined by berbert , et al.2 ( 1996 ) . the process started using a manual size 40 k - file adapted to a ultrasound device ( enac ; osada electric co , japan ) , energized for 1 min . from this point on , smaller files were progressively used to ensure that the desired canal working length was reached . for all groups , canals were flushed with 1.8 ml 2.5% sodium hypochlorite ( chemical institute of marilia , unimar , marilia , sp , brazil ) at each change of file . the canals were then dried using paper points ( tanari industrial ltda , manaus , am , brazil ) and the teeth were decoronated length - wise in a buccolingual direction using a diamond disk . the efficiency of each instrumentation technique under study was measured by assessing quantitatively the amount of dye that remained on the root canal walls . halves containing the root canal dentin walls were digitally captured using a desk scanner ( genius colorpage - vivid pro ii ) coupled to a pentium iv computer equipped with the mgi photosuit software . once resolution , brightness and contrast features were standardized , images were accessed using the sigma scan software ( jandel scientific corporation , san rafael , ca ) . total canal area was obtained ( mm ) and the uninstrumented canal areas that remained stained with the dye were determined . data were entered into a windows excell 2000 spreadsheet , affording the quantification of dye that remained after instrumentation by the percentile difference between total root canal area and the dye - stained uninstrumented canal area . as data met the requirements of normal distribution and variance homogeneity , one - way anova was used to compare statistically the four experimental groups at 5% significance level . the quantitative analysis of the data obtained for the total root canal area and the dye - stained uninstrumented canal areas , as well as the percentile difference between them are shown on figure 1 . table 3 shows the results of the f distribution for the percentile difference between the total root canal area and the dye - stained uninstrumented canal area . no statistically significant difference were found between the groups ( p = 0.101 and f = 2.23 ) . root canal instrumentation is not a simple procedure and demands manual skills as well as deep knowledge of the internal root canal system . the preparation of curved root canals and root canals with isthmuses is more complex because the instrument suffers the tensions and deformations imposed by the procedure , which can ultimately influence the outcomes of the root canal treatment . the experimental model adopted in the present study , using natural human teeth , was chosen because their root canal anatomy is remarkably flattened and irregular . this aspect of human root canals may pose additional difficulties to instrumentation and challenge the treatment outcomes because complete removal of remaining tissues from isthmus areas is sometimes hard to achieve9,13,23 . different methods to evaluate instrumentation techniques and their results have been reviewed in the literature . among the methodologies adopted to investigate root canal preparation methods are scanning electronic microscopy3,8,10,23 , simulated polyester resin root canals22 , computer software5 and clearing17 . gonalves jr9 ( 2003 ) and silva , et al.19 ( 2004 ) used root canal dye techniques to compare instrumentation protocols with respect to efficiency and instrumented area . in the present study it is important to mention that the dye used was proved to be insoluble in water , which makes each instrumentation technique the sole dye removal agent in the study . image digitization of the halves and dye - stained areas on the root canal walls allowed the in - depth analysis of experimental results . although these are twodimensional images , the fact that the specimens present well - defined mesiodistal flattened root canals , with minimal depth9 validates the use of the two - dimensional image approach . table 3 and figure 1 show how the extension of the dye - stained root canal area increases from group iv ( ultrasonic ) to group ii ( protaper ) , iii ( oregon ) and i ( k3 ) , in this sequence . these results allowed observing that none of the adopted techniques was efficient enough to completely remove the dye adhered to the canal walls . similar results have been reported by barbizam , et al.1 ( 2002 ) and zmener , et al.23 ( 2005 ) . table 3 shows that , although no statistically significant difference was observed between the groups , the instruments that reached the root canal walls and isthmuses more effectively produced better results in canal preparation . nevertheless , given that rotary instrumentation systems tend to operate more centrally inside the root canal7,9 and considering that the root canals prepared in the present study were flattened , the performance of the protaper system ( group ii , table 3 ) is worth mentioning . the protaper system produced significantly better instrumentation than the k3 system ( group i , table 3 ) . this is contrary to what is ordinarily expected of oregon and ultrasonic techniques . k3 and protaper techniques have differently tapered rotary instruments to operate on the cervical third and produced considerably different results in the present study . while the k3 system exhibited the worst result among the four groups ( mean= 39.76 ) , the protaper system produced a good instrumentation ( mean = 23.31 ) ( table 3 ) . this is probably due to the gradually tapered instrument design of protaper files , as tapering ranges from 0.02 to 0.12 mm from its end d1 to base d2 . , the active instruments ( protaper ) appeared to remove the smear layer more effectively than instruments with radial lands ( k3 ) . the use of the ultrasonic technique ( group iv , table 3 ) produced a satisfactory result for removal of the dye - stained dentin . this is due to the adoption of the crown - down instrumentation , which started with greater file sizes . it is worth mentioning that thicker instruments tend to be more efficient , removing larger amounts of dentin within shorter times . these findings agree with those of esberard , et al.6 ( 1987 ) , who suggested the need for a closer contact between the instrument and canal walls for optimal dentin removal . in the present study , this procedure was adopted to allow a better comparison between techniques , as these techniques do not intrinsically use irrigation . the results also showed that the ultrasonic technique without constant irrigation proved to be the most efficient technique . the enlargement of the cervical third before instrumentation of the apical region had a positive effect on the performance of the techniques because the influence of dentin thickness is lessened , thus favoring the full - length direct action of instruments along the canal walls . furthermore , shovelton18 ( 1964 ) demonstrated that in addition to dentin contamination , large bacterial counts are found along this portion of the canal . in the present study , the cervical region of all teeth was prepared , by either rotary instruments with accentuate taper or gates - glidden drills ( for the modified oregon technique ) , or even files with larger sizes , as those used for the ultrasonic technique . the use of gates - glidden drills has a beneficial effect on root canal preparation and plays an important role in the cleaning of the cervical third of the canal . the results of the present contradict those of previous investigations in which the modified oregon technique ( group iii ) was shown to produce better results than ultrasonic , canal finder and profile rotary techniques9,16 , when the canal were preflared with gates - glidden drills . in the present study , the progression of instrumentation without apical pressure had little contribution on the cleaning of polar and isthmus zones . progressive instrumentation works smoothly and harmoniously for the benefit of root canal preparation as a whole . root canal enlargement allows introducing the files further into the canal , in contact with the dentin walls . yet , it is not possible to direct the action of the instruments , differently from what is observed for the filing movement . based on the number of remaining dye - stained areas on the canal walls , the outcomes of this study demonstrated that none of the techniques produced an appropriate canal preparation . portions of the canal walls that were untouched by the instruments , such as isthmuses , were often found in all groups . this is imputable to both the inefficiency of the technique and the anatomic abnormalities , even in a technologic scenario that affords a variety of rotary instruments of diverse characteristics10 . in a comparative investigation that assessed manual instrumentation , sonic and ultrasonic techniques , langeland , et al.12 ( 1985 ) affirmed that canal anatomy plays a more important role in endodontic treatment than any instrumentation technique . the authors reported that although the techniques cleaned straight , tubular canals , they failed to reach the same efficiency for curved , irregular canals . therefore , it is important to take into account that all instrumentation techniques demand more attention in the treatment of teeth with pulp necrosis and periapical lesion , in which contamination is wide - ranging and intense , with consequent demand for intracanal dressings and sealing . in the light of the results obtained in the present study , it was observed that : ( i ) no instrumentation technique fully removed all dye - stained areas from the canal walls ; ( ii ) the quantitative assessment of dye removal from the canal walls did not show statistically significant difference between the studied groups ( p<0.05 ) ; ( ii ) the ideal instrumentation technique for curved and irregular root canals has not yet been made available .

purpose . to assess visual function using optical quality analysis system ( oqas ) at varying levels of contrast in pseudophakic eyes . methods . the study included patients admitted to seoul st . mary 's hospital between january and february 2012 : 143 pseudophakic eyes with one of five intraocular lens types , examined 26 months after cataract surgery , and 93 normal eyes ( enhanced visual acuity ( va ) < 0.1 logmar ) in age - matched controls . subjects were examined at three contrast levels using the oqas . results . at 100% , 20% , and 9% contrast , simulated mean va was 0.16 0.18 logmar , 0.30 0.18 logmar , and 0.52 0.17 logmar , in normal eyes , and 0.16 0.12 logmar , 0.33 0.20 logmar , and 0.56 0.21 logmar , respectively , in pseudophakic eyes . simulated va decreased significantly when contrast was reduced , regardless of ocular status , age group , and lens type ( p < 0.05 ) . there were no significant differences between normal and pseudophakic eyes among subjects aged 5069 ( p > 0.05 ) . among subjects aged 7079 , pseudophakic eyes showed improved simulated va ( p = 0.000 ) and objective scattering index values ( p = 0.008 ) . conclusions . patients with intraocular lenses have similar or superior visual function when compared to those with normal eyes at 26 months after cataract surgery , even under low - contrast conditions . objective : the efficiency of rotary , manual and ultrasonic root canal instrumentation techniques was investigated in proximally flattened root canals.material and methods : forty human mandibular left and right central incisors , lateral incisors and premolars were used . the pulp tissue was removed and the root canals were filled with red die . teeth were instrumented using three techniques : ( i ) k3 and protaper rotary systems ; ( ii ) ultrasonic crown - down technique ; and ( iii ) progressive manual technique . roots were bisected longitudinally in a buccolingual direction . the instrumented canal walls were digitally captured and the images obtained were analyzed using the sigma scan software . canal walls were evaluated for total canal wall area versus noninstrumented area on which dye remained.results:no statistically significant difference was found between the instrumentation techniques studied ( p<0.05).conclusion : the findings of this study showed that no instrumentation technique was 100% efficient to remove the dye .